Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

NASA Technical Reports Server (NTRS)

Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

1997-01-01

A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice1[OPEN

PubMed Central

Cermak, Tomas; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Voytas, Daniel F.

2016-01-01

We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

Spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus.

PubMed

Mandl, C W; Holzmann, H; Meixner, T; Rauscher, S; Stadler, P F; Allison, S L; Heinz, F X

1998-03-01

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3' NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3' NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3' NCR and never extended into the conserved 3'-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3'-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.

The Flagellar Hook Protein, FlgE, of Salmonella enterica Serovar Typhimurium Is Posttranscriptionally Regulated in Response to the Stage of Flagellar Assembly

PubMed Central

Bonifield, Heather R.; Yamaguchi, Shigeru; Hughes, Kelly T.

2000-01-01

We investigated the posttranscriptional regulation of flgE, a class 2 gene that encodes the hook subunit protein of the flagella. RNase protection assays demonstrated that the flgE gene was transcribed at comparable levels in numerous strains defective in known steps of flagellar assembly. However, Western analyses of these strains demonstrated substantial differences in FlgE protein levels. Although wild-type FlgE levels were observed in strains with deletions of genes encoding components of the switch complex and the flagellum-specific secretion apparatus, no protein was detected in a strain with deletions of the rod, ring, and hook-associated proteins. To determine whether FlgE levels were affected by the stage of hook–basal-body assembly, Western analysis was performed on strains with mutations at individual loci encompassed by the deletion. FlgE protein was undetectable in rod mutants, intermediate in ring mutants, and wild type in hook-associated protein mutants. The lack of negative regulation in switch complex and flagellum-specific secretion apparatus deletion mutants blocked for flagellar construction prior to rod assembly suggests that these structures play a role in the negative regulation of FlgE. Quantitative Western analyses of numerous flagellar mutants indicate that FlgE levels reflect the stage at which flagellar assembly is blocked. These data provide evidence for negative posttranscriptional regulation of FlgE in response to the stage of flagellar assembly. PMID:10869084

Caenorhabditis elegans syndecan (SDN-1) is required for normal egg laying and associates with the nervous system and the vulva.

PubMed

Minniti, Alicia N; Labarca, Mariana; Hurtado, Claudia; Brandan, Enrique

2004-10-01

In Caenorhabditis elegans, the identification of many enzymes involved in the synthesis and modification of glycosaminoglycans (GAGs), essential components of proteoglycans, has attained special attention in recent years. Mutations in all the genes that encode for GAG biosynthetic enzymes show defects in the development of the vulva, specifically in the invagination of the vulval epithelium. Mutants for certain heparan sulfate modifying enzymes present axonal and cellular guidance defects in specific neuronal classes. Although most of the enzymes involved in the biosynthesis and modification of heparan sulfate have been characterized in C. elegans, little is known regarding the core proteins to which these GAGs covalently bind in proteoglycans. A single syndecan homologue (sdn-1) has been identified in the C. elegans genome through sequence analysis. In the present study, we show that C. elegans synthesizes sulfated proteoglycans, seen as three distinct species in western blot analysis. In the sdn-1 (ok449) deletion mutant allele we observed the lack of one species, which corresponds to a 50 kDa product after heparitinase treatment. The expression of sdn-1 mRNA and sequencing revealed that sdn-1 (ok449) deletion mutants lack two glycosylation sites. Hence, the missing protein in the western blot analysis probably corresponds to SDN-1. In addition, we show that SDN-1 localizes to the C. elegans nerve ring, nerve cords and to the vulva. SDN-1 is found specifically phosphorylated in nerve ring neurons and in the vulva, in both wild-type worms and sdn-1 (ok449) deletion mutants. These mutants show a defective egg-laying phenotype. Our results show for the first time, the identification, localization and some functional aspects of syndecan in the nematode C. elegans.

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.

PubMed

Ikegami, Tetsuro; Won, Sungyong; Peters, C J; Makino, Shinji

2006-03-01

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.

A partial deletion within foot-and-mouth disease virus non-structural protein 3A causes clinical attenuation in cattle but does not prevent subclinical infection

USDA-ARS?s Scientific Manuscript database

Deletions within the 3A coding region of foot-and-mouth disease virus (FMDV) are associated with decreased virulence in cattle; however, the mechanisms are unknown. We compared experimental infection of cattle with virulent FMDV O1Campos (O1Ca) and a mutant derivative (O1Ca-delta3A) lacking residues...

Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling.

PubMed

Mundell, S J; Matharu, A-L; Nisar, S; Palmer, T M; Benovic, J L; Kelly, E

2010-02-01

We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.

Overlap of copper and iron uptake systems in mitochondria in Saccharomyces cerevisiae

PubMed Central

Wang, Jing; Gammon, Micah G.; Maynard, Margaret K.; White, Olivia L.; Cobine, Jai A.; Mahone, Wilkerson K.

2016-01-01

In Saccharomyces cerevisiae, the mitochondrial carrier family protein Pic2 imports copper into the matrix. Deletion of PIC2 causes defects in mitochondrial copper uptake and copper-dependent growth phenotypes owing to decreased cytochrome c oxidase activity. However, copper import is not completely eliminated in this mutant, so alternative transport systems must exist. Deletion of MRS3, a component of the iron import machinery, also causes a copper-dependent growth defect on non-fermentable carbon. Deletion of both PIC2 and MRS3 led to a more severe respiratory growth defect than either individual mutant. In addition, MRS3 expressed from a high copy number vector was able to suppress the oxygen consumption and copper uptake defects of a strain lacking PIC2. When expressed in Lactococcus lactis, Mrs3 mediated copper and iron import. Finally, a PIC2 and MRS3 double mutant prevented the copper-dependent activation of a heterologously expressed copper sensor in the mitochondrial intermembrane space. Taken together, these data support a role for the iron transporter Mrs3 in copper import into the mitochondrial matrix. PMID:26763345

Chitin synthase III: synthetic lethal mutants and "stress related" chitin synthesis that bypasses the CSD3/CHS6 localization pathway.

PubMed

Osmond, B C; Specht, C A; Robbins, P W

1999-09-28

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Delta, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that "stress response" chitin synthesis proceeds through an alternate Chs3p targeting pathway.

Chitin synthase III: Synthetic lethal mutants and “stress related” chitin synthesis that bypasses the CSD3/CHS6 localization pathway

PubMed Central

Osmond, Barbara C.; Specht, Charles A.; Robbins, Phillips W.

1999-01-01

We screened Saccharomyces strains for mutants that are synthetically lethal with deletion of the major chitin synthase gene CHS3. In addition to finding, not surprisingly, that mutations in major cell wall-related genes such as FKS1 (glucan synthase) and mutations in any of the Golgi glycosylation complex genes (MNN9 family) are lethal in combination with chs3Δ, we found that a mutation in Srv2p, a bifunctional regulatory gene, is notably lethal in the chs3 deletion. In extending studies of fks1-chitin synthase 3 interactions, we made the surprising discovery that deletion of CSD3/CHS6, a gene normally required for Chs3p delivery and activity in vivo, was not lethal with fks1 and, in fact, that lack of Csd3p/Chs6p did not decrease the high level of stress-related chitin made in the fks1 mutant. This finding suggests that “stress response” chitin synthesis proceeds through an alternate Chs3p targeting pathway. PMID:10500155

Schmallenberg virus non-structural protein NSm: Intracellular distribution and role of non-hydrophobic domains.

PubMed

Kraatz, Franziska; Wernike, Kerstin; Reiche, Sven; Aebischer, Andrea; Reimann, Ilona; Beer, Martin

2018-03-01

Schmallenberg virus (SBV) induces fetal malformation, abortions and stillbirth in ruminants. While the non-structural protein NSs is a major virulence factor, the biological function of NSm, the second non-structural protein which consists of three hydrophobic transmembrane (I, III, V) and two non-hydrophobic regions (II, IV), is still unknown. Here, a series of NSm mutants displaying deletions of nearly the entire NSm or of the non-hydrophobic domains was generated and the intracellular distribution of NSm was assessed. SBV-NSm is dispensable for the generation of infectious virus and mutants lacking domains II - V showed growth properties similar to the wild-type virus. In addition, a comparable intracellular distribution of SBV-NSm was observed in mammalian cells infected with domain II mutants or wild-type virus. In both cases, NSm co-localized with the glycoprotein Gc in the Golgi compartment. However, domain IV-deletion mutants showed an altered distribution pattern and no co-localization of NSm and Gc. Copyright © 2018 Elsevier Inc. All rights reserved.

Potential complications when developing gene deletion clones in Xylella fastidiosa.

PubMed

Johnson, Kameka L; Cursino, Luciana; Athinuwat, Dusit; Burr, Thomas J; Mowery, Patricia

2015-04-16

The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce's disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers. Site-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations. We have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present. We have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

Roles of Catalase and Trehalose in the Protection from Hydrogen Peroxide Toxicity in Saccharomyces cerevisiae.

PubMed

Nishimoto, Takuto; Watanabe, Takeru; Furuta, Masakazu; Kataoka, Michihiko; Kishida, Masao

2016-01-01

The roles of catalase and trehalose in Saccharomyces cerevisiae subject to hydrogen peroxide (H2O2) treatment were examined by measuring the catalase activity and intracellular trehalose levels in mutants lacking catalase or trehalose synthetase. Intracellular trehalose was elevated but the survival rate after H2O2 treatment remained low in mutants with deletion of the Catalase T gene. On the other hand, deletion of the trehalose synthetase gene increased the catalase activity in mutated yeast to levels higher than those in the wild-type strain, and these mutants exhibited some degree of tolerance to H2O2 treatment. These results suggest that Catalase T is critical in the yeast response to oxidative damage caused by H2O2 treatment, but trehalose also plays a role in protection against H2O2 treatment.

Lysis delay and burst shrinkage of coliphage T7 by deletion of terminator Tφ reversed by deletion of early genes.

PubMed

Nguyen, Huong Minh; Kang, Changwon

2014-02-01

Bacteriophage T7 terminator Tϕ is a class I intrinsic terminator coding for an RNA hairpin structure immediately followed by oligo(U), which has been extensively studied in terms of its transcription termination mechanism, but little is known about its physiological or regulatory functions. In this study, using a T7 mutant phage, where a 31-bp segment of Tϕ was deleted from the genome, we discovered that deletion of Tϕ from T7 reduces the phage burst size but delays lysis timing, both of which are disadvantageous for the phage. The burst downsizing could directly result from Tϕ deletion-caused upregulation of gene 17.5, coding for holin, among other Tϕ downstream genes, because infection of gp17.5-overproducing Escherichia coli by wild-type T7 phage showed similar burst downsizing. However, the lysis delay was not associated with cellular levels of holin or lysozyme or with rates of phage adsorption. Instead, when allowed to evolve spontaneously in five independent adaptation experiments, the Tϕ-lacking mutant phage, after 27 or 29 passages, recovered both burst size and lysis time reproducibly by deleting early genes 0.5, 0.6, and 0.7 of class I, among other mutations. Deletion of genes 0.5 to 0.7 from the Tϕ-lacking mutant phage decreased expression of several Tϕ downstream genes to levels similar to that of the wild-type phage. Accordingly, phage T7 lysis timing is associated with cellular levels of Tϕ downstream gene products. This suggests the involvement of unknown factor(s) besides the known lysis proteins, lysozyme and holin, and that Tϕ plays a role of optimizing burst size and lysis time during T7 infection. IMPORTANCE Bacteriophages are bacterium-infecting viruses. After producing numerous progenies inside bacteria, phages lyse bacteria using their lysis protein(s) to get out and start a new infection cycle. Normally, lysis is tightly controlled to ensure phage progenies are maximally produced and released at an optimal time. Here, we have discovered that phage T7, besides employing its known lysis proteins, additionally uses its transcription terminator Tϕ to guarantee the optimal lysis of the E. coli host. Tϕ, positioned in the middle of the T7 genome, must be inactivated at least partially to allow for transcription-driven translocation of T7 DNA into hosts and expression of Tϕ downstream but promoter-lacking genes. What role is played by Tϕ before inactivation? Without Tϕ, not only was lysis time delayed but also the number of progenies was reduced in this study. Furthermore, T7 can overcome Tϕ deletion by further deleting some genes, highlighting that a phage has multiple strategies for optimizing lysis.

The Amino-Terminal PrP Domain Is Crucial to Modulate Prion Misfolding and Aggregation

PubMed Central

Cordeiro, Yraima; Kraineva, Julia; Gomes, Mariana P. B.; Lopes, Marilene H.; Martins, Vilma R.; Lima, Luís M. T. R.; Foguel, Débora; Winter, Roland; Silva, Jerson L.

2005-01-01

The main hypothesis for prion diseases is that the cellular protein (PrPC) can be altered into a misfolded, β-sheet-rich isoform (PrPSc), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we investigate the effects of amino-terminal deletion mutations, rPrPΔ51–90 and rPrPΔ32–121, on the stability and the packing properties of recombinant murine PrP. The region lacking in rPrPΔ51–90 is involved physiologically in copper binding and the other construct lacks more amino-terminal residues (from 32 to 121). The pressure stability is dramatically reduced with decreasing N-domain length and the process is not reversible for rPrPΔ51–90 and rPrPΔ32–121, whereas it is completely reversible for the wild-type form. Decompression to atmospheric pressure triggers immediate aggregation for the mutants in contrast to a slow aggregation process for the wild-type, as observed by Fourier-transform infrared spectroscopy. The temperature-induced transition leads to aggregation of all rPrPs, but the unfolding temperature is lower for the rPrP amino-terminal deletion mutants. The higher susceptibility to pressure of the amino-terminal deletion mutants can be explained by a change in hydration and cavity distribution. Taken together, our results show that the amino-terminal region has a pivotal role on the development of prion misfolding and aggregation. PMID:16040743

Novel functions of prototype foamy virus Gag glycine- arginine-rich boxes in reverse transcription and particle morphogenesis.

PubMed

Müllers, Erik; Uhlig, Tobias; Stirnnagel, Kristin; Fiebig, Uwe; Zentgraf, Hanswalter; Lindemann, Dirk

2011-02-01

Prototype foamy virus (PFV) Gag lacks the characteristic orthoretroviral Cys-His motifs that are essential for various steps of the orthoretroviral replication cycle, such as RNA packaging, reverse transcription, infectivity, integration, and viral assembly. Instead, it contains three glycine-arginine-rich boxes (GR boxes) in its C terminus that putatively represent a functional equivalent. We used a four-plasmid replication-deficient PFV vector system, with uncoupled RNA genome packaging and structural protein translation, to analyze the effects of deletion and various substitution mutations within each GR box on particle release, particle-associated protein composition, RNA packaging, DNA content, infectivity, particle morphology, and intracellular localization. The degree of viral particle release by all mutants was similar to that of the wild type. Only minimal effects on Pol encapsidation, exogenous reverse transcriptase (RT) activity, and genomic viral RNA packaging were observed. In contrast, particle-associated DNA content and infectivity were drastically reduced for all deletion mutants and were undetectable for all alanine substitution mutants. Furthermore, GR box I mutants had significant changes in particle morphology, and GR box II mutants lacked the typical nuclear localization pattern of PFV Gag. Finally, it could be shown that GR boxes I and III, but not GR box II, can functionally complement each other. It therefore appears that, similar to the orthoretroviral Cys-His motifs, the PFV Gag GR boxes are important for RNA encapsidation, genome reverse transcription, and virion infectivity as well as for particle morphogenesis.

Interaction of metronidazole with DNA repair mutants of Escherichia coli.

PubMed

Yeung, T C; Beaulieu, B B; McLafferty, M A; Goldman, P

1984-01-01

It has been proposed that one of metronidazole's partially reduced intermediates interacts either with DNA to exert a bactericidal effect or with water to form acetamide. To test this hypothesis we have examined the effect of metronidazole on several mutants of Escherichia coli that are defective in DNA repair. UV-susceptible RecA- and UvrB- point mutants have an increased susceptibility to metronidazole as manifested by both a decreased minimal inhibitory concentration and a greater bactericidal response to metronidazole in resting cultures. By these criteria, however, we find that UvrB- deletion mutants, which lack the ability to reduce nitrate and chlorate, are no more susceptible to metronidazole than is the wild type. We find, however, that these deletion mutants also lack the ability to reduce metronidazole and thus possibly to form its reactive species. When metronidazole's bactericidal effect is expressed in terms of the concurrent accumulation of acetamide derived from metronidazole, then all RecA- and UvrB- mutants are killed more efficiently than their wild types. The data are consistent, therefore, with metronidazole's lethal effect being mediated by a partially reduced intermediate on the metabolic pathway between metronidazole and acetamide. Defects in other aspects of the DNA repair system do not confer this increased susceptibility to the proposed intermediate. A Tag- mutant, for example, which is defective in 3-methyl-adenine-DNA glycosylase, does not have this increased susceptibility to the presumed precursor of acetamide. Thus, these results provide further support for the hypothesis that the bactericidal effect of metronidazole is mediated by a partially reduced intermediate in the metabolic conversion of metronidazole to acetamide and suggest that this intermediate interacts with DNA to produce a lesion similar to that caused by UV light.

Interaction of metronidazole with DNA repair mutants of Escherichia coli.

PubMed Central

Yeung, T C; Beaulieu, B B; McLafferty, M A; Goldman, P

1984-01-01

It has been proposed that one of metronidazole's partially reduced intermediates interacts either with DNA to exert a bactericidal effect or with water to form acetamide. To test this hypothesis we have examined the effect of metronidazole on several mutants of Escherichia coli that are defective in DNA repair. UV-susceptible RecA- and UvrB- point mutants have an increased susceptibility to metronidazole as manifested by both a decreased minimal inhibitory concentration and a greater bactericidal response to metronidazole in resting cultures. By these criteria, however, we find that UvrB- deletion mutants, which lack the ability to reduce nitrate and chlorate, are no more susceptible to metronidazole than is the wild type. We find, however, that these deletion mutants also lack the ability to reduce metronidazole and thus possibly to form its reactive species. When metronidazole's bactericidal effect is expressed in terms of the concurrent accumulation of acetamide derived from metronidazole, then all RecA- and UvrB- mutants are killed more efficiently than their wild types. The data are consistent, therefore, with metronidazole's lethal effect being mediated by a partially reduced intermediate on the metabolic pathway between metronidazole and acetamide. Defects in other aspects of the DNA repair system do not confer this increased susceptibility to the proposed intermediate. A Tag- mutant, for example, which is defective in 3-methyl-adenine-DNA glycosylase, does not have this increased susceptibility to the presumed precursor of acetamide. Thus, these results provide further support for the hypothesis that the bactericidal effect of metronidazole is mediated by a partially reduced intermediate in the metabolic conversion of metronidazole to acetamide and suggest that this intermediate interacts with DNA to produce a lesion similar to that caused by UV light. PMID:6367636

Effect of the deletion of the C region on the structure and hydration of insulin-like growth factor 1: a molecular dynamics investigation

NASA Astrophysics Data System (ADS)

Degreve, Leo; Silva, Luciene B.

The structure and hydration of insulin-like growth factor 1 and an inactive mutant lacking the C region have been investigated in aqueous solution by molecular dynamics simulation. The overall structures of the two polypeptide resemble those determined by NMR spectroscopy. The deletion of the C region in the wild polypeptide introduces structural stability in the mutant, leading to a better definition of the secondary structure elements. A detailed hydration analysis was performed using the radial distribution functions and energy distributions. The backbone of the mutant is in general more solvent accessible than the wild polypeptide backbone. The structural rearrangements induced in the mutant led to changes in the solvent exposition of Tyr24 and Tyr60, which are residues important for ligand-receptor complex formation. Tyr24 exhibited a similar degree of solvent exposition in both IGF-1 and in the mutant; however, its hydroxyl group in the wild polypeptide is better solvated than in the mutant. Tyr60 was found to be solvent exposed in the wild protein, while in the mutant the involvement of its hydroxyl group in intramolecular hydrogen bonds led to it being buried away from the solvent.

Combined action of the major secreted exo- and endopolygalacturonases is required for full virulence of Fusarium oxysporum.

PubMed

Bravo Ruiz, Gustavo; Di Pietro, Antonio; Roncero, M Isabel G

2016-04-01

The genome of the tomato pathogen Fusarium oxysporum f. sp. lycopersici encodes eight different polygalacturonases (PGs): four endoPGs and four exoPGs. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed that endoPGs pg1 and pg5 and exoPGs pgx4 and pgx6 are expressed at significant levels during growth on citrus pectin, polygalacturonic acid or the monomer galacturonic acid, as well as during the infection of tomato plants. The remaining PG genes exhibit low expression levels under all the conditions tested. Secreted PG activity was decreased significantly during growth on pectin in the single deletion mutants lacking either pg1 or pgx6, as well as in the double mutant. Although the single deletion mutants did not display a significant virulence reduction on tomato plants, the Δpg1Δpgx6 double mutant was significantly attenuated in virulence. The combined action of exoPGs and endoPGs is thus essential for plant infection by the vascular wilt fungus F. oxysporum. © 2015 BSPP and John Wiley & Sons Ltd.

Deletion of the gene encoding the glycolytic enzyme triosephosphate isomerase (tpi) alters morphology of Salmonella enterica serovar Typhimurium and decreases fitness in mice.

PubMed

Paterson, Gavin K; Cone, Danielle B; Northen, Helen; Peters, Sarah E; Maskell, Duncan J

2009-05-01

The glycolytic enzyme triosephosphate isomerase (tpi) (EC 5.3.1.1) plays a key role in central carbon metabolism yet few studies have characterized isogenic bacterial mutants lacking this enzyme and none have examined its role in the in vivo fitness of a bacterial pathogen. Here we have deleted tpiA in Salmonella enterica serovar Typhimurium and found that the mutant had an altered morphology, displaying an elongated shape compared with the wild type. In a mouse model of typhoid fever the tpiA mutant was attenuated for growth as assessed by bacterial counts in the livers and spleens of infected mice. However, this attenuation was not deemed sufficient for consideration of a tpiA mutant as a live attenuated vaccine strain. These phenotypes were complemented by provision of tpiA on pBR322. We therefore provide the first demonstration that tpiA is required for full in vivo fitness of a bacterial pathogen, and that it has a discernable impact on cell morphology.

Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium.

PubMed

Porwollik, Steffen; Santiviago, Carlos A; Cheng, Pui; Long, Fred; Desai, Prerak; Fredlund, Jennifer; Srikumar, Shabarinath; Silva, Cecilia A; Chu, Weiping; Chen, Xin; Canals, Rocío; Reynolds, M Megan; Bogomolnaya, Lydia; Shields, Christine; Cui, Ping; Guo, Jinbai; Zheng, Yi; Endicott-Yazdani, Tiana; Yang, Hee-Jeong; Maple, Aimee; Ragoza, Yury; Blondel, Carlos J; Valenzuela, Camila; Andrews-Polymenis, Helene; McClelland, Michael

2014-01-01

We constructed two collections of targeted single gene deletion (SGD) mutants and two collections of targeted multi-gene deletion (MGD) mutants in Salmonella enterica sv Typhimurium 14028s. The SGD mutant collections contain (1), 3517 mutants in which a single gene is replaced by a cassette containing a kanamycin resistance (KanR) gene oriented in the sense direction (SGD-K), and (2), 3376 mutants with a chloramphenicol resistance gene (CamR) oriented in the antisense direction (SGD-C). A combined total of 3773 individual genes were deleted across these SGD collections. The MGD collections contain mutants bearing deletions of contiguous regions of three or more genes and include (3), 198 mutants spanning 2543 genes replaced by a KanR cassette (MGD-K), and (4), 251 mutants spanning 2799 genes replaced by a CamR cassette (MGD-C). Overall, 3476 genes were deleted in at least one MGD collection. The collections with different antibiotic markers permit construction of all viable combinations of mutants in the same background. Together, the libraries allow hierarchical screening of MGDs for different phenotypic followed by screening of SGDs within the target MGD regions. The mutants of these collections are stored at BEI Resources (www.beiresources.org) and publicly available.

The YPR153W gene is essential for the pressure tolerance of tryptophan permease Tat2 in the yeast Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Kurosaka, Goyu; Abe, Fumiyoshi

2018-01-01

In the yeast Saccharomyces cerevisiae, hydrostatic pressure at 25 MPa is known to be nonlethal but significantly impairs the uptake of tryptophan by the permease Tat2, thereby inhibiting the growth of strains that require tryptophan from the medium. Here, we found that the lack of the YPR153W gene, so far poorly characterized for its role in yeast, caused a serious adverse effect on the growth at 10-25 MPa in the strain that required tryptophan. Deletion for YPR153W resulted in an increased rate of pressure-induced degradation of Tat2, suggesting that Tat2 is destabilized in the YPR153W deletion mutant at 25 MPa. Overexpression of the TAT2 gene enabled the deletion mutant to grow at 25 MPa. These results suggest that Ypr153w is essential for the stability and proper transport function of Tat2 under pressure at 10-25 MPa.

Phosphoribosyl diphosphate synthetase-independent NAD de novo synthesis in Escherichia coli: a new phenotype of phosphate regulon mutants.

PubMed Central

Hove-Jensen, B

1996-01-01

Phosphoribosyl diphosphate-lacking (delta prs) mutant strains of Escherichia coli require NAD, guanosine, uridine, histidine, and tryptophan for growth. NAD is required by phosphoribosyl diphosphate-lacking mutants because of lack of one of the substrates for the quinolinate phosphoribosyltransferase reaction, an enzyme of the NAD de novo pathway. Several NAD-independent mutants of a host from which prs had been deleted were isolated; all of them were shown to have lesions in the pstSCAB-phoU operon, in which mutations lead to derepression of the Pho regulon. In addition NAD-independent growth was dependent on a functional quinolinate phosphoribosyltransferase. The prs suppressor mutations led to the synthesis of a new phosphoryl compound that may act as a precursor for a new NAD biosynthetic pathway. This compound may be synthesized by the product of an unknown phosphate starvation-inducible gene of the Pho regulon because the ability of pst or phoU mutations to suppress the NAD requirement requires PhoB, the transcriptional activator of the Pho regulon. PMID:8550505

Functional characterization of mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 lacking short domains within the large, lumen-exposed loop of the chlorophyll protein CP47 in photosystem II.

PubMed

Gleiter, H M; Haag, E; Shen, J R; Eaton-Rye, J J; Inoue, Y; Vermaas, W F; Renger, G

1994-10-11

Several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions or a single-site mutation within the large, lumen-exposed loop (loop E) of the chlorophyll a-binding photosystem II core protein, CP47, are analyzed for their functional properties by measuring the flash-induced pattern of thermoluminescence, oxygen yield, and fluorescence quantum yield. A physiological and biochemical characterization of these mutant strains has been given in two previous reports [Eaton-Rye, J.J., & Vermaas, W.F.J. (1991) Plant Mol. Biol. 17, 1165-1177; Haag, E., Eaton-Rye, J.J., Renger, G., & Vermaas, S. F.J. (1993) Biochemistry 32, 4444-4454]. The results of the present study show that deletion of charged and conserved amino acids in a region roughly located between residues 370 and 390 decreases the binding affinity of the extrinsic PS II-O protein to photosystem II. Marked differences with PSII-O deletion mutants are observed with respect to Ca2+ requirement and the flash-induced pattern of oxygen evolution. Under conditions where a sufficient light activation is provided, the psbB mutants assayed in this study reveal normal S-state parameters and lifetimes. The results bear two basic implications: (i) the manganese involved in water oxidation can still be bound in a functionally normal or only slightly distorted manner, and (ii) the binding of the extrinsic PS II-O protein to photosystem II is impaired in mutants carrying a deletion in the domain between residues 370 and 390, but the presence of the PS II-O protein is still of functional relevance for the PS II complex, e.g., for maintenance of a high-affinity binding site for Ca2+ and/or involvement during the process of photoactivation.

Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

PubMed

Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

2016-04-15

Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae. Copyright © 2016 Elsevier B.V. All rights reserved.

Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuwasako, Kenji, E-mail: kuwasako@fc.miyazaki-u.ac.jp; Kitamura, Kazuo; Nagata, Sayaka

2010-02-12

Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [{sup 125}I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2more » complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser{sup 449} to Ser{sup 467} were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.« less

A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

PubMed

Somaraju Chalasani, Madhavi Latha; Muppirala, Madhavi; G Ponnam, Surya Prakash; Kannabiran, Chitra; Swarup, Ghanshyam

2013-01-01

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231-294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

Detection of three-base deletion by exciplex formation with perylene derivatives.

PubMed

Kashida, Hiromu; Kondo, Nobuyo; Sekiguchi, Koji; Asanuma, Hiroyuki

2011-06-14

Here, we synthesized fluorescent DNA probes labeled with two perylene derivatives for the detection of a three-base deletion mutant. One such probe discriminated the three-base deletion mutant from the wild-type sequence by exciplex emission, and the deletion mutant was identifiable even by the naked eye. This journal is © The Royal Society of Chemistry 2011

A proteomic analysis of ferulic acid metabolism in Amycolatopsis sp. ATCC 39116.

PubMed

Meyer, Florian; Netzer, Julius; Meinert, Christina; Voigt, Birgit; Riedel, Katharina; Steinbüchel, Alexander

2018-05-16

The pseudonocardiate Amycolatopsis sp. ATCC 39116 is used for the biotechnical production of natural vanillin from ferulic acid. Our laboratory has performed genetic modifications of this strain previously, but there are still many gaps in our knowledge regarding its vanillin tolerance and the general metabolism. We performed cultivations with this bacterium and compared the proteomes of stationary phase cells before ferulic acid feeding with those during ferulic acid feeding. Thereby, we identified 143 differently expressed proteins. Deletion mutants were constructed and characterized to analyze the function of nine corresponding genes. Using these mutants, we identified an active ferulic acid β-oxidation pathway and the enzymes which constitute this pathway. A combined deletion mutant in which the β-oxidation as well as non-β-oxidation pathways of ferulic acid degradation were deleted was unable to grow on ferulic acid as the sole source of carbon and energy. This mutant differs from the single deletion mutants and was unable to grow on ferulic acid. Furthermore, we showed that the non-β-oxidation pathway is involved in caffeic acid degradation; however, its deletion is complemented even in the double deletion mutant. This shows that both pathways can complement each other. The β-oxidation deletion mutant produced significantly reduced amounts of vanillic acid (0.12 instead of 0.35 g/l). Therefore, the resulting mutant could be used as an improved production strain. The quinone oxidoreductase deletion mutant (ΔytfG) degraded ferulic acid slower at first but produced comparable amounts of vanillin and significantly less vanillyl alcohol when compared to the parent strain.

Novel model to study virulence determinants of Escherichia coli K1.

PubMed

Khan, Naveed Ahmed; Goldsworthy, Graham John

2007-12-01

It is shown here for the first time that locusts can be used as a model to study Escherichia coli K1 pathogenesis. E. coli K-12 strain HB101 has very low pathogenicity to locusts and does not invade the locust brain, whereas the injection of 2 x 10(6) E. coli K1 strain RS218 (O18:K1:H7) kills almost 100% of locusts within 72 h and invades the brain within 24 h of injection. Both mortality and invasion of the brain in locusts after injection of E. coli K1 require at least two of the known virulence determinants shown for mammals. Thus, deletion mutants that lack outer membrane protein A or cytotoxic necrotizing factor 1 have reduced abilities to kill locusts and to invade the locust brain compared to the parent E. coli K1. Interestingly, deletion mutants lacking FimH or the NeuDB gene cluster are still able to cause high mortality. It is argued that the likely existence of additional virulence determinants can be investigated in vivo by using this insect system.

Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis

PubMed Central

Williams, Monique J.; Wiid, Ian J.; Hiten, Nicholas F.; Viljoen, Albertus J.; Pietersen, Ray-Dean D.; van Helden, Paul D.

2013-01-01

Ergothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle of Mycobacterium smegmatis revealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant of M. smegmatis lacking egtD (MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion of egtD from wild-type M. smegmatis and an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protecting M. smegmatis against oxidative stress and that ERG is able to partly compensate for the loss of MSH. PMID:23629716

Molecular architecture of the hsp70 promoter after deletion of the TATA box or the upstream regulation region.

PubMed Central

Weber, J A; Taxman, D J; Lu, Q; Gilmour, D S

1997-01-01

GAGA factor, TFIID, and paused polymerase are present on the hsp70 promoter in Drosophila melanogaster prior to transcriptional activation. In order to investigate the interplay between these components, mutant constructs were analyzed after they had been transformed into flies on P elements. One construct lacked the TATA box and the other lacked the upstream regulatory region where GAGA factor binds. Transcription of each mutant during heat shock was at least 50-fold less than that of a normal promoter construct. Before and after heat shock, both mutant promoters were found to adopt a DNase I hypersensitive state that included the region downstream from the transcription start site. High-resolution analysis of the DNase I cutting pattern identified proteins that could be contributing to the hypersensitivity. GAGA factor footprints were clearly evident in the upstream region of the TATA deletion construct, and a partial footprint possibly caused by TFIID was evident on the TATA box of the upstream deletion construct. Permanganate treatment of intact salivary glands was used to further characterize each promoter construct. Paused polymerase and TFIID were readily detected on the normal promoter construct, whereas both deletions exhibited reduced levels of each of these factors. Hence both the TATA box and the upstream region are required to efficiently recruit TFIID and a paused polymerase to the promoter prior to transcriptional activation. In contrast, GAGA factor appears to be capable of binding and establishing a DNase I hypersensitive region in the absence of TFIID and polymerase. Interestingly, purified GAGA factor was found to bind near the transcription start site, and the strength of this interaction was increased by the presence of the upstream region. GAGA factor alone might be capable of establishing an open chromatin structure that encompasses the upstream regulatory region as well as the core promoter region, thus facilitating the binding of TFIID. PMID:9199313

Enhanced recognition memory following glycine transporter 1 deletion in forebrain neurons.

PubMed

Singer, Philipp; Boison, Detlev; Möhler, Hanns; Feldon, Joram; Yee, Benjamin K

2007-10-01

Selective deletion of glycine transporter 1 (GlyT1) in forebrain neurons enhances N-methyl-D-aspartate receptor (NMDAR)-dependent neurotransmission and facilitates associative learning. These effects are attributable to increases in extracellular glycine availability in forebrain neurons due to reduced glycine re-uptake. Using a forebrain- and neuron-specific GlyT1-knockout mouse line (CamKIIalphaCre; GlyT1tm1.2fl/fI), the authors investigated whether this molecular intervention can affect recognition memory. In a spontaneous object recognition memory test, enhanced preference for a novel object was demonstrated in mutant mice relative to littermate control subjects at a retention interval of 2 hr, but not at 2 min. Furthermore, mutants were responsive to a switch in the relative spatial positions of objects, whereas control subjects were not. These potential procognitive effects were demonstrated against a lack of difference in contextual novelty detection: Mutant and control subjects showed equivalent preference for a novel over a familiar context. Results therefore extend the possible range of potential promnesic effects of specific forebrain neuronal GlyT1 deletion from associative learning to recognition memory and further support the possibility that mnemonic functions can be enhanced by reducing GlyT1 function. (PsycINFO Database Record (c) 2007 APA, all rights reserved).

aroA-Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant

PubMed Central

Frahm, Michael; Kocijancic, Dino; Rohde, Manfred; Eckweiler, Denitsa; Bielecka, Agata; Bueno, Emilio; Cava, Felipe; Abraham, Wolf-Rainer; Curtiss, Roy; Häussler, Susanne; Erhardt, Marc; Weiss, Siegfried

2016-01-01

ABSTRACT Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that ΔaroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, ΔaroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of ΔaroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine. PMID:27601574

Characterization of a Spontaneous Nonmagnetic Mutant of Magnetospirillum gryphiswaldense Reveals a Large Deletion Comprising a Putative Magnetosome Island

PubMed Central

Schübbe, Sabrina; Kube, Michael; Scheffel, André; Wawer, Cathrin; Heyen, Udo; Meyerdierks, Anke; Madkour, Mohamed H.; Mayer, Frank; Reinhardt, Richard; Schüler, Dirk

2003-01-01

Frequent spontaneous loss of the magnetic phenotype was observed in stationary-phase cultures of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR-1. A nonmagnetic mutant, designated strain MSR-1B, was isolated and characterized. The mutant lacked any structures resembling magnetosome crystals as well as internal membrane vesicles. The growth of strain MSR-1B was impaired under all growth conditions tested, and the uptake and accumulation of iron were drastically reduced under iron-replete conditions. A large chromosomal deletion of approximately 80 kb was identified in strain MSR-1B, which comprised both the entire mamAB and mamDC clusters as well as further putative operons encoding a number of magnetosome-associated proteins. A bacterial artificial chromosome clone partially covering the deleted region was isolated from the genomic library of wild-type M. gryphiswaldense. Sequence analysis of this fragment revealed that all previously identified mam genes were closely linked with genes encoding other magnetosome-associated proteins within less than 35 kb. In addition, this region was remarkably rich in insertion elements and harbored a considerable number of unknown gene families which appeared to be specific for magnetotactic bacteria. Overall, these findings suggest the existence of a putative large magnetosome island in M. gryphiswaldense and other magnetotactic bacteria. PMID:13129949

Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

PubMed Central

Sánchez-Sampedro, Lucas; Gómez, Carmen Elena; Mejías-Pérez, Ernesto; Pérez-Jiménez, Eva; Oliveros, Juan Carlos

2013-01-01

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4+ and CD8+ T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4+ whereas DNA-LACK/M101-LACK preferentially induced CD8+ T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors. PMID:23596295

Vital and dispensable roles of Plasmodium multidrug resistance transporters during blood- and mosquito-stage development.

PubMed

Rijpma, Sanna R; van der Velden, Maarten; Annoura, Takeshi; Matz, Joachim M; Kenthirapalan, Sanketha; Kooij, Taco W A; Matuschewski, Kai; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Siebelink-Stoter, Rianne; Graumans, Wouter; Ramesar, Jai; Klop, Onny; Russel, Frans G M; Sauerwein, Robert W; Janse, Chris J; Franke-Fayard, Blandine M; Koenderink, Jan B

2016-07-01

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species. © 2016 John Wiley & Sons Ltd.

The non-essential UL50 gene of avian infectious laryngotracheitis virus encodes a functional dUTPase which is not a virulence factor.

PubMed

Fuchs, W; Ziemann, K; Teifke, J P; Werner, O; Mettenleiter, T C

2000-03-01

The DNA sequence of the infectious laryngotracheitis virus (ILTV) UL50, UL51 and UL52 gene homologues was determined. Although the deduced UL50 protein lacks the first of five conserved domains of the corresponding proteins of mammalian alphaherpesviruses, the ILTV gene product was also shown to possess dUTPase activity. The generation of UL50-negative ILTV mutants was facilitated by recombination plasmids encoding green fluorescent protein (GFP), and expression constructs of predicted transactivator proteins of ILTV (alphaTIF, ICP4) were successfully used to increase the infectivity of viral genomic DNA. A GFP-expressing UL50-deletion mutant of ILTV showed reduced cell-to-cell spread in vitro, and was attenuated in vivo. A similar deletion mutant without the foreign gene, however, propagated like wild-type ILTV in cell culture and was pathogenic in chickens. We conclude that the viral dUTPase is not required for efficient replication of ILTV in the respiratory tract of infected animals. The replication defect of the GFP-expressing ILTV recombinant is most likely caused by toxic effects of the reporter gene product, since spontaneously occurring inactivation mutants exhibited wild-type-like growth.

The novel ER membrane protein PRO41 is essential for sexual development in the filamentous fungus Sordaria macrospora.

PubMed

Nowrousian, Minou; Frank, Sandra; Koers, Sandra; Strauch, Peter; Weitner, Thomas; Ringelberg, Carol; Dunlap, Jay C; Loros, Jennifer J; Kück, Ulrich

2007-05-01

The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41-EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41-EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi.

The novel ER membrane protein PRO41 is essential for sexual development in the filamentous fungus Sordaria macrospora

PubMed Central

Nowrousian, Minou; Frank, Sandra; Koers, Sandra; Strauch, Peter; Weitner, Thomas; Ringelberg, Carol; Dunlap, Jay C.; Loros, Jennifer J.; Kück, Ulrich

2013-01-01

Summary The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41–EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41–EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi. PMID:17501918

Lack of tau proteins rescues neuronal cell death and decreases amyloidogenic processing of APP in APP/PS1 mice.

PubMed

Leroy, Karelle; Ando, Kunie; Laporte, Vincent; Dedecker, Robert; Suain, Valérie; Authelet, Michèle; Héraud, Céline; Pierrot, Nathalie; Yilmaz, Zehra; Octave, Jean-Noël; Brion, Jean-Pierre

2012-12-01

Lack of tau expression has been reported to protect against excitotoxicity and to prevent memory deficits in mice expressing mutant amyloid precursor protein (APP) identified in familial Alzheimer disease. In APP mice, mutant presenilin 1 (PS1) enhances generation of Aβ42 and inhibits cell survival pathways. It is unknown whether the deficient phenotype induced by concomitant expression of mutant PS1 is rescued by absence of tau. In this study, we have analyzed the effect of tau deletion in mice expressing mutant APP and PS1. Although APP/PS1/tau(+/+) mice had a reduced survival, developed spatial memory deficits at 6 months and motor impairments at 12 months, these deficits were rescued in APP/PS1/tau(-/-) mice. Neuronal loss and synaptic loss in APP/PS1/tau(+/+) mice were rescued in the APP/PS1/tau(-/-) mice. The amyloid plaque burden was decreased by roughly 50% in the cortex and the spinal cord of the APP/PS1/tau(-/-) mice. The levels of soluble and insoluble Aβ40 and Aβ42, and the Aβ42/Aβ40 ratio were reduced in APP/PS1/tau(-/-) mice. Levels of phosphorylated APP, of β-C-terminal fragments (CTFs), and of β-secretase 1 (BACE1) were also reduced, suggesting that β-secretase cleavage of APP was reduced in APP/PS1/tau(-/-) mice. Our results indicate that tau deletion had a protective effect against amyloid induced toxicity even in the presence of mutant PS1 and reduced the production of Aβ. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Characterization of a double deletion mutant of Fusarium verticillioides lacking two putative trehalose-6-phosphate phosphatase genes

USDA-ARS?s Scientific Manuscript database

Fusarium verticillioides is a fungal pathogen that commonly infects the stalk, ear, and kernels of corn and can produce fumonisins, a family of mycotoxins linked to disease in livestock and humans. Our goal is to characterize the role of the disaccharide trehalose in growth and stress response in F....

Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de

1994-04-01

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-pointmore » sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.« less

endodermal-amyloplast less 1 is a novel allele of SHORT-ROOT

NASA Astrophysics Data System (ADS)

Morita, Miyo T.; Saito, Chieko; Nakano, Akihiko; Tasaka, Masao

Plants can sense the direction of gravity and change the growth orientation of their organs. Arabidopsis mutants have been isolated and characterized in order to elucidate the molecular mechanisms of gravitropism. endodermal-amyloplast less 1 ( eal1) is a unique mutant that completely lacks gravitropism in inflorescence stems and exhibits reduced gravitropism in hypocotyls, whereas its roots showed normal gravitropism. Previously, it was suggested that differentiation or development of amyloplasts in shoot statocytes (endodermal cells) is affected by the eal1 mutation. Here, we have identified EAL1 as a SHORT-ROOT ( SHR) allele based on map position. Three nucleotides in the SHR coding region were deleted in the eal1 mutant, resulting in the deletion of just one amino acid. The protein encoded by the novel allele of SHR appears to have retained its function as a transcription factor since the endodermal cell layer was formed both in roots and in shoots of eal1. SCARECROW (SCR) promoter activity monitored by reporter protein expression was significantly decreased in eal1, suggesting that the activity of SHR lacking one amino acid is reduced. In addition, transcription levels of SHOOT GRAVITROPISM 5 (SGR5), which is mainly expressed in the endodermis of inflorescence stems, was markedly decreased. Together with the presence of abnormal endodermal amyloplasts in eal1, these results strongly suggest that the endodermis observed in eal1 is not sufficiently differentiated to execute shoot gravitropism.

Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2018-02-01

In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Assessment of the Toxicity of CuO Nanoparticles by Using Saccharomyces cerevisiae Mutants with Multiple Genes Deleted

PubMed Central

Bao, Shaopan; Lu, Qicong; Dai, Heping; Zhang, Chao

2015-01-01

To develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on various Saccharomyces cerevisiae (S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P < 0.05). When the toxicity of the CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles. PMID:26386067

The effects of disruption of phosphoglucose isomerase gene on carbon utilisation and cellulase production in Trichoderma reesei Rut-C30

PubMed Central

2011-01-01

Background Cellulase and hemicellulase genes in the fungus Trichoderma reesei are repressed by glucose and induced by lactose. Regulation of the cellulase genes is mediated by the repressor CRE1 and the activator XYR1. T. reesei strain Rut-C30 is a hypercellulolytic mutant, obtained from the natural strain QM6a, that has a truncated version of the catabolite repressor gene, cre1. It has been previously shown that bacterial mutants lacking phosphoglucose isomerase (PGI) produce more nucleotide precursors and amino acids. PGI catalyzes the second step of glycolysis, the formation of fructose-6-P from glucose-6-P. Results We deleted the gene pgi1, encoding PGI, in the T. reesei strain Rut-C30 and we introduced the cre1 gene in a Δpgi1 mutant. Both Δpgi1 and cre1+Δpgi1 mutants showed a pellet-like and growth as well as morphological alterations compared with Rut-C30. None of the mutants grew in media with fructose, galactose, xylose, glycerol or lactose but they grew in media with glucose, with fructose and glucose, with galactose and fructose or with lactose and fructose. No growth was observed in media with xylose and glucose. On glucose, Δpgi1 and cre1+Δpgi1 mutants showed higher cellulase activity than Rut-C30 and QM6a, respectively. But in media with lactose, none of the mutants improved the production of the reference strains. The increase in the activity did not correlate with the expression of mRNA of the xylanase regulator gene, xyr1. Δpgi1 mutants were also affected in the extracellular β-galactosidase activity. Levels of mRNA of the glucose 6-phosphate dehydrogenase did not increase in Δpgi1 during growth on glucose. Conclusions The ability to grow in media with glucose as the sole carbon source indicated that Trichoderma Δpgi1 mutants were able to use the pentose phosphate pathway. But, they did not increase the expression of gpdh. Morphological characteristics were the result of the pgi1 deletion. Deletion of pgi1 in Rut-C30 increased cellulase production, but only under repressing conditions. This increase resulted partly from the deletion itself and partly from a genetic interaction with the cre1-1 mutation. The lower cellulase activity of these mutants in media with lactose could be attributed to a reduced ability to hydrolyse this sugar but not to an effect on the expression of xyr1. PMID:21609467

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

PubMed Central

Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Suárez, Cristina; Salas, José

2013-01-01

The function of the African swine fever virus (ASFV) reparative DNA polymerase, Pol X, was investigated in the context of virus infection. Pol X is a late structural protein that localizes at cytoplasmic viral factories during DNA replication. Using an ASFV deletion mutant lacking the Pol X gene, we have shown that Pol X is not required for virus growth in Vero cells or swine macrophages under one-step growth conditions. However, at a low multiplicity of infection, when multiple rounds of replication occur, the growth of the mutant virus is impaired in swine macrophages but not in Vero cells, suggesting that Pol X is needed to repair the accumulated DNA damage. The replication of the mutant virus in Vero cells presents sensitivity to oxidative damage, and mutational analysis of viral DNA shows that deletion of Pol X results in an increase in the mutation frequency in macrophages. Therefore, our data reveal a biological role for ASFV Pol X in the context of the infected cell in the preservation of viral genetic information. PMID:23824796

Use of a nonhomologous end joining deficient strain (Deltaku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis.

PubMed

Haarmann, Thomas; Lorenz, Nicole; Tudzynski, Paul

2008-01-01

The ergot fungus Claviceps purpurea uses mainly the nonhomologous-end-joining (NHEJ) system for integration of exogenous DNA, leading to a low frequency of homologous integration (1-2%). To improve gene targeting efficiency we deleted the C. purpurea ku70 gene in two different strains: the pathogenic strain 20.1 and the apathogenic, ergot alkaloid producing strain P1. The mutants were not impaired in vegetative and pathogenic development nor alkaloid production. Gene targeting efficiency was significantly increased (50-60%) in the Deltaku70 mutants. The P1 Deltaku70 strain (producing ergotamine and ergocryptine) was used for targeted deletion of lpsA1, one of the two trimodular NRPS genes present in the alkaloid gene cluster, encoding D-lysergyl peptide synthetases involved in formation of the tripeptide moiety of ergopeptines. Mutants lacking the lpsA1 gene were shown to be incapable of producing ergotamine but were still able to produce ergocryptine, proving that LpsA1 is involved in ergotamine biosynthesis.

Construction and characterization of a double deletion mutant of Fusarium verticillioides lacking two putative trehalose-6-phosphate phosphatase genes

USDA-ARS?s Scientific Manuscript database

Fusarium verticillioides is a fungal pathogen that commonly infects the stalk, ear, and kernels of corn and can produce fumonisins, a family of mycotoxins linked to disease in livestock and humans. Our goal is to characterize the role of the disaccharide trehalose in growth and stress response in F....

Secondary metabolites of the grapevine pathogen Eutypa lata inhibit mitochondrial respiration, based on a model bioassay using the yeast Saccharomyces cerevisiae.

PubMed

Kim, Jong H; Mahoney, Noreen; Chan, Kathleen L; Molyneux, Russell J; Campbell, Bruce C

2004-10-01

Acetylenic phenols and a chromene isolated from the grapevine fungal pathogen Eutypa lata were examined for mode of toxicity. The compounds included eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl aldehyde), eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), eulatachromene, 2- isoprenyl-5-formyl-benzofuran, siccayne, and eulatinol. A bioassay using the yeast Saccharomyces cerevisiae showed that all compounds were either lethal or inhibited growth. A respiratory assay using 2,3,5-triphenyltetrazolium (TTC) indicated that eutypinol and eulatachromene inhibited mitochondrial respiration in wild-type yeast. Bioassays also showed that 2- isoprenyl-5-formyl-benzofuran and siccayne inhibited mitochondrial respiration in the S. cerevisiae deletion mutant vph2Delta, lacking a vacuolar type H (+) ATPase (V-ATPase) assembly protein. Cell growth of tsa1Delta, a deletion mutant of S. cerevisiae lacking a thioredoxin peroxidase (cTPx I), was greatly reduced when grown on media containing eutypinol or eulatachromene and exposed to hydrogen peroxide (H(2)O(2)) as an oxidative stress. This reduction in growth establishes the toxic mode of action of these compounds through inhibition of mitochondrial respiration.

Na+/H+ antiport is essential for Yersinia pestis virulence.

PubMed

Minato, Yusuke; Ghosh, Amit; Faulkner, Wyatt J; Lind, Erin J; Schesser Bartra, Sara; Plano, Gregory V; Jarrett, Clayton O; Hinnebusch, B Joseph; Winogrodzki, Judith; Dibrov, Pavel; Häse, Claudia C

2013-09-01

Na(+)/H(+) antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na(+)/H(+) antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na(+)/H(+) antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na(+) levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na(+)/H(+) antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.

Lethal factor is not required for Bacillus anthracis virulence in guinea pigs and rabbits.

PubMed

Levy, Haim; Weiss, Shay; Altboum, Zeev; Schlomovitz, Josef; Rothschild, Nili; Blachinsky, Eran; Kobiler, David

2011-11-01

The major virulence factor of Bacillus anthracis is the tripartite anthrax toxin, comprising the protective antigen (PA), lethal factor (LF) and edema factor (EF). The LF of B. anthracis is a metalloprotease that has been shown to play an important role in pathogenicity. Deletion of this gene (lef) in the Sterne strain was reported to dramatically reduce the pathogenicity of this strain in mice, and was reported to be as dramatic as the deletion of PA. We evaluated the effect on pathogenicity of the lef deletion in the fully virulent Vollum strain in guinea pigs and NZW rabbits by either subcutaneous injection or intranasal instillation. In guinea pigs, no major differences between the mutant strain and the wild type could be detected in the LD(50) or mean time to death values. On the other hand, the lef deletion caused death of 50-70% of all rabbits infected with the mutant spores at doses equivalent or higher than the wild type LD(50). The surviving rabbits, which were infected with spore doses higher than the wild type LD(50), developed a protective immune response that conferred resistance to challenge with the wild type strain. These findings may indicate that the mutant lacking the LF is capable of host colonization which causes death in 50-70% of the animals and a protective immune response in the others. These results indicate that unlike the data obtained in mice, the LF mutation does not abolish B. anthracis pathogenicity. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mimicking the BIM BH3 domain overcomes resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer

PubMed Central

Xia, Jinjing; Bai, Hao; Yan, Bo; Li, Rong; Shao, Minhua; Xiong, Liwen; Han, Baohui

2017-01-01

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are widely applied to treat EGFR-mutant non-small cell lung cancer (NSCLC). BIM is a BH3 domain-containing protein encoded by BCL2L11. Some EGFR-mutant NSCLC patients showing BIM deletion polymorphism are resistant to EGFR TKIs. We retrospectively investigated BIM deletion polymorphism in NSCLC patients, its correlation with EGFR TKI (erlotinib) resistance, and the mechanism underlying the drug resistance. Among 245 EGFR-mutant NSCLC patients examined, BIM deletion polymorphism was detected in 43 (12.24%). Median progression-free and overall survival was markedly shorter in patients with BIM deletion polymorphism than with BIM wide-type. Moreover, NSCLC cells expressing EGFR-mutant harboring BIM polymorphism were more resistant to erlotinib-induced apoptosis than BIM wide-type cells. However, combined use of erlotinib and the BH3-mimetic ABT-737 up-regulated BIM expression and overcame erlotinib resistance in EGFR-mutant NSCLC cells harboring BIM deletion polymorphism. In vivo, erlotinib suppressed growth of BIM wide-type NSCLC cell xenographs by inducing apoptosis. Combined with ABT-737, erlotinib also suppressed NSCLC xenographs expressing EGFR-mutant harboring BIM deletion polymorphism. These results indicate that BIM polymorphism is closely related to a poor clinical response to EGFR TKIs in EGFR-mutant NSCLC patients, and that the BH3-mimetic ABT-737 restores BIM functionality and EGFR-TKI sensitivity. PMID:29312548

Mimicking the BIM BH3 domain overcomes resistance to EGFR tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer.

PubMed

Xia, Jinjing; Bai, Hao; Yan, Bo; Li, Rong; Shao, Minhua; Xiong, Liwen; Han, Baohui

2017-12-12

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are widely applied to treat EGFR-mutant non-small cell lung cancer (NSCLC). BIM is a BH3 domain-containing protein encoded by BCL2L11. Some EGFR-mutant NSCLC patients showing BIM deletion polymorphism are resistant to EGFR TKIs. We retrospectively investigated BIM deletion polymorphism in NSCLC patients, its correlation with EGFR TKI (erlotinib) resistance, and the mechanism underlying the drug resistance. Among 245 EGFR-mutant NSCLC patients examined, BIM deletion polymorphism was detected in 43 (12.24%). Median progression-free and overall survival was markedly shorter in patients with BIM deletion polymorphism than with BIM wide-type. Moreover, NSCLC cells expressing EGFR-mutant harboring BIM polymorphism were more resistant to erlotinib-induced apoptosis than BIM wide-type cells. However, combined use of erlotinib and the BH3-mimetic ABT-737 up-regulated BIM expression and overcame erlotinib resistance in EGFR-mutant NSCLC cells harboring BIM deletion polymorphism. In vivo , erlotinib suppressed growth of BIM wide-type NSCLC cell xenographs by inducing apoptosis. Combined with ABT-737, erlotinib also suppressed NSCLC xenographs expressing EGFR-mutant harboring BIM deletion polymorphism. These results indicate that BIM polymorphism is closely related to a poor clinical response to EGFR TKIs in EGFR-mutant NSCLC patients, and that the BH3-mimetic ABT-737 restores BIM functionality and EGFR-TKI sensitivity.

The YJR127C/ZMS1 gene product is involved in glycerol-based respiratory growth of the yeast Saccharomyces cerevisiae.

PubMed

Lu, Lin; Roberts, George G; Oszust, Cynthia; Hudson, Alan P

2005-10-01

A putative yeast mitochondrial upstream activating sequence (UAS) was used in a one-hybrid screening procedure that identified the YJR127C ORF on chromosome X. This gene was previously designated ZMS1 and is listed as a transcription factor on the SGD website. Real time RT-PCR assays showed that expression of YJR127C/ZMS1 was glucose-repressible, and a deletion mutant for the gene showed a growth defect on glycerol-based but not on glucose- or ethanol-based medium. Real time RT-PCR analyses identified severely attenuated transcript levels from GUT1 and GUT2 to be the source of that growth defect, the products of GUT1 and GUT2 are required for glycerol utilization. mRNA levels from a large group of mitochondria- and respiration-related nuclear genes also were shown to be attenuated in the deletion mutant. Importantly, transcript levels from the mitochondrial OLI1 gene, which has an associated organellar UAS, were attenuated in the DeltaYJR127C mutant during glycerol-based growth, but those from COX3 (OXI2), which lacks an associated mitochondrial UAS, were not. Transcriptome analysis of the glycerol-grown deletion mutant showed that genes in several metabolic and other categories are affected by loss of this gene product, including protein transport, signal transduction, and others. Thus, the product of YJR127C/ZMS1 is involved in transcriptional control for genes in both cellular genetic compartments, many of which specify products required for glycerol-based growth, respiration, and other functions.

Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit.

PubMed

Liu, Cheng-Qian; Hu, Kang-Di; Li, Ting-Ting; Yang, Ying; Yang, Feng; Li, Yan-Hong; Liu, He-Ping; Chen, Xiao-Yan; Zhang, Hua

2017-01-01

Aspergillus niger, a saprophytic fungus, is widely distributed in soil, air and cereals, and can cause postharvest diseases in fruit. Polygalacturonase (PG) is one of the main enzymes in fungal pathogens to degrade plant cell wall. To evaluate whether the deletion of an exo-polygalacturonase gene pgxB would influence fungal pathogenicity to fruit, pgxB gene was deleted in Aspergillus niger MA 70.15 (wild type) via homologous recombination. The ΔpgxB mutant showed similar growth behavior compared with the wild type. Pectin medium induced significant higher expression of all pectinase genes in both wild type and ΔpgxB in comparison to potato dextrose agar medium. However, the ΔpgxB mutant was less virulent on apple fruits as the necrosis diameter caused by ΔpgxB mutant was significantly smaller than that of wild type. Results of quantitive-PCR showed that, in the process of infection in apple fruit, gene expressions of polygalacturonase genes pgaI, pgaII, pgaA, pgaC, pgaD and pgaE were enhanced in ΔpgxB mutant in comparison to wild type. These results prove that, despite the increased gene expression of other polygalacturonase genes in ΔpgxB mutant, the lack of pgxB gene significantly reduced the virulence of A. niger on apple fruit, suggesting that pgxB plays an important role in the infection process on the apple fruit.

Identification of a functional capsule locus in Streptococcus mitis.

PubMed

Rukke, H V; Hegna, I K; Petersen, F C

2012-04-01

The polysaccharide capsule of Streptococcus pneumoniae is a hallmark for virulence in humans. In its close relative Streptococcus mitis, a common human commensal, analysis of the sequenced genomes of six strains revealed the presence of a putative capsule locus in four of them. We constructed an isogenic S. mitis mutant from the type strain that lacked the 19 open reading frames in the capsule locus (Δcps mutant), using a deletion strategy similar to previous capsule functional studies in S. pneumoniae. Transmission electron microscopy and atomic force microscopy revealed a capsule-like structure in the S. mitis type strain that was absent or reduced in the Δcps mutant. Since S. mitis are predominant oral colonizers of tooth surfaces, we addressed the relevance of the capsule locus for the S. mitis overall surface properties, autoaggregation and biofilm formation. The capsule deletion resulted in a mutant with approximately two-fold increase in hydrophobicity. Binding to the Stains-all cationic dye was reduced by 40%, suggesting a reduction in the overall negative surface charge of the mutant. The mutant exhibited also increased autoaggregation in coaggregation buffer, and up to six-fold increase in biofilm levels. The results suggested that the capsule locus is associated with production of a capsule-like structure in S. mitis and indicated that the S. mitis capsule-like structure may confer surface attributes similar to those associated with the capsule in S. pneumoniae. © 2011 John Wiley & Sons A/S.

Intracistronic complementation in the simian virus 40 A gene.

PubMed Central

Tornow, J; Cole, C N

1983-01-01

A set of eight simian virus 40 mutants was constructed with lesions in the A gene, which encodes the large tumor (T) antigen. These mutants have small deletions (3-20 base pairs) at either 0.497, 0.288, or 0.243 map units. Mutants having both in-phase and frameshift mutations at each site were isolated. Neither plaque formation nor replication of the mutant DNAs could be detected after transfection of monkey kidney cells. Another nonviable mutant, dlA2459, had a 14-base-pair deletion at 0.193 map unit and was positive for viral DNA replication. Each of the eight mutants were tested for ability to form plaques after cotransfection with dlA2459 DNA. The four mutants that had in-phase deletions were able to complement dlA2459. The other four, which had frameshift deletions, did not. No plaques were formed after cotransfection of cells with any other pair of group A mutants. This suggests that the defect in dlA2459 defines a distinct functional domain of simian virus 40 T antigen. Images PMID:6312452

Headbobber: A Combined Morphogenetic and Cochleosaccular Mouse Model to Study 10qter Deletions in Human Deafness

PubMed Central

Buniello, Annalisa; Hardisty-Hughes, Rachel E.; Pass, Johanna C.; Bober, Eva; Smith, Richard J.; Steel, Karen P.

2013-01-01

The recessive mouse mutant headbobber (hb) displays the characteristic behavioural traits associated with vestibular defects including headbobbing, circling and deafness. This mutation was caused by the insertion of a transgene into distal chromosome 7 affecting expression of native genes. We show that the inner ear of hb/hb mutants lacks semicircular canals and cristae, and the saccule and utricle are fused together in a single utriculosaccular sac. Moreover, we detect severe abnormalities of the cochlear sensory hair cells, the stria vascularis looks severely disorganised, Reissner's membrane is collapsed and no endocochlear potential is detected. Myo7a and Kcnj10 expression analysis show a lack of the melanocyte-like intermediate cells in hb/hb stria vascularis, which can explain the absence of endocochlear potential. We use Trp2 as a marker of melanoblasts migrating from the neural crest at E12.5 and show that they do not interdigitate into the developing strial epithelium, associated with abnormal persistence of the basal lamina in the hb/hb cochlea. We perform array CGH, deep sequencing as well as an extensive expression analysis of candidate genes in the headbobber region of hb/hb and littermate controls, and conclude that the headbobber phenotype is caused by: 1) effect of a 648 kb deletion on distal Chr7, resulting in the loss of three protein coding genes (Gpr26, Cpmx2 and Chst15) with expression in the inner ear but unknown function; and 2) indirect, long range effect of the deletion on the expression of neighboring genes on Chr7, associated with downregulation of Hmx3, Hmx2 and Nkx1.2 homeobox transcription factors. Interestingly, deletions of the orthologous region in humans, affecting the same genes, have been reported in nineteen patients with common features including sensorineural hearing loss and vestibular problems. Therefore, we propose that headbobber is a useful model to gain insight into the mechanisms underlying deafness in human 10qter deletion syndrome. PMID:23457544

[Correlation of chromosome 1p and 19q status and expression of R132H mutant IDH1 protein in oligodendroglial tumors].

PubMed

Yao, Kun; Duan, Zejun; Hu, Zeliang; Bian, Yu; Qi, Xueling

2014-10-01

To correlate the presence of chromosome 1p/19q deletion with the expression of R132H mutant IDH1 status in oligodendroglial tumors, and to explore molecular markers for predicting chemosensitivity of oligodendroglial tumors. The study included 75 oligodendroglial tumors (38 oligodendrogliomas and 37 oligoastrocytomas). Immunohistochemistry was used to detect the expression of R132H mutant IDH1 protein, and fluorescence in situ hybridization (FISH) was employed to detect 1p/19q deletion. Deletion of chromosome 1p and/or 19q was detected in 37 cases (37/75, 49.3%), among which co-deletion of 1p and 19q was seen in 34 cases (closely correlated, P < 0.01). Oligodendrogliomas WHOIIhad a slightly higher deletion rate than oligodendrogliomas WHO III, although without statistical significance. Oligodendrogliomas WHO IIand WHO III had a significantly higher deletion rate of chromosome 1p/19q than oligoastrocytomas WHO II and WHO III (P < 0.05). While combined loss of 1p/19q was always detected in oligodendrogliomas when FISH was positive, isolated 1p or 19q deletion was only found in oligoastrocytomas. The expression of R132H mutant IDH1 was detected in 51 of 75 cases (68.0%), in which oligodendrogliomas had a higher positive rate than oligoastrocytomas. Statistical analysis demonstrated a significant correlation between the expression of R132H mutant IDH1 protein and the presence of combined 1p/19q deletion in oligodendrogliomas (P < 0.05). A significant correlation was observed between the expression of R132H mutant protein and 1p/19q LOH.Expression of 132H mutant IDH1 protein is the potential biomarker for predicating the presence of 1p/19q deletion in oligodendrogliomas.

Replacement of Lipopolysaccharide with Free Lipid A Molecules in Escherichia coli Mutants Lacking All Core Sugars

PubMed Central

Reynolds, C. Michael; Raetz, Christian R. H.

2009-01-01

Escherichia coli mutants deficient in 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo) biosynthesis are conditionally lethal, but their phenotypes are bypassed by certain suppressor mutations or by over-expression of MsbA, the inner membrane flippase for core-lipid A. These strains grow on broth with the tetra-acylated precursor lipid IVA replacing lipopolysaccharide (Meredith, T. C. et al. ACS Chem. Biol. 1, 33–42, 2006). Deletion of kdtA, which encodes the Kdo transferase, is possible under these conditions. We now show that lipid IVA reaches the outer surface of the outer membrane in these strains, as judged by its accessibility to the lipase PagL. On the assumption that MsbA is optimized to transport penta- or hexa-acylated lipid A, we over-expressed the lauroyl or the myristoyl transferase of lipid A biosynthesis, encoded by lpxL and lpxM respectively, and demonstrated that kdtA deletion mutants were also viable in this setting. Although E. coli LpxL is stimulated by the presence of the Kdo-disaccharide in its acceptor substrate, LpxL does slowly acylate lipid IVA. Over-expression of LpxL from a plasmid suppressed the lethality of kdtA deletions on nutrient broth at 30 or 37 °C without the need for MsbA over-production. These strains accumulated penta- and hexa-acylated free lipid A containing a secondary laurate chain, or a laurate and a myristate chain, respectively. Deletion of kdtA in strains over-expressing LpxM accumulated penta-acylated lipid A with a secondary myristate moiety. None of the strains lacking kdtA grew in the presence of bile salts at any temperature or on nutrient broth at 42 °C. Our findings show that the main function of Kdo is to provide the right substrates for the acyltransferases LpxL and LpxM, resulting in the synthesis of penta- and hexa-acylated lipid A, which is optimal for the MsbA flippase. PMID:19754149

Identification of a basic helix-loop-helix-type transcription regulator gene in Aspergillus oryzae by systematically deleting large chromosomal segments.

PubMed

Jin, Feng Jie; Takahashi, Tadashi; Machida, Masayuki; Koyama, Yasuji

2009-09-01

We previously developed two methods (loop-out and replacement-type recombination) for generating large-scale chromosomal deletions that can be applied to more effective chromosomal engineering in Aspergillus oryzae. In this study, the replacement-type method is used to systematically delete large chromosomal DNA segments to identify essential and nonessential regions in chromosome 7 (2.93 Mb), which is the smallest A. oryzae chromosome and contains a large number of nonsyntenic blocks. We constructed 12 mutants harboring deletions that spanned 16- to 150-kb segments of chromosome 7 and scored phenotypic changes in the resulting mutants. Among the deletion mutants, strains designated Delta5 and Delta7 displayed clear phenotypic changes involving growth and conidiation. In particular, the Delta5 mutant exhibited vigorous growth and conidiation, potentially beneficial characteristics for certain industrial applications. Further deletion analysis allowed identification of the AO090011000215 gene as the gene responsible for the Delta5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loop-helix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes.

Seipin is involved in the regulation of phosphatidic acid metabolism at a subdomain of the nuclear envelope in yeast.

PubMed

Wolinski, Heimo; Hofbauer, Harald F; Hellauer, Klara; Cristobal-Sarramian, Alvaro; Kolb, Dagmar; Radulovic, Maja; Knittelfelder, Oskar L; Rechberger, Gerald N; Kohlwein, Sepp D

2015-11-01

Yeast Fld1 and Ldb16 resemble mammalian seipin, implicated in neutral lipid storage. Both proteins form a complex at the endoplasmic reticulum-lipid droplet (LD) interface. Malfunction of this complex either leads to LD clustering or to the generation of supersized LD (SLD) in close vicinity to the nuclear envelope, in response to altered phospholipid (PL) composition. We show that similar to mutants lacking Fld1, deletion of LDB16 leads to abnormal proliferation of a subdomain of the nuclear envelope, which is tightly associated with clustered LD. The human lipin-1 ortholog, the PAH1 encoded phosphatidic acid (PA) phosphatase, and its activator Nem1 are highly enriched at this site. The specific accumulation of PA-binding marker proteins indicates a local enrichment of PA in the fld1 and ldb16 mutants. Furthermore, we demonstrate that clustered LD in fld1 or ldb16 mutants are transformed to SLD if phosphatidylcholine synthesis is compromised by additional deletion of the phosphatidylethanolamine methyltransferase, Cho2. Notably, treatment of wild-type cells with oleate induced a similar LD clustering and nuclear membrane proliferation phenotype as observed in fld1 and ldb16 mutants. These data suggest that the Fld1-Ldb16 complex affects PA homeostasis at an LD-forming subdomain of the nuclear envelope. Lack of Fld1-Ldb16 leads to locally elevated PA levels that induce an abnormal proliferation of nER membrane structures and the clustering of associated LD. We suggest that the formation of SLD is a consequence of locally altered PL metabolism at this site. Copyright © 2015. Published by Elsevier B.V.

Systematic Phenotyping of a Large-Scale Candida glabrata Deletion Collection Reveals Novel Antifungal Tolerance Genes

PubMed Central

Hiller, Ekkehard; Istel, Fabian; Tscherner, Michael; Brunke, Sascha; Ames, Lauren; Firon, Arnaud; Green, Brian; Cabral, Vitor; Marcet-Houben, Marina; Jacobsen, Ilse D.; Quintin, Jessica; Seider, Katja; Frohner, Ingrid; Glaser, Walter; Jungwirth, Helmut; Bachellier-Bassi, Sophie; Chauvel, Murielle; Zeidler, Ute; Ferrandon, Dominique; Gabaldón, Toni; Hube, Bernhard; d'Enfert, Christophe; Rupp, Steffen; Cormack, Brendan; Haynes, Ken; Kuchler, Karl

2014-01-01

The opportunistic fungal pathogen Candida glabrata is a frequent cause of candidiasis, causing infections ranging from superficial to life-threatening disseminated disease. The inherent tolerance of C. glabrata to azole drugs makes this pathogen a serious clinical threat. To identify novel genes implicated in antifungal drug tolerance, we have constructed a large-scale C. glabrata deletion library consisting of 619 unique, individually bar-coded mutant strains, each lacking one specific gene, all together representing almost 12% of the genome. Functional analysis of this library in a series of phenotypic and fitness assays identified numerous genes required for growth of C. glabrata under normal or specific stress conditions, as well as a number of novel genes involved in tolerance to clinically important antifungal drugs such as azoles and echinocandins. We identified 38 deletion strains displaying strongly increased susceptibility to caspofungin, 28 of which encoding proteins that have not previously been linked to echinocandin tolerance. Our results demonstrate the potential of the C. glabrata mutant collection as a valuable resource in functional genomics studies of this important fungal pathogen of humans, and to facilitate the identification of putative novel antifungal drug target and virulence genes. PMID:24945925

Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.

PubMed

Lin, Hsin Hung; Pan, Yih Jiuan; Hsu, Shen Hsing; Van, Ru Chuan; Hsiao, Yi Yuong; Chen, Jiun Hsien; Pan, Rong Long

2005-10-15

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.

Enhanced oxygen consumption in Herbaspirillum seropedicae fnr mutants leads to increased NifA mediated transcriptional activation.

PubMed

Batista, Marcelo Bueno; Wassem, Roseli; Pedrosa, Fábio de Oliveira; de Souza, Emanuel Maltempi; Dixon, Ray; Monteiro, Rose Adele

2015-05-07

Orthologous proteins of the Crp/Fnr family have been previously implicated in controlling expression and/or activity of the NifA transcriptional activator in some diazotrophs. This study aimed to address the role of three Fnr-like proteins from H. seropedicae SmR1 in controlling NifA activity and consequent NifA-mediated transcription activation. The activity of NifA-dependent transcriptional fusions (nifA::lacZ and nifB::lacZ) was analysed in a series of H. seropedicae fnr deletion mutant backgrounds. We found that combined deletions in both the fnr1 and fnr3 genes lead to higher expression of both the nifA and nifB genes and also an increased level of nifH transcripts. Expression profiles of nifB under different oxygen concentrations, together with oxygen consumption measurements suggest that the triple fnr mutant has higher respiratory activity when compared to the wild type, which we believe to be responsible for greater stability of the oxygen sensitive NifA protein. This conclusion was further substantiated by measuring the levels of NifA protein and its activity in fnr deletion strains in comparison with the wild-type. Fnr proteins are indirectly involved in controlling the activity of NifA in H. seropedicae, probably as a consequence of their influence on respiratory activity in relation to oxygen availability. Additionally we can suggest that there is some redundancy in the physiological function of the three Fnr paralogs in this organism, since altered respiration and effects on NifA activity are only observed in deletion strains lacking both fnr1 and fnr3.

miR-11 regulates pupal size of Drosophila melanogaster via directly targeting Ras85D.

PubMed

Li, Yao; Li, Shengjie; Jin, Ping; Chen, Liming; Ma, Fei

2017-01-01

MicroRNAs play diverse roles in various physiological processes during Drosophila development. In the present study, we reported that miR-11 regulates pupal size during Drosophila metamorphosis via targeting Ras85D with the following evidences: pupal size was increased in the miR-11 deletion mutant; restoration of miR-11 in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant; ectopic expression of miR-11 in brain insulin-producing cells (IPCs) and whole body shows consistent alteration of pupal size; Dilps and Ras85D expressions were negatively regulated by miR-11 in vivo; miR-11 targets Ras85D through directly binding to Ras85D 3'-untranslated region in vitro; removal of one copy of Ras85D in the miR-11 deletion mutant rescued the increased pupal size phenotype observed in the miR-11 deletion mutant. Thus, our current work provides a novel mechanism of pupal size determination by microRNAs during Drosophila melanogaster metamorphosis. Copyright © 2017 the American Physiological Society.

RipA, a Cytoplasmic Membrane Protein Conserved among Francisella Species, Is Required for Intracellular Survival▿

PubMed Central

Fuller, James R.; Craven, Robin R.; Hall, Joshua D.; Kijek, Todd M.; Taft-Benz, Sharon; Kawula, Thomas H.

2008-01-01

Francisella tularensis is a highly virulent bacterial pathogen that invades and replicates within numerous host cell types, including macrophages and epithelial cells. In an effort to better understand this process, we screened a transposon insertion library of the F. tularensis live vaccine strain (LVS) for mutant strains that invaded but failed to replicate within alveolar epithelial cell lines. One such strain isolated from this screen contained an insertion in the gene FTL_1914, which is conserved among all sequenced Francisella species yet lacks significant homology to any gene with known function. A deletion strain lacking FTL_1914 was constructed. This strain did not replicate in either epithelial or macrophage-like cells, and intracellular replication was restored by the wild-type allele in trans. Based on the deletion mutant phenotype, FTL_1914 was termed ripA (required for intracellular proliferation, factor A). Following uptake by J774.A1 cells, F. tularensis LVS ΔripA colocalized with LAMP-1 then escaped the phagosome at the same rate and frequency as wild-type LVS-infected cells. Electron micrographs of the F. tularensis LVS ΔripA mutant demonstrated the reentry of the mutant bacteria into double membrane vacuoles characteristic of autophagosomes in a process that was not dependent on replication. The F. tularensis LVS ΔripA mutant was significantly impaired in its ability to persist in the lung and in its capacity to disseminate and colonize the liver and spleen in a mouse model of pulmonary tularemia. The RipA protein was expressed during growth in laboratory media and localized to the cytoplasmic membrane. Thus, RipA is a cytoplasmic membrane protein conserved among Francisella species that is required for intracellular replication within the host cell cytoplasm as well as disease progression, dissemination, and virulence. PMID:18765722

Identification of aflatoxin biosynthesis genes by genetic complementation in an Aspergillus flavus mutant lacking the aflatoxin gene cluster.

PubMed Central

Prieto, R; Yousibova, G L; Woloshuk, C P

1996-01-01

Aspergillus flavus mutant strain 649, which has a genomic DNA deletion of at least 120 kb covering the aflatoxin biosynthesis cluster, was transformed with a series of overlapping cosmids that contained DNA harboring the cluster of genes. The mutant phenotype of strain 649 was rescued by transformation with a combination of cosmid clones 5E6, 8B9, and 13B9, indicating that the cluster of genes involved in aflatoxin biosynthesis resides in the 90 kb of A. flavus genomic DNA carried by these clones. Transformants 5E6 and 20B11 and transformants 5E6 and 8B9 accumulated intermediate metabolites of the aflatoxin pathway, which were identified as averufanin and/or averufin, respectively.These data suggest that avf1, which is involved in the conversion of averufin to versiconal hemiacetal acetate, was present in the cosmid 13B9. Deletion analysis of 13B9 located the gene on a 7-kb DNA fragment of the cosmid. Transformants containing cosmid 8B9 converted exogenously supplied O-methylsterigmatocystin to aflatoxin, indicating that the oxidoreductase gene (ord1), which mediates the conversion of O-methylsterigmatocystin to aflatoxin, is carried by this cosmid. The analysis of transformants containing deletions of 8B9 led to the localization of ord1 on a 3.3-kb A. flavus genomic DNA fragment of the cosmid. PMID:8967772

E3L and F1L Gene Functions Modulate the Protective Capacity of Modified Vaccinia Virus Ankara Immunization in Murine Model of Human Smallpox.

PubMed

Volz, Asisa; Jany, Sylvia; Freudenstein, Astrid; Lantermann, Markus; Ludwig, Holger; Sutter, Gerd

2018-01-04

The highly attenuated Modified Vaccinia virus Ankara (MVA) lacks most of the known vaccinia virus (VACV) virulence and immune evasion genes. Today MVA can serve as a safety-tested next-generation smallpox vaccine. Yet, we still need to learn about regulatory gene functions preserved in the MVA genome, such as the apoptosis inhibitor genes F1L and E3L . Here, we tested MVA vaccine preparations on the basis of the deletion mutant viruses MVA-ΔF1L and MVA-ΔE3L for efficacy against ectromelia virus (ECTV) challenge infections in mice. In non-permissive human tissue culture the MVA deletion mutant viruses produced reduced levels of the VACV envelope antigen B5. Upon mousepox challenge at three weeks after vaccination, MVA-ΔF1L and MVA-ΔE3L exhibited reduced protective capacity in comparison to wildtype MVA. Surprisingly, however, all vaccines proved equally protective against a lethal ECTV infection at two days after vaccination. Accordingly, the deletion mutant MVA vaccines induced high levels of virus-specific CD8+ T cells previously shown to be essential for rapidly protective MVA vaccination. These results suggest that inactivation of the anti-apoptotic genes F1L or E3L modulates the protective capacity of MVA vaccination most likely through the induction of distinct orthopoxvirus specific immunity in the absence of these viral regulatory proteins.

Role of mannitol dehydrogenases in osmoprotection of Gluconobacter oxydans.

PubMed

Zahid, Nageena; Deppenmeier, Uwe

2016-12-01

Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP + -dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD + as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450 mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP + -dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP + -dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.

Polygalacturonase gene pgxB in Aspergillus niger is a virulence factor in apple fruit

PubMed Central

Yang, Ying; Yang, Feng; Li, Yan-Hong; Liu, He-Ping; Chen, Xiao-Yan

2017-01-01

Aspergillus niger, a saprophytic fungus, is widely distributed in soil, air and cereals, and can cause postharvest diseases in fruit. Polygalacturonase (PG) is one of the main enzymes in fungal pathogens to degrade plant cell wall. To evaluate whether the deletion of an exo-polygalacturonase gene pgxB would influence fungal pathogenicity to fruit, pgxB gene was deleted in Aspergillus niger MA 70.15 (wild type) via homologous recombination. The ΔpgxB mutant showed similar growth behavior compared with the wild type. Pectin medium induced significant higher expression of all pectinase genes in both wild type and ΔpgxB in comparison to potato dextrose agar medium. However, the ΔpgxB mutant was less virulent on apple fruits as the necrosis diameter caused by ΔpgxB mutant was significantly smaller than that of wild type. Results of quantitive-PCR showed that, in the process of infection in apple fruit, gene expressions of polygalacturonase genes pgaI, pgaII, pgaA, pgaC, pgaD and pgaE were enhanced in ΔpgxB mutant in comparison to wild type. These results prove that, despite the increased gene expression of other polygalacturonase genes in ΔpgxB mutant, the lack of pgxB gene significantly reduced the virulence of A. niger on apple fruit, suggesting that pgxB plays an important role in the infection process on the apple fruit. PMID:28257463

LMOf2365_0442 Encoding for a Fructose Specific PTS Permease IIA May Be Required for Virulence in L. monocytogenes Strain F2365

PubMed Central

Liu, Yanhong; Yoo, Brian B.; Hwang, Cheng-An; Suo, Yujuan; Sheen, Shiowshuh; Khosravi, Parvaneh; Huang, Lihan

2017-01-01

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, which is a major public health concern due to the high fatality rate. LMOf2365_0442, 0443, and 0444 encode for fructose-specific EIIABC components of phosphotransferase transport system (PTS) permease that is responsible for sugar transport. In previous studies, in-frame deletion mutants of a putative fructose-specific PTS permease (LMOf2365_0442, 0443, and 0444) were constructed and analyzed. However, the virulence potential of these deletion mutants has not been studied. In this study, two in vitro methods were used to analyze the virulence potential of these L. monocytogenes deletion mutants. First, invasion assays were used to measure the invasion efficiencies to host cells using the human HT-29 cell line. Second, plaque forming assays were used to measure cell-to-cell spread in host cells. Our results showed that the deletion mutant ΔLMOf2365_0442 had reduced invasion and cell-to-cell spread efficiencies in human cell line compared to the parental strain LMOf2365, indicating that LMOf2365_0442 encoding for a fructose specific PTS permease IIA may be required for virulence in L. monocytogenes strain F2365. In addition, the gene expression levels of 15 virulence and stress-related genes were analyzed in the stationary phase cells of the deletion mutants using RT-PCR assays. Virulence-related gene expression levels were elevated in the deletion mutants ΔLMOf2365_0442-0444 compared to the wild type parental strain LMOf2365, indicating the down-regulation of virulence genes by this PTS permease in L. monocytogenes. Finally, stress-related gene clpC expression levels were also increased in all of the deletion mutants, suggesting the involvement of this PTS permease in stress response. Furthermore, these deletion mutants displayed the same pressure tolerance and the same capacity for biofilm formation compared to the wild-type parental strain LMOf2365. In summary, our findings suggest that the LMOf2365_0442 gene can be used as a potential target to develop inhibitors for new therapeutic and pathogen control strategies for public health. PMID:28900418

Deletion of zmp1 improves Mycobacterium bovis BCG-mediated protection in a guinea pig model of tuberculosis.

PubMed

Sander, Peter; Clark, Simon; Petrera, Agnese; Vilaplana, Cristina; Meuli, Michael; Selchow, Petra; Zelmer, Andrea; Mohanan, Deepa; Andreu, Nuria; Rayner, Emma; Dal Molin, Michael; Bancroft, Gregory J; Johansen, Pål; Cardona, Pere-Joan; Williams, Ann; Böttger, Erik C

2015-03-10

Having demonstrated previously that deletion of zinc metalloprotease zmp1 in Mycobacterium bovis BCG increased immunogenicity of BCG vaccines, we here investigated the protective efficacy of BCG zmp1 deletion mutants in a guinea pig model of tuberculosis infection. zmp1 deletion mutants of BCG provided enhanced protection by reducing the bacterial load of tubercle bacilli in the lungs of infected guinea pigs. The increased efficacy of BCG due to zmp1 deletion was demonstrated in both BCG Pasteur and BCG Denmark indicating that the improved protection by zmp1 deletion is independent from the BCG sub-strain. In addition, unmarked BCG Δzmp1 mutant strains showed a better safety profile in a CB-17 SCID mouse survival model than the parental BCG strains. Together, these results support the further development of BCG Δzmp1 for use in clinical trials. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of Novel Hepatitis B Virus PreS/S-Gene Mutations in a Patient with Occult Hepatitis B Virus Infection

PubMed Central

Xu, Zhihui; Chen, Rongjuan; Si, Lanlan; Lu, Shanshan; Li, Xiaodong; Wang, Shuai; Zhang, Kai; Li, Jin; Han, Juqiang; Xu, Dongping

2016-01-01

Objective The impact of hepatitis B virus (HBV) preS/S-gene mutations on occult HBV infection (OBI) is not fully understood. This study characterized multiple novel HBV preS/S-gene mutants obtained from an OBI patient. Methods PreS/S-gene mutants were analyzed by clonal sequencing. Viral replication and expression were analyzed by transfecting HBV genomic recombinants into HepG2 cells. Results Twenty-one preS/S-gene mutants were cloned from four sequential serum samples, including 13 mutants that were not previously documented: (1) sI/T126V+sG145R; (2) preS1 nt 3014−3198 deletion; (3) preS1 nt 3046−3177 deletion; (4) preS1 nt 3046−3177 deletion+s115−116 “INGTST” insertion; (5) preS1 nt 3046−3177 deletion+s115−116 “INGTST” insertion+sG145R; (6) preS1 nt 3115−3123 deletion+sQ129N; (7) preS1 nt 3115−3123 deletion+s126−127 “RPCMNCTI” insertion; (8) s115−116 “INGTST” insertion; (9) s115−116 “INGTST” insertion+sG145R; (10) s126−127 “RPCMNCTI” insertion; (11) preS1 nt 2848−2862 deletion+preS2 initiation codon M→I; (12) s122−123 “KSTGLCK” insertion+sQ129N; and (13) preS2 initiation codon M→I+s131−133TSM→NST. The proportion of preS1 nt 3046−3177 deletion and preS2 initiation codon M→I+s131−133TSM→NST mutants increased in the viral pool with prolonged disease. The 13 novel OBI-related mutants showed a 51.2−99.9% decrease in HBsAg levels compared with that of the wild type. Additional N-glycosylation-associated mutations, sQ129N and s131−133TSM→NST, but not s126−127 “RPCMNCTI,” greatly attenuated anti-HBs binding to HBsAg. Compared with the wild type, replication and surface antigen promoter II activity of the preS1 nt 3046−3177 deletion mutant decreased by 43.3% and 97.0%, respectively. Conclusion PreS/S-gene mutations may play coordinated roles in the presentation of OBI and might be associated with disease progression. This has implications for HBV diagnosis and vaccine improvement. PMID:27182775

A Population of Deletion Mutants and an Integrated Mapping and Exome-seq Pipeline for Gene Discovery in Maize

PubMed Central

Jia, Shangang; Li, Aixia; Morton, Kyla; Avoles-Kianian, Penny; Kianian, Shahryar F.; Zhang, Chi; Holding, David

2016-01-01

To better understand maize endosperm filling and maturation, we used γ-irradiation of the B73 maize reference line to generate mutants with opaque endosperm and reduced kernel fill phenotypes, and created a population of 1788 lines including 39 Mo17 × F2s showing stable, segregating, and viable kernel phenotypes. For molecular characterization of the mutants, we developed a novel functional genomics platform that combined bulked segregant RNA and exome sequencing (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. To exemplify the utility of the mutants and provide proof-of-concept for the bioinformatics platform, we present detailed characterization of line 937, an opaque mutant harboring a 6203 bp in-frame deletion covering six exons within the Opaque-1 gene. In addition, we describe mutant line 146 which contains a 4.8 kb intragene deletion within the Sugary-1 gene and line 916 in which an 8.6 kb deletion knocks out a Cyclin A2 gene. The publically available algorithm developed in this work improves the identification of causative deletions and its corresponding gaps within mapping peaks. This study demonstrates the utility of γ-irradiation for forward genetics in large nondense genomes such as maize since deletions often affect single genes. Furthermore, we show how this classical mutagenesis method becomes applicable for functional genomics when combined with state-of-the-art genomics tools. PMID:27261000

Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains.

PubMed

Armas, Pablo; Agüero, Tristán H; Borgognone, Mariana; Aybar, Manuel J; Calcaterra, Nora B

2008-10-17

Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.

The Actinomyces oris Type 2 Fimbrial Shaft FimA Mediates Coaggregation with Oral Streptococci, Adherence to RBC and Biofilm Development

PubMed Central

Mishra, Arunima; Wu, Chenggang; Yang, Jinghua; Cisar, John O.; Das, Asis; Ton-That, Hung

2010-01-01

Interbacterial interactions between oral streptococci and actinomyces and their adherence to tooth surface and the associated host cells are key early events that promote development of the complex oral biofilm referred to as dental plaque. These interactions depend largely on a lectin-like activity associated with the Actinomyces oris type 2 fimbria, a surface structure assembled by sortase (SrtC2)-dependent polymerization of the shaft and tip fimbrillins, FimA and FimB, respectively. To dissect the function of specific fimbrillins in various adherence processes, we have developed a convenient new technology for generating unmarked deletion mutants of A. oris. Here, we show that the fimB mutant, which produced type 2 fimbriae composed only of FimA, like the wild type coaggregated strongly with receptor-bearing streptococci, agglutinated with sialidase-treated RBC, and formed monospecies biofilm. In contrast, the fimA and srtC2 mutants lacked type 2 fimbriae and were non-adherent in each of these assays. Plasmidbased expression of the deleted gene in respective mutants restored adherence to wild-type levels. These findings uncover the importance of the lectin-like activity of the polymeric FimA shaft rather than the tip. The multivalent adhesive function of FimA makes it an ideal molecule for exploring novel intervention strategies to control plaque biofilm formation. PMID:20545853

Deletion of vanilloid receptor (TRPV1) in mice alters behavioral effects of ethanol

PubMed Central

Blednov, Y.A.; Harris, R.A.

2009-01-01

The vanilloid receptor TRPV1 is activated by ethanol and this may be important for some of the central and peripheral actions of ethanol. To determine if this receptor has a role in ethanol-mediated behaviors, we studied null mutant mice in which the Trpv1 gene was deleted. Mice lacking this gene showed significantly higher preference for ethanol and consumed more ethanol in a two-bottle choice test as compared with wild type littermates. Null mutant mice showed shorter duration of loss of righting reflex induced by low doses of ethanol (3.2 and 3.4 g/kg) and faster recovery from motor incoordination induced by ethanol (2 g/kg). However, there were no differences between null mutant and wild type mice in severity of ethanol-induced acute withdrawal (4 g/kg) or conditioned taste aversion to ethanol (2.5 g/kg). Two behavioral phenotypes (decreased sensitivity to ethanol-induced sedation and faster recovery from ethanol-induced motor incoordination) seen in null mutant mice were reproduced in wild type mice by injection of a TRPV1 antagonist, capsazepine (10 mg/kg). These two ethanol behaviors were changed in the opposite direction after injection of capsaicin, a selective TRPV1 agonist, in wild type mice. The studies provide the first evidence that TRPV1 is important for specific behavioral actions of ethanol. PMID:19705551

Deletion of a dehydratase important for intracellular growth and cording renders rough Mycobacterium abscessus avirulent

PubMed Central

Halloum, Iman; Carrère-Kremer, Séverine; Blaise, Mickael; Viljoen, Albertus; Bernut, Audrey; Le Moigne, Vincent; Vilchèze, Catherine; Guérardel, Yann; Lutfalla, Georges; Herrmann, Jean-Louis; Jacobs, William R.; Kremer, Laurent

2016-01-01

Mycobacterium abscessus (Mabs) is a rapidly growing Mycobacterium and an emerging pathogen in humans. Transitioning from a smooth (S) high-glycopeptidolipid (GPL) producer to a rough (R) low-GPL producer is associated with increased virulence in zebrafish, which involves the formation of massive serpentine cords, abscesses, and rapid larval death. Generating a cord-deficient Mabs mutant would allow us to address the contribution of cording in the physiopathological signs of the R variant. Herein, a deletion mutant of MAB_4780, encoding a dehydratase, distinct from the β-hydroxyacyl-ACP dehydratase HadABC complex, was constructed in the R morphotype. This mutant exhibited an alteration of the mycolic acid composition and a pronounced defect in cording. This correlated with an extremely attenuated phenotype not only in wild-type but also in immunocompromised zebrafish embryos lacking either macrophages or neutrophils. The abolition of granuloma formation in embryos infected with the dehydratase mutant was associated with a failure to replicate in macrophages, presumably due to limited inhibition of the phagolysosomal fusion. Overall, these results indicate that MAB_4780 is required for Mabs to successfully establish acute and lethal infections. Therefore, targeting MAB_4780 may represent an attractive antivirulence strategy to control Mabs infections, refractory to most standard chemotherapeutic interventions. The combination of a dehydratase assay with a high-resolution crystal structure of MAB_4780 opens the way to identify such specific inhibitors. PMID:27385830

Reduced alcohol consumption in mice lacking preprodynorphin.

PubMed Central

Blednov, Yuri A.; Walker, Danielle; Martinez, Marni; Harris., R. Adron

2007-01-01

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the κ-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 hours) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest thath this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability). PMID:17307643

Reduced alcohol consumption in mice lacking preprodynorphin.

PubMed

Blednov, Yuri A; Walker, Danielle; Martinez, Marni; Harris, R Adron

2006-10-01

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the kappa-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 h) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin, but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild-type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference, or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest that this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability).

Evidence for transceptor function of cellodextrin transporters in Neurospora crassa.

PubMed

Znameroski, Elizabeth A; Li, Xin; Tsai, Jordan C; Galazka, Jonathan M; Glass, N Louise; Cate, Jamie H D

2014-01-31

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as "transceptors" with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.

Modulating the folding of P-glycoprotein and cystic fibrosis transmembrane conductance regulator truncation mutants with pharmacological chaperones.

PubMed

Wang, Ying; Loo, Tip W; Bartlett, M Claire; Clarke, David M

2007-03-01

Cystic fibrosis transmembrane conductance regulator (CFTR) and P-glycoprotein (P-gp) are ATP-binding cassette (ABC) transporters that have two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Defective folding of CFTR lacking phenylalanine 508 (DeltaPhe508) in NBD1 is the most common cause of cystic fibrosis. The Phe508 position seems to be universally important in ABC transporters because deletion of the equivalent residue (Tyr490) in P-gp also inhibits maturation of the protein. The pharmacological chaperone VRT-325 can repair the DeltaPhe508-type folding defects in P-gp or CFTR. VRT-325 may repair the folding defects by promoting dimerization of the two NBDs or by promoting folding of the TMDs. To distinguish between these two mechanisms, we tested the ability of VRT-325 to promote folding of truncation mutants lacking one or both NBDs. Sensitivity to glycosidases was used as an indirect indicator of folding. It was found that VRT-325 could promote maturation of truncation mutants lacking NBD2. Truncation mutants of CFTR or P-gp lacking both NBDs showed deficiencies in core-glycosylation that could be partially reversed by carrying out expression in the presence of VRT-325. The results show that dimerization of the two NBDs to form a "nucleotide-sandwich" structure or NBD interactions with the TMDs are not essential for VRT-325 enhancement of folding. Instead, VRT-325 can promote folding of the TMDs alone. The ability of VRT-325 to promote core-glycosylation of the NBD-less truncation mutants suggests that one mechanism whereby the compound enhances folding is by promoting proper insertion of TM segments attached to the glycosylated loops so that they adopt an orientation favorable for glycosylation.

Survival of a bacterioferritin deletion mutant of Brucella melitensis 16M in human monocyte-derived macrophages.

PubMed Central

Denoel, P A; Crawford, R M; Zygmunt, M S; Tibor, A; Weynants, V E; Godfroid, F; Hoover, D L; Letesson, J J

1997-01-01

A bacterioferritin (BFR) deletion mutant of Brucella melitensis 16M was generated by gene replacement. The deletion was complemented with a broad-host-range vector carrying the wild-type bfr gene, pBBR-bfr. The survival and growth of the mutant, B. melitensis PAD 2-78, were similar to those of its parental strain in human monocyte-derived macrophages (MDM). These results suggest that BFR is not essential for the intracellular survival of B. melitensis in human MDM. PMID:9317046

Phase I study of combined therapy with vorinostat and gefitinib to treat BIM deletion polymorphism-associated resistance in EGFR-mutant lung cancer (VICTROY-J): a study protocol.

PubMed

Takeuchi, Shinji; Yoshimura, Kenichi; Fujiwara, Tadami; Ando, Masahiko; Shimizu, Shinobu; Nagase, Katsuhiko; Hasegawa, Yoshinori; Takahashi, Toshiaki; Katakami, Nobuyuki; Inoue, Akira; Yano, Seiji

2017-01-01

The BIM deletion polymorphism is reported to be associated with poor outcomes of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) treated with EGFR-TKIs, including gefitinib. We have shown that a histone deacetylase inhibitor, vorinostat, can epigenetically restore BIM function and apoptosis sensitivity to EGFR-TKIs in EGFR-mutant NSCLC cells with BIM deletion polymorphisms. The purpose of this study is to determine the feasibility of combined treatment of vorinostat with gefitinib in BIM deletion polymorphism positive EGFR-mutant NSCLC patients. BIM deletion polymorphism positive EGFR-mutant NSCLC patients treated with at least one EGFR-TKI and one regimen of chemotherapy are being recruited to this study. Vorinostat (200-400 mg) will be administered orally once daily on days 1-7, and gefitinib 250 mg orally once daily on days 1-14. With a fixed dose of gefitinib, the dose of vorinostat will be escalated following a conventional 3+3 design. The primary endpoint is to define the maximum tolerated dose (MTD) of vorinostat combined with 250 mg of gefitinib. This is the first phase I study of combined therapy with vorinostat and gefitinib for NSCLC patients double selected for an EGFR mutation and BIM deletion polymorphism. J. Med. Invest. 64: 321-325, August, 2017.

[Changes of biological behavioral of E. coli K1 after ppk1 gene deletion].

PubMed

Peng, Liang; Pan, Jiayun; Luo, Su; Yang, Zhenghui; Huang, Mufang; Cao, Hong

2014-06-01

To study the changes in biological behaviors of meningitis E. coli K1 strain E44 after deletion of polyphosphate kinase 1 (ppk1) gene and explore the role of ppk1 in the pathogenesis of E. coli K1-induced meningitis. The wild-type strain E. coli K1 and ppk1 deletion mutant were exposed to heat at 56 degrees celsius; for 6 min, and their survival rates were determined. The adhesion and invasion of the bacteria to human brain microvascular endothelial cells (HBMECs) were observed using electron microscopy and quantitative tests. HBMECs were co-incubated with wild-type strain or ppk1 deletion mutant, and the cytoskeleton rearrangement was observed under laser scanning confocal microscope. The survival rate of the ppk1 deletion mutant was significantly lower than that of the wild-type strain after heat exposure. The ppk1 deletion mutant also showed lowered cell adhesion and invasion abilities and weakened ability to induce cytoskeleton rearrangement in HBMECs. ppk1 gene is important for E.coli K1 for heat resistance, cell adhesion and invasion, and for inducing cytoskeletal rearrangement in HBMECs.

Co-ordination of NDH and Cup proteins in CO2 uptake in cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Han, Xunling; Sun, Nan; Xu, Min; Mi, Hualing

2017-06-01

High and low affinity CO2-uptake systems containing CupA (NDH-1MS) and CupB (NDH-1MS'), respectively, have been identified in Synechocystis sp. PCC 6803, but it is yet unknown how the complexes function in CO2 uptake. In this work, we found that deletion of cupB significantly lowered the growth of cells, and deletion of both cupA and cupB seriously suppressed the growth below pH 7.0 even under 3% CO2. The rate of photosynthetic oxygen evolution was decreased slightly by deletion of cupA but significantly by deletion of cupB and more severely by deletion of both cupA and cupB, especially in response to changed pH conditions under 3% CO2. Furthermore, we found that assembly of CupB into NDH-1MS' was dependent on NdhD4 and NdhF4. NDH-1MS' was not affected in the NDH-1MS-degradation mutant and NDH-1MS was not affected in the NDH-1MS'-degradation mutants, indicating the existence of independent CO2-uptake systems under high CO2 conditions. The light-induced proton gradient across thylakoid membranes was significantly inhibited in ndhD-deletion mutants, suggesting that NdhDs functions in proton pumping. The carbonic anhydrase activity was suppressed partly in the cupA- or cupB-deletion mutant but severely in the mutant with both cupA and cupB deletion, indicating that CupA and CupB function in conversion of CO2 to HCO3-. In turn, deletion of cup genes lowered the transthylakoid membrane proton gradient and deletion of ndhDs decreased the CO2 hydration. Our results suggest that NDH-1M provides an alkaline region to activate Cup proteins involved in CO2 uptake. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation.

PubMed

Carrión, Javier; Folgueira, Cristina; Soto, Manuel; Fresno, Manuel; Requena, Jose M

2011-07-27

Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

A mechanistic model of tau amyloid aggregation based on direct observation of oligomers

NASA Astrophysics Data System (ADS)

Shammas, Sarah L.; Garcia, Gonzalo A.; Kumar, Satish; Kjaergaard, Magnus; Horrocks, Mathew H.; Shivji, Nadia; Mandelkow, Eva; Knowles, Tuomas P. J.; Mandelkow, Eckhard; Klenerman, David

2015-04-01

Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate constants, have been difficult to determine due to lack of suitable methods to identify and follow the low concentration of oligomers over time. Here we use single-molecule fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L. Our kinetic analysis reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quantitatively determine how these mutations alter the aggregation energy landscape.

Deletion of the N-terminal Domain (NTD) Alters the Ethanol Inhibition of NMDA Receptors in a Subunit-Dependent Manner

PubMed Central

Smothers, C. Thetford; Jin, Chun; Woodward, John J.

2013-01-01

Background Ethanol inhibition of NMDA receptors is poorly understood due in part to the organizational complexity of the receptor that provides ample locations for sites of action. Among these the N-terminal domain of NMDA receptor subunits contains binding sites for a variety of modulatory agents including zinc, protons and GluN2B selective antagonists such as ifenprodil or Ro-25–6981. Ethanol inhibition of neuronal NMDA receptors expressed in some brain areas has been reported to be occluded by the presence of ifenprodil or similar compounds suggesting that the N-terminal domain may be important in regulating the ethanol sensitivity of NMDA receptors. Methods Wild-type GluN1 and GluN2 subunits and those in which the coding sequence for the N-terminal domain was deleted were expressed in HEK293 cells. Whole-cell voltage-clamp recording was used to assess ethanol inhibition of wild-type and mutant receptors lacking the N-terminal domain. Results As compared to wild-type GluN1/GluN2A receptors, ethanol inhibition was slightly greater in cells expressing GluN2A subunits lacking the N-terminal domain. In contrast, GluN2B N-terminal deletion mutants showed normal ethanol inhibition while those lacking the N-terminal domain in both GluN1 and GluN2B subunits had decreased ethanol inhibition as compared to wild-type receptors. N-terminal domain lacking GluN2B receptors were insensitive to ifenprodil but retained normal sensitivity to ethanol. Conclusions These findings indicate that the N-terminal domain modestly influences the ethanol sensitivity of NMDA receptors in a subunit-dependent manner. They also show that ifenprodil’s actions on GluN2B containing receptors can be dissociated from those of ethanol. These results suggest that while the N-terminal domain is not a primary site of action for ethanol on NMDA receptors, it likely affects sensitivity via actions on intrinsic channel properties. PMID:23905549

An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geller, A.I.; Keyomarsi, K.; Bryan, J.

1990-11-01

The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli {beta}-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; tsmore » mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses {beta}-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system.« less

90K Glycoprotein Promotes Degradation of Mutant β-Catenin Lacking the ISGylation or Phosphorylation Sites in the N-terminus.

PubMed

Park, So-Yeon; Yoon, Somy; Kim, Hangun; Kim, Kyung Keun

2016-10-01

β-Catenin is a major transducer of the Wnt signaling pathway, which is aberrantly expressed in colorectal and other cancers. Previously, we showed that β-catenin is downregulated by the 90K glycoprotein via ISGylation-dependent degradation. However, the further mechanisms of β-catenin degradation by 90K-mediated ISGylation pathway were not investigated. This study aimed to identify the β-catenin domain responsible for the action of 90K and to compare the mechanism of 90K on β-catenin degradation with phosphorylation-dependent ubiquitinational degradation of β-catenin. The deletion mutants of β-catenin lacking N- or C-terminal domain or mutating the N-terminal lysine or nonlysine residue were employed to delineate the characteristics of β-catenin degradation by 90K-mediated ISGylation pathway. 90K induced Herc5 and ISG15 expression and reduced β-catenin levels in HeLa and CSC221 cells. The N-terminus of β-catenin is required for 90K-induced β-catenin degradation, but the N-terminus of β-catenin is not essential for interaction with Herc5. However, substituting lysine residues in the N-terminus of β-catenin with arginine or deleting serine or threonine residue containing domains from the N-terminus does not affect 90K-induced β-catenin degradation, indicating that the N-terminal 86 amino acids of β-catenin are crucial for 90K-mediated ISGylation/degradation of β-catenin in which the responsible lysine or nonlysine residues were not identified. Our present results highlight the action of 90K on promoting degradation of mutant β-catenin lacking the phosphorylation sites in the N-terminus. It provides further insights into the discrete pathway downregulating the stabilized β-catenin via acquiring mutations at the serine/threonine residues in the N-terminus. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Expression of the Flp proteins by Haemophilus ducreyi is necessary for virulence in human volunteers.

PubMed

Janowicz, Diane M; Cooney, Sean A; Walsh, Jessica; Baker, Beth; Katz, Barry P; Fortney, Kate R; Zwickl, Beth W; Ellinger, Sheila; Munson, Robert S

2011-09-22

Haemophilus ducreyi, the causative agent of the sexually transmitted disease chancroid, contains a flp (fimbria like protein) operon that encodes proteins predicted to contribute to adherence and pathogenesis. H. ducreyi mutants that lack expression of Flp1 and Flp2 or TadA, which has homology to NTPases of type IV secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts (HFF). A tadA mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a flp1flp2 mutant is virulent in rabbits. Whether a flp deletion mutant would cause disease in humans is not clear. We constructed 35000HPΔflp1-3, a deletion mutant that lacks expression of all three Flp proteins but has an intact tad secretion system. 35000HPΔflp1-3 was impaired in its ability to form microcolonies and to attach to HFF in vitro when compared to its parent (35000HP). Complementation of the mutant with flp1-3 in trans restored the parental phenotype. To test whether expression of Flp1-3 was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000HP (ranging from 54 to 67 CFU) on one arm and three doses of 35000HPΔflp1-3 (ranging from 63 to 961 CFU) on the other arm. The overall papule formation rate for the parent was 80% (95% confidence interval, CI, 55.2%-99.9%) and for the mutant was 70.0% (95% CI, 50.5%-89.5%) (P = 0.52). Mutant papules were significantly smaller (mean, 11.2 mm2) than were parent papules (21.8 mm2) 24 h after inoculation (P = 0.018). The overall pustule formation rates were 46.7% (95% CI 23.7-69.7%) at 30 parent sites and 6.7% (95% CI, 0.1-19.1%) at 30 mutant sites (P = 0.001). These data suggest that production and secretion of the Flp proteins contributes to microcolony formation and attachment to HFF cells in vitro. Expression of flp1-3 is also necessary for H. ducreyi to initiate disease and progress to pustule formation in humans. Future studies will focus on how Flp proteins contribute to microcolony formation and attachment in vivo. © 2011 Janowicz et al; licensee BioMed Central Ltd.

Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression.

PubMed

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2014-01-01

In filamentous fungi, the expression of secretory glycoside hydrolase encoding genes, such as those for amylases, cellulases, and xylanases, is generally repressed in the presence of glucose. CreA and CreB have been observed to be regulating factors for carbon catabolite repression. In this study, we generated single and double deletion creA and/or creB mutants in Aspergillus oryzae. The α-amylase activities of each strain were compared under various culture conditions. For the wild-type strain, mRNA levels of α-amylase were markedly decreased in the later stage of submerged culture under inducing conditions, whereas this reduced expression was not observed for single creA and double creA/creB deletion mutants. In addition, α-amylase activity of the wild-type strain was reduced in submerged culture containing high concentrations of inducing sugars, whereas all constructed mutants showed higher α-amylase activities. In particular, the α-amylase activity of the double deletion mutant in a medium containing 5% starch was >10-fold higher than that of the wild-type strain under the same culture conditions. In solid-state cultures using wheat bran as a substrate, the α-amylase activities of single creA and double deletion mutants were >2-fold higher than that of the wild-type strain. These results suggested that deleting both creA and creB resulted in dramatic improvements in the production of secretory glycoside hydrolases in filamentous fungi.

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

PubMed Central

Yang, Hee-Jeong; Bogomolnaya, Lydia M.; Elfenbein, Johanna R.; Endicott-Yazdani, Tiana; Reynolds, M. Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin

2016-01-01

Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

A selective EP4 PGE2 receptor agonist alleviates disease in a new mouse model of X-linked nephrogenic diabetes insipidus

PubMed Central

Li, Jian Hua; Chou, Chung-Lin; Li, Bo; Gavrilova, Oksana; Eisner, Christoph; Schnermann, Jürgen; Anderson, Stasia A.; Deng, Chu-Xia; Knepper, Mark A.; Wess, Jürgen

2009-01-01

X-linked nephrogenic diabetes insipidus (XNDI) is a severe kidney disease caused by inactivating mutations in the V2 vasopressin receptor (V2R) gene that result in the loss of renal urine-concentrating ability. At present, no specific pharmacological therapy has been developed for XNDI, primarily due to the lack of suitable animal models. To develop what we believe to be the first viable animal model of XNDI, we generated mice in which the V2R gene could be conditionally deleted during adulthood by administration of 4-OH-tamoxifen. Radioligand-binding studies confirmed the lack of V2R-binding sites in kidneys following 4-OH-tamoxifen treatment, and further analysis indicated that upon V2R deletion, adult mice displayed all characteristic symptoms of XNDI, including polyuria, polydipsia, and resistance to the antidiuretic actions of vasopressin. Gene expression analysis suggested that activation of renal EP4 PGE2 receptors might compensate for the lack of renal V2R activity in XNDI mice. Strikingly, both acute and chronic treatment of the mutant mice with a selective EP4 receptor agonist greatly reduced all major manifestations of XNDI, including changes in renal morphology. These physiological improvements were most likely due to a direct action on EP4 receptors expressed on collecting duct cells. These findings illustrate the usefulness of the newly generated V2R mutant mice for elucidating and testing new strategies for the potential treatment of humans with XNDI. PMID:19729836

Motor impairments, striatal degeneration, and altered dopamine-glutamate interplay in mice lacking PSD-95.

PubMed

Zhang, Jingping; Saur, Taixiang; Duke, Angela N; Grant, Seth G N; Platt, Donna M; Rowlett, James K; Isacson, Ole; Yao, Wei-Dong

2014-01-01

Excessive activation of the N-methyl-d-aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32-positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor-mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more susceptible to NMDA excitotoxicity, causing neuronal damage and neurological impairments. Understanding PSD-95-dependent neuroprotective mechanisms may help elucidate processes underlying neurodegeneration in HD and other neurological disorders.

Genetics of the Blue Light-Dependent Signal Cascade That Controls Phototaxis in the Cyanobacterium Synechocystis sp. PCC6803.

PubMed

Sugimoto, Yuki; Nakamura, Hiroshi; Ren, Shukun; Hori, Koichi; Masuda, Shinji

2017-03-01

The Synechocystis sp. PCC6803 can move on a solid surface in response to light, a phenomenon called phototaxis. Although many of the photoreceptors involved in phototaxis have been identified, the mechanisms that regulate directional motility of Synechocystis are not well understood. Previous studies showed that a mutant lacking the blue light-using flavin (BLUF) photoreceptor PixD exhibits negative phototaxis under conditions where the wild type responds positively. PixD interacts with the pseudo-response regulator-like protein PixE in a light-dependent manner, suggesting that this intermolecular interaction is important for phototaxis regulation, although genetic evidence has been lacking. To gain further insight into phototaxis regulation by PixD-PixE signaling, we constructed the deletion mutants ΔPixE and ΔPixD-ΔPixE, and characterized their phenotypes, which matched those of the wild type (positive phototaxis). Because ΔPixD exhibited negative phototaxis, PixE must function downstream of PixD. Under intense blue light (>100 μmol m-2 s-1; 470 nm) the wild type exhibited negative phototaxis, but ΔPixD-PixE exhibited positive phototaxis toward low-intensity blue light (∼0.8 μmol m-2 s-1; 470 nm). These results suggest that an unknown light-sensing system(s), that is necessary for directional cell movement, can be activated by low-intensity blue light; on the other hand, PixD needs high-intensity blue light to be activated. We also isolated spontaneous mutants that compensated for the pixE deletion. Genome-wide sequencing of the mutants revealed that the uncharacterized gene sll2003 regulates positive and negative phototaxis in response to light intensity. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Dual Function of a Tip Fimbrillin of Actinomyces in Fimbrial Assembly and Receptor Binding▿

PubMed Central

Wu, Chenggang; Mishra, Arunima; Yang, Jinghua; Cisar, John O.; Das, Asis; Ton-That, Hung

2011-01-01

Interaction of Actinomyces oris with salivary proline-rich proteins (PRPs), which serve as fimbrial receptors, involves type 1 fimbriae. Encoded by the gene locus fimQ-fimP-srtC1, the type 1 fimbria is comprised of the fimbrial shaft FimP and the tip fimbrillin FimQ. Fimbrial polymerization requires the fimbria-specific sortase SrtC1, which catalyzes covalent linkage of fimbrial subunits. Using genetics, biochemical methods, and electron microscopy, we provide evidence that the tip fimbrillin, FimQ, is involved in fimbrial assembly and interaction with PRPs. Specifically, while deletion of fimP completely abolished the type 1 fimbrial structures, surface display of monomeric FimQ was not affected by this mutation. Surprisingly, deletion of fimQ significantly reduced surface assembly of the type 1 fimbriae. This defect was rescued by recombinant FimQ ectopically expressed from a plasmid. In agreement with the role of type 1 fimbriae in binding to PRPs, aggregation of A. oris with PRP-coated beads was abrogated in cells lacking srtC1 or fimP. This aggregation defect of the ΔfimP mutant was mainly due to significant reduction of FimQ on the bacterial surface, as the aggregation was not observed in a strain lacking fimQ. Increasing expression of FimQ in the ΔfimP mutant enhanced aggregation, while overexpression of FimP in the ΔfimQ mutant did not. Furthermore, recombinant FimQ, not FimP, bound surface-associated PRPs in a dose-dependent manner. Thus, not only does FimQ function as the major adhesin of the type 1 fimbriae, it also plays an important role in fimbrial assembly. PMID:21531799

Contribution of NMDA receptor hypofunction in prefrontal and cortical excitatory neurons to schizophrenia-like phenotypes.

PubMed

Rompala, Gregory R; Zsiros, Veronika; Zhang, Shuqin; Kolata, Stefan M; Nakazawa, Kazu

2013-01-01

Pharmacological and genetic studies support a role for NMDA receptor (NMDAR) hypofunction in the etiology of schizophrenia. We have previously demonstrated that NMDAR obligatory subunit 1 (GluN1) deletion in corticolimbic interneurons during early postnatal development is sufficient to confer schizophrenia-like phenotypes in mice. However, the consequence of NMDAR hypofunction in cortical excitatory neurons is not well delineated. Here, we characterize a conditional knockout mouse strain (CtxGluN1 KO mice), in which postnatal GluN1 deletion is largely confined to the excitatory neurons in layer II/III of the medial prefrontal cortex and sensory cortices, as evidenced by the lack of GluN1 mRNA expression in in situ hybridization immunocytochemistry as well as the lack of NMDA currents with in vitro recordings. Mutants were impaired in prepulse inhibition of the auditory startle reflex as well as object-based short-term memory. However, they did not exhibit impairments in additional hallmarks of schizophrenia-like phenotypes (e.g. spatial working memory, social behavior, saccharine preference, novelty and amphetamine-induced hyperlocomotion, and anxiety-related behavior). Furthermore, upon administration of the NMDA receptor antagonist, MK-801, there were no differences in locomotor activity versus controls. The mutant mice also showed negligible levels of reactive oxygen species production following chronic social isolation, and recording of miniature-EPSC/IPSCs from layer II/III excitatory neurons in medial prefrontal cortex suggested no alteration in GABAergic activity. All together, the mutant mice displayed cognitive deficits in the absence of additional behavioral or cellular phenotypes reflecting schizophrenia pathophysiology. Thus, NMDAR hypofunction in prefrontal and cortical excitatory neurons may recapitulate only a cognitive aspect of human schizophrenia symptoms.

Thioredoxin A Is Essential for Motility and Contributes to Host Infection of Listeria monocytogenes via Redox Interactions.

PubMed

Cheng, Changyong; Dong, Zhimei; Han, Xiao; Wang, Hang; Jiang, Li; Sun, Jing; Yang, Yongchun; Ma, Tiantian; Shao, Chunyan; Wang, Xiaodu; Chen, Zhongwei; Fang, Weihuan; Freitag, Nancy E; Huang, Huarong; Song, Houhui

2017-01-01

Microbes employ the thioredoxin system to defend against oxidative stress and ensure correct disulfide bonding to maintain protein function. Listeria monocytogenes has been shown to encode a putative thioredoxin, TrxA, but its biological roles and underlying mechanisms remain unknown. Here, we showed that expression of L. monocytogenes TrxA is significantly induced in bacteria treated with the thiol-specific oxidizing agent, diamide. Deletion of trxA markedly compromised tolerance of the pathogen to diamide, and mainly impaired early stages of infection in human intestinal epithelial Caco-2 cells. In addition, most trxA mutant bacteria were not associated with polymerized actin, and the rare bacteria that were associated with polymerized actin displayed very short tails or clouds during infection. Deletion or constitutive overexpression of TrxA, which was regulated by SigH, severely attenuated the virulence of the pathogen. Transcriptome analysis of L. monocytogenes revealed over 270 genes that were differentially transcribed in the Δ trxA mutant compared to the wild-type, especially for the virulence-associated genes plcA, mpl, hly, actA , and plcB . Particularly, deletion of TrxA completely reduced LLO expression, and thereby led to a thoroughly impaired hemolytic activity. Expression of these virulence factors are positively regulated by the master regulator PrfA that was found here to use TrxA to maintain its reduced forms for activation. Interestingly, the trxA deletion mutant completely lacked flagella and was non-motile. We further confirmed that this deficiency is attributable to TrxA in maintaining the reduced intracellular monomer status of MogR, the key regulator for flagellar formation, to ensure correct dimerization. In summary, we demonstrated for the first time that L. monocytogenes thioredoxin A as a vital cellular reductase is essential for maintaining a highly reducing environment in the bacterial cytosol, which provides a favorable condition for protein folding and activation, and therefore contributes to bacterial virulence and motility.

The NH2 terminus regulates voltage-dependent gating of CALHM ion channels.

PubMed

Tanis, Jessica E; Ma, Zhongming; Foskett, J Kevin

2017-08-01

Calcium homeostasis modulator protein-1 (CALHM1) and its Caenorhabditis elegans (ce) homolog, CLHM-1, belong to a new family of physiologically important ion channels that are regulated by voltage and extracellular Ca 2+ (Ca 2+ o ) but lack a canonical voltage-sensing domain. Consequently, the intrinsic voltage-dependent gating mechanisms for CALHM channels are unknown. Here, we performed voltage-clamp experiments on ceCLHM-1 chimeric, deletion, insertion, and point mutants to assess the role of the NH 2 terminus (NT) in CALHM channel gating. Analyses of chimeric channels in which the ceCLHM-1 and human (h)CALHM1 NH 2 termini were interchanged showed that the hCALHM1 NT destabilized channel-closed states, whereas the ceCLHM-1 NT had a stabilizing effect. In the absence of Ca 2+ o , deletion of up to eight amino acids from the ceCLHM-1 NT caused a hyperpolarizing shift in the conductance-voltage relationship with little effect on voltage-dependent slope. However, deletion of nine or more amino acids decreased voltage dependence and induced a residual conductance at hyperpolarized voltages. Insertion of amino acids into the NH 2 -terminal helix also decreased voltage dependence but did not prevent channel closure. Mutation of ceCLHM-1 valine 9 and glutamine 13 altered half-maximal activation and voltage dependence, respectively, in 0 Ca 2+ In 2 mM Ca 2+ o , ceCLHM-1 NH 2 -terminal deletion and point mutant channels closed completely at hyperpolarized voltages with apparent affinity for Ca 2+ o indistinguishable from wild-type ceCLHM-1, although the ceCLHM-1 valine 9 mutant exhibited an altered conductance-voltage relationship and kinetics. We conclude that the NT plays critical roles modulating voltage dependence and stabilizing the closed states of CALHM channels. Copyright © 2017 the American Physiological Society.

Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15.

PubMed

Brandt, Artur; Löhers, Katharina; Beier, Manfred; Leube, Barbara; de Torres, Carmen; Mora, Jaume; Arora, Parineeta; Jat, Parmjit S; Royer-Pokora, Brigitte

2016-01-01

We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells.

Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15

PubMed Central

Brandt, Artur; Löhers, Katharina; Beier, Manfred; Leube, Barbara; de Torres, Carmen; Mora, Jaume; Arora, Parineeta; Jat, Parmjit S.; Royer-Pokora, Brigitte

2016-01-01

We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD) limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD) 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT) in conjunction with a novel thermolabile mutant (U19dl89-97tsA58) of SV40 large T antigen (LT). This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells. PMID:27213811

Fine structure of OXI1, the mitochondrial gene coding for subunit II of yeast cytochrome c oxidase.

PubMed

Weiss-Brummer, B; Guba, R; Haid, A; Schweyen, R J

1979-12-01

Genetic and biochemical studies have been performed with 110 mutants which are defective in cytochrome a·a3 and map in the regions on mit DNA previously designated OXI1 and OXI2. With 88 mutations allocated to OXI1 fine structure mapping was achieved by the analysis of rho (-) deletions. The order of six groups of mutational sites (A 1, A2, B 1, B2, C 1, C2) thus determined was confirmed by oxi i x oxi j recombination analysis.Analysis of mitochondrially translated polypeptides of oxil mutants by SDS-polyacrylamide electrophoresis reveals three classes of mutant patterns: i) similar to wild-tpye (19 mutants); ii) lacking SU II of cytochrome c oxidase (53 mutants); iii) lacking this subunit and exhibiting a single new polypeptide of lower Mr (16 mutants). Mutations of each of these classes are scattered over the OXI1 region without any detectable clustering; this is consistent with the assumption that all oxil mutations studied are within the same gene.New polypeptides observed in oxil mutants of class iii) vary in Mr in the range from 10,500 to 33,000. Those of Mr 17,000 to 33,000 are shown to be antigenically related to subunit II of cytochrome c oxidase. Colinearity is established between the series of new polypeptides of Mr values increasing from 10,500 to 31,500 and the order of the respective mutational sites on the map, e.g. mutations mapping in A 1 generate the smallest and mutations mapping in C2 the largest mutant fragments.From these data we conclude that i) all mutations allocated to the OXI1 region are in the same gene; ii) this gene codes for subunit II of cytochrome c oxidase; iii) the direction of translation is from CAP to 0X12. Out of 19 mutants allocated to OXI2 three exhibit a new polypeptide; these and all the other oxi2 mutants lack subunit III of cytochrome oxidase. This result provides preliminary evidence that the OXI2 region harbours the structural gene for this subunit III.

Lactose-induced cell death of beta-galactosidase mutants in Kluyveromyces lactis.

PubMed

Lodi, Tiziana; Donnini, Claudia

2005-05-01

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.

Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

NASA Astrophysics Data System (ADS)

Parra-Belky, Karlett

2002-11-01

A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

Wave2 activates serum response element via its VCA region and functions downstream of Rac.

PubMed

Ishiguro, Kazuhiro; Cao, Zhifang; Ilasca, Marco Lopez; Ando, Takafumi; Xavier, Ramnik

2004-12-10

WAVE2 is a member of the WASP/WAVE family of protein effectors of actin reorganization and cell movement. In this report, we demonstrate that WAVE2 overexpression induces serum response element (SRE) activation through serum response factor. A WAVE2 mutant lacking the VCA region did not induce SRE activation and actin polymerization. WAVE2-induced SRE activation was blocked by exposure of cells to Latrunculin A, or overexpression of actin mutant R62D. The DeltaVCA mutant inhibited Rac V12-induced SRE activation, suggesting that WAVE2 lies downstream of Rac. Similar deletion of the VCA domain of WASP attenuated Cdc42 V12-mediated SRE activation, suggesting that WAVE2 acts in relation to Rac as WASP acts in relation to Cdc42. WAVE2 overexpression did not activate NF-kappaB.

Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation.

PubMed

Du, Yu; Xie, Guizhen; Yang, Chunfa; Fang, Baishan; Chen, Hongwen

2014-06-01

pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Understanding the role of argininosuccinate lyase transcript variants in the clinical and biochemical variability of the urea cycle disorder argininosuccinic aciduria.

PubMed

Hu, Liyan; Pandey, Amit V; Eggimann, Sandra; Rüfenacht, Véronique; Möslinger, Dorothea; Nuoffer, Jean-Marc; Häberle, Johannes

2013-11-29

Argininosuccinic aciduria (ASA) is an autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with a wide clinical spectrum from asymptomatic to severe hyperammonemic neonatal onset life-threatening courses. We investigated the role of ASL transcript variants in the clinical and biochemical variability of ASA. Recombinant proteins for ASL wild type, mutant p.E189G, and the frequently occurring transcript variants with exon 2 or 7 deletions were (co-)expressed in human embryonic kidney 293T cells. We found that exon 2-deleted ASL forms a stable truncated protein with no relevant activity but a dose-dependent dominant negative effect on enzymatic activity after co-expression with wild type or mutant ASL, whereas exon 7-deleted ASL is unstable but seems to have, nevertheless, a dominant negative effect on mutant ASL. These findings were supported by structural modeling predictions for ASL heterotetramer/homotetramer formation. Illustrating the physiological relevance, the predominant occurrence of exon 7-deleted ASL was found in two patients who were both heterozygous for the ASL mutant p.E189G. Our results suggest that ASL transcripts can contribute to the highly variable phenotype in ASA patients if expressed at high levels. Especially, the exon 2-deleted ASL variant may form a heterotetramer with wild type or mutant ASL, causing markedly reduced ASL activity.

Behavioral Abnormalities and Circuit Defects in the Basal Ganglia of a Mouse Model of 16p11.2 Deletion Syndrome

PubMed Central

Portmann, Thomas; Ellegood, Jacob; Dolen, Gul; Bader, Patrick L.; Grueter, Brad A.; Goold, Carleton; Fisher, Elaine; Clifford, Katherine; Rengarajan, Pavitra; Kalikhman, David; Loureiro, Darren; Saw, Nay L.; Zhengqui, Zhou; Miller, Michael A.; Lerch, Jason P.; Henkelman, Mark; Shamloo, Mehrdad; Malenka, Robert C.; Crawley, Jacqueline N.; Dolmetsch, Ricardo E.

2014-01-01

Summary A deletion on human chromosome 16p11.2 is associated with autism spectrum disorders. We deleted the syntenic region on mouse chromosome 7F3. MRI and high-throughput single-cell transcriptomics revealed anatomical and cellular abnormalities, particularly in cortex and striatum of juvenile mutant mice (16p11+/−). We found elevated numbers of striatal medium spiny neurons (MSNs) expressing the dopamine D2 receptor (Drd2+) and fewer dopamine-sensitive (Drd1+) neurons in deep layers of cortex. Electrophysiological recordings of Drd2+ MSN revealed synaptic defects, suggesting abnormal basal ganglia circuitry function in 16p11+/− mice. This is further supported by behavioral experiments showing hyperactivity, circling, and deficits in movement control. Strikingly, 16p11+/− mice showed a complete lack of habituation reminiscent of what is observed in some autistic individuals. Our findings unveil a fundamental role of genes affected by the 16p11.2 deletion in establishing the basal ganglia circuitry and provide insights in the pathophysiology of autism. PMID:24794428

The BIM Deletion Polymorphism and its Clinical Implication in Patients with EGFR-Mutant Non-Small-Cell Lung Cancer Treated with EGFR Tyrosine Kinase Inhibitors.

PubMed

Lee, Ji Yun; Ku, Bo Mi; Lim, Sung Hee; Lee, Min-Young; Kim, Haesu; Kim, Moonjin; Kim, Sungmin; Jung, Hyun Ae; Sun, Jong-Mu; Ahn, Jin Seok; Park, Keunchil; Ahn, Myung-Ju

2015-06-01

A germline BIM deletion polymorphism has been proposed to predict poor treatment response to certain kinase inhibitors. The purpose of this study was to explore whether the BIM deletion polymorphism predicts treatment efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in Korean patients with EGFR-mutant non-small-cell lung cancer (NSCLC). Peripheral blood samples from a total of 205 patients with EGFR-mutant NSCLC who were treated with EGFR TKIs between July 2008 and April 2013 were included. The incidence of BIM deletions in these samples was detected by polymerase chain reaction. We compared the clinical outcomes in patients with and without the polymorphism after treatment with EGFR TKIs (gefitinib or erlotinib). The BIM deletion polymorphism was present in 15.6% (32 of 205) of patients. One patient was homozygous for the deletion, and the remaining 31 had heterozygous deletions. The majority of patients were younger than 65 years (74%), female (68%), never smokers (76%), and had stage IV NSCLC (67%). There were no associations between the BIM deletion polymorphism and clinicopathological features including gender, age, smoking status, histology, stage, and number of metastasis sites. Patients with and without the BIM deletion polymorphism had similar objective response rates (91 vs. 84%, p = 0.585). Progression-free survival and overall survival did not differ significantly between patients with and without the BIM deletion polymorphism (median progression-free survival 12 vs. 11 months, p = 0.160; median overall survival 31 vs. 30 months, p = 0.452). Multivariate analysis identified significantly predictive markers for clinical outcomes of EGFR TKIs including Eastern Cooperative Oncology Group performance status 0-1, adenocarcinoma histology, recurrent disease, and EGFR mutation type. The results were validated in an independent cohort of 69 NSCLC patients. It remains to be determined whether the BIM deletion polymorphism provides intrinsic resistance or decreased sensitivity to EGFR TKIs in EGFR-mutant NSCLC patients.

Deletion of PdMit1, a homolog of yeast Csg1, affects growth and Ca(2+) sensitivity of the fungus Penicillium digitatum, but does not alter virulence.

PubMed

Zhu, Congyi; Wang, Weili; Wang, Mingshuang; Ruan, Ruoxin; Sun, Xuepeng; He, Meixian; Mao, Cungui; Li, Hongye

2015-04-01

GDP-mannose:inositol-phosphorylceramide (MIPC) and its derivatives are important for Ca(2+) sensitization of Saccharomyces cerevisiae and for the virulence of Candida albicans, but its role in the virulence of plant fungal pathogens remains unclear. In this study, we report the identification and functional characterization of PdMit1, the gene encoding MIPC synthase in Penicillium digitatum, one of the most important pathogens of postharvest citrus fruits. To understand the function of PdMit1, a PdMit1 deletion mutant was generated. Compared to its wild-type control, the PdMit1 deletion mutant exhibited slow radial growth, decreased conidia production and delayed conidial germination, suggesting that PdMit1 is important for the growth of mycelium, sporulation and conidial germination. The PdMit1 deletion mutant also showed hypersensitivity to Ca(2+). Treatment with 250 mmol/l Ca(2+) induced vacuole fusion in the wild-type strain, but not in the PdMit1 deletion mutant. Treatment with 250mmol/lCaCl2 upregulated three Ca(2+)-ATPase genes in the wild-type strain, and this was significantly inhibited in the PdMit1 deletion mutant. These results suggest that PdMit1 may have a role in regulating vacuole fusion and expression of Ca(2+)-ATPase genes by controlling biosynthesis of MIPC, and thereby imparts P. digitatum Ca(2+) tolerance. However, we found that PdMit1 is dispensable for virulence of P. digitatum. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Computer-Aided Resolution of an Experimental Paradox in Bacterial Chemotaxis

PubMed Central

Abouhamad, Walid N.; Bray, Dennis; Schuster, Martin; Boesch, Kristin C.; Silversmith, Ruth E.; Bourret, Robert B.

1998-01-01

Escherichia coli responds to its environment by means of a network of intracellular reactions which process signals from membrane-bound receptors and relay them to the flagellar motors. Although characterization of the reactions in the chemotaxis signaling pathway is sufficiently complete to construct computer simulations that predict the phenotypes of mutant strains with a high degree of accuracy, two previous experimental investigations of the activity remaining upon genetic deletion of multiple signaling components yielded several contradictory results (M. P. Conley, A. J. Wolfe, D. F. Blair, and H. C. Berg, J. Bacteriol. 171:5190–5193, 1989; J. D. Liu and J. S. Parkinson, Proc. Natl. Acad. Sci. USA 86:8703–8707, 1989). For example, “building up” the pathway by adding back CheA and CheY to a gutted strain lacking chemotaxis genes resulted in counterclockwise flagellar rotation whereas “breaking down” the pathway by deleting chemotaxis genes except cheA and cheY resulted in alternating episodes of clockwise and counterclockwise flagellar rotation. Our computer simulation predicts that trace amounts of CheZ expressed in the gutted strain could account for this difference. We tested this explanation experimentally by constructing a mutant containing a new deletion of the che genes that cannot express CheZ and verified that the behavior of strains built up from the new deletion does in fact conform to both the phenotypes observed for breakdown strains and computer-generated predictions. Our findings consolidate the present view of the chemotaxis signaling pathway and highlight the utility of molecularly based computer models in the analysis of complex biochemical networks. PMID:9683468

Determination of the promoter region of mouse ribosomal RNA gene by an in vitro transcription system.

PubMed Central

Yamamoto, O; Takakusa, N; Mishima, Y; Kominami, R; Muramatsu, M

1984-01-01

Sequences required for a faithful and efficient transcription of a cloned mouse ribosomal RNA gene (rDNA) are determined by testing a series of deletion mutants in an in vitro transcription system utilizing two kinds of mouse cellular extract. Deletion of sequences upstream of -40 or downstream of +52 causes only slight reduction in promoter activity as compared with the "wild-type" template. For upstream deletion mutants, the removal of a sequence between -40 and -35 causes a significant decrease in the capacity to direct efficient initiation. This decrease becomes more pronounced when the deletion reaches -32 and the sequence A-T-C-T-T-T, conserved among mouse, rat, and human rDNAs, is lost. Residual template activity is further reduced as more upstream sequence is deleted and finally becomes undetectable when the deletion is extended from -22 down to -17, corresponding to the loss of the conserved sequence T-A-T-T-G. As for downstream deletion mutants, the removal of the sequence downstream of +23 causes some (and further deletions up to +11 cause a more) serious decrease in template activity in vitro. These deletions involve other conserved sequences downstream of the transcription start site. However, the removal of the original transcription start site does not abolish the transcription initiation completely, provided that the whole upstream sequence is intact. Images PMID:6320178

The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

PubMed Central

Benedet, Mattia; Falchi, Federica A.; Puccio, Simone; Di Benedetto, Cristiano; Peano, Clelia; Polissi, Alessandra

2016-01-01

The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in γ-Proteobacteria. LptBFG constitute the IM ABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable ΔlptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptFSupC). In complementation tests, lptFSupC mutants suppress lethality of both ΔlptC and lptC conditional expression mutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine. PMID:27529623

The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter.

PubMed

Benedet, Mattia; Falchi, Federica A; Puccio, Simone; Di Benedetto, Cristiano; Peano, Clelia; Polissi, Alessandra; Dehò, Gianni

2016-01-01

The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in γ-Proteobacteria. LptBFG constitute the IM ABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable ΔlptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptFSupC). In complementation tests, lptFSupC mutants suppress lethality of both ΔlptC and lptC conditional expression mutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein.

PubMed

Duncan, R; Horne, D; Strong, J E; Leone, G; Pon, R T; Yeung, M C; Lee, P W

1991-06-01

We have been investigating structure-function relationships in the reovirus cell attachment protein sigma 1 using various deletion mutants and protease analysis. In the present study, a series of deletion mutants were constructed which lacked 90, 44, 30, 12, or 4 amino acids from the C-terminus of the 455-amino acid-long reovirus type 3 (T3) sigma 1 protein. The full-length and truncated sigma 1 proteins were expressed in an in vitro transcription/translation system and assayed for L cell binding activity. It was found that the removal of as few as four amino acids from the C-terminus drastically affected the cell binding function of the sigma 1 protein. The C-terminal-truncated proteins were further characterized using trypsin, chymotrypsin, and monoclonal and polyclonal antibodies. Our results indicated that the C-terminal portions of the mutant proteins were misfolded, leading to a loss in cell binding function. The N-terminal fibrous tail of the proteins was unaffected by the deletions as was sigma 1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of sigma 1. In an attempt to identify smaller, functional peptides, full-length sigma 1 expressed in vitro was digested with trypsin and subsequently with chymotrypsin under various conditions. The results clearly demonstrated the highly stable nature of the C-terminal globular head of sigma 1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of sigma 1 exists as a compact, protease-resistant oligomeric structure; (2) an intact C-terminus is required for proper head folding and generation of the conformationally dependent cell binding domain.

A dual role of p53 in the control of autophagy.

PubMed

Tasdemir, Ezgi; Chiara Maiuri, M; Morselli, Eugenia; Criollo, Alfredo; D'Amelio, Marcello; Djavaheri-Mergny, Mojgan; Cecconi, Francesco; Tavernarakis, Nektarios; Kroemer, Guido

2008-08-01

Genotoxic stress can induce autophagy in a p53-dependent fashion and p53 can transactivate autophagy-inducing genes. We have observed recently that inactivation of p53 by deletion, depletion or inhibition can trigger autophagy. Thus, human and mouse cells subjected to knockout, knockdown or pharmacological inhibition of p53 manifest signs of autophagy such as depletion of p62/SQSTM1, LC3 lipidation, redistribution of GFP-LC3 in cytoplasmic puncta, and accumulation of autophagosomes and autolysosomes, both in vitro and in vivo. Inhibition of p53 causes autophagy in enucleated cells, indicating that the cytoplasmic, non-nuclear pool of p53 can regulate autophagy. Accordingly, retransfection of p53(-/-) cells with wild-type p53 as well as a p53 mutant that is excluded from the nucleus (due to the deletion of the nuclear localization sequence) can inhibit autophagy, whereas retransfection with a nucleus-restricted p53 mutant (in which the nuclear localization sequence has been deleted) does not inhibit autophagy. Several distinct autophagy inducers (e.g., starvation, rapamycin, lithium, tunicamycin and thapsigargin) stimulate the rapid degradation of p53. In these conditions, inhibition of the p53-specific E3 ubiquitin ligase HDM2 can avoid p53 depletion and simultaneously prevent the activation of autophagy. Moreover, a p53 mutant that lacks the HDM2 ubiquitinylation site and hence is more stable than wild-type p53 is particularly efficient in suppressing autophagy. In conclusion, p53 plays a dual role in the control of autophagy. On the one hand, nuclear p53 can induce autophagy through transcriptional effects. On the other hand, cytoplasmic p53 may act as a master repressor of autophagy.

NMDA receptor subunits and associated signaling molecules mediating antidepressant-related effects of NMDA-GluN2B antagonism

PubMed Central

Kiselycznyk, Carly; Jury, Nicholas; Halladay, Lindsay; Nakazawa, Kazu; Mishina, Masayoshi; Sprengel, Rolf; Grant, Seth G.N.; Svenningsson, Per; Holmes, Andrew

2015-01-01

Drugs targeting the glutamate N-methyl-D-aspartate receptor (NMDAR) may be efficacious for treating mood disorders, as exemplified by the rapid antidepressant effects produced by single administration of the NMDAR antagonist ketamine. Though the precise mechanisms underlying the antidepressant-related effects of NMDAR antagonism remain unclear, recent studies implicate specific NMDAR subunits, including GluN2A and GluN2B, as well as the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit glutamate receptor interacting molecule, PSD-95. Here, integrating mutant and pharmacological in mice, we investigated the contribution of these subunits and molecules to antidepressant-related behaviors and the antidepressant-related effects of the GluN2B blocker, Ro 25-6981. We found that global deletion of GluA1 or PSD-95 reduced forced swim test (FST) immobility, mimicking the antidepressant-related effect produced by systemically administered Ro 25-6981 in C57BL/6J mice. Moreover, the FST antidepressant-like effects of systemic Ro 25-6981 were intact in mutants with global GluA1 deletion or GluN1 deletion in forebrain interneurons, but were absent in mutants constitutively lacking GluN2A or PSD-95. Next, we found that microinfusing Ro 25-6981 into the medial prefrontal cortex (mPFC), but not basolateral amygdala, of C57BL/6J mice was sufficient to produce an antidepressant-like effect. Together, these findings extend and refine current understanding of the mechanisms mediating antidepressant-like effects produced by NMDAR-GluN2B antagonists, and may inform the development of a novel class of medications for treating depression that target the GluN2B subtype of NMDAR. PMID:25800971

Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, Ambystoma mexicanum.

PubMed

Woodcock, M Ryan; Vaughn-Wolfe, Jennifer; Elias, Alexandra; Kump, D Kevin; Kendall, Katharina Denise; Timoshevskaya, Nataliya; Timoshevskiy, Vladimir; Perry, Dustin W; Smith, Jeramiah J; Spiewak, Jessica E; Parichy, David M; Voss, S Randal

2017-01-31

The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning-candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyr a ) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyr a has a 142 bp deletion and similar engineered alleles recapitulated the albino phenotype. Finally, we show that historical introgression of tyr a significantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl.

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

PubMed Central

2011-01-01

Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis. PMID:21794145

Basal exon skipping and nonsense-associated altered splicing allows bypassing complete CEP290 loss-of-function in individuals with unusually mild retinal disease.

PubMed

Barny, Iris; Perrault, Isabelle; Michel, Christel; Soussan, Mickael; Goudin, Nicolas; Rio, Marlène; Thomas, Sophie; Attié-Bitach, Tania; Hamel, Christian; Dollfus, Hélène; Kaplan, Josseline; Rozet, Jean-Michel; Gerard, Xavier

2018-05-16

CEP290 mutations cause a spectrum of ciliopathies from Leber congenital amaurosis type 10 (LCA10) to embryo-lethal Meckel syndrome (MKS). Using panel-based molecular diagnosis testing for inherited retinal diseases, we identified two individuals with some preserved vision despite biallelism for presumably truncating CEP290 mutations. The first one carried a homozygous 1 base-pair deletion in exon 17, introducing a premature termination codon (PTC) in exon 18 (c.1666del; p.Ile556Phefs*17). mRNA analysis revealed a basal exon skipping (BES) of exon 18, providing mutant cells with the ability to escape protein truncation, while disrupting the reading frame in controls. The second individual harbored compound heterozygous nonsense mutations in exon 8 (c.508A>T, p.Lys170*) and exon 32 (c.4090G>T, p.Glu1364*), respectively. Some CEP290 lacking exon 8 were detected in mutant fibroblasts but not in controls whereas some skipping of exon 32 occurred in both lines, but with higher amplitude in the mutant. Considering that the deletion of either exon maintains the reading frame in either line, skipping in mutant cells likely involves nonsense-associated altered splicing (NAS) alone (exon 8), or with BES (exon 32). Skipping of PTC-containing exons in mutant cells allowed production of CEP290 isoforms with preserved ability to assemble into a high molecular weight complex and to interact efficiently with proteins important for cilia formation and intraflagellar trafficking. In contrast, studying LCA10 and MKS fibroblasts we show moderate to severe cilia alterations, providing support for a correlation between disease severity and the ability of cells to express shortened, yet functional, CEP290 isoforms.

Structure and Expression of Hybrid Dysgenesis-Induced Alleles of the Ovarian Tumor (Otu) Gene in Drosophila Melanogaster

PubMed Central

Sass, G. L.; Mohler, J. D.; Walsh, R. C.; Kalfayan, L. J.; Searles, L. L.

1993-01-01

Mutations at the ovarian tumor (otu) gene of Drosophila melanogaster cause female sterility and generate a range of ovarian phenotypes. Quiescent (QUI) mutants exhibit reduced germ cell proliferation; in oncogenic (ONC) mutants germ cells undergo uncontrolled proliferation generating excessive numbers of undifferentiated cells; the egg chambers of differentiated (DIF) mutants differentiate to variable degrees but fail to complete oogenesis. We have examined mutations caused by insertion and deletion of P elements at the otu gene. The P element insertion sites are upstream of the major otu transcription start sites. In deletion derivatives, the P element, regulatory regions and/or protein coding sequences have been removed. In both insertion and deletion mutants, the level of otu expression correlates directly with the severity of the phenotype: the absence of otu function produces the most severe QUI phenotype while the ONC mutants express lower levels of otu than those which are DIF. The results of this study demonstrate that the diverse mutant phenotypes of otu are the consequence of different levels of otu function. PMID:8436274

Using genic sequence capture in combination with a syntenic pseudo genome to map a deletion mutant in a wheat species.

PubMed

Gardiner, Laura-Jayne; Gawroński, Piotr; Olohan, Lisa; Schnurbusch, Thorsten; Hall, Neil; Hall, Anthony

2014-12-01

Mapping-by-sequencing analyses have largely required a complete reference sequence and employed whole genome re-sequencing. In species such as wheat, no finished genome reference sequence is available. Additionally, because of its large genome size (17 Gb), re-sequencing at sufficient depth of coverage is not practical. Here, we extend the utility of mapping by sequencing, developing a bespoke pipeline and algorithm to map an early-flowering locus in einkorn wheat (Triticum monococcum L.) that is closely related to the bread wheat genome A progenitor. We have developed a genomic enrichment approach using the gene-rich regions of hexaploid bread wheat to design a 110-Mbp NimbleGen SeqCap EZ in solution capture probe set, representing the majority of genes in wheat. Here, we use the capture probe set to enrich and sequence an F2 mapping population of the mutant. The mutant locus was identified in T. monococcum, which lacks a complete genome reference sequence, by mapping the enriched data set onto pseudo-chromosomes derived from the capture probe target sequence, with a long-range order of genes based on synteny of wheat with Brachypodium distachyon. Using this approach we are able to map the region and identify a set of deleted genes within the interval. © 2014 The Authors.The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes

PubMed Central

Veesenmeyer, Jeff L.; Andersen, Aaron W.; Lu, Xiaojun; Hussa, Elizabeth A.; Murfin, Kristen E.; Chaston, John M.; Dillman, Adler R.; Wassarman, Karen M.; Sternberg, Paul W.; Goodrich-Blair, Heidi

2014-01-01

Summary The bacterium Xenorhabdus nematophila is a mutualist of entomopathogenic Steinernema carpocapsae nematodes and facilitates infection of insect hosts. X. nematophila colonizes the intestine of S. carpocapsae which carries it between insects. In the X. nematophila colonization-defective mutant nilD6::Tn5, the transposon is inserted in a region lacking obvious coding potential. We demonstrate that the transposon disrupts expression of a single CRISPR RNA, NilD RNA. A variant NilD RNA also is expressed by X. nematophila strains from S. anatoliense and S. websteri nematodes. Only nilD from the S. carpocapsae strain of X. nematophila rescued the colonization defect of the nilD6::Tn5 mutant, and this mutant was defective in colonizing all three nematode host species. NilD expression depends on the presence of the associated Cas6e but not Cas3, components of the Type I-E CRISPR-associated machinery. While cas6e deletion in the complemented strain abolished nematode colonization, its disruption in the wild-type parent did not. Likewise, nilD deletion in the parental strain did not impact colonization of the nematode, revealing that the requirement for NilD is evident only in certain genetic backgrounds. Our data demonstrate that NilD RNA is conditionally necessary for mutualistic host colonization and suggest that it functions to regulate endogenous gene expression. PMID:25041533

Histone deacetylases 1 and 2 regulate the transcriptional programs of nephron progenitors and renal vesicles.

PubMed

Liu, Hongbing; Chen, Shaowei; Yao, Xiao; Li, Yuwen; Chen, Chao-Hui; Liu, Jiao; Saifudeen, Zubaida; El-Dahr, Samir S

2018-05-18

Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a , as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles. © 2018. Published by The Company of Biologists Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asai, Hirohide; Hirano, Makito; Kiriyama, Takao

Intranuclear events due to mutations in the Parkin gene remain elusive in autosomal recessive juvenile parkinsonism (ARJP). We identified a mutant PARKIN protein in fibroblast cultures from a pair of siblings with ARJP who were homozygous for the exon 4-deleted Parkin gene. Disease was mild in one patient and debilitating in the other. The detected mutant, encoded by a transcript lacking exon 3 as well as exon 4, is an in-frame deletion that removes 121 aa, resulting in a 344-aa protein (PaDel3,4). Cell culture and transfection studies revealed negative correlations between expression levels of PaDel3,4 and those of cell cyclemore » proteins, including cyclin E, CDK2, ppRb, and E2F-1, and demonstrated that GFP-PaDel3,4 entered nucleus and ubiquitinated cyclin E as a part of SCF{sup hSel-10} ligase complex in the patient cells. In addition, nuclear localization signal-tagged PaDel3,4 expressed in the transfected patient cells most effectively ubiquitinated cyclin E and reduced DNA damage, protecting cells from oxidative stress. Antisense-oligonucleotide treatment promoted skipping of exon 3 and thus generated PaDel3,4, increasing cell survival. Collectively, we propose that naturally- and experimentally-induced exon skipping at least partly restores the mutant Parkin gene deficit, providing a molecular basis for the development of therapeutic exon skipping.« less

Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids.

PubMed Central

Arthur, A K; Höss, A; Fanning, E

1988-01-01

The genomic coding sequence of the large T antigen of simian virus 40 (SV40) was cloned into an Escherichia coli expression vector by joining new restriction sites, BglII and BamHI, introduced at the intron boundaries of the gene. Full-length large T antigen, as well as deletion and amino acid substitution mutants, were inducibly expressed from the lac promoter of pUC9, albeit with different efficiencies and protein stabilities. Specific interaction with SV40 origin DNA was detected for full-length T antigen and certain mutants. Deletion mutants lacking T-antigen residues 1 to 130 and 260 to 708 retained specific origin-binding activity, demonstrating that the region between residues 131 and 259 must carry the essential binding domain for DNA-binding sites I and II. A sequence between residues 302 and 320 homologous to a metal-binding "finger" motif is therefore not required for origin-specific binding. However, substitution of serine for either of two cysteine residues in this motif caused a dramatic decrease in origin DNA-binding activity. This region, as well as other regions of the full-length protein, may thus be involved in stabilizing the DNA-binding domain and altering its preference for binding to site I or site II DNA. Images PMID:2835505

Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants.

PubMed Central

Dolja, V V; Haldeman, R; Robertson, N L; Dougherty, W G; Carrington, J C

1994-01-01

Tobacco etch potyvirus engineered to express the reporter protein beta-glucuronidase (TEV-GUS) was used for direct observation and quantitation of virus translocation in plants. Four TEV-GUS mutants were generated containing capsid proteins (CPs) with single amino acid substitutions (R154D and D198R), a double substitution (DR), or a deletion of part of the N-terminal domain (delta N). Each modified virus replicated as well as the parental virus in protoplasts, but was defective in cell-to-cell movement through inoculated leaves. The R154D, D198R and DR mutants were restricted essentially to single, initially infected cells. The delta N variant exhibited slow cell-to-cell movement in inoculated leaves, but was unable to move systemically due to a lack of entry into or replication in vascular-associated cells. Both cell-to-cell and systemic movement defects of each mutant were rescued in transgenic plants expressing wild-type TEV CP. Cell-to-cell movement, but not systemic movement, of the DR mutant was rescued partially in transgenic plants expressing TEV CP lacking the C-terminal domain, and in plants expressing CP from the heterologous potyvirus, potato virus Y. Despite comparable levels of accumulation of parental virus and each mutant in symptomatic tissue of TEV CP-expressing transgenic plants, virions were detected only in parental virus- and delta N mutant-infected plants, as revealed using three independent assays. These data suggest that the potyvirus CP possesses distinct, separable activities required for virion assembly, cell-to-cell movement and long-distance transport. Images PMID:7511101

Deletion of HAPS_2096 Increases Sensitivity to Cecropin B in Haemophilus parasuis.

PubMed

Chen, Fanjie; Hu, Han; Li, Zhonghua; Huang, Jiacheng; Cai, Xuwang; Wang, Chunmei; He, Qigai; Cao, Jiyue

2015-01-01

Cecropin B (CB) is a very effective natural antimicrobial peptide that has shown great potential for future antimicrobial drug development. HAPS_2096 is a Haemophilus parasuis gene that encodes the periplasmic substrate-binding protein of an ATP-binding cassette-type amino acid transporter. In this research, we constructed and verified an HAPS_2096 deletion mutant and a complementary HAPS_2096 mutant of H. parasuis JS0135. A bactericidal assay revealed that the HAPS_2096 deletion mutant was significantly more sensitive than the wild-type strain to 0.25-0.5 µg/ml CB. However, the gene complementation alleviated the CB sensitivity of the mutant. Immunoelectron microscopy observation following a 30-min treatment with a sublethal concentration of CB (0.25 μg/ml) revealed more extensive morphological damage in the mutant strain than in the wild-type strain. Hence, our results suggest that the HAPS_2096 gene contributes to H. parasuis resistance to CB. © 2015 S. Karger AG, Basel.

Chromosomal instability in Streptomyces avermitilis: major deletion in the central region and stable circularized chromosome

PubMed Central

2010-01-01

Background The chromosome of Streptomyces has been shown to be unstable, frequently undergoing gross chromosomal rearrangements. However, the mechanisms underlying this phenomenon remain unclear, with previous studies focused on two chromosomal ends as targets for rearrangements. Here we investigated chromosomal instability of Streptomyces avermitilis, an important producer of avermectins, and characterized four gross chromosomal rearrangement events, including a major deletion in the central region. The present findings provide a valuable contribution to the mechanistic study of genetic instability in Streptomyces. Results Thirty randomly-selected "bald" mutants derived from the wild-type strain all contained gross chromosomal rearrangements of various types. One of the bald mutants, SA1-8, had the same linear chromosomal structure as the high avermectin-producing mutant 76-9. Chromosomes of both strains displayed at least three independent chromosomal rearrangements, including chromosomal arm replacement to form new 88-kb terminal inverted repeats (TIRs), and two major deletions. One of the deletions eliminated the 36-kb central region of the chromosome, but surprisingly did not affect viability of the cells. The other deletion (74-kb) was internal to the right chromosomal arm. The chromosome of another bald mutant, SA1-6, was circularized with deletions at both ends. No obvious homology was found in all fusion sequences. Generational stability analysis showed that the chromosomal structure of SA1-8 and SA1-6 was stable. Conclusions Various chromosomal rearrangements, including chromosomal arm replacement, interstitial deletions and chromosomal circularization, occurred in S. avermitilis by non-homologous recombination. The finding of an inner deletion involving in the central region of S. avermitilis chromosome suggests that the entire Streptomyces chromosome may be the target for rearrangements, which are not limited, as previously reported, to the two chromosomal ends. PMID:20653985

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed Central

Seif, R; Martin, R G

1979-01-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation. Images PMID:229274

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed

Seif, R; Martin, R G

1979-12-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.

Selection of recombinant MVA by rescue of the essential D4R gene.

PubMed

Ricci, Patricia S; Schäfer, Birgit; Kreil, Thomas R; Falkner, Falko G; Holzer, Georg W

2011-12-12

Modified vaccinia virus Ankara (MVA) has become a promising vaccine vector due to its immunogenicity and its proven safety in humans. As a general approach for stringent and rapid selection of recombinant MVA, we assessed marker rescue of the essential viral D4R gene in an engineered deletion mutant that is fully replication defective in wild-type cells. Recombinant, replicating virus was obtained by re-introduction of the deleted viral gene as a dominant selection marker into the deletion mutant.

Bombyx mori nucleopolyhedrovirus ORF54, a viral desmoplakin gene, is associated with the infectivity of budded virions.

PubMed

Zhang, Min-Juan; Tian, Cai-Hong; Fan, Xiao-Ying; Lou, Yi-Han; Cheng, Ruo-Lin; Zhang, Chuan-Xi

2012-07-01

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF54 (Bm54), a member of the viral desmoplakin N-terminus superfamily, is homologous to Autographa californica nucleopolyhedrovirus (AcMNPV) ORF66, which is required for the efficient egress of nucleocapsids from the nucleus and occlusion body formation. In this paper, we generated a bacmid with the Bm54 gene deleted via homologous recombination in Escherichia coli and characterized the mutant virus using a transfection-infection assay and transmission electron microscopy analysis. Our results demonstrated that the cells transfected with viral DNA lacking Bm54 produced non-infectious budded viruses (BVs). Electron microscopy showed that although the deletion of Bm54 did not affect assembly and release of nucleocapsids, it severely affected polyhedron formation. In conclusion, deletion of Bm54 resulted in non-infectious BV and defective polyhedra. Although the sequences of Bm54 and Ac66 are very similar, the two genes function quite differently in the regulation of viral life cycle.

Riboflavin Is an Active Redox Cofactor in the Na+-pumping NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

PubMed Central

Juárez, Oscar; Nilges, Mark J.; Gillespie, Portia; Cotton, Jennifer; Barquera, Blanca

2008-01-01

Here we present new evidence that riboflavin is present as one of four flavins in Na+-NQR. In particular, we present conclusive evidence that the source of the neutral radical is not one of the FMNs and that riboflavin is the center that gives rise to the neutral flavosemiquinone. The riboflavin is a bona fide redox cofactor and is likely to be the last redox carrier of the enzyme, from which electrons are donated to quinone. We have constructed a double mutant that lacks both covalently bound FMN cofactors (NqrB-T236Y/NqrC-T225Y) and have studied this mutant together with the two single mutants (NqrB-T236Y and NqrC-T225Y) and a mutant that lacks the noncovalently bound FAD in NqrF (NqrF-S246A). The double mutant contains riboflavin and FAD in a 0.6:1 ratio, as the only flavins in the enzyme; noncovalently bound flavins were detected. In the oxidized form, the double mutant exhibits an EPR signal consistent with a neutral flavosemiquinone radical, which is abolished on reduction of the enzyme. The same radical can be observed in the FAD deletion mutant. Furthermore, when the oxidized enzyme reacts with ubiquinol (the reduced form of the usual electron acceptor) in a process that reverses the physiological direction of the electron flow, a single kinetic phase is observed. The kinetic difference spectrum of this process is consistent with one-electron reduction of a neutral flavosemiquinone. The presence of riboflavin in the role of a redox cofactor is thus far unique to Na+-NQR. PMID:18832377

Genome-wide screen of Saccharomyces cerevisiae for killer toxin HM-1 resistance.

PubMed

Miyamoto, Masahiko; Furuichi, Yasuhiro; Komiyama, Tadazumi

2011-01-01

We screened a set of Saccharomyces cerevisiae deletion mutants for resistance to killer toxin HM-1, which kills susceptible yeasts through inhibiting 1,3-beta-glucan synthase. By using HM-1 plate assay, we found that eight gene-deletion mutants had higher HM-1-resistance compared with the wild-type. Among these eight genes, five--ALG3, CAX4, MNS1, OST6 and YBL083C--were associated with N-glycan formation and maturation. The ALG3 gene has been shown before to be highly resistant to HM-1. The YBL083C gene may be a dubious open reading frame that overlaps partially the ALG3 gene. The deletion mutant of the MNS1 gene that encodes 1,2-alpha-mannosidase showed with a 13-fold higher HM-1 resistance compared with the wild-type. By HM-1 binding assay, the yeast plasma membrane fraction of alg3 and mns1 cells had less binding ability compared with wild-type cells. These results indicate that the presence of the terminal 1,3-alpha-linked mannose residue of the B-chain of the N-glycan structure is essential for interaction with HM-1. A deletion mutant of aquaglyceroporin Fps1p also showed increased HM-1 resistance. A deletion mutant of osmoregulatory mitogen-activated protein kinase Hog1p was more sensitive to HM-1, suggesting that high-osmolarity glycerol pathways plays an important role in the compensatory response to HM-1 action. Copyright © 2010 John Wiley & Sons, Ltd.

Flavobacterium johnsoniae gldN and gldO Are Partially Redundant Genes Required for Gliding Motility and Surface Localization of SprB▿ †

PubMed Central

Rhodes, Ryan G.; Samarasam, Mudiarasan Napoleon; Shrivastava, Abhishek; van Baaren, Jessica M.; Pochiraju, Soumya; Bollampalli, Sreelekha; McBride, Mark J.

2010-01-01

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. Mutations in gldN cause a partial defect in gliding. A novel bacteriophage selection strategy was used to aid construction of a strain with a deletion spanning gldN and the closely related gene gldO in an otherwise wild-type F. johnsoniae UW101 background. Bacteriophage transduction was used to move a gldN mutation into F. johnsoniae UW101 to allow phenotypic comparison with the gldNO deletion mutant. Cells of the gldN mutant formed nonspreading colonies on agar but retained some ability to glide in wet mounts. In contrast, cells of the gldNO deletion mutant were completely nonmotile, indicating that cells require GldN, or the GldN-like protein GldO, to glide. Recent results suggest that Porphyromonas gingivalis PorN, which is similar in sequence to GldN, has a role in protein secretion across the outer membrane. Cells of the F. johnsoniae gldNO deletion mutant were defective in localization of the motility protein SprB to the cell surface, suggesting that GldN may be involved in secretion of components of the motility machinery. Cells of the gldNO deletion mutant were also deficient in chitin utilization and were resistant to infection by bacteriophages, phenotypes that may also be related to defects in protein secretion. PMID:20038590

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.

PubMed

Matsuoka, Hiroyuki; Hashimoto, Kazuya; Saijo, Aki; Takada, Yuki; Kondo, Akihiko; Ueda, Mitsuyoshi; Ooshima, Hiroshi; Tachibana, Taro; Azuma, Masayuki

2014-02-01

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.

Forward genetics in Candida albicans that reveals the Arp2/3 complex is required for hyphal formation, but not endocytosis

PubMed Central

Epp, Elias; Walther, Andrea; Guylaine, Lépine; Leon, Zully; Mullick, Alaka; Raymond, Martine; Wendland, Jürgen; Whiteway, Malcolm

2014-01-01

Summary Candida albicans is a diploid fungal pathogen lacking a defined complete sexual cycle, and thus has been refractory to standard forward genetic analysis. Instead, transcription profiling and reverse genetic strategies based on Saccharomyces cerevisiae have typically been used to link genes to functions. To overcome restrictions inherent in such indirect approaches, we have investigated a forward genetic mutagenesis strategy based on the UAU1 technology. We screened 4700 random insertion mutants for defects in hyphal development and linked two new genes (ARP2 and VPS52) to hyphal growth. Deleting ARP2 abolished hyphal formation, generated round and swollen yeast phase cells, disrupted cortical actin patches and blocked virulence in mice. The mutants also showed a global lack of induction of hyphae-specific genes upon the yeast-to-hyphae switch. Surprisingly, both arp2Δ/Δ and arp2Δ/Δarp3Δ/Δ mutants were still able to endocytose FM4-64 and Lucifer Yellow, although as shown by time-lapse movies internalization of FM4-64 was somewhat delayed in mutant cells. Thus the non-essential role of the Arp2/3 complex discovered by forward genetic screening in C. albicans showed that uptake of membrane components from the plasma membrane to vacuolar structures is not dependent on this actin nucleating machinery. PMID:20141603

Old yellow enzymes protect against acrolein toxicity in the yeast Saccharomyces cerevisiae.

PubMed

Trotter, Eleanor W; Collinson, Emma J; Dawes, Ian W; Grant, Chris M

2006-07-01

Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but particularly affected gene expression, metabolism, and cellular signaling. Surprisingly, the screen did not identify any antioxidants or similar stress-protective molecules, indicating that acrolein toxicity may not be mediated via reactive oxygen species. Most strikingly, a mutant lacking an old yellow enzyme (OYE2) was identified as being acrolein sensitive. Old yellow enzymes are known to reduce alpha,beta-unsaturated carbonyl compounds in vitro, but their physiological roles have remained uncertain. We show that mutants lacking OYE2, but not OYE3, are sensitive to acrolein, and overexpression of both isoenzymes increases acrolein tolerance. Our data indicate that OYE2 is required for basal levels of tolerance, whereas OYE3 expression is particularly induced following acrolein stress. Despite the range of alpha,beta-unsaturated carbonyl compounds that have been identified as substrates of old yellow enzymes in vitro, we show that old yellow enzymes specifically mediate resistance to small alpha,beta-unsaturated carbonyl compounds, such as acrolein, in vivo.

Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants

NASA Technical Reports Server (NTRS)

Davenport, K. D.; Williams, K. E.; Ullmann, B. D.; Gustin, M. C.; McIntire, L. V. (Principal Investigator)

1999-01-01

Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.

Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants.

PubMed Central

Davenport, K D; Williams, K E; Ullmann, B D; Gustin, M C

1999-01-01

Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype. PMID:10545444

[Gene deletion and functional analysis of the heptyl glycosyltransferase (waaF) gene in Vibrio parahemolyticus O-antigen cluster].

PubMed

Zhao, Feng; Meng, Songsong; Zhou, Deqing

2016-02-04

To construct heptyl glycosyltransferase gene II (waaF) gene deletion mutant of Vibrio parahaemolyticus, and explore the function of the waaF gene in Vibrio parahaemolyticus. The waaF gene deletion mutant was constructed by chitin-based transformation technology using clinical isolates, and then the growth rate, morphology and serotypes were identified. The different sources (O3, O5 and O10) waaF gene complementations were constructed through E. coli S17λpir strains conjugative transferring with Vibrio parahaemolyticus, and the function of the waaF gene was further verified by serotypes. The waaF gene deletion mutant strain was successfully constructed and it grew normally. The growth rate and morphology of mutant were similar with the wild type strains (WT), but the mutant could not occurred agglutination reaction with O antisera. The O3 and O5 sources waaF gene complementations occurred agglutination reaction with O antisera, but the O10 sources waaF gene complementations was not. The waaF gene was related with O-antigen synthesis and it was the key gene of O-antigen synthesis pathway in Vibrio parahaemolyticus. The function of different sources waaF gene were not the same.

Hepatitis B Virus Core Gene Mutations Which Block Nucleocapsid Envelopment

PubMed Central

Koschel, Matthias; Oed, Daniela; Gerelsaikhan, Tudevdagwa; Thomssen, Reiner; Bruss, Volker

2000-01-01

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment. PMID:10590084

Conditional poliovirus mutants made by random deletion mutagenesis of infectious cDNA.

PubMed Central

Kirkegaard, K; Nelsen, B

1990-01-01

Small deletions were introduced into DNA plasmids bearing cDNA copies of Mahoney type 1 poliovirus RNA. The procedure used was similar to that of P. Hearing and T. Shenk (J. Mol. Biol. 167:809-822, 1983), with modifications designed to introduce only one lesion randomly into each DNA molecule. Methods to map small deletions in either large DNA or RNA molecules were employed. Two poliovirus mutants, VP1-101 and VP1-102, were selected from mutagenized populations on the basis of their host range phenotype, showing a large reduction in the relative numbers of plaques on CV1 and HeLa cells compared with wild-type virus. The deletions borne by the mutant genomes were mapped to the region encoding the amino terminus of VP1. That these lesions were responsible for the mutant phenotypes was substantiated by reintroduction of the sequenced lesions into a wild-type poliovirus cDNA by deoxyoligonucleotide-directed mutagenesis. The deletion of nucleotides encoding amino acids 8 and 9 of VP1 was responsible for the VP1-101 phenotype; the VP1-102 defect was caused by the deletion of the sequences encoding the first four amino acids of VP1. The peptide sequence at the VP1-VP3 proteolytic cleavage site was altered from glutamine-glycine to glutamine-methionine in VP1-102; this apparently did not alter the proteolytic cleavage pattern. The biochemical defects resulting from these mutations are discussed in the accompanying report. Images PMID:2152811

A proof of the DBRF-MEGN method, an algorithm for deducing minimum equivalent gene networks

PubMed Central

2011-01-01

Background We previously developed the DBRF-MEGN (difference-based regulation finding-minimum equivalent gene network) method, which deduces the most parsimonious signed directed graphs (SDGs) consistent with expression profiles of single-gene deletion mutants. However, until the present study, we have not presented the details of the method's algorithm or a proof of the algorithm. Results We describe in detail the algorithm of the DBRF-MEGN method and prove that the algorithm deduces all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants. Conclusions The DBRF-MEGN method provides all of the exact solutions of the most parsimonious SDGs consistent with expression profiles of gene deletion mutants. PMID:21699737

Requirements and effects of palmitoylation of rat PLD1.

PubMed

Xie, Z; Ho, W T; Exton, J H

2001-03-23

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs (HXKX(4)D), denoted HKD, located in the N- and C-terminal halves, which are required for phospholipase D activity. The two halves of rPLD1 can associate in vivo, and the association is essential for catalytic activity and Ser/Thr phosphorylation of the enzyme. In this study, we found that this association is also required for palmitoylation of rPLD1, which occurs on cysteines 240 and 241. In addition, palmitoylation of rPLD1 requires the N-terminal sequence but not the conserved C-terminal sequence, since rPLD1 that lacks the first 168 amino acids is not palmitoylated in vivo, while the inactive C-terminal deletion mutant is. Palmitoylation of rPLD1 is not necessary for catalytic activity, since N-terminal truncation mutants lacking the first 168 or 319 amino acids exhibit high basal activity although they cannot be stimulated by protein kinase C (PKC). The lack of response to PKC is not due to the lack of palmitoylation, since mutation of both Cys(240) and Cys(241) to alanine in full-length rPLD1 abolishes palmitoylation, but the mutant still retains basal activity and responds to PKC. Palmitoylation-deficient rPLD1 can associate with crude membranes; however, the association is weakened. Wild type rPLD1 remains membrane-associated when extracted with 1 m NaCl or Na(2)CO(3) (pH 11), while rPLD1 mutants that lack palmitoylation are partially released. In addition, we found that palmitoylation-deficient mutants are much less modified by Ser/Thr phosphorylation compared with wild type rPLD1. Characterization of the other cysteine mutations of rPLD1 showed that mutation of cysteine 310 or 612 to alanine increased basal phospholipase D activity 2- and 4-fold, respectively. In summary, palmitoylation of rPLD1 requires interdomain association and the presence of the N-terminal 168 amino acids. Mutations of cysteines 240 and 241 to alanine abolish the extensive Ser/Thr phosphorylation of the enzyme and weaken its association with membranes.

Cloning and expression of the tabtoxin biosynthetic region from Pseudomonas syringae.

PubMed Central

Kinscherf, T G; Coleman, R H; Barta, T M; Willis, D K

1991-01-01

Pseudomonas syringae BR2, a causal agent of bean wildfire, was subjected to Tn5 mutagenesis in an effort to isolate mutants unable to produce the beta-lactam antibiotic tabtoxin. Three of the tabtoxin-minus (Tox-) mutants generated appeared to have physically linked Tn5 insertions and retained their resistance to the active toxin form, tabtoxnine-beta-lactam (T beta L). The wild-type DNA corresponding to the mutated region was cloned and found to restore the Tn5 mutants to toxin production. The use of cloned DNA from the region as hybridization probes revealed that the region is highly conserved among tabtoxin-producing pathovars of P. syringae and that the region deletes at a relatively high frequency (10(-3)/CFU) in BR2. The Tox- deletion mutants also lost resistance to tabtoxinine-beta-lactam. A cosmid designated pRTBL823 restored toxin production and resistance to BR2 deletion mutants. This cosmid also converted the tabtoxin-naive P. syringae epiphyte Cit7 to toxin production and resistance, indicating that pRTBL823 contains a complete set of biosynthetic and resistance genes. Tox- derivatives of BR2 did not produce disease symptoms on bean. Clones that restored toxin production to both insertion and deletion mutants also restored the ability to cause disease. However, tabtoxin-producing Cit7 derivatives remained nonpathogenic on bean and tobacco, suggesting that tabtoxin production alone is not sufficient to cause disease. Images PMID:1648077

Appearance of the bona fide spiral tubule of ORF virus is dependent on an intact 10-kilodalton viral protein.

PubMed

Spehner, D; De Carlo, S; Drillien, R; Weiland, F; Mildner, K; Hanau, D; Rziha, H-J

2004-08-01

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the Parapoxvirus genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.

A test of the transcription model for biased inheritance of yeast mitochondrial DNA.

PubMed

Lorimer, H E; Brewer, B J; Fangman, W L

1995-09-01

Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.

The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis.

PubMed

Moolla, Nabiela; Goosens, Vivianne J; Kana, Bavesh D; Gordhan, Bhavna G

2014-01-01

The increased prevalence of drug resistant strains of Mycobacterium tuberculosis (Mtb) indicates that significant mutagenesis occurs during tuberculosis disease in humans. DNA damage by host-derived reactive oxygen/nitrogen species is hypothesized to be critical for the mutagenic process in Mtb thus, highlighting an important role for DNA repair enzymes in maintenance of genome fidelity. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases and together with EndonucleaseIII (Nth) are central to the base excision repair pathway in bacteria. In this study we assess the contribution of Nei and Nth DNA repair enzymes in Mycobacterium smegmatis (Msm), which retains a single nth homologue and duplications of the Fpg (fpg1 and fpg2) and Nei (nei1 and nei2) homologues. Using an Escherichia coli nth deletion mutant, we confirm the functionality of the mycobacterial nth gene in the base excision repair pathway. Msm mutants lacking nei1, nei2 and nth individually or in combination did not display aberrant growth in broth culture. Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth together with the fpg homolgues did not result in any growth/survival defects or changes in mutation rate. Furthermore, no differential emergence of the common rifampicin resistance conferring genotypes were noted. Collectively, these data confirm a role for Nth in base excision repair in mycobacteria and further highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Parallel analysis of tagged deletion mutants efficiently identifies genes involved in endoplasmic reticulum biogenesis.

PubMed

Wright, Robin; Parrish, Mark L; Cadera, Emily; Larson, Lynnelle; Matson, Clinton K; Garrett-Engele, Philip; Armour, Chris; Lum, Pek Yee; Shoemaker, Daniel D

2003-07-30

Increased levels of HMG-CoA reductase induce cell type- and isozyme-specific proliferation of the endoplasmic reticulum. In yeast, the ER proliferations induced by Hmg1p consist of nuclear-associated stacks of smooth ER membranes known as karmellae. To identify genes required for karmellae assembly, we compared the composition of populations of homozygous diploid S. cerevisiae deletion mutants following 20 generations of growth with and without karmellae. Using an initial population of 1,557 deletion mutants, 120 potential mutants were identified as a result of three independent experiments. Each experiment produced a largely non-overlapping set of potential mutants, suggesting that differences in specific growth conditions could be used to maximize the comprehensiveness of similar parallel analysis screens. Only two genes, UBC7 and YAL011W, were identified in all three experiments. Subsequent analysis of individual mutant strains confirmed that each experiment was identifying valid mutations, based on the mutant's sensitivity to elevated HMG-CoA reductase and inability to assemble normal karmellae. The largest class of HMG-CoA reductase-sensitive mutations was a subset of genes that are involved in chromatin structure and transcriptional regulation, suggesting that karmellae assembly requires changes in transcription or that the presence of karmellae may interfere with normal transcriptional regulation. Copyright 2003 John Wiley & Sons, Ltd.

FitSearch: a robust way to interpret a yeast fitness profile in terms of drug's mode-of-action.

PubMed

Lee, Minho; Han, Sangjo; Chang, Hyeshik; Kwak, Youn-Sig; Weller, David M; Kim, Dongsup

2013-01-01

Yeast deletion-mutant collections have been successfully used to infer the mode-of-action of drugs especially by profiling chemical-genetic and genetic-genetic interactions on a genome-wide scale. Although tens of thousands of those profiles are publicly available, a lack of an accurate method for mining such data has been a major bottleneck for more widespread use of these useful resources. For general usage of those public resources, we designed FitRankDB as a general repository of fitness profiles, and developed a new search algorithm, FitSearch, for identifying the profiles that have a high similarity score with statistical significance for a given fitness profile. We demonstrated that our new repository and algorithm are highly beneficial to researchers who attempting to make hypotheses based on unknown modes-of-action of bioactive compounds, regardless of the types of experiments that have been performed using yeast deletion-mutant collection in various types of different measurement platforms, especially non-chip-based platforms. We showed that our new database and algorithm are useful when attempting to construct a hypothesis regarding the unknown function of a bioactive compound through small-scale experiments with a yeast deletion collection in a platform independent manner. The FitRankDB and FitSearch enhance the ease of searching public yeast fitness profiles and obtaining insights into unknown mechanisms of action of drugs. FitSearch is freely available at http://fitsearch.kaist.ac.kr.

FitSearch: a robust way to interpret a yeast fitness profile in terms of drug's mode-of-action

PubMed Central

2013-01-01

Background Yeast deletion-mutant collections have been successfully used to infer the mode-of-action of drugs especially by profiling chemical-genetic and genetic-genetic interactions on a genome-wide scale. Although tens of thousands of those profiles are publicly available, a lack of an accurate method for mining such data has been a major bottleneck for more widespread use of these useful resources. Results For general usage of those public resources, we designed FitRankDB as a general repository of fitness profiles, and developed a new search algorithm, FitSearch, for identifying the profiles that have a high similarity score with statistical significance for a given fitness profile. We demonstrated that our new repository and algorithm are highly beneficial to researchers who attempting to make hypotheses based on unknown modes-of-action of bioactive compounds, regardless of the types of experiments that have been performed using yeast deletion-mutant collection in various types of different measurement platforms, especially non-chip-based platforms. Conclusions We showed that our new database and algorithm are useful when attempting to construct a hypothesis regarding the unknown function of a bioactive compound through small-scale experiments with a yeast deletion collection in a platform independent manner. The FitRankDB and FitSearch enhance the ease of searching public yeast fitness profiles and obtaining insights into unknown mechanisms of action of drugs. FitSearch is freely available at http://fitsearch.kaist.ac.kr. PMID:23368702

A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1

PubMed Central

de Berardinis, Véronique; Vallenet, David; Castelli, Vanina; Besnard, Marielle; Pinet, Agnès; Cruaud, Corinne; Samair, Sumitta; Lechaplais, Christophe; Gyapay, Gabor; Richez, Céline; Durot, Maxime; Kreimeyer, Annett; Le Fèvre, François; Schächter, Vincent; Pezo, Valérie; Döring, Volker; Scarpelli, Claude; Médigue, Claudine; Cohen, Georges N; Marlière, Philippe; Salanoubat, Marcel; Weissenbach, Jean

2008-01-01

We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches. PMID:18319726

Remote intracranial recurrence of IDH mutant gliomas is associated with TP53 mutations and an 8q gain

PubMed Central

Nakae, Shunsuke; Kato, Takema; Murayama, Kazuhiro; Sasaki, Hikaru; Abe, Masato; Kumon, Masanobu; Kumai, Tadashi; Yamashiro, Kei; Inamasu, Joji; Hasegawa, Mitsuhiro; Kurahashi, Hiroki; Hirose, Yuichi

2017-01-01

Most IDH mutant gliomas harbor either 1p/19q co-deletions or TP53 mutation; 1p/19q co-deleted tumors have significantly better prognoses than tumors harboring TP53 mutations. To investigate the clinical factors that contribute to differences in tumor progression of IDH mutant gliomas, we classified recurrent tumor patterns based on MRI and correlated these patterns with their genomic characterization. Accordingly, in IDH mutant gliomas (N = 66), 1p/19 co-deleted gliomas only recurred locally, whereas TP53 mutant gliomas recurred both locally and in remote intracranial regions. In addition, diffuse tensor imaging suggested that remote intracranial recurrence in the astrocytomas, IDH-mutant with TP53 mutations may occur along major fiber bundles. Remotely recurrent tumors resulted in a higher mortality and significantly harbored an 8q gain; astrocytomas with an 8q gain resulted in significantly shorter overall survival than those without an 8q gain. OncoScan® arrays and next-generation sequencing revealed specific 8q regions (i.e., between 8q22 and 8q24) show a high copy number. In conclusion, only tumors with TP53 mutations showed patterns of remote recurrence in IDH mutant gliomas. Furthermore, an 8q gain was significantly associated with remote intracranial recurrence and can be considered a poor prognostic factor in astrocytomas, IDH-mutant. PMID:29156679

Transcriptional response to deletion of the phosphatidylserine decarboxylase Psd1p in the yeast Saccharomyces cerevisiae.

PubMed

Gsell, Martina; Mascher, Gerald; Schuiki, Irmgard; Ploier, Birgit; Hrastnik, Claudia; Daum, Günther

2013-01-01

In the yeast, Saccharomyces cerevisiae, the synthesis of the essential phospholipid phosphatidylethanolamine (PE) is accomplished by a network of reactions which comprises four different pathways. The enzyme contributing most to PE formation is the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) which catalyzes conversion of phosphatidylserine (PS) to PE. To study the genome wide effect of an unbalanced cellular and mitochondrial PE level and in particular the contribution of Psd1p to this depletion we performed a DNA microarray analysis with a ∆psd1 deletion mutant. This approach revealed that 54 yeast genes were significantly up-regulated in the absence of PSD1 compared to wild type. Surprisingly, marked down-regulation of genes was not observed. A number of different cellular processes in different subcellular compartments were affected in a ∆psd1 mutant. Deletion mutants bearing defects in all 54 candidate genes, respectively, were analyzed for their growth phenotype and their phospholipid profile. Only three mutants, namely ∆gpm2, ∆gph1 and ∆rsb1, were affected in one of these parameters. The possible link of these mutations to PE deficiency and PSD1 deletion is discussed.

Deletion of the β-Propeller Protein Gene Rv1057 Reduces ESAT-6 Secretion and Intracellular Growth of Mycobacterium tuberculosis.

PubMed

Fu, Jiafang; Zong, Gongli; Zhang, Peipei; Gu, Yuanxin; Cao, Guangxiang

2018-04-01

Rv1057 is the only β-propeller protein in Mycobacterium tuberculosis, but its biological function is still unclear. In this study, we generated a deletion mutant of Rv1057 (D1057) in the virulent M. tuberculosis strain H37Rv and examined the characteristics of the mutant in vitro and in macrophages. We found that deletion of Rv1057 reduces secretion of the major virulence factor ESAT-6 and ESAT-6 stops in the cell envelope fraction during secretion, although ESAT-6 levels were similar in lysates of the mutant and control strains. In infected macrophages, Rv1057 deletion significantly reduced the secretion levels of cytokines IL-1β, IL-10, TNF-α, and INF-γ, but did not affect IL-4 and IL-8. D1057-infected macrophages also release less LDH and produce more nitric oxide (NO) than H37Rv- and D1057com (Rv1057 complemented strain of D1057com)-infected macrophages, indicating that D1057 has the decreased cytotoxicity compared to H37Rv or D1057com. In addition, the capacity of the Rv1057 deletion mutant to grow in macrophages was significantly lower than that of H37Rv and D1057com. Our findings support a role for Rv1057 in ESAT-6 secretion and in modulating the interactions between M. tuberculosis and macrophages.

High-molecular-weight glutenin subunit-deficient mutants induced by ion beam and the effects of Glu-1 loci deletion on wheat quality properties.

PubMed

Zhang, Lujun; Chen, Qiufang; Su, Mingjie; Yan, Biao; Zhang, Xiangqi; Jiao, Zhen

2016-03-15

High-molecular-weight glutenin subunits (HMW-GSs) play a critical role in determining the viscoelastic properties of wheat. Mutations induced by ion beam radiation have been applied to improve the yield and quality of crop. In this study, HMW-GS-deficient mutant lines were selected and the effects of Glu-1 loci deletion on wheat quality properties were illustrated according to the analysis of dry seeds of common wheat (Triticum aestivum L.) Xiaoyan 81 treated with a nitrogen ion beam. Three HMW-GS-deficient mutant lines were obtained and then detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Large-chromosome-fragment deletion resulted in specific deficiencies, and the deleted region sizes were determined using molecular markers. Agronomic characters, quantity and proportion of glutenins and dough microstructure of the deletion lines all proved to be quite different from those of wild-type Xiaoyan 81. Analysis of quality properties suggested that GluA1(-) had superior property parameters, while GluB1(-) and GluD1(-) both showed a significant decrease in quality properties compared with Xiaoyan 81. The effects of the three Glu-1 loci on flour and dough quality-related parameters should be Glu-D1 > Glu-B1 > Glu-A1. Ion beam radiation can be used as a mutagen to create new crop mutants. © 2015 Society of Chemical Industry.

Effect of apoA-I Mutations in the Capacity of Reconstituted HDL to Promote ABCG1-Mediated Cholesterol Efflux.

PubMed

Daniil, Georgios; Zannis, Vassilis I; Chroni, Angeliki

2013-01-01

ATP binding cassette transporter G1 (ABCG1) mediates the cholesterol transport from cells to high-density lipoprotein (HDL), but the role of apolipoprotein A-I (apoA-I), the main protein constituent of HDL, in this process is not clear. To address this, we measured cholesterol efflux from HEK293 cells or J774 mouse macrophages overexpressing ABCG1 using as acceptors reconstituted HDL (rHDL) containing wild-type or various mutant apoA-I forms. It was found that ABCG1-mediated cholesterol efflux was severely reduced (by 89%) when using rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185-243)]. ABCG1-mediated cholesterol efflux was not affected or moderately decreased by rHDL containing amino-terminal deletion mutants and several mid-region deletion or point apoA-I mutants, and was restored to 69-99% of control by double deletion mutants apoA-I[Δ(1-41)Δ(185-243)] and apoA-I[Δ(1-59)Δ(185-243)]. These findings suggest that the central helices alone of apoA-I associated to rHDL can promote ABCG1-mediated cholesterol efflux. Further analysis showed that rHDL containing the carboxyl-terminal deletion mutant apoA-I[Δ(185-243)] only slightly reduced (by 22%) the ABCG1-mediated efflux of 7-ketocholesterol, indicating that depending on the sterol type, structural changes in rHDL-associated apoA-I affect differently the ABCG1-mediated efflux of cholesterol and 7-ketocholesterol. Overall, our findings demonstrate that rHDL-associated apoA-I structural changes affect the capacity of rHDL to accept cellular cholesterol by an ABCG1-mediated process. The structure-function relationship seen here between rHDL-associated apoA-I mutants and ABCG1-mediated cholesterol efflux closely resembles that seen before in lipid-free apoA-I mutants and ABCA1-dependent cholesterol efflux, suggesting that both processes depend on the same structural determinants of apoA-I.

Development of a Markerless Genetic Exchange System in Desulfovibrio vulgaris Hildenborough and Its Use in Generating a Strain with Increased Transformation Efficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keller, Kimberly L.; Bender, Kelly S.; Wall, Judy D.

2009-07-21

In recent years, the genetic manipulation of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough has seen enormous progress. In spite of this progress, the current marker exchange deletion method does not allow for easy selection of multiple sequential gene deletions in a single strain because of the limited number of selectable markers available in D. vulgaris. To broaden the repertoire of genetic tools for manipulation, an in-frame, markerless deletion system has been developed. The counterselectable marker that makes this deletion system possible is the pyrimidine salvage enzyme, uracil phosphoribosyltransferase, encoded by upp. In wild-type D. vulgaris, growth was shown to bemore » inhibited by the toxic pyrimidine analog 5-fluorouracil (5-FU); whereas, a mutant bearing a deletion of the upp gene was resistant to 5-FU. When a plasmid containing the wild-type upp gene expressed constitutively from the aph(3')-II promoter (promoter for the kanamycin resistance gene in Tn5) was introduced into the upp deletion strain, sensitivity to 5-FU was restored. This observation allowed us to develop a two-step integration and excision strategy for the deletion of genes of interest. Since this inframe deletion strategy does not retain an antibiotic cassette, multiple deletions can be generated in a single strain without the accumulation of genes conferring antibiotic resistances. We used this strategy to generate a deletion strain lacking the endonuclease (hsdR, DVU1703) of a type I restriction-modification system, that we designated JW7035. The transformation efficiency of the JW7035 strain was found to be 100 to 1000 times greater than that of the wild-type strain when stable plasmids were introduced via electroporation.« less

The role of RNase H2 in processing ribonucleotides incorporated during DNA replication.

PubMed

Williams, Jessica S; Gehle, Daniel B; Kunkel, Thomas A

2017-05-01

Saccharomyces cerevisiae RNase H2 resolves RNA-DNA hybrids formed during transcription and it incises DNA at single ribonucleotides incorporated during nuclear DNA replication. To distinguish between the roles of these two activities in maintenance of genome stability, here we investigate the phenotypes of a mutant of yeast RNase H2 (rnh201-RED; ribonucleotide excision defective) that retains activity on RNA-DNA hybrids but is unable to cleave single ribonucleotides that are stably incorporated into the genome. The rnh201-RED mutant was expressed in wild type yeast or in a strain that also encodes a mutant allele of DNA polymerase ε (pol2-M644G) that enhances ribonucleotide incorporation during DNA replication. Similar to a strain that completely lacks RNase H2 (rnh201Δ), the pol2-M644G rnh201-RED strain exhibits replication stress and checkpoint activation. Moreover, like its null mutant counterpart, the double mutant pol2-M644G rnh201-RED strain and the single mutant rnh201-RED strain delete 2-5 base pairs in repetitive sequences at a high rate that is topoisomerase 1-dependent. The results highlight an important role for RNase H2 in maintaining genome integrity by removing single ribonucleotides incorporated during DNA replication. Published by Elsevier B.V.

Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jinsil; Ha, Hye-Jeong; Kim, Sujin

Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that anmore » rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.« less

Construction of a Recyclable Genetic Marker and Serial Gene Deletions in the Human Pathogenic Mucorales Mucor circinelloides.

PubMed

Garcia, Alexis; Adedoyin, Gloria; Heitman, Joseph; Lee, Soo Chan

2017-07-05

Mucor circinelloides is a human pathogen, biofuel producer, and model system that belongs to a basal fungal lineage; however, the genetics of this fungus are limited. In contrast to ascomycetes and basidiomycetes, basal fungal lineages have been understudied. This may be caused by a lack of attention given to these fungi, as well as limited tools for genetic analysis. Nonetheless, the importance of these fungi as pathogens and model systems has increased. M. circinelloides is one of a few genetically tractable organisms in the basal fungi, but it is far from a robust genetic system when compared to model fungi in the subkingdom Dikarya. One problem is the organism is resistant to drugs utilized to select for dominant markers in other fungal transformation systems. Thus, we developed a blaster recyclable marker system by using the pyrG gene (encoding an orotidine-5'-phosphate decarboxylase, ortholog of URA3 in Saccharomyces cerevisiae ). A 237-bp fragment downstream of the pyrG gene was tandemly incorporated into the upstream region of the gene, resulting in construction of a pyrG-dpl237 marker. To test the functionality of the pyrG-dpl237 marker, we disrupted the carRP gene that is involved in carotenoid synthesis in pyrG - mutant background. The resulting carRP :: pyrG-dpl237 mutants exhibit a white colony phenotype due to lack of carotene, whereas wild type displays yellowish colonies. The pyrG marker was then successfully excised, generating carRP-dpl237 on 5-FOA medium. The mutants became auxotrophic and required uridine for growth. We then disrupted the calcineurin B regulatory subunit cnbR gene in the carRP :: dpl237 strain, generating mutants with the alleles carRP :: dpl237 and cnbR :: pyrG These results demonstrate that the recyclable marker system is fully functional, and therefore the pyrG-dpl237 marker can be used for sequential gene deletions in M. circinelloides . Copyright © 2017 Garcia et al.

Circularized Chromosome with a Large Palindromic Structure in Streptomyces griseus Mutants

PubMed Central

Uchida, Tetsuya; Ishihara, Naoto; Zenitani, Hiroyuki; Hiratsu, Keiichiro; Kinashi, Haruyasu

2004-01-01

Streptomyces linear chromosomes display various types of rearrangements after telomere deletion, including circularization, arm replacement, and amplification. We analyzed the new chromosomal deletion mutants Streptomyces griseus 301-22-L and 301-22-M. In these mutants, chromosomal arm replacement resulted in long terminal inverted repeats (TIRs) at both ends; different sizes were deleted again and recombined inside the TIRs, resulting in a circular chromosome with an extremely large palindrome. Short palindromic sequences were found in parent strain 2247, and these sequences might have played a role in the formation of this unique structure. Dynamic structural changes of Streptomyces linear chromosomes shown by this and previous studies revealed extraordinary strategies of members of this genus to keep a functional chromosome, even if it is linear or circular. PMID:15150216

Essential role of the HMG domain in the function of yeast mitochondrial histone HM: functional complementation of HM by the nuclear nonhistone protein NHP6A.

PubMed

Kao, L R; Megraw, T L; Chae, C B

1993-06-15

The yeast mitochondrial histone protein HM is required for maintenance of the mitochondrial genome, and disruption of the gene encoding HM (HIM1/ABF2) results in formation of a respiration-deficient petite mutant phenotype. HM contains two homologous regions, which share sequence similarity with the eukaryotic nuclear nonhistone protein, HMG-1. Experiments with various deletion mutants of HM show that a single HMG domain of HM is functional and can restore respiration competency to cells that lack HM protein (him1 mutant cells). The gene encoding the putative yeast nuclear HMG-1 homolog, the NHP6A protein, can functionally complement the him1 mutation. These results suggest that the HMG domain is the basic unit for the function of HM in mitochondria and that the function of HMG-1 proteins in the nucleus and HM in the mitochondrion may be equivalent.

Lack of galactose or galacturonic acid in Bradyrhizobium japonicum USDA 110 exopolysaccharide leads to different symbiotic responses in soybean.

PubMed

Quelas, Juan Ignacio; Mongiardini, Elías J; Casabuono, Adriana; López-García, Silvina L; Althabegoiti, M Julia; Covelli, Julieta M; Pérez-Giménez, Julieta; Couto, Alicia; Lodeiro, Aníbal R

2010-12-01

Exopolysaccharide (EPS) and lipopolysaccharide (LPS) from Bradyrhizobium japonicum are important for infection and nodulation of soybean (Glycine max), although their roles are not completely understood. To better understand this, we constructed mutants in B. japonicum USDA 110 impaired in galactose or galacturonic acid incorporation into the EPS without affecting the LPS. The derivative LP 3010 had a deletion of lspL-ugdH and produced EPS without galacturonic acid whereas LP 3013, with an insertion in exoB, produced EPS without galactose. In addition, the strain LP 3017, with both mutations, had EPS devoid of both galactosides. The missing galactosides were not replaced by other sugars. The defects in EPS had different consequences. LP 3010 formed biofilms and nodulated but was defective in competitiveness for nodulation; and, inside nodules, the peribacteroid membranes tended to fuse, leading to the merging of symbiosomes. Meanwhile, LP 3013 and LP 3017 were unable to form biofilms and produced empty pseudonodules but exoB suppressor mutants were obtained when LP 3013 plant inoculation was supplemented with wild-type EPS. Similar phenotypes were observed with all these mutants in G. soja. Therefore, the lack of each galactoside in the EPS has a different functional effect on the B. japonicum-soybean symbiosis.

Increased frequency of DNA deletions in pink-eyed unstable mice carrying a mutation in the Werner syndrome gene homologue.

PubMed

Lebel, Michel

2002-01-01

Werner syndrome (WS) is a rare autosomal recessive disorder characterized by genomic instability and the premature onset of a number of age-related diseases, including cancers. Accumulating evidence indicates that the WS gene product is involved in resolving aberrant DNA structures that may arise during the process of DNA replication and/or transcription. To estimate the frequency of DNA deletions directly in the skin of mouse embryos, mice with a deletion of part of the murine WRN helicase domain were created. These mutant mice were then crossed to the pink-eyed unstable animals, which have a 70 kb internal duplication at the pink-eyed dilution (p) gene. This report indicates that the frequency of deletion of the duplicated sequence at the p locus is elevated in mice with a mutation in the WRN allele when compared with wild-type mice. In addition, the inhibitor of topoisomerase I camptothecin also increases the frequency of deletion at the p locus. This frequency is even more elevated in WRN mutant mice treated with camptothecin. In contrast, while the inhibition of poly(ADP-ribose) polymerase (PARP) activity by 3-aminobenzamide increases the frequency of DNA deletion, mutant WRN mice are not significantly more sensitive to the inhibition of PARP activity than wild-type animals.

The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.

PubMed

Fujinami, Shun; Ito, Masahiro

2018-01-01

It is well known that the Na + cycle and the cell wall are essential for alkaline adaptation of Na + -dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na + concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na + concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.

GABAA Receptors Containing ρ1 Subunits Contribute to In Vivo Effects of Ethanol in Mice

PubMed Central

Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Leiter, Courtney R.; Osterndorff-Kahanek, Elizabeth; Johnson, David; Borghese, Cecilia M.; Hanrahan, Jane R.; Johnston, Graham A. R.; Chebib, Mary; Harris, R. Adron

2014-01-01

GABAA receptors consisting of ρ1, ρ2, or ρ3 subunits in homo- or hetero-pentamers have been studied mainly in retina but are detected in many brain regions. Receptors formed from ρ1 are inhibited by low ethanol concentrations, and family-based association analyses have linked ρ subunit genes with alcohol dependence. We determined if genetic deletion of ρ1 in mice altered in vivo ethanol effects. Null mutant male mice showed reduced ethanol consumption and preference in a two-bottle choice test with no differences in preference for saccharin or quinine. Null mutant mice of both sexes demonstrated longer duration of ethanol-induced loss of righting reflex (LORR), and males were more sensitive to ethanol-induced motor sedation. In contrast, ρ1 null mice showed faster recovery from acute motor incoordination produced by ethanol. Null mutant females were less sensitive to ethanol-induced development of conditioned taste aversion. Measurement of mRNA levels in cerebellum showed that deletion of ρ1 did not change expression of ρ2, α2, or α6 GABAA receptor subunits. (S)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ1” antagonist), when administered to wild type mice, mimicked the changes that ethanol induced in ρ1 null mice (LORR and rotarod tests), but the ρ1 antagonist did not produce these effects in ρ1 null mice. In contrast, (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (“ρ2” antagonist) did not change ethanol actions in wild type but produced effects in mice lacking ρ1 that were opposite of the effects of deleting (or inhibiting) ρ1. These results suggest that ρ1 has a predominant role in two in vivo effects of ethanol, and a role for ρ2 may be revealed when ρ1 is deleted. We also found that ethanol produces similar inhibition of function of recombinant ρ1 and ρ2 receptors. These data indicate that ethanol action on GABAA receptors containing ρ1/ρ2 subunits may be important for specific effects of ethanol in vivo. PMID:24454882

A Genetic System for Clostridium ljungdahlii: a Chassis for Autotrophic Production of Biocommodities and a Model Homoacetogen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leang, C; Ueki, T; Nevin, KP

Methods for genetic manipulation of Clostridium ljungdahlii are of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies of C. ljungdahlii have not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation in C. ljungdahlii were identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vectormore » was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility in C. ljungdahlii. Deletion of fliA yielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases, adhE1 and adhE2, were deleted individually or together. Deletion of adhE1, but not adhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of the adhE1-deficient strain. Expression of adhE1 in trans partially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation of C. ljungdahlii to optimize autotrophic biocommodity production.« less

Zebrafish foxP2 Zinc Finger Nuclease Mutant Has Normal Axon Pathfinding

PubMed Central

Xing, Lingyan; Hoshijima, Kazuyuki; Grunwald, David J.; Fujimoto, Esther; Quist, Tyler S.; Sneddon, Jacob; Chien, Chi-Bin; Stevenson, Tamara J.; Bonkowsky, Joshua L.

2012-01-01

foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA) of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2nd coding exon: a 17 base-pair (bp) deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development. PMID:22937139

Zebrafish foxP2 zinc finger nuclease mutant has normal axon pathfinding.

PubMed

Xing, Lingyan; Hoshijima, Kazuyuki; Grunwald, David J; Fujimoto, Esther; Quist, Tyler S; Sneddon, Jacob; Chien, Chi-Bin; Stevenson, Tamara J; Bonkowsky, Joshua L

2012-01-01

foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA) of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2(nd) coding exon: a 17 base-pair (bp) deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.

Pheromones and Pheromone Receptors Are Required for Proper Sexual Development in the Homothallic Ascomycete Sordaria macrospora

PubMed Central

Mayrhofer, Severine; Weber, Jan M.; Pöggeler, Stefanie

2006-01-01

The homothallic, filamentous ascomycete Sordaria macrospora is self-fertile and produces sexual fruiting bodies (perithecia) without a mating partner. Even so, S. macrospora transcriptionally expresses two pheromone-precursor genes (ppg1 and ppg2) and two pheromone-receptor genes (pre1 and pre2). The proteins encoded by these genes are similar to α-factor-like and a-factor-like pheromones and to G-protein-coupled pheromone receptors of the yeast Saccharomyces cerevisiae. It has been suggested that in S. macrospora, PPG1/PRE2 and PPG2/PRE1 form two cognate pheromone–receptor pairs. To investigate their function, we deleted (Δ) pheromone-precursor genes (Δppg1, Δppg2) and receptor genes (Δpre1, Δpre2) and generated single- as well as double-knockout strains. No effect on vegetative growth, fruiting-body, and ascospore development was seen in the single pheromone-mutant and receptor-mutant strains, respectively. However, double-knockout strains lacking any compatible pheromone-receptor pair (Δpre2/Δppg2, Δpre1/Δppg1) and the double-pheromone mutant (Δppg1/Δppg2) displayed a drastically reduced number of perithecia and sexual spores, whereas deletion of both receptor genes (Δpre1/Δpre2) completely eliminated fruiting-body and ascospore formation. The results suggest that pheromones and pheromone receptors are required for optimal sexual reproduction of the homothallic S. macrospora. PMID:16387884

Pheromones and pheromone receptors are required for proper sexual development in the homothallic ascomycete Sordaria macrospora.

PubMed

Mayrhofer, Severine; Weber, Jan M; Pöggeler, Stefanie

2006-03-01

The homothallic, filamentous ascomycete Sordaria macrospora is self-fertile and produces sexual fruiting bodies (perithecia) without a mating partner. Even so, S. macrospora transcriptionally expresses two pheromone-precursor genes (ppg1 and ppg2) and two pheromone-receptor genes (pre1 and pre2). The proteins encoded by these genes are similar to alpha-factor-like and a-factor-like pheromones and to G-protein-coupled pheromone receptors of the yeast Saccharomyces cerevisiae. It has been suggested that in S. macrospora, PPG1/PRE2 and PPG2/PRE1 form two cognate pheromone-receptor pairs. To investigate their function, we deleted (delta) pheromone-precursor genes (delta ppg1, delta ppg2) and receptor genes (delta pre1, delta pre2) and generated single- as well as double-knockout strains. No effect on vegetative growth, fruiting-body, and ascospore development was seen in the single pheromone-mutant and receptor-mutant strains, respectively. However, double-knockout strains lacking any compatible pheromone-receptor pair (delta pre2/delta ppg2, delta pre1/delta ppg1) and the double-pheromone mutant (delta ppg1/delta ppg2) displayed a drastically reduced number of perithecia and sexual spores, whereas deletion of both receptor genes (delta pre1/delta pre2) completely eliminated fruiting-body and ascospore formation. The results suggest that pheromones and pheromone receptors are required for optimal sexual reproduction of the homothallic S. macrospora.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Xiaohong; Zhang Shuhui; Lin Jing

The role of the hepatitis B virus X protein (HBx) in hepatocarcinogenesis remains controversial. To investigate the biological impact of hepatitis B virus x gene (HBx) mutation on hepatoma cells, plasmids expressing the full-length HBx or HBx deletion mutants were constructed. The biological activities in these transfectants were analyzed by a series of assays. Results showed that HBx3'-20 and HBx3'-40 amino acid deletion mutants exhibited an increase in cellular proliferation, focus formation, tumorigenicity, and invasive growth and metastasis through promotion of the cell cycle from G0/G1 to the S phase, when compared with the full-length HBx. In contrast, HBx3'-30 aminomore » acid deletion mutant repressed cell proliferation by blocking in G1 phase. The expression of P53, p21{sup WAF1}, p14{sup ARF}, and MDM2 proteins was regulated by expression of HBx mutants. In conclusions, HBx variants showed different effects and functions on cell proliferation and invasion by regulation of the cell cycle progression and its associated proteins expression.« less

Ghrelin restoration of function in vitro in somatotropes from male mice lacking the Janus kinase (JAK)-binding site of the leptin receptor.

PubMed

Syed, Mohsin; Cozart, Michael; Haney, Anessa C; Akhter, Noor; Odle, Angela K; Allensworth-James, Melody; Crane, Christopher; Syed, Farhan M; Childs, Gwen V

2013-04-01

Deletion of the signaling domain of leptin receptors selectively in somatotropes, with Cre-loxP technology, reduced the percentage of immunolabeled GH cells and serum GH. We hypothesized that the deficit occurred when leptin's postnatal surge failed to stimulate an expansion in the cell population. To learn more about the deficiency in GH cells, we tested their expression of GHRH receptors and GH mRNA and the restorative potential of secretagogue stimulation in vitro. In freshly plated dissociated pituitary cells from control male mice, GHRH alone (0.3 nM) increased the percentage of immunolabeled GH cells from 27 ± 0.05% (vehicle) to 42 ± 1.8% (P < .002) and the secretion of GH 1.8-3×. Deletion mutant pituitary cells showed a 40% reduction in percentages of immunolabeled GH cells (16.7 ± 0.4%), which correlated with a 47% reduction in basal GH levels (50 ng/mL control; 26.7 ng/mL mutants P = .01). A 50% reduction in the percentage of mutant cells expressing GHRH receptors (to 12%) correlated with no or reduced responses to GHRH. Ghrelin alone (10 nM) stimulated more GH cells in mutants (from 16.7-23%). When added with 1-3 nM GHRH, ghrelin restored GH cell percentages and GH secretion to levels similar to those of stimulated controls. Counts of somatotropes labeled for GH mRNA confirmed normal percentages of somatotropes in the population. These discoveries suggest that leptin may optimize somatotrope function by facilitating expression of membrane GHRH receptors and the production or maintenance of GH stores.

A eukaryotic-acquired gene by a biotrophic phytopathogen allows prolonged survival on the host by counteracting the shut-down of plant photosynthesis.

PubMed

Garavaglia, Betiana S; Thomas, Ludivine; Gottig, Natalia; Dunger, Germán; Garofalo, Cecilia G; Daurelio, Lucas D; Ndimba, Bongani; Orellano, Elena G; Gehring, Chris; Ottado, Jorgelina

2010-01-28

Xanthomonas citri pv. citri, the bacteria responsible for citrus canker posses a biological active plant natriuretic peptide (PNP)-like protein, not present in any other bacteria. PNPs are a class of extracellular, systemically mobile peptides that elicit a number of plant responses important in homeostasis and growth. Previously, we showed that a Xanthomonas citri pv. citri mutant lacking the PNP-like protein XacPNP produced more necrotic lesions in citrus leaves than wild type infections and suggested a role for XacPNP in the regulation of host homeostasis. Here we have analyzed the proteome modifications observed in citrus leaves infected with the wild type and XacPNP deletion mutant bacteria. While both of them cause down-regulation of enzymes related to photosynthesis as well as chloroplastic ribosomal proteins, proteins related to defense responses are up-regulated. However, leaves infiltrated with the XacPNP deletion mutant show a more pronounced decrease in photosynthetic proteins while no reduction in defense related proteins as compared to the wild-type pathogen. This suggests that XacPNP serves the pathogen to maintain host photosynthetic efficiency during pathogenesis. The results from the proteomics analyses are consistent with our chlorophyll fluorescence data and transcript analyses of defense genes that show a more marked reduction in photosynthesis in the mutant but no difference in the induction of genes diagnostic for biotic-stress responses. We therefore conclude that XacPNP counteracts the shut-down of host photosynthesis during infection and in that way maintains the tissue in better conditions, suggesting that the pathogen has adapted a host gene to modify its natural host and render it a better reservoir for prolonged bacterial survival and thus for further colonization.

Trehalose synthesis in Aspergillus niger: characterization of six homologous genes, all with conserved orthologs in related species

PubMed Central

2014-01-01

Background The disaccharide trehalose is a major component of fungal spores and is released upon germination. Moreover, the sugar is well known for is protective functions, e.g. against thermal stress and dehydration. The properties and synthesis of trehalose have been well investigated in the bakers’ yeast Saccharomyces cerevisiae. In filamentous fungi, such knowledge is limited, although several gene products have been identified. Results Using Aspergillus niger as a model fungus, the aim of this study was to provide an overview of all genes involved in trehalose synthesis. This fungus has three potential trehalose-6-phosphate synthase encoding genes, tpsA-C, and three putative trehalose phosphate phosphatase encoding genes, tppA-C, of which two have not previously been identified. Expression of all six genes was confirmed using real-time PCR, and conserved orthologs could be identified in related Aspergilli. Using a two-hybrid approach, there is a strong indication that four of the proteins physically interact, as has previously been shown in S. cerevisiae. When creating null mutants of all the six genes, three of them, ΔtpsA, ΔtppA and ΔtppB, had lower internal trehalose contents. The only mutant with a pronounced morphological difference was ΔtppA, in which sporulation was severely reduced with abnormal conidiophores. This was also the only mutant with accumulated levels of trehalose-6-phosphate, indicating that the encoded protein is the main phosphatase under normal conditions. Besides ΔtppA, the most studied deletion mutant in this work was ΔtppB. This gene encodes a protein conserved in filamentous Ascomycota. The ΔtppB mutant displayed a low, but not depleted, internal trehalose content, and conidia were more susceptible to thermal stress. Conclusion A. niger contains at least 6 genes putatively involved in trehalose synthesis. Gene expressions related to germination have been quantified and deletion mutants characterized: Mutants lacking tpsA, tppA or tppB have reduced internal trehalose contents. Furthermore, tppA, under normal conditions, encodes the functional trehalose-6-phosphate-phosphatase. PMID:24725382

Genetic Separation of Hypoxanthine and Guanine-Xanthine Phosphoribosyltransferase Activities by Deletion Mutations in Salmonella typhimurium

PubMed Central

Gots, Joseph S.; Benson, Charles E.; Shumas, Susan R.

1972-01-01

Certain proAB deletion mutants of Salmonella typhimurium were found to be simultaneously deleted in a gene required for the utilization of guanine and xanthine (designated gxu). These mutants were resistant to 8-azaguanine and when carrying an additional pur mutation were unable to use guanine or xanthine as a purine source. The defect was correlated with deficiencies in the uptake and phosphoribosyltransferase activities for guanine and xanthine. Hypoxanthine and adenine activities were unaltered. The deficiency was restored to normal by transduction to pro+ and in F′ merodiploids. PMID:4563984

The mouse lymphoma assay detects recombination, deletion, and aneuploidy.

PubMed

Wang, Jianyong; Sawyer, Jeffrey R; Chen, Ling; Chen, Tao; Honma, Masamitsu; Mei, Nan; Moore, Martha M

2009-05-01

The mouse lymphoma assay (MLA) uses the thymidine kinase (Tk) gene of the L5178Y/Tk(+/-)-3.7.2C mouse lymphoma cell line as a reporter gene to evaluate the mutagenicity of chemical and physical agents. The MLA is recommended by both the United States Food and Drug Administration and the United States Environmental Protection Agency as the preferred in vitro mammalian cell mutation assay for genetic toxicology screening because it detects a wide range of genetic alterations, including both point mutations and chromosomal mutations. However, the specific types of chromosomal mutations that can be detected by the MLA need further clarification. For this purpose, three chemicals, including two clastogens and an aneugen (3'-azido-3'-deoxythymidine, mitomycin C, and taxol), were used to induce Tk mutants. Loss of heterozygosity (LOH) analysis was used to select mutants that could be informative as to whether they resulted from deletion, mitotic recombination, or aneuploidy. A combination of additional methods, G-banding analysis, chromosome painting, and a real-time PCR method to detect the copy number (CN) of the Tk gene was then used to provide a detailed analysis. LOH involving at least 25% of chromosome 11, a normal karyotype, and a Tk CN of 2 would indicate that the mutant resulted from recombination, whereas LOH combined with a karyotypically visible deletion of chromosome 11 and a Tk CN of 1 would indicate a deletion. Aneuploidy was confirmed using G-banding combined with chromosome painting analysis for mutants showing LOH at every microsatellite marker on chromosome 11. From this analysis, it is clear that mouse lymphoma Tk mutants can result from recombination, deletion, and aneuploidy.

Roles of Two Shewanella oneidensis MR-1 Extracellular Endonucleases ▿ †

PubMed Central

Gödeke, Julia; Heun, Magnus; Bubendorfer, Sebastian; Paul, Kristina; Thormann, Kai M.

2011-01-01

The dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 is capable of using extracellular DNA (eDNA) as the sole source of carbon, phosphorus, and nitrogen. In addition, we recently demonstrated that S. oneidensis MR-1 requires eDNA as a structural component during all stages of biofilm formation. In this study, we characterize the roles of two Shewanella extracellular endonucleases, ExeS and ExeM. While ExeS is likely secreted into the medium, ExeM is predicted to remain associated with the cell envelope. Both exeM and exeS are highly expressed under phosphate-limited conditions. Mutants lacking exeS and/or exeM exhibit decreased eDNA degradation; however, the capability of S. oneidensis MR-1 to use DNA as the sole source of phosphorus is only affected in mutants lacking exeM. Neither of the two endonucleases alleviates toxic effects of increased eDNA concentrations. The deletion of exeM and/or exeS significantly affects biofilm formation of S. oneidensis MR-1 under static conditions, and expression of exeM and exeS drastically increases during static biofilm formation. Under hydrodynamic conditions, a deletion of exeM leads to altered biofilms that consist of densely packed structures which are covered by a thick layer of eDNA. Based on these results, we hypothesize that a major role of ExeS and, in particular, ExeM of S. oneidensis MR-1, is to degrade eDNA as a matrix component during biofilm formation to improve nutrient supply and to enable detachment. PMID:21705528

Disruption of non-anchored cell wall protein NCW-1 promotes cellulase production by increasing cellobiose uptake in Neurospora crassa.

PubMed

Lin, Liangcai; Chen, Yong; Li, Jingen; Wang, Shanshan; Sun, Wenliang; Tian, Chaoguang

2017-04-01

To elucidate the mechanism of cellulase signal transduction in filamentous fungi including the components of the cellulase induction pathway. Neurospora crassa ncw-1 encodes a non-anchored cell wall protein. The absence of ncw-1 increased cellulase gene expression and this is not due to relieving carbon catabolite repression mediated by the cre-1 pathway. A mutant lacking genes encoding both three major β-glucosidase enzymes and NCW-1 (Δ3βGΔncw-1) was constructed. Transcriptome analysis of the quadruple mutant demonstrated enhanced expression of cellodextrin transporters after ncw-1 deletion, indicating that ncw-1 affects cellulase expression and production by inhibiting the uptake of the cellodextrin. NCW-1 is a novel component that plays a critical role in the cellulase induction signaling pathway.

Flies lacking all synapsins are unexpectedly healthy but are impaired in complex behaviour.

PubMed

Godenschwege, Tanja A; Reisch, Dietmar; Diegelmann, Sören; Eberle, Kai; Funk, Natalja; Heisenberg, Martin; Hoppe, Viviane; Hoppe, Jürgen; Klagges, Bert R E; Martin, Jean-René; Nikitina, Ekaterina A; Putz, Gabi; Reifegerste, Rita; Reisch, Natascha; Rister, Jens; Schaupp, Michael; Scholz, Henrike; Schwärzel, Martin; Werner, Ursula; Zars, Troy D; Buchner, Sigrid; Buchner, Erich

2004-08-01

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.

Evidence for a Key Role of Cytochrome bo3 Oxidase in Respiratory Energy Metabolism of Gluconobacter oxydans

PubMed Central

Richhardt, Janine; Luchterhand, Bettina; Büchs, Jochen

2013-01-01

The obligatory aerobic acetic acid bacterium Gluconobacter oxydans oxidizes a variety of substrates in the periplasm by membrane-bound dehydrogenases, which transfer the reducing equivalents to ubiquinone. Two quinol oxidases, cytochrome bo3 and cytochrome bd, then catalyze transfer of the electrons from ubiquinol to molecular oxygen. In this study, mutants lacking either of these terminal oxidases were characterized. Deletion of the cydAB genes for cytochrome bd had no obvious influence on growth, whereas the lack of the cyoBACD genes for cytochrome bo3 severely reduced the growth rate and the cell yield. Using a respiration activity monitoring system and adjusting different levels of oxygen availability, hints of a low-oxygen affinity of cytochrome bd oxidase were obtained, which were supported by measurements of oxygen consumption in a respirometer. The H+/O ratio of the ΔcyoBACD mutant with mannitol as the substrate was 0.56 ± 0.11 and more than 50% lower than that of the reference strain (1.26 ± 0.06) and the ΔcydAB mutant (1.31 ± 0.16), indicating that cytochrome bo3 oxidase is the main component for proton extrusion via the respiratory chain. Plasmid-based overexpression of cyoBACD led to increased growth rates and growth yields, both in the wild type and the ΔcyoBACD mutant, suggesting that cytochrome bo3 might be a rate-limiting factor of the respiratory chain. PMID:23852873

Characterization of hemizygous deletions in Citrus using array-Comparative Genomic Hybridization and microsynteny comparisons with the poplar genome

PubMed Central

Ríos, Gabino; Naranjo, Miguel A; Iglesias, Domingo J; Ruiz-Rivero, Omar; Geraud, Marion; Usach, Antonio; Talón, Manuel

2008-01-01

Background Many fruit-tree species, including relevant Citrus spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and self-incompatibility prevent the production of homozygous cultivars. Genomic technology may provide citrus researchers with a new set of tools to address these various restrictions. In this work, we report a valuable genomics-based protocol for the structural analysis of deletion mutations on an heterozygous background. Results Two independent fast neutron mutants of self-incompatible clementine (Citrus clementina Hort. Ex Tan. cv. Clemenules) were the subject of the study. Both mutants, named 39B3 and 39E7, were expected to carry DNA deletions in hemizygous dosage. Array-based Comparative Genomic Hybridization (array-CGH) using a Citrus cDNA microarray allowed the identification of underrepresented genes in these two mutants. Subsequent comparison of citrus deleted genes with annotated plant genomes, especially poplar, made possible to predict the presence of a large deletion in 39B3 of about 700 kb and at least two deletions of approximately 100 and 500 kb in 39E7. The deletion in 39B3 was further characterized by PCR on available Citrus BACs, which helped us to build a partial physical map of the deletion. Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues. Conclusion In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina. The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana. This work corroborates the potential of Citrus genomic resources to assist mutagenesis-based approaches for functional genetics, structural studies and comparative genomics, and hence to facilitate citrus variety improvement. PMID:18691431

Molecular analysis and comparison of radiation-induced large deletions of the HPRT locus in primary human skin fibroblasts

NASA Technical Reports Server (NTRS)

Yamada, Y.; Park, M. S.; Okinaka, R. T.; Chen, D. J.

1996-01-01

Genetic alterations in gamma-ray- and alpha-particle-induced HPRT mutants were examined by multiplex polymerase chain reaction (PCR) analysis. A total of 39-63% of gamma-ray-induced and 31-57% of alpha-particle-induced mutants had partial or total deletions of the HPRT gene. The proportion of these deletion events was dependent on radiation dose, and at the resolution limits employed there were no significant differences between the spectra induced by equitoxic doses of alpha particles (0.2-0.4 Gy) and gamma rays (3 Gy). The molecular nature of the deletions was analyzed by the use of sequence tagged site (STS) primers and PCR amplification as a "probe" for specific regions of the human X chromosome within the Xq26 region. These STSs were closely linked and spanned regions approximately 1.7 Mbp from the telomeric side and 1.7 Mbp from the centromeric side of the HPRT gene. These markers include: DXS53, 299R, DXS79, yH3L, 3/19, PR1, PR25, H2, yH3R, 1/44, 1/67, 1/1, DXS86, D8C6, DXS10 and DXS144. STS analyses indicated that the maximum size of total deletions in radiation-induced HPRT mutants can be greater than 2.7 Mbp and deletion size appears to be dependent on radiation dose. There were no apparent differences in the sizes of the deletions induced by alpha particles or gamma rays. On the other hand, deletions containing portions of the HPRT gene were observed to be 800 kbp or less, and the pattern of the partial deletion induced by alpha particles appeared to be different from that induced by gamma rays.

Saccharomyces cerevisiae Msh2-Msh3 acts in repair of base-base mispairs.

PubMed

Harrington, Jill M; Kolodner, Richard D

2007-09-01

DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding.

Saccharomyces cerevisiae Msh2-Msh3 Acts in Repair of Base-Base Mispairs▿ †

PubMed Central

Harrington, Jill M.; Kolodner, Richard D.

2007-01-01

DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding. PMID:17636021

GSK-3 signaling in developing cortical neurons is essential for radial migration and dendritic orientation.

PubMed

Morgan-Smith, Meghan; Wu, Yaohong; Zhu, Xiaoqin; Pringle, Julia; Snider, William D

2014-07-29

GSK-3 is an essential mediator of several signaling pathways that regulate cortical development. We therefore created conditional mouse mutants lacking both GSK-3α and GSK-3β in newly born cortical excitatory neurons. Gsk3-deleted neurons expressing upper layer markers exhibited striking migration failure in all areas of the cortex. Radial migration in hippocampus was similarly affected. In contrast, tangential migration was not grossly impaired after Gsk3 deletion in interneuron precursors. Gsk3-deleted neurons extended axons and developed dendritic arbors. However, the apical dendrite was frequently branched while basal dendrites exhibited abnormal orientation. GSK-3 regulation of migration in neurons was independent of Wnt/β-catenin signaling. Importantly, phosphorylation of the migration mediator, DCX, at ser327, and phosphorylation of the semaphorin signaling mediator, CRMP-2, at Thr514 were markedly decreased. Our data demonstrate that GSK-3 signaling is essential for radial migration and dendritic orientation and suggest that GSK-3 mediates these effects by phosphorylating key microtubule regulatory proteins.DOI: http://dx.doi.org/10.7554/eLife.02663.001. Copyright © 2014, Morgan-Smith et al.

Metabolic engineering of Corynebacterium glutamicum ATCC13869 for L-valine production.

PubMed

Chen, Cheng; Li, Yanyan; Hu, Jinyu; Dong, Xunyan; Wang, Xiaoyuan

2015-05-01

In this study, an L-valine-producing strain was developed from Corynebacterium glutamicum ATCC13869 through deletion of the three genes aceE, alaT and ilvA combined with the overexpression of six genes ilvB, ilvN, ilvC, lrp1, brnF and brnE. Overexpression of lrp1 alone increased L-valine production by 16-fold. Deletion of the aceE, alaT and ilvA increased L-valine production by 44-fold. Overexpression of the six genes ilvB, ilvN, ilvC, lrp1, brnE and brnF in the triple deletion mutant WCC003 further increased L-valine production. The strain WCC003/pJYW-4-ilvBNC1-lrp1-brnFE produced 243mM L-valine in flask cultivation and 437mM (51g/L) L-valine in fed-batch fermentation and lacked detectable amino-acid byproduct such as l-alanine and l-isoleucine that are usually found in the fermentation of L-valine-producing C. glutamicum. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

The type III secretion system is involved in the invasion and intracellular survival of Escherichia coli K1 in human brain microvascular endothelial cells.

PubMed

Yao, Yufeng; Xie, Yi; Perace, Donna; Zhong, Yi; Lu, Jie; Tao, Jing; Guo, Xiaokui; Kim, Kwang Sik

2009-11-01

Type III secretion systems (T3SSs) have been documented in many Gram-negative bacteria, including enterohemorrhagic Escherichia coli. We have previously shown the existence of a putative T3SS in meningitis-causing E. coli K1 strains, referred to as E. coli type III secretion 2 (ETT2). The sequence of ETT2 in meningitis-causing E. coli K1 strain EC10 (O7:K1) revealed that ETT2 comprises the epr, epa and eiv genes, but bears mutations, deletions and insertions. We constructed the EC10 mutants deleted of ETT2 or eivA gene, and their contributions to bacterial pathogenesis were evaluated in human brain microvascular endothelial cells (HBMECs). The deletion mutant of ETT2 exhibited defects in invasion and intracellular survival compared with the parental E. coli K1 strain EC10. The mutant deleted of eivA within ETT2 was also significantly defective in invasion and intracellular survival in HBMECs, and the defects of the eiv mutant were restored to the levels of the parent strain EC10 by transcomplementation. These findings suggest that ETT2 plays a role in the pathogenesis of E. coli K1 infection, including meningitis.

Elimination of the last reactions in ergosterol biosynthesis alters the resistance of Saccharomyces cerevisiae to multiple stresses.

PubMed

Liu, Guodong; Chen, Yun; Færgeman, Nils J; Nielsen, Jens

2017-09-01

The sterol composition of membranes is known to influence many phenotypes of yeast. However, a systematic study of the relationship between sterol composition and stress resistances has not been conducted. Here, we therefore constructed single or double gene deletion mutants of the last four enzymes in ergosterol biosynthesis in a prototrophic genetic background of Saccharomyces cerevisiae. Identification of the sterol composition of these mutants revealed a high flexibility of the sterol-processing steps instead of the previously proposed sequential conversion. Compared with the wild type, the mutants showed altered resistances to different exogenous stresses regarding the specific growth rate and duration of lag phase. The erg5 deletion mutant whose sterol has a saturated side chain exhibited overall robust growth under the tested stress conditions. The thermotolerant phenotype of erg5 deletion mutant was reproduced in filamentous fungus Penicillium oxalicum. These results highlight the important role of sterols in the response of yeast cells to environmental stresses, and suggest the possibility of improving the robustness of industrial yeast strains by engineering their sterol composition. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production.

PubMed

Jung, Moo-Young; Ng, Chiam Yu; Song, Hyohak; Lee, Jinwon; Oh, Min-Kyu

2012-07-01

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.

Structure and biosynthesis of two exopolysaccharides produced by Lactobacillus johnsonii FI9785.

PubMed

Dertli, Enes; Colquhoun, Ian J; Gunning, A Patrick; Bongaerts, Roy J; Le Gall, Gwénaëlle; Bonev, Boyan B; Mayer, Melinda J; Narbad, Arjan

2013-11-01

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1-3)-β-Glcp-(1-5)-β-Galf-(1-6)-α-Glcp-(1-4)-β-Galp-(1-4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.

Wt1 dictates the fate of fetal and adult Leydig cells during development in the mouse testis.

PubMed

Wen, Qing; Zheng, Qiao-Song; Li, Xi-Xia; Hu, Zhao-Yuan; Gao, Fei; Cheng, C Yan; Liu, Yi-Xun

2014-12-15

Wilms' tumor 1 (Wt1) is a tumor suppressor gene encoding ∼24 zinc finger transcription factors. In the mammalian testis, Wt1 is expressed mostly by Sertoli cells (SCs) involved in testis development, spermatogenesis, and adult Leydig cell (ALC) steroidogenesis. Global knockout (KO) of Wt1 is lethal in mice due to defects in embryogenesis. Herein, we showed that Wt1 is involved in regulating fetal Leydig cell (FLC) degeneration and ALC differentiation during testicular development. Using Wt1(-/flox);Amh-Cre mice that specifically deleted Wt1 in the SC vs. age-matched wild-type (WT) controls, FLC-like-clusters were found in Wt1-deficient testes that remained mitotically active from postnatal day 1 (P1) to P56, and no ALC was detected at these ages. Leydig cells in mutant adult testes displayed morphological features of FLC. Also, FLC-like cells in adult mutant testes had reduced expression in ALC-associated genes Ptgds, Sult1e1, Vcam1, Hsd11b1, Hsd3b6, and Hsd17b3 but high expression of FLC-associated genes Thbs2 and Hsd3b1. Whereas serum LH and testosterone level in mutant mice were not different from controls, intratesticular testosterone level was significantly reduced. Deletion of Wt1 gene also perturbed the expression of steroidogenic enzymes Star, P450c17, Hsd3b6, Hsd3b1, Hsd17b1, and Hsd17b3. FLCs in adult mutant testes failed to convert androstenedione to testosterone due to a lack of Hsd17b3, and this defect was rescued by coculturing with fetal SCs. In summary, FLC-like cells in mutant testes are putative FLCs that remain mitotically active in adult mice, illustrating that Wt1 dictates the fate of FLC and ALC during postnatal testis development. Copyright © 2014 the American Physiological Society.

Mutations in the Cytoplasmic Domain of the Newcastle Disease Virus Fusion Protein Confer Hyperfusogenic Phenotypes Modulating Viral Replication and Pathogenicity

PubMed Central

Samal, Sweety; Khattar, Sunil K.; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L.

2013-01-01

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis. PMID:23843643

Mutations in the cytoplasmic domain of the Newcastle disease virus fusion protein confer hyperfusogenic phenotypes modulating viral replication and pathogenicity.

PubMed

Samal, Sweety; Khattar, Sunil K; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L; Samal, Siba K

2013-09-01

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis.

Structure and Biosynthesis of Two Exopolysaccharides Produced by Lactobacillus johnsonii FI9785*

PubMed Central

Dertli, Enes; Colquhoun, Ian J.; Gunning, A. Patrick; Bongaerts, Roy J.; Le Gall, Gwénaëlle; Bonev, Boyan B.; Mayer, Melinda J.; Narbad, Arjan

2013-01-01

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1–3)-β-Glcp-(1–5)-β-Galf-(1–6)-α-Glcp-(1–4)-β-Galp-(1–4)-β-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host. PMID:24019531

Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein.

PubMed

Wartenberg, Dirk; Lapp, Katrin; Jacobsen, Ilse D; Dahse, Hans-Martin; Kniemeyer, Olaf; Heinekamp, Thorsten; Brakhage, Axel A

2011-11-01

Surface-associated and secreted proteins represent primarily exposed components of Aspergillus fumigatus during host infection. Several secreted proteins are known to be involved in defense mechanisms or immune evasion, thus, probably contributing to pathogenicity. Furthermore, several secreted antigens were identified as possible biomarkers for the verification of diseases caused by Aspergillus species. Nevertheless, there is only limited knowledge about the composition of the secretome and about molecular functions of particular proteins. To identify secreted proteins potentially essential for virulence, the core secretome of A. fumigatus grown in minimal medium was determined. Two-dimensional gel electrophoretic separation and subsequent MALDI-TOF-MS/MS analyses resulted in the identification of 64 different proteins. Additionally, secretome analyses of A. fumigatus utilizing elastin, collagen or keratin as main carbon and nitrogen source were performed. Thereby, the alkaline serine protease Alp1 was identified as the most abundant protein and hence presumably represents an important protease during host infection. Interestingly, the Asp-hemolysin (Asp-HS), which belongs to the protein family of aegerolysins and which was often suggested to be involved in fungal virulence, was present in the secretome under all growth conditions tested. In addition, a second, non-secreted protein with an aegerolysin domain annotated as Asp-hemolysin-like (HS-like) protein can be found to be encoded in the genome of A. fumigatus. Generation and analysis of Asp-HS and HS-like deletion strains revealed no differences in phenotype compared to the corresponding wild-type strain. Furthermore, hemolysis and cytotoxicity was not altered in both single-deletion and double-deletion mutants lacking both aegerolysin genes. All mutant strains showed no attenuation in virulence in a mouse infection model for invasive pulmonary aspergillosis. Overall, this study provides a comprehensive analysis of secreted proteins of A. fumigatus and a detailed characterization of hemolysin mutants. Copyright © 2011 Elsevier GmbH. All rights reserved.

Deletion and overexpression studies on DacB2, a putative low molecular mass penicillin binding protein from Mycobacterium tuberculosis H(37)Rv.

PubMed

Bourai, Neema; Jacobs, William R; Narayanan, Sujatha

2012-02-01

Mycobacterium tuberculosis genome encodes several high and low molecular mass penicillin binding proteins. One such low molecular mass protein is DacB2 encoded by open reading frame Rv2911 of M. tuberculosis which is predicted to play a role in peptidoglycan synthesis. In this study we have tried to gain an insight into the role of this accessory cell division protein in mycobacterial physiology by performing overexpression and deletion studies. The overproduction of DacB2 in non-pathogenic, fast growing mycobacterium Mycobacterium smegmatis mc(2)155 resulted in reduced growth, an altered colony morphology, a defect in sliding motility and biofilm formation. A point mutant of DacB2 was made wherein the active site serine residue was mutated to cysteine to abolish the penicillin binding function of protein. The overexpression of mutant protein showed similar results indicating that the effects produced were independent of protein's penicillin binding function. The gene encoding DacB2 was deleted in M. tuberculosis by specialized transduction method. The deletion mutant showed reduced growth in Sauton's medium under acidic and low oxygen availability. The in vitro infection studies with THP-1 cells showed increased intracellular survival of dacB2 mutant compared to parent and complemented strains. The colony morphology and antibiotic sensitivity of mutant and wild-type strains were similar. Copyright © 2011 Elsevier Ltd. All rights reserved.

A Temperature-Independent Cold-Shock Protein Homolog Acts as a Virulence Factor in Xylella fastidiosa.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2016-05-01

Xylella fastidiosa, causal agent of Pierce's disease (PD) of grapevine, is a fastidious organism that requires very specific conditions for replication and plant colonization. Cold temperatures reduce growth and survival of X. fastidiosa both in vitro and in planta. However, little is known regarding physiological responses of X. fastidiosa to temperature changes. Cold-shock proteins (CSP), a family of nucleic acid-binding proteins, act as chaperones facilitating translation at low temperatures. Bacterial genomes often encode multiple CSP, some of which are strongly induced following exposure to cold. Additionally, CSP contribute to the general stress response through mRNA stabilization and posttranscriptional regulation. A putative CSP homolog (Csp1) with RNA-binding activity was identified in X. fastidiosa Stag's Leap. The csp1 gene lacked the long 5' untranslated region characteristic of cold-inducible genes and was expressed in a temperature-independent manner. As compared with the wild type, a deletion mutant of csp1 (∆csp1) had decreased survival rates following cold exposure and salt stress in vitro. The deletion mutant also was significantly less virulent in grapevine, as compared with the wild type, in the absence of cold stress. These results suggest an important function of X. fastidiosa Csp1 in response to cellular stress and during plant colonization.

Reduction of Aspergillus niger Virulence in Apple Fruits by Deletion of the Catalase Gene cpeB.

PubMed

Zhang, Meng-Ke; Tang, Jun; Huang, Zhong-Qin; Hu, Kang-Di; Li, Yan-Hong; Han, Zhuo; Chen, Xiao-Yan; Hu, Lan-Ying; Yao, Gai-Fang; Zhang, Hua

2018-05-30

Aspergillus niger, a common saprophytic fungus, causes rot in many fruits. We studied the role of a putative catalase-peroxidase-encoding gene, cpeB, in oxidative stress and virulence in fruit. The cpeB gene was deleted in A. niger by homologous recombination, and the Δ cpeB mutant showed decreased CAT activity compared with that of the wild type. The cpeB gene deletion caused increased sensitivity to H 2 O 2 stress, and spore germination was significantly reduced; in addition, the reactive-oxygen-species (ROS) metabolites superoxide anions (·O 2 - ), hydrogen peroxide (H 2 O 2 ), and malondialdehyde (MDA) accumulated in the Δ cpeB mutant during H 2 O 2 stress. Furthermore, ROS metabolism in A. niger infected apples was determined, and our results showed that the Δ cpeB mutant induced an attenuated response in apple fruit during the fruit-pathogen interaction; the cpeB gene deletion significantly reduced the development of lesions, suggesting that the cpeB gene in A. niger is essential for full virulence in apples.

Mechanisms of mutagenesis in human cells exposed to 55 MeV protons

NASA Technical Reports Server (NTRS)

Gauny, S.; Wiese, C.; Kronenberg, A.

2001-01-01

Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from <1 cM to 64 cM, while mutations that arose by allelic recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pallan, Pradeep S.; Marshall, William S.; Harp, Joel

To understand the role of structural elements of RNA pseudoknots in controlling the extent of -1-type ribosomal frameshifting, we determined the crystal structure of a high-efficiency frameshifting mutant of the pseudoknot from potato leaf roll virus (PLRV). Correlations of the structure with available in vitro frameshifting data for PLRV pseudoknot mutants implicate sequence and length of a stem-loop linker as modulators of frameshifting efficiency. Although the sequences and overall structures of the RNA pseudoknots from PLRV and beet western yellow virus (BWYV) are similar, nucleotide deletions in the linker and adjacent minor groove loop abolish frameshifting only with the latter.more » Conversely, mutant PLRV pseudoknots with up to four nucleotides deleted in this region exhibit nearly wild-type frameshifting efficiencies. The crystal structure helps rationalize the different tolerances for deletions in the PLRV and BWYV RNAs, and we have used it to build a three-dimensional model of the PRLV pseudoknot with a four-nucleotide deletion. The resulting structure defines a minimal RNA pseudoknot motif composed of 22 nucleotides capable of stimulating -1-type ribosomal frameshifts.« less

Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in Castellaniella defragrans

PubMed Central

2012-01-01

Background Monoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic β-myrcene degradation pathway in Castellaniella defragrans strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase ldi/LDI catalyzes the hydration of β-myrcene to (S)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase geoA/GeDH and a geranial dehydrogenase geoB/GaDH contribute to the formation of geranic acid. A genetic system was for the first time applied for the betaproteobacterium to prove in vivo the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events. Results Polar effects were absent in the in-frame deletion mutants C. defragrans Δldi and C. defragrans ΔgeoA. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of geoA resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were in trans complemented with the broad-host range expression vector pBBR1MCS-4ldi and pBBR1MCS-2geoA, restoring in both cases the wild type phenotype. Conclusions In-frame deletion mutants of genes in the anaerobic β-myrcene degradation revealed novel insights in the in vivo function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the β-myrcene degradation pathway. Growth on cyclic monoterpenes independent of the initial enzyme LDI suggests the presence of a second enzyme system activating unsaturated hydrocarbons. PMID:22947208

Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System.

PubMed

Wang, Shaohua; Dong, Sheng; Wang, Pixiang; Tao, Yong; Wang, Yi

2017-05-15

Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk , two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P J23119 , we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the pta buk double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations. IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production. Copyright © 2017 American Society for Microbiology.

In Silico Analysis of Usher Encoding Genes in Klebsiella pneumoniae and Characterization of Their Role in Adhesion and Colonization

PubMed Central

Khater, Fida; Balestrino, Damien; Charbonnel, Nicolas; Dufayard, Jean François; Brisse, Sylvain; Forestier, Christiane

2015-01-01

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiella pneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and ϭ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches. PMID:25751658

Farewell to GBM-O: Genomic and transcriptomic profiling of glioblastoma with oligodendroglioma component reveals distinct molecular subgroups.

PubMed

Hinrichs, Benjamin H; Newman, Scott; Appin, Christina L; Dunn, William; Cooper, Lee; Pauly, Rini; Kowalski, Jeanne; Rossi, Michael R; Brat, Daniel J

2016-01-13

Glioblastoma with oligodendroglioma component (GBM-O) was recognized as a histologic pattern of glioblastoma (GBM) by the World Health Organization (WHO) in 2007 and is distinguished by the presence of oligodendroglioma-like differentiation. To better understand the genetic underpinnings of this morphologic entity, we performed a genome-wide, integrated copy number, mutational and transcriptomic analysis of eight (seven primary, primary secondary) cases. Three GBM-O samples had IDH1 (p.R132H) mutations; two of these also demonstrated 1p/19q co-deletion and had a proneural transcriptional profile, a molecular signature characteristic of oligodendroglioma. The additional IDH1 mutant tumor lacked 1p/19q co-deletion, harbored a TP53 mutation, and overall, demonstrated features most consistent with IDH mutant (secondary) GBM. Finally, five tumors were IDH wild-type (IDHwt) and had chromosome seven gains, chromosome 10 losses, and homozygous 9p deletions (CDKN2A), alterations typical of IDHwt (primary) GBM. IDHwt GBM-Os also demonstrated EGFR and PDGFRA amplifications, which correlated with classical and proneural expression subtypes, respectively. Our findings demonstrate that GBM-O is composed of three discrete molecular subgroups with characteristic mutations, copy number alterations and gene expression patterns. Despite displaying areas that morphologically resemble oligodendroglioma, the current results indicate that morphologically defined GBM-O does not correspond to a particular genetic signature, but rather represents a collection of genetically dissimilar entities. Ancillary testing, especially for IDH and 1p/19q, should be used for determining these molecular subtypes.

A Conserved Acidic Motif in the N-Terminal Domain of Nitrate Reductase Is Necessary for the Inactivation of the Enzyme in the Dark by Phosphorylation and 14-3-3 Binding1

PubMed Central

Pigaglio, Emmanuelle; Durand, Nathalie; Meyer, Christian

1999-01-01

It has previously been shown that the N-terminal domain of tobacco (Nicotiana tabacum) nitrate reductase (NR) is involved in the inactivation of the enzyme by phosphorylation, which occurs in the dark (L. Nussaume, M. Vincentz, C. Meyer, J.P. Boutin, and M. Caboche [1995] Plant Cell 7: 611–621). The activity of a mutant NR protein lacking this N-terminal domain was no longer regulated by light-dark transitions. In this study smaller deletions were performed in the N-terminal domain of tobacco NR that removed protein motifs conserved among higher plant NRs. The resulting truncated NR-coding sequences were then fused to the cauliflower mosaic virus 35S RNA promoter and introduced in NR-deficient mutants of the closely related species Nicotiana plumbaginifolia. We found that the deletion of a conserved stretch of acidic residues led to an active NR protein that was more thermosensitive than the wild-type enzyme, but it was relatively insensitive to the inactivation by phosphorylation in the dark. Therefore, the removal of this acidic stretch seems to have the same effects on NR activation state as the deletion of the N-terminal domain. A hypothetical explanation for these observations is that a specific factor that impedes inactivation remains bound to the truncated enzyme. A synthetic peptide derived from this acidic protein motif was also found to be a good substrate for casein kinase II. PMID:9880364

Identification of mutant phenotypes associated with loss of individual microRNAs in sensitized genetic backgrounds in Caenorhabditis elegans

PubMed Central

Brenner, John L.; Jasiewicz, Kristen L.; Fahley, Alisha F.; Kemp, Benedict J.; Abbott, Allison L.

2010-01-01

Summary MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the translation and/or the stability of their mRNA targets. Previous work showed that for most miRNA genes of C. elegans, single gene knockouts did not result in detectable mutant phenotypes [1]. This may be due, in part, to functional redundancy between miRNAs. However, in most cases, worms carrying deletions of all members of a miRNA family do not display strong mutant phenotypes [2]. They may function together with unrelated miRNAs or with non-miRNA genes in regulatory networks, possibly to ensure the robustness of developmental mechanisms. To test this, we examined worms lacking individual miRNAs in genetically sensitized backgrounds. These include genetic backgrounds with reduced processing and activity of all miRNAs or with reduced activity of a wide array of regulatory pathways [3]. Using these two approaches, mutant phenotypes were identified for 25 out of 31 miRNAs included in this analysis. Our findings describe biological roles for individual miRNAs and suggest that use of sensitized genetic backgrounds provides an efficient approach for miRNA functional analysis. PMID:20579881

Regulation of Saccharomyces cerevisiae genetic engineering on the production of acetate esters and higher alcohols during Chinese Baijiu fermentation.

PubMed

Li, Wei; Wang, Jian-Hui; Zhang, Cui-Ying; Ma, Hong-Xia; Xiao, Dong-Guang

2017-06-01

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.

Systematic deletion of homeobox genes in Podospora anserina uncovers their roles in shaping the fruiting body.

PubMed

Coppin, Evelyne; Berteaux-Lecellier, Véronique; Bidard, Frédérique; Brun, Sylvain; Ruprich-Robert, Gwenaël; Espagne, Eric; Aït-Benkhali, Jinane; Goarin, Anne; Nesseir, Audrey; Planamente, Sara; Debuchy, Robert; Silar, Philippe

2012-01-01

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures.

Systematic Deletion of Homeobox Genes in Podospora anserina Uncovers Their Roles in Shaping the Fruiting Body

PubMed Central

Coppin, Evelyne; Berteaux-Lecellier, Véronique; Bidard, Frédérique; Brun, Sylvain; Ruprich-Robert, Gwenaël; Espagne, Eric; Aït-Benkhali, Jinane; Goarin, Anne; Nesseir, Audrey; Planamente, Sara; Debuchy, Robert; Silar, Philippe

2012-01-01

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures. PMID:22662159

Combinational Deletion of Three Membrane Protein-Encoding Genes Highly Attenuates Yersinia pestis while Retaining Immunogenicity in a Mouse Model of Pneumonic Plague

PubMed Central

Tiner, Bethany L.; Kirtley, Michelle L.; Erova, Tatiana E.; Popov, Vsevolod L.; Baze, Wallace B.; van Lier, Christina J.; Ponnusamy, Duraisamy; Andersson, Jourdan A.; Motin, Vladimir L.; Chauhan, Sadhana

2015-01-01

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection. PMID:25605764

Combinational deletion of three membrane protein-encoding genes highly attenuates yersinia pestis while retaining immunogenicity in a mouse model of pneumonic plague.

PubMed

Tiner, Bethany L; Sha, Jian; Kirtley, Michelle L; Erova, Tatiana E; Popov, Vsevolod L; Baze, Wallace B; van Lier, Christina J; Ponnusamy, Duraisamy; Andersson, Jourdan A; Motin, Vladimir L; Chauhan, Sadhana; Chopra, Ashok K

2015-04-01

Previously, we showed that deletion of genes encoding Braun lipoprotein (Lpp) and MsbB attenuated Yersinia pestis CO92 in mouse and rat models of bubonic and pneumonic plague. While Lpp activates Toll-like receptor 2, the MsbB acyltransferase modifies lipopolysaccharide. Here, we deleted the ail gene (encoding the attachment-invasion locus) from wild-type (WT) strain CO92 or its lpp single and Δlpp ΔmsbB double mutants. While the Δail single mutant was minimally attenuated compared to the WT bacterium in a mouse model of pneumonic plague, the Δlpp Δail double mutant and the Δlpp ΔmsbB Δail triple mutant were increasingly attenuated, with the latter being unable to kill mice at a 50% lethal dose (LD50) equivalent to 6,800 LD50s of WT CO92. The mutant-infected animals developed balanced TH1- and TH2-based immune responses based on antibody isotyping. The triple mutant was cleared from mouse organs rapidly, with concurrent decreases in the production of various cytokines and histopathological lesions. When surviving animals infected with increasing doses of the triple mutant were subsequently challenged on day 24 with the bioluminescent WT CO92 strain (20 to 28 LD50s), 40 to 70% of the mice survived, with efficient clearing of the invading pathogen, as visualized in real time by in vivo imaging. The rapid clearance of the triple mutant, compared to that of WT CO92, from animals was related to the decreased adherence and invasion of human-derived HeLa and A549 alveolar epithelial cells and to its inability to survive intracellularly in these cells as well as in MH-S murine alveolar and primary human macrophages. An early burst of cytokine production in macrophages elicited by the triple mutant compared to WT CO92 and the mutant's sensitivity to the bactericidal effect of human serum would further augment bacterial clearance. Together, deletion of the ail gene from the Δlpp ΔmsbB double mutant severely attenuated Y. pestis CO92 to evoke pneumonic plague in a mouse model while retaining the required immunogenicity needed for subsequent protection against infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Genomic anatomy of the Tyrp1 (brown) deletion complex

PubMed Central

Smyth, Ian M.; Wilming, Laurens; Lee, Angela W.; Taylor, Martin S.; Gautier, Phillipe; Barlow, Karen; Wallis, Justine; Martin, Sancha; Glithero, Rebecca; Phillimore, Ben; Pelan, Sarah; Andrew, Rob; Holt, Karen; Taylor, Ruth; McLaren, Stuart; Burton, John; Bailey, Jonathon; Sims, Sarah; Squares, Jan; Plumb, Bob; Joy, Ann; Gibson, Richard; Gilbert, James; Hart, Elizabeth; Laird, Gavin; Loveland, Jane; Mudge, Jonathan; Steward, Charlie; Swarbreck, David; Harrow, Jennifer; North, Philip; Leaves, Nicholas; Greystrong, John; Coppola, Maria; Manjunath, Shilpa; Campbell, Mark; Smith, Mark; Strachan, Gregory; Tofts, Calli; Boal, Esther; Cobley, Victoria; Hunter, Giselle; Kimberley, Christopher; Thomas, Daniel; Cave-Berry, Lee; Weston, Paul; Botcherby, Marc R. M.; White, Sharon; Edgar, Ruth; Cross, Sally H.; Irvani, Marjan; Hummerich, Holger; Simpson, Eleanor H.; Johnson, Dabney; Hunsicker, Patricia R.; Little, Peter F. R.; Hubbard, Tim; Campbell, R. Duncan; Rogers, Jane; Jackson, Ian J.

2006-01-01

Chromosome deletions in the mouse have proven invaluable in the dissection of gene function. The brown deletion complex comprises >28 independent genome rearrangements, which have been used to identify several functional loci on chromosome 4 required for normal embryonic and postnatal development. We have constructed a 172-bacterial artificial chromosome contig that spans this 22-megabase (Mb) interval and have produced a contiguous, finished, and manually annotated sequence from these clones. The deletion complex is strikingly gene-poor, containing only 52 protein-coding genes (of which only 39 are supported by human homologues) and has several further notable genomic features, including several segments of >1 Mb, apparently devoid of a coding sequence. We have used sequence polymorphisms to finely map the deletion breakpoints and identify strong candidate genes for the known phenotypes that map to this region, including three lethal loci (l4Rn1, l4Rn2, and l4Rn3) and the fitness mutant brown-associated fitness (baf). We have also characterized misexpression of the basonuclin homologue, Bnc2, associated with the inversion-mediated coat color mutant white-based brown (Bw). This study provides a molecular insight into the basis of several characterized mouse mutants, which will allow further dissection of this region by targeted or chemical mutagenesis. PMID:16505357

Identification of a classical mutant in the industrial host Aspergillus niger by systems genetics: LaeA is required for citric acid production and regulates the formation of some secondary metabolites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Jing; Arentshorst, Mark; Nair, Deepa

Rapid acidification of the culture medium by the production of organic acids and the production of acid-induced proteases are key characteristics of the filamentous fungus Aspergillus niger. The D15 mutant of A. niger is non-acidifying mutant and used often for the expression of recombinant proteins in A. niger, because of its reduced production of extracellular proteases under non-acidic conditions. In this study, the D15 mutant is characterized in detail. Strongly reduced levels of citric and oxalic acid were observed in the D15 mutant both in shake flask cultures and in controlled batch cultivations. To identify the mutation in the D15more » mutant, we successfully combined high-throughput sequencing (Illumina) with bulk segregant analysis. Because of the lack of a sexual cycle for A. niger, the parasexual cycle was used to generate a pool of segregants. From the 52 single nucleotide polymorphisms (SNPs) between the parental strains, three SNPs were homozygous in the genomic DNA of pool of segregants. These three SNPs mapped to all the right arm of chromosome II, indicating that this region contains the genetic locus affecting the phenotype related to acid production. Of the three SNPs, one mutation resulted in a missense mutation in the gene encoding the A. niger homologue of the A. nidulans methyltransferase gene laeA. Complementation analysis of the original mutant with the laeA gene and targeted disruption of laeA further confirmed that LaeA is involved in citric acid production in A. niger lab (N402) and citric acid production strains (ATCC 11414). Analysis of the secondary metabolite (SM) profile of the laeA mutants indicate that LaeA is required for the production of several SMs (asperrubrol, atromentin and JBIR86), but deletion of laeA also resulted in the presence of SMs (aspernigrin A/B and BMS-192548) that were not detected in the wild-type strain. The levels of ten other SMs were not strongly affected as a result of laeA deletion indicating that only a limited number of SM gene clusters are controlled by LaeA activity.« less

Evaluation of the effects of sdiA, a luxR homologue, on adherence and motility of Escherichia coli O157 : H7.

PubMed

Sharma, Vijay K; Bearson, Shawn M D; Bearson, Bradley L

2010-05-01

Quorum-sensing (QS) signalling pathways are important regulatory networks for controlling the expression of genes promoting adherence of enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 to epithelial cells. A recent study has shown that EHEC O157 : H7 encodes a luxR homologue, called sdiA, which upon overexpression reduces the expression of genes encoding flagellar and locus of enterocyte effacement (LEE) proteins, thus negatively impacting on the motility and intimate adherence phenotypes, respectively. Here, we show that the deletion of sdiA from EHEC O157 : H7 strain 86-24, and from a hha (a negative regulator of ler) mutant of this strain, enhanced bacterial adherence to HEp-2 epithelial cells of the sdiA mutant strains relative to the strains containing a wild-type copy of sdiA. Quantitative reverse transcription PCR showed that the expression of LEE-encoded genes ler, espA and eae in strains with the sdiA deletions was not significantly different from that of the strains wild-type for sdiA. Similarly, no additional increases in the expression of LEE genes were observed in a sdiA hha double mutant strain relative to that observed in the hha deletion mutant. While the expression of fliC, which encodes flagellin, was enhanced in the sdiA mutant strain, the expression of fliC was reduced by several fold in the hha mutant strain, irrespective of the presence or absence of sdiA, indicating that the genes sdiA and hha exert opposing effects on the expression of fliC. The strains with deletions in sdiA or hha showed enhanced expression of csgA, encoding curlin of the curli fimbriae, with the expression of csgA highest in the sdiA hha double mutant, suggesting an additive effect of these two gene deletions on the expression of csgA. No significant differences were observed in the expression of the genes lpfA and fimA of the operons encoding long polar and type 1 fimbriae in the sdiA mutant strain. These data indicate that SdiA has no significant effect on the expression of LEE genes, but that it appears to act as a strong repressor of genes encoding flagella and curli fimbriae, and the alleviation of the SdiA-mediated repression of these genes in an EHEC O157 : H7 sdiA mutant strain contributes to enhanced bacterial motility and increased adherence to HEp-2 epithelial cells.

Zinc oxide and silver nanoparticles toxicity in the baker's yeast, Saccharomyces cerevisiae.

PubMed

Galván Márquez, Imelda; Ghiyasvand, Mergan; Massarsky, Andrey; Babu, Mohan; Samanfar, Bahram; Omidi, Katayoun; Moon, Thomas W; Smith, Myron L; Golshani, Ashkan

2018-01-01

Engineered nanomaterials (ENMs) are increasingly incorporated into a variety of commercial applications and consumer products; however, ENMs may possess cytotoxic properties due to their small size. This study assessed the effects of two commonly used ENMs, zinc oxide nanoparticles (ZnONPs) and silver nanoparticles (AgNPs), in the model eukaryote Saccharomyces cerevisiae. A collection of ≈4600 S. cerevisiae deletion mutant strains was used to deduce the genes, whose absence makes S. cerevisiae more prone to the cytotoxic effects of ZnONPs or AgNPs. We demonstrate that S. cerevisiae strains that lack genes involved in transmembrane and membrane transport, cellular ion homeostasis, and cell wall organization or biogenesis exhibited the highest sensitivity to ZnONPs. In contrast, strains that lack genes involved in transcription and RNA processing, cellular respiration, and endocytosis and vesicular transport exhibited the highest sensitivity to AgNPs. Secondary assays confirmed that ZnONPs affected cell wall function and integrity, whereas AgNPs exposure decreased transcription, reduced endocytosis, and led to a dysfunctional electron transport system. This study supports the use of S. cerevisiae Gene Deletion Array as an effective high-throughput technique to determine cellular targets of ENM toxicity.

Embryonic epithelial Pten deletion through Nkx2.1-cre leads to thyroid tumorigenesis in a strain-dependent manner

PubMed Central

Tiozzo, Caterina; Danopoulos, Soula; Lavarreda-Pearce, Maria; Baptista, Sheryl; Varimezova, Radka; Al Alam, Denise; Warburton, David; Virender, Rehan; De Langhe, Stijn; Di Cristofano, Antonio

2014-01-01

Even though the role of the tyrosine phosphatase Pten as a tumor suppressor gene has been well established in thyroid cancer, its role during thyroid development is still elusive. We therefore targeted Pten deletion in the thyroid epithelium by crossing Ptenflox/flox with a newly developed Nkx2.1-cre driver line in the BALB/c and C57BL/6 genetic backgrounds. C57BL/6 homozygous Pten mutant mice died around 2 weeks of age due to tracheal and esophageal compression by a hyperplasic thyroid. By contrast, BALB/c homozygous Pten mutant mice survived up to 2 years, but with a slightly increased thyroid volume. Characterization of the thyroid glands from C57BL/6 homozygous Pten mutant mice at postnatal day 14 (PN14) showed abnormally enlarged tissue with areas of cellular hyperplasia, disruption of the normal architecture, and follicular degeneration. In addition, differing degrees of hypothyroidism, thyroxine (T4) decrease, and thyroid-stimulating hormone elevation between the strains in the mutants and the heterozygous mutant were detected at PN14. Finally, C57BL/6 heterozygous Pten mutant mice developed thyroid tumors after 2 years of age. Our results indicate that Pten has a pivotal role in thyroid development and its deletion results in thyroid tumor formation, with the timing and severity of the tumor depending on the particular genetic background. PMID:22167068

Msh1p counteracts oxidative lesion-induced instability of mtDNA and stimulates mitochondrial recombination in Saccharomyces cerevisiae.

PubMed

Kaniak, Aneta; Dzierzbicki, Piotr; Rogowska, Agata T; Malc, Ewa; Fikus, Marta; Ciesla, Zygmunt

2009-03-01

The proximity of the mitochondrial genome to the respiratory chain, a major source of ROS (radical oxygen species), makes mtDNA more vulnerable to oxidative damage than nuclear DNA. Mitochondrial BER (base excision repair) is generally considered to be the main pathway involved in the prevention of oxidative lesion-induced mutations in mtDNA. However, we previously demonstrated that the increased frequency of mitochondrial Oli(r) mutants in an ogg1Delta strain, lacking the activity of a crucial mtBER glycosylase, is reduced in the presence of plasmids encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS. This finding suggested that Msh1p might be involved in the prevention of mitochondrial mutagenesis induced by oxidative stress. Here we show that a double mutant carrying the msh1-R813W allele, encoding a variant of the protein defective in the ATP hydrolysis activity, combined with deletion of SOD2, encoding the mitochondrial superoxide dismutase, displays a synergistic effect on the frequency of Oli(r) mutants, indicating that Msh1p prevents generation of oxidative lesion-induced mitochondrial mutations. We also show that double mutants carrying the msh1-R813W allele, combined with deletion of either OGG1 or APN1, the latter resulting in deficiency of the Apn1 endonuclease, exhibit a synergistic effect on the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. This suggests that Msh1p, Ogg1p and Apn1p play overlapping functions in maintaining the stability of mtDNA. In addition, we demonstrate, using a novel ARG8(m) recombination assay, that a surplus of Msh1p results in enhanced mitochondrial recombination. Interestingly, the mutant forms of the protein, msh1p-R813W and msh1p-G776D, fail to stimulate recombination. We postulate that the Msh1p-enhanced homologous recombination may play an important role in the prevention of oxidative lesion-induced rearrangements of the mitochondrial genome.

Inheritance of the lysozyme inhibitor Ivy was an important evolutionary step by Yersinia pestis to avoid the host innate immune response.

PubMed

Derbise, Anne; Pierre, François; Merchez, Maud; Pradel, Elizabeth; Laouami, Sabrina; Ricard, Isabelle; Sirard, Jean-Claude; Fritz, Jill; Lemaître, Nadine; Akinbi, Henry; Boneca, Ivo G; Sebbane, Florent

2013-05-15

Yersinia pestis (the plague bacillus) and its ancestor, Yersinia pseudotuberculosis (which causes self-limited bowel disease), encode putative homologues of the periplasmic lysozyme inhibitor Ivy and the membrane-bound lysozyme inhibitor MliC. The involvement of both inhibitors in virulence remains subject to debate. Mutants lacking ivy and/or mliC were generated. We evaluated the mutants' ability to counter lysozyme, grow in serum, and/or counter leukocytes; to produce disease in wild-type, neutropenic, or lysozyme-deficient rodents; and to induce host inflammation. MliC was not required for lysozyme resistance and the development of plague. Deletion of ivy decreased Y. pestis' ability to counter lysozyme and polymorphonuclear neutrophils, but it did not affect the bacterium's ability to grow in serum or resist macrophages. Y. pestis lacking Ivy had attenuated virulence, unless animals were neutropenic or lysozyme deficient. The Ivy mutant induced inflammation to a degree similar to that of the parental strain. Last, Y. pseudotuberculosis did not require Ivy to counter lysozyme and for virulence. Ivy is required to counter lysozyme during infection, but its role as a virulence factor is species dependent. Our study also shows that a gene that is not necessary for the virulence of an ancestral bacterium may become essential in the emergence of a new pathogen.

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

PubMed Central

Oguri, Tamiko; Schneider, Barbara

2012-01-01

Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motility. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because the genes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the gene for a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a divergently transcribed gene that regulates cdsH expression, which we designated cutR (formerly ybaO, or STM0459). CdsH appears to be the major cysteine-degrading and sulfide-producing enzyme aerobically but not anaerobically. Mutants with deletions of cdsH and ybaO exhibited increased sensitivity to cysteine toxicity and altered swarming motility but unaltered cysteine-enhanced antibiotic resistance and survival in macrophages. PMID:22685283

Assessment of Mycobacterium bovis deleted in p27-p55 virulence operon as candidate vaccine against tuberculosis in animal models.

PubMed

Bianco, María V; Clark, Simon; Blanco, Federico C; Garbaccio, Sergio; García, Elizabeth; Cataldi, Angel A; Williams, Ann; Bigi, Fabiana

2014-01-01

A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.

Bile system morphogenesis defects and liver dysfunction upon targeted deletion of HNF1beta.

PubMed

Coffinier, Catherine; Gresh, Lionel; Fiette, Laurence; Tronche, François; Schütz, Günther; Babinet, Charles; Pontoglio, Marco; Yaniv, Moshe; Barra, Jacqueline

2002-04-01

The inactivation of the Hnf1beta gene identified an essential role in epithelial differentiation of the visceral endoderm and resulted in early embryonic death. In the present study, we have specifically inactivated this gene in hepatocytes and bile duct cells using the Cre/loxP system. Mutant animals exhibited severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD). The paucity of small IHBD was linked to a failure in the organization of duct structures during liver organogenesis, suggesting an essential function of Hnf1b in bile duct morphogenesis. Mutant mice also lacked interlobular arteries. As HNF1beta is not expressed in these cells, it further emphasizes the link between arterial and biliary formation. Hepatocyte metabolism was also affected and we identified hepatocyte-specific HNF1beta target genes involved in bile acids sensing and in fatty acid oxidation.

Choline catabolism to glycine betaine contributes to Pseudomonas aeruginosa survival during murine lung infection.

PubMed

Wargo, Matthew J

2013-01-01

Pseudomonas aeruginosa can acquire and metabolize a variety of molecules including choline, an abundant host-derived molecule. In P. aeruginosa, choline is oxidized to glycine betaine which can be used as an osmoprotectant, a sole source of carbon and nitrogen, and as an inducer of the virulence factor, hemolytic phospholipase C (PlcH) via the transcriptional regulator GbdR. The primary objective was to determine the contribution of choline conversion to glycine betaine to P. aeruginosa survival during mouse lung infection. A secondary objective was to gain insight into the relative contributions of the different roles of glycine betaine to P. aeruginosa survival during infection. Using a model of acute murine pneumonia, we determined that deletion of the choline oxidase system (encoded by betBA) decreased P. aeruginosa survival in the mouse lung. Deletion of the glycine betaine demethylase genes (gbcA-B), required for glycine betaine catabolism, did not impact P. aeruginosa survival in the lung. Thus, the defect of the betBA mutant was not due to a requirement for glycine betaine catabolism or dependence on a downstream metabolite. Deletion of betBA decreased the abundance of plcH transcript during infection, which suggested a role for PlcH in the betBA survival defect. To test the contribution of plcH to the betBA mutant phenotype a betBAplcHR double deletion mutant was generated. The betBA and betBAplcHR double mutant had a small but significant survival defect compared to the plcHR single mutant, suggesting that regulation of plcH expression is not the only role for glycine betaine during infection. The conclusion was that choline acquisition and its oxidation to glycine betaine contribute to P. aeruginosa survival in the mouse lung. While defective plcH induction can explain a portion of the betBA mutant phenotype, the exact mechanisms driving the betBA mutant survival defect remain unknown.

Choline Catabolism to Glycine Betaine Contributes to Pseudomonas aeruginosa Survival during Murine Lung Infection

PubMed Central

Wargo, Matthew J.

2013-01-01

Pseudomonas aeruginosa can acquire and metabolize a variety of molecules including choline, an abundant host-derived molecule. In P. aeruginosa, choline is oxidized to glycine betaine which can be used as an osmoprotectant, a sole source of carbon and nitrogen, and as an inducer of the virulence factor, hemolytic phospholipase C (PlcH) via the transcriptional regulator GbdR. The primary objective was to determine the contribution of choline conversion to glycine betaine to P. aeruginosa survival during mouse lung infection. A secondary objective was to gain insight into the relative contributions of the different roles of glycine betaine to P. aeruginosa survival during infection. Using a model of acute murine pneumonia, we determined that deletion of the choline oxidase system (encoded by betBA) decreased P. aeruginosa survival in the mouse lung. Deletion of the glycine betaine demethylase genes (gbcA-B), required for glycine betaine catabolism, did not impact P. aeruginosa survival in the lung. Thus, the defect of the betBA mutant was not due to a requirement for glycine betaine catabolism or dependence on a downstream metabolite. Deletion of betBA decreased the abundance of plcH transcript during infection, which suggested a role for PlcH in the betBA survival defect. To test the contribution of plcH to the betBA mutant phenotype a betBAplcHR double deletion mutant was generated. The betBA and betBAplcHR double mutant had a small but significant survival defect compared to the plcHR single mutant, suggesting that regulation of plcH expression is not the only role for glycine betaine during infection. The conclusion was that choline acquisition and its oxidation to glycine betaine contribute to P. aeruginosa survival in the mouse lung. While defective plcH induction can explain a portion of the betBA mutant phenotype, the exact mechanisms driving the betBA mutant survival defect remain unknown. PMID:23457628

Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility.

PubMed Central

Geber, A; Hitchcock, C A; Swartz, J E; Pullen, F S; Marsden, K E; Kwon-Chung, K J; Bennett, J E

1995-01-01

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae. PMID:8593007

Insm1 promotes the transition of olfactory progenitors from apical and proliferative to basal, terminally dividing and neuronogenic.

PubMed

Rosenbaum, Jason N; Duggan, Anne; García-Añoveros, Jaime

2011-02-01

Insm1 is a zinc-finger transcription factor transiently expressed throughout the developing nervous system in late progenitors and nascent neurons. Insm1 is also highly expressed in medulloblastomas and other neuroendocrine tumors. We generated mice lacking the Insm1 gene and used them to elucidate its role in neurogenic proliferation of the embryonic olfactory epithelium. We found that deletion of Insm1 results in more apical cells and fewer nascent and mature neurons. In the embryonic olfactory epithelium of Insm1 mutants we detect fewer basal progenitors, which produce neurons, and more apical progenitors, which at this stage produce additional progenitors. Furthermore, in the mutants we detect fewer progenitors expressing NEUROD1, a marker of terminally dividing, neuronogenic (neuron-producing) progenitors (immediate neuronal precursors), and more progenitors expressing ASCL1, a marker of the transit amplifying progenitors that migrate from the apical to the basal edges of the epithelium while dividing to generate the terminal, neuronogenic progenitors. Finally, with timed administration of nucleoside analogs we demonstrate that the Insm1 mutants contain fewer terminally dividing progenitors at embryonic day 12.5. Altogether, these results suggest a role for Insm1 in promoting the transition of progenitors from apical and proliferative to basal, terminal and neuronogenic. This role appears partially conserved with that of its nematode ortholog, egl-46. The similar effects of Insm1 deletion on progenitors of embryonic olfactory epithelium and cortex point to striking parallels in the development of these neuroepithelia, and particularly between the basal progenitors of olfactory epithelium and the subventricular zone progenitors of cortex.

Overexpression of the active diacylglycerol acyltransferase variant transforms Saccharomyces cerevisiae into an oleaginous yeast.

PubMed

Kamisaka, Yasushi; Kimura, Kazuyoshi; Uemura, Hiroshi; Yamaoka, Masakazu

2013-08-01

Lipid production by Saccharomyces cerevisiae was improved by overexpression of the yeast diacylglycerol acyltransferase Dga1p lacking the N-terminal 29 amino acids (Dga1∆Np), which was previously found to be an active form in the ∆snf2 mutant. Overexpression of Dga1∆Np in the ∆snf2 mutant, however, did not increase lipid content as expected, which prompted us to search for a more suitable strain in which to study the role of Dga1∆Np in lipid accumulation. We found that the overexpression of Dga1∆Np in the ∆dga1 mutant effectively increased the lipid content up to about 45 % in the medium containing 10 % glucose. The high lipid content of the transformant was dependent on glucose concentration, nitrogen limitation, and active leucine biosynthesis. To better understand the effect of dga1 disruption on the ability of Dga1∆Np to stimulate lipid accumulation, the ∆dga1-1 mutant, in which the 3'-terminal 36 bp of the dga1 open reading frame (ORF) remained, and the ∆dga1-2 mutant, in which the 3'-terminal 36 bp were also deleted, were prepared with URA3 disruption cassettes. Surprisingly, the overexpression of Dga1∆Np in the ∆dga1-1 mutant had a lower lipid content than the original ∆dga1 mutant, whereas overexpression in the ∆dga1-2 mutant led to a high lipid content of about 45 %. These results indicated that deletion of the 3' terminal region of the dga1 ORF, rather than abrogation of genomic Dga1p expression, was crucial for the effect of Dga1∆Np on lipid accumulation. To investigate whether dga1 disruption affected gene expression adjacent to DGA1, we found that the overexpression of Esa1p together with Dga1∆Np in the ∆dga1 mutant reverted the lipid content to the level of the wild-type strain overexpressing Dga1∆Np. In addition, RT-qPCR analysis revealed that ESA1 mRNA expression in the ∆dga1 mutant was decreased compared to the wild-type strain at the early stages of culture, suggesting that lowered Esa1p expression is involved in the effect of dga1 disruption on Dga1∆Np-dependent lipid accumulation. These results provide a new strategy to engineer S. cerevisiae for optimal lipid production.

Involvement of Escherichia coli K1 ibeT in bacterial adhesion that is associated with the entry into human brain microvascular endothelial cells.

PubMed

Zou, Yanming; He, Lina; Chi, Feng; Jong, Ambrose; Huang, Sheng-He

2008-12-01

IbeT is a downstream gene of the invasion determinant ibeA in the chromosome of a clinical isolate of Escherichia coli K1 strain RS218 (serotype 018:K1:H7). Both ibeT and ibeA are in the same operon. Our previous mutagenesis and complementation studies suggested that ibeT may coordinately contribute to E. coli K1 invasion with ibeA. An isogenic in-frame deletion mutant of ibeT has been made by chromosomal gene replacement with a recombinant suicide vector carrying a fragment with an ibeT internal deletion. The characteristics of the mutant in meningitic E. coli infection were examined in vitro [cell culture of human brain microvascular endothelial cells (HBMEC)] and in vivo (infant rat model of E. coli meningitis) in comparison with the parent strain. The ibeT deletion mutant was significantly less adhesive and invasive than its parent strain E. coli E44 in vitro, and the adhesion- and invasion-deficient phenotypes of the mutant can be complemented by the ibeT gene. Recombinant IbeT protein is able to block E. coli E44 invasion of HBMEC. Furthermore, the ibeT deletion mutant is less capable of colonizing intestine and less virulent in bacterial translocation across the blood-brain barrier (BBB) than its parent E. coli E44 in vivo. These data suggest that ibeT-mediated E. coli K1 adhesion is associated with the bacterial invasion process.

Sporulation-specific cell division defects in ylmE mutants of Streptomyces coelicolor are rescued by additional deletion of ylmD.

PubMed

Zhang, Le; Willemse, Joost; Hoskisson, Paul A; van Wezel, Gilles P

2018-05-09

Cell division during the reproductive phase of the Streptomyces life-cycle requires tight coordination between synchronous formation of multiple septa and DNA segregation. One remarkable difference with most other bacterial systems is that cell division in Streptomyces is positively controlled by the recruitment of FtsZ by SsgB. Here we show that deletion of ylmD (SCO2081) or ylmE (SCO2080), which lie in operon with ftsZ in the dcw cluster of actinomycetes, has major consequences for sporulation-specific cell division in Streptomyces coelicolor. Electron and fluorescence microscopy demonstrated that ylmE mutants have a highly aberrant phenotype with defective septum synthesis, and produce very few spores with low viability and high heat sensitivity. FtsZ-ring formation was also highly disturbed in ylmE mutants. Deletion of ylmD had a far less severe effect on sporulation. Interestingly, the additional deletion of ylmD restored sporulation to the ylmE null mutant. YlmD and YlmE are not part of the divisome, but instead localize diffusely in aerial hyphae, with differential intensity throughout the sporogenic part of the hyphae. Taken together, our work reveals a function for YlmD and YlmE in the control of sporulation-specific cell division in S. coelicolor, whereby the presence of YlmD alone results in major developmental defects.

Loss of Either Rac1 or Rac3 GTPase Differentially Affects the Behavior of Mutant Mice and the Development of Functional GABAergic Networks

PubMed Central

Pennucci, Roberta; Talpo, Francesca; Astro, Veronica; Montinaro, Valentina; Morè, Lorenzo; Cursi, Marco; Castoldi, Valerio; Chiaretti, Sara; Bianchi, Veronica; Marenna, Silvia; Cambiaghi, Marco; Tonoli, Diletta; Leocani, Letizia; Biella, Gerardo; D'Adamo, Patrizia; de Curtis, Ivan

2016-01-01

Rac GTPases regulate the development of cortical/hippocampal GABAergic interneurons by affecting the early development and migration of GABAergic precursors. We have addressed the function of Rac1 and Rac3 proteins during the late maturation of hippocampal interneurons. We observed specific phenotypic differences between conditional Rac1 and full Rac3 knockout mice. Rac1 deletion caused greater generalized hyperactivity and cognitive impairment compared with Rac3 deletion. This phenotype matched with a more evident functional impairment of the inhibitory circuits in Rac1 mutants, showing higher excitability and reduced spontaneous inhibitory currents in the CA hippocampal pyramidal neurons. Morphological analysis confirmed a differential modification of the inhibitory circuits: deletion of either Rac caused a similar reduction of parvalbumin-positive inhibitory terminals in the pyramidal layer. Intriguingly, cannabinoid receptor-1-positive terminals were strongly increased only in the CA1 of Rac1-depleted mice. This increase may underlie the stronger electrophysiological defects in this mutant. Accordingly, incubation with an antagonist for cannabinoid receptors partially rescued the reduction of spontaneous inhibitory currents in the pyramidal cells of Rac1 mutants. Our results show that Rac1 and Rac3 have independent roles in the formation of GABAergic circuits, as highlighted by the differential effects of their deletion on the late maturation of specific populations of interneurons. PMID:26582364

Dicer-Like Proteins Regulate Sexual Development via the Biogenesis of Perithecium-Specific MicroRNAs in a Plant Pathogenic Fungus Fusarium graminearum

PubMed Central

Zeng, Wenping; Wang, Jie; Wang, Ying; Lin, Jing; Fu, Yanping; Xie, Jiatao; Jiang, Daohong; Chen, Tao; Liu, Huiquan; Cheng, Jiasen

2018-01-01

Ascospores act as the primary inoculum of Fusarium graminearum, which causes the destructive disease Fusarium head blight (FHB), or scab. MicroRNAs (miRNAs) have been reported in the F. graminearum vegetative stage, and Fgdcl2 is involved in microRNA-like RNA (milRNA) biogenesis but has no major impact on vegetative growth, abiotic stress or pathogenesis. In the present study, we found that ascospore discharge was decreased in the Fgdcl1 deletion mutant, and completely blocked in the double-deletion mutant of Fgdcl1 and Fgdcl2. Besides, more immature asci were observed in the double-deletion mutant. Interestingly, the up-regulated differentially expressed genes (DEGs) common to ΔFgdcl1 and ΔFgdcl1/2 were related to ion transmembrane transporter and membrane components. The combination of small RNA and transcriptome sequencing with bioinformatics analysis predicted 143 novel milRNAs in wild-type perithecia, and 138 of these milRNAs partly or absolutely depended on Fgdcl1, while only 5 novel milRNAs were still obtained in the Fgdcl1 and Fgdcl2 double-deletion mutant. Furthermore, 117 potential target genes were predicted. Overall, Fgdcl1 and Fgdcl2 genes were partly functionally redundant in ascospore discharge and perithecium-specific milRNA generation in F. graminearum, and these perithecium-specific milRNAs play potential roles in sexual development. PMID:29755439

PBP deletion mutants of Escherichia coli exhibit irregular distribution of MreB at the deformed zones.

PubMed

Vijayan, Saptha; Mallick, Sathi; Dutta, Mouparna; Narayani, M; Ghosh, Anindya S

2014-02-01

MreB is a cytoskeletal protein, which is responsible for maintaining proper cellular morphology and is essential for cell survival. Likewise, penicillin-binding protein 5 (PBP5) helps in maintaining cell shape, though non-essential for survival. The contradicting feature of these two proteins paves the way for this study, wherein we attempt to draw a relation on the nature of distribution of MreB in PBP deletion mutants. The study revealed that the uniform MreB helices/patches were destabilized/disturbed at the zone of deformities of the PBP mutants, whereas the helical patterns were retained at the regions maintaining a rod shape. We interpret that MreB remains functional irrespective of its distribution being misguided by the aberrant shapes of PBP mutants.

Southern Analysis of Genomic Alterations in Gamma-Ray-Induced Aprt- Hamster Cell Mutants

PubMed Central

Grosovsky, Andrew J.; Drobetsky, Elliot A.; deJong, Pieter J.; Glickman, Barry W.

1986-01-01

The role of genomic alterations in mutagenesis induced by ionizing radiation has been the subject of considerable speculation. By Southern blotting analysis we show here that 9 of 55 (approximately 1/6) gamma-ray-induced mutants at the adenine phosphoribosyl transferase (aprt) locus of Chinese hamster ovary (CHO) cells have a detectable genomic rearrangement. These fall into two classes: intragenic deletions and chromosomal rearrangements. In contrast, no major genomic alterations were detected among 67 spontaneous mutants, although two restriction site loss events were observed. Three gamma-ray-induced mutants were found to be intragenic deletions; all may have identical break-points. The remaining six gamma-ray-induced mutants demonstrating a genomic alteration appear to be the result of chromosomal rearrangements, possibly translocation or inversion events. None of the remaining gamma-ray-induced mutants showed any observable alteration in blotting pattern indicating a substantial role for point mutation in gamma-ray-induced mutagenesis at the aprt locus. PMID:3013724

Role of the CipA Scaffoldin Protein in Cellulose Solubilization, as Determined by Targeted Gene Deletion and Complementation in Clostridium thermocellum

PubMed Central

Olson, Daniel G.; Giannone, Richard J.; Hettich, Robert L.

2013-01-01

The CipA scaffoldin protein plays a key role in the Clostridium thermocellum cellulosome. Previous studies have revealed that mutants deficient in binding or solubilizing cellulose also exhibit reduced expression of CipA. To confirm that CipA is, in fact, necessary for rapid solubilization of crystalline cellulose, the gene was deleted from the chromosome using targeted gene deletion technologies. The CipA deletion mutant exhibited a 100-fold reduction in cellulose solubilization rate, although it was eventually able to solubilize 80% of the 5 g/liter cellulose initially present. The deletion mutant was complemented by a copy of cipA expressed from a replicating plasmid. In this strain, Avicelase activity was restored, although the rate was 2-fold lower than that in the wild type and the duration of the lag phase was increased. The cipA coding sequence is located at the beginning of a gene cluster containing several other genes thought to be responsible for the structural organization of the cellulosome, including olpB, orf2p, and olpA. Tandem mass spectrometry revealed a 10-fold reduction in the expression of olpB, which may explain the lower growth rate. This deletion experiment adds further evidence that CipA plays a key role in cellulose solubilization by C. thermocellum, and it raises interesting questions about the differential roles of the anchor scaffoldin proteins OlpB, Orf2p, and SdbA. PMID:23204466

An attenuated Shigella mutant lacking the RNA-binding protein Hfq provides cross-protection against Shigella strains of broad serotype.

PubMed

Mitobe, Jiro; Sinha, Ritam; Mitra, Soma; Nag, Dhrubajyoti; Saito, Noriko; Shimuta, Ken; Koizumi, Nobuo; Koley, Hemanta

2017-07-01

Few live attenuated vaccines protect against multiple serotypes of bacterial pathogen because host serotype-specific immune responses are limited to the serotype present in the vaccine strain. Here, immunization with a mutant of Shigella flexneri 2a protected guinea pigs against subsequent infection by S. dysenteriae type 1 and S. sonnei strains. This deletion mutant lacked the RNA-binding protein Hfq leading to increased expression of the type III secretion system via loss of regulation, resulting in attenuation of cell viability through repression of stress response sigma factors. Such increased antigen production and simultaneous attenuation were expected to elicit protective immunity against Shigella strains of heterologous serotypes. Thus, the vaccine potential of this mutant was tested in two guinea pig models of shigellosis. Animals vaccinated in the left eye showed fewer symptoms upon subsequent challenge via the right eye, and even survived subsequent intestinal challenge. In addition, oral vaccination effectively induced production of immunoglobulins without severe side effects, again protecting all animals against subsequent intestinal challenge with S. dysenteriae type 1 or S. sonnei strains. Antibodies against common virulence proteins and the O-antigen of S. flexneri 2a were detected by immunofluorescence microscopy. Reaction of antibodies with various strains, including enteroinvasive Escherichia coli, suggested that common virulence proteins induced protective immunity against a range of serotypes. Therefore, vaccination is expected to cover not only the most prevalent serotypes of S. sonnei and S. flexneri 2a, but also various Shigella strains, including S. dysenteriae type 1, which produces Shiga toxin.

HDAC4 preserves skeletal muscle structure following long-term denervation by mediating distinct cellular responses.

PubMed

Pigna, Eva; Renzini, Alessandra; Greco, Emanuela; Simonazzi, Elena; Fulle, Stefania; Mancinelli, Rosa; Moresi, Viviana; Adamo, Sergio

2018-02-24

Denervation triggers numerous molecular responses in skeletal muscle, including the activation of catabolic pathways and oxidative stress, leading to progressive muscle atrophy. Histone deacetylase 4 (HDAC4) mediates skeletal muscle response to denervation, suggesting the use of HDAC inhibitors as a therapeutic approach to neurogenic muscle atrophy. However, the effects of HDAC4 inhibition in skeletal muscle in response to long-term denervation have not been described yet. To further study HDAC4 functions in response to denervation, we analyzed mutant mice in which HDAC4 is specifically deleted in skeletal muscle. After an initial phase of resistance to neurogenic muscle atrophy, skeletal muscle with a deletion of HDAC4 lost structural integrity after 4 weeks of denervation. Deletion of HDAC4 impaired the activation of the ubiquitin-proteasome system, delayed the autophagic response, and dampened the OS response in skeletal muscle. Inhibition of the ubiquitin-proteasome system or the autophagic response, if on the one hand, conferred resistance to neurogenic muscle atrophy; on the other hand, induced loss of muscle integrity and inflammation in mice lacking HDAC4 in skeletal muscle. Moreover, treatment with the antioxidant drug Trolox prevented loss of muscle integrity and inflammation in in mice lacking HDAC4 in skeletal muscle, despite the resistance to neurogenic muscle atrophy. These results reveal new functions of HDAC4 in mediating skeletal muscle response to denervation and lead us to propose the combined use of HDAC inhibitors and antioxidant drugs to treat neurogenic muscle atrophy.

CO 2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

DOE PAGES

Xiong, Wei; Lin, Paul P.; Magnusson, Lauren; ...

2016-10-28

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO 2. This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO 2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO 2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO 2 to formate serves as a CO 2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO 2 via two biochemical reactions:more » the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO 2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO 2 uptake, and provided physical evidence of a distinct in vivo 'rPFOR-PFL shunt' to reduce CO 2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO 2 fixation via the reductive C1 metabolic pathway in C. thermocellum. Lastly, these findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO 2 as well.« less

Activation of PI3K/Akt signaling by n-terminal SH2 domain mutants of the p85α regulatory subunit of PI3K is enhanced by deletion of its c-terminal SH2 domain.

PubMed

Hofmann, Bianca T; Jücker, Manfred

2012-10-01

The phosphoinositide 3-kinase (PI3K) is frequently activated in human cancer cells due to gain of function mutations in the catalytic (p110) and the regulatory (p85) subunits. The regulatory subunit consists of an SH3 domain and two SH2 domains. An oncogenic form of p85α named p65 lacking the c-terminal SH2 domain (cSH2) has been cloned from an irradiation-induced murine thymic lymphoma and transgenic mice expressing p65 in T lymphocytes develop a lymphoproliferative disorder. We have recently detected a c-terminal truncated form of p85α named p76α in a human lymphoma cell line lacking most of the cSH2 domain due to a frame shift mutation. Here, we report that the deletion of the cSH2 domain enhances the activating effects of the n-terminal SH2 domain (nSH2) mutants K379E and R340E on the PI3K/Akt pathway and micro tumor formation in a focus assay. Further analysis revealed that this transforming effect is mediated by activation of the catalytic PI3K isoform p110α and downstream signaling through mTOR. Our data further support a mechanistic model in which mutations of the cSH2 domain of p85α can abrogate its negative regulatory function on PI3K activity via the nSH2 domain of p85α. Copyright © 2012 Elsevier Inc. All rights reserved.

CO 2-fixing one-carbon metabolism in a cellulose-degrading bacterium Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Wei; Lin, Paul P.; Magnusson, Lauren

Clostridium thermocellum can ferment cellulosic biomass to formate and other end products, including CO 2. This organism lacks formate dehydrogenase (Fdh), which catalyzes the reduction of CO 2 to formate. However, feeding the bacterium 13C-bicarbonate and cellobiose followed by NMR analysis showed the production of 13C-formate in C. thermocellum culture, indicating the presence of an uncharacterized pathway capable of converting CO 2 to formate. Combining genomic and experimental data, we demonstrated that the conversion of CO 2 to formate serves as a CO 2 entry point into the reductive one-carbon (C1) metabolism, and internalizes CO 2 via two biochemical reactions:more » the reversed pyruvate:ferredoxin oxidoreductase (rPFOR), which incorporates CO 2 using acetyl-CoA as a substrate and generates pyruvate, and pyruvate-formate lyase (PFL) converting pyruvate to formate and acetyl-CoA. We analyzed the labeling patterns of proteinogenic amino acids in individual deletions of all five putative PFOR mutants and in a PFL deletion mutant. We identified two enzymes acting as rPFOR, confirmed the dual activities of rPFOR and PFL crucial for CO 2 uptake, and provided physical evidence of a distinct in vivo 'rPFOR-PFL shunt' to reduce CO 2 to formate while circumventing the lack of Fdh. Such a pathway precedes CO 2 fixation via the reductive C1 metabolic pathway in C. thermocellum. Lastly, these findings demonstrated the metabolic versatility of C. thermocellum, which is thought of as primarily a cellulosic heterotroph but is shown here to be endowed with the ability to fix CO 2 as well.« less

A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast

PubMed Central

Björk, Glenn R.; Huang, Bo; Persson, Olof P.; Byström, Anders S.

2007-01-01

Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm5s2U) as wobble nucleoside. These tRNAs read the A- and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm5-) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm5). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm5 side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm5s2U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm5s2U-lacking tRNALys alone was sufficient to restore viability of the double mutant. PMID:17592039

Absence of mutation at the 5'-upstream promoter region of the TPM4 gene from cardiac mutant axolotl (Ambystoma mexicanum).

PubMed

Denz, Christopher R; Zhang, Chi; Jia, Pingping; Du, Jianfeng; Huang, Xupei; Dube, Syamalima; Thomas, Anish; Poiesz, Bernard J; Dube, Dipak K

2011-09-01

Tropomyosins are a family of actin-binding proteins that show cell-specific diversity by a combination of multiple genes and alternative RNA splicing. Of the 4 different tropomyosin genes, TPM4 plays a pivotal role in myofibrillogenesis as well as cardiac contractility in amphibians. In this study, we amplified and sequenced the upstream regulatory region of the TPM4 gene from both normal and mutant axolotl hearts. To identify the cis-elements that are essential for the expression of the TPM4, we created various deletion mutants of the TPM4 promoter DNA, inserted the deleted segments into PGL3 vector, and performed promoter-reporter assay using luciferase as the reporter gene. Comparison of sequences of the promoter region of the TPM4 gene from normal and mutant axolotl revealed no mutations in the promoter sequence of the mutant TPM4 gene. CArG box elements that are generally involved in controlling the expression of several other muscle-specific gene promoters were not found in the upstream regulatory region of the TPM4 gene. In deletion experiments, loss of activity of the reporter gene was noted upon deletion which was then restored upon further deletion suggesting the presence of both positive and negative cis-elements in the upstream regulatory region of the TPM4 gene. We believe that this is the first axolotl promoter that has ever been cloned and studied with clear evidence that it functions in mammalian cell lines. Although striated muscle-specific cis-acting elements are absent from the promoter region of TPM4 gene, our results suggest the presence of positive and negative cis-elements in the promoter region, which in conjunction with positive and negative trans-elements may be involved in regulating the expression of TPM4 gene in a tissue-specific manner.

Construction of a psb C deletion strain in Synechocystis 6803.

PubMed

Goldfarb, N; Knoepfle, N; Putnam-Evans, C

1997-01-01

Synechocystis 6803 is a cyanobacterium that carries out-oxygenic photosynthesis. We are interested in the introduction of mutations in the large extrinsic loop region of the CP43 protein of Photosystem II (PSII). CP43 appears to be required for the stable assembly of the PSII complex and also appears to play a role in photosynthetic oxygen evolution. Deletion of short segments of the large extrinsic loop results in mutants incapable of evolving oxygen. Alterations in psbC, the gene encoding CP43, are introduced into Synechocystis 6803 by transformation and homologous recombination. Specifically, plasmid constructs bearing the site-directed mutations are introduced into a deletion strain where the portion of the gene encoding the area of mutation has been deleted and replaced by a gene conferring antibiotic resistance. We have constructed a deletion strain of Synechocystis appropriate for the introduction of mutations in the large extrinsic loop of CP43 and have used it successfully to produce site-directed mutants.

Deletion of acetate transporter gene ADY2 improved tolerance of Saccharomyces cerevisiae against multiple stresses and enhanced ethanol production in the presence of acetic acid.

PubMed

Zhang, Mingming; Zhang, Keyu; Mehmood, Muhammad Aamer; Zhao, Zongbao Kent; Bai, Fengwu; Zhao, Xinqing

2017-12-01

The aim of this work was to study the effects of deleting acetate transporter gene ADY2 on growth and fermentation of Saccharomyces cerevisiae in the presence of inhibitors. Comparative transcriptome analysis revealed that three genes encoding plasma membrane carboxylic acid transporters, especially ADY2, were significantly downregulated under the zinc sulfate addition condition in the presence of acetic acid stress, and the deletion of ADY2 improved growth of S. cerevisiae under acetic acid, ethanol and hydrogen peroxide stresses. Consistently, a concomitant increase in ethanol production by 14.7% in the presence of 3.6g/L acetic acid was observed in the ADY2 deletion mutant of S. cerevisiae BY4741. Decreased intracellular acetic acid, ROS accumulation, and plasma membrane permeability were observed in the ADY2 deletion mutant. These findings would be useful for developing robust yeast strains for efficient ethanol production. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineering the bacterial shapes for enhanced inclusion bodies accumulation.

PubMed

Jiang, Xiao-Ran; Wang, Huan; Shen, Rui; Chen, Guo-Qiang

2015-05-01

Many bacteria can accumulate inclusion bodies such as sulfur, polyphosphate, glycogen, proteins or polyhydroxyalkanoates. To exploit bacteria as factories for effective production of inclusion bodies, a larger intracellular space is needed for more inclusion body accumulation. In this study, polyhydroxybutyrate (PHB) was investigated as an inclusion bodies representative to be accumulated by Escherichia coli JM109SG. Various approaches were taken to increase the bacterial cell sizes including deletion on actin-like protein gene mreB, weak expression of mreB in mreB deletion mutant, and weak expression of mreB in mreB deletion mutant under inducible expression of SulA, the inhibitor of division ring protein FtsZ. All of the methods resulted in different levels of increases in bacterial sizes and PHB granules accumulation. Remarkably, an increase of over 100% PHB accumulation was observed in recombinant E. coli overexpressing mreB in an mreB deletion mutant under inducible expression of FtsZ inhibiting protein SulA. The molecular mechanism of enlarged bacterial size was found to be directly relate to weakened cytoskeleton which was the result of broken skeleton helix. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Genetic and Genomic Insights into the Role of Benzoate-Catabolic Pathway Redundancy in Burkholderia xenovorans LB400†

PubMed Central

Denef, V. J.; Klappenbach, J. A.; Patrauchan, M. A.; Florizone, C.; Rodrigues, J. L. M.; Tsoi, T. V.; Verstraete, W.; Eltis, L. D.; Tiedje, J. M.

2006-01-01

Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (boxC) and the megaplasmid (boxM). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the ΔbenABCD::kan mutant), the boxC pathway (the ΔboxABC::kan mutant), and both pathways (the ΔbenABCDΔ boxABC::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the ΔbenABCD::kan and ΔbenABCD ΔboxABC::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the ΔbenABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400. PMID:16391095

Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis.

PubMed

Jiao, Yang; Guo, Rongxian; Tang, Peipei; Kang, Xilong; Yin, Junlei; Wu, Kaiyue; Geng, Shizhong; Li, Qiuchun; Sun, Jun; Xu, Xiulong; Zhou, Xiaohui; Gan, Junji; Jiao, Xinan; Liu, Xiufan; Pan, Zhiming

2017-03-03

Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O 9 antigen (O 9 MAb) and O 9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD 50 ) of the rfbG deletion mutant strain was 10 6.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

Single-gene deletions that restore mating competence to diploid yeast.

PubMed

Schmidlin, Tom; Kaeberlein, Matt; Kudlow, Brian A; MacKay, Vivian; Lockshon, Daniel; Kennedy, Brian K

2008-03-01

Using the Saccharomyces cerevisiae MATa/MATalpha ORF deletion collection, homozygous deletion strains were identified that undergo mating with MATa or MATalpha haploids. Seven homozygous deletions were identified that confer enhanced mating. Three of these, lacking CTF8, CTF18, and DCC1, mate at a low frequency with either MATa or MATalpha haploids. The products of these genes form a complex involved in sister chromatid cohesion. Each of these strains also exhibits increased chromosome loss rates, and mating likely occurs due to loss of one copy of chromosome III, which bears the MAT locus. Three other homozygous diploid deletion strains, ylr193cDelta/ylr193cDelta, yor305wDelta/yor305wDelta, and ypr170cDelta/ypr170cDelta, mate at very low frequencies with haploids of either or both mating types. However, an ist3Delta/ist3Delta strain mates only with MATa haploids. It is shown that IST3, previously linked to splicing, is required for efficient processing of the MATa1 message, particularly the first intron. As a result, the ist3Delta/ist3Delta strain expresses unbalanced ratios of Matalpha to Mata proteins and therefore mates with MATa haploids. Accordingly, mating in this diploid can be repressed by introduction of a MATa1 cDNA. In summary, this study underscores and elaborates upon predicted pathways by which mutations restore mating function to yeast diploids and identifies new mutants warranting further study.

Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

PubMed

Nallapareddy, Sreedhar R; Singh, Kavindra V; Murray, Barbara E

2006-01-01

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality

PubMed Central

van der Geize, R.; de Jong, W.; Hessels, G. I.; Grommen, A. W. F.; Jacobs, A. A. C.; Dijkhuizen, L.

2008-01-01

A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated ΔsupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the ΔsupAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive. PMID:18984616

Boosting Anaplerotic Reactions by Pyruvate Kinase Gene Deletion and Phosphoenolpyruvate Carboxylase Desensitization for Glutamic Acid and Lysine Production in Corynebacterium glutamicum.

PubMed

Yokota, Atsushi; Sawada, Kazunori; Wada, Masaru

In the 1980s, Shiio and coworkers demonstrated using random mutagenesis that the following three phenotypes were effective for boosting lysine production by Corynebacterium glutamicum: (1) low-activity-level citrate synthase (CS L ), (2) phosphoenolpyruvate carboxylase (PEPC) resistant to feedback inhibition by aspartic acid (PEPC R ), and (3) pyruvate kinase (PYK) deficiency. Here, we reevaluated these phenotypes and their interrelationship in lysine production using recombinant DNA techniques.The pyk deletion and PEPC R (D299N in ppc) independently showed marginal effects on lysine production, but both phenotypes synergistically increased lysine yield, demonstrating the importance of PEPC as an anaplerotic enzyme in lysine production. Similar effects were also found for glutamic acid production. CS L (S252C in gltA) further increased lysine yield. Thus, using molecular techniques, the combination of these three phenotypes was reconfirmed to be effective for lysine production. However, a simple CS L mutant showed instabilities in growth and lysine yield.Surprisingly, the pyk deletion was found to increase biomass production in wild-type C. glutamicum ATCC13032 under biotin-sufficient conditions. The mutant showed a 37% increase in growth (based on OD 660 ) compared with the ATCC13032 strain in a complex medium containing 100 g/L glucose. Metabolome analysis revealed the intracellular accumulation of excess precursor metabolites. Thus, their conversion into biomass was considered to relieve the metabolic distortion in the pyk-deleted mutant. Detailed physiological studies of various pyk-deleted mutants also suggested that malate:quinone oxidoreductase (MQO) is important to control both the intracellular oxaloacetic acid (OAA) level and respiration rate. These findings may facilitate the rational use of C. glutamicum in fermentation industries.

A deletion in the chromosome of Bacteroides thetaiotaomicron that abolishes production of chondroitinase II does not affect survival of the organism in gastrointestinal tracts of exgermfree mice.

PubMed Central

Salyers, A A; Guthrie, E P

1988-01-01

Bacteroides thetaiotaomicron, an obligate anaerobe normally found in high concentrations in the human colon, is one of the few colon bacteria that can ferment host mucopolysaccharides such as chondroitin sulfate. Previously, we found that a directed insertional mutation in the gene that codes for the chondroitinase II gene of B. thetaiotaomicron did not affect growth on chondroitin sulfate despite the fact that chondroitinase II accounts for 70% of the total cellular chondroitinase activity. Thus, the chondroitinase II gene did not seem to contribute significantly to growth on chondroitin sulfate when the bacteria were grown in laboratory medium. To determine whether this enzyme is important for bacteria growing in the intestinal tract, we tested the ability of a strain that does not produce chondroitinase II to colonize the intestinal tracts of germfree mice and to compete with wild-type B. thetaiotaomicron. The mutant used in these experiments carried a 0.5-kilobase deletion in the chondroitinase II gene and was constructed so that, unlike the original insertion mutant, it contained no exogenous DNA. The deletion mutant colonized the intestinal tracts of germfree mice at the same levels as the wild type. When a mixture of the deletion mutant and wild type was used to colonize germfree mice, the percent wild type, measured by colony hybridization with the deleted 0.5-kilobase fragment as the hybridization probe, did not rise to 100% even after periods as long as 9 weeks. In most experiments, the percent wild type did not rise significantly above the percent in the original mixture.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:3140726

Increased metabolite production by deletion of an HDA1-type histone deacetylase in the phytopathogenic fungi, Magnaporthe oryzae (Pyricularia oryzae) and Fusarium asiaticum.

PubMed

Maeda, K; Izawa, M; Nakajima, Y; Jin, Q; Hirose, T; Nakamura, T; Koshino, H; Kanamaru, K; Ohsato, S; Kamakura, T; Kobayashi, T; Yoshida, M; Kimura, M

2017-11-01

Histone deacetylases (HDACs) play an important role in the regulation of chromatin structure and gene expression. We found that dark pigmentation of Magnaporthe oryzae (anamorph Pyricularia oryzae) ΔMohda1, a mutant strain in which an orthologue of the yeast HDA1 was disrupted by double cross-over homologous recombination, was significantly stimulated in liquid culture. Analysis of metabolites in a ΔMohda1 mutant culture revealed that the accumulation of shunt products of the 1,8-dihydroxynaphthalene melanin and ergosterol pathways were significantly enhanced compared to the wild-type strain. Northern blot analysis of the ΔMohda1 mutant revealed transcriptional activation of three melanin genes that are dispersed throughout the genome of M. oryzae. The effect of deletion of the yeast HDA1 orthologue was also observed in Fusarium asiaticum from the Fusarium graminearum species complex; the HDF2 deletion mutant produced increased levels of nivalenol-type trichothecenes. These results suggest that histone modification via HDA1-type HDAC regulates the production of natural products in filamentous fungi. Natural products of fungi have significant impacts on human welfare, in both detrimental and beneficial ways. Although HDA1-type histone deacetylase is not essential for vegetative growth, deletion of the gene affects the expression of clustered secondary metabolite genes in some fungi. Here, we report that such phenomena are also observed in physically unlinked genes required for melanin biosynthesis in the rice blast fungus. In addition, production of Fusarium trichothecenes, previously reported to be unaffected by HDA1 deletion, was significantly upregulated in another Fusarium species. Thus, the HDA1-inactivation strategy may be regarded as a general approach for overproduction and/or discovery of fungal metabolites. © 2017 The Society for Applied Microbiology.

Calpains are involved in asexual and sexual development, cell wall integrity and pathogenicity of the rice blast fungus

PubMed Central

Liu, Xiao-Hong; Ning, Guo-Ao; Huang, Lu-Yao; Zhao, Ya-Hui; Dong, Bo; Lu, Jian-Ping; Lin, Fu-Cheng

2016-01-01

Calpains are ubiquitous and well-conserved proteins that belong to the calcium-dependent, non-lysosomal cysteine protease family. In this study, 8 putative calpains were identified using Pfam domain analysis and BlastP searches in M. oryzae. Three single gene deletion mutants (ΔMocapn7, ΔMocapn9 and ΔMocapn14) and two double gene deletion mutants (ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7) were obtained using the high-throughput gene knockout system. The calpain disruption mutants showed defects in colony characteristics, conidiation, sexual reproduction and cell wall integrity. The mycelia of the ΔMocapn7, ΔMocapn4ΔMocapn7 and ΔMocapn9ΔMocapn7 mutants showed reduced pathogenicity on rice and barley. PMID:27502542

The killing speed of egt-inactivated Bombyx mori nucleopolyhedrovirus depends on the developmental stage of B. mori larvae.

PubMed

Katsuma, Susumu; Shimada, Toru

2015-03-01

Several lines of evidence have shown that the deletion of the ecdysteroid UDP-glucosyltransferase gene (egt) from the nucleopolyhedrovirus (NPV) genome increases the killing speed of host lepidopteran larvae. However, it has not been investigated in detail whether the effects of egt deletion depend on the larval stages of the host insect. In this study, we performed bioassays using 10 continuous larval stages of the 4th- or 5th-instar Bombyx mori larvae and B. mori NPV egt mutants. The fast-killing phenotype was observed in the egt mutants only when the infection process progressed through larval-larval transition. All day-2 4th-instar larvae infected with the egt mutants entered the molting stage and died much earlier than wild-type-infected larvae. Bodies of egt mutant-infected larvae were filled with excessive fluid immediately after head capsule slippage, owing presumably to the degeneration of Malpighian tubules. Fourth- or 5th-instar larvae infected with the egt mutants at early stages of each instar died similarly to those infected with the wild-type virus. Under infection in the middle stages of the 5th-instar, the survival time of egt mutant-infected larvae was significantly longer than that of the wild-type virus-infected larvae. These results clearly show that the effects of egt deletion on killing speed of NPV are largely dependent on the developmental stage of the host larvae infected by the virus. Copyright © 2015 Elsevier Inc. All rights reserved.

Deletion of the Braun Lipoprotein-Encoding Gene and Altering the Function of Lipopolysaccharide Attenuate the Plague Bacterium

PubMed Central

Sha, Jian; Kirtley, Michelle L.; van Lier, Christina J.; Wang, Shaofei; Erova, Tatiana E.; Kozlova, Elena V.; Cao, Anthony; Cong, Yingzi; Fitts, Eric C.; Rosenzweig, Jason A.

2013-01-01

Braun (murein) lipoprotein (Lpp) and lipopolysaccharide (LPS) are major components of the outer membranes of Enterobacteriaceae family members that are capable of triggering inflammatory immune responses by activating Toll-like receptors 2 and 4, respectively. Expanding on earlier studies that demonstrated a role played by Lpp in Yersinia pestis virulence in mouse models of bubonic and pneumonic plague, we characterized an msbB in-frame deletion mutant incapable of producing an acyltransferase that is responsible for the addition of lauric acid to the lipid A moiety of LPS, as well as a Δlpp ΔmsbB double mutant of the highly virulent Y. pestis CO92 strain. Although the ΔmsbB single mutant was minimally attenuated, the Δlpp single mutant and the Δlpp ΔmsbB double mutant were significantly more attenuated than the isogenic wild-type (WT) bacterium in bubonic and pneumonic animal models (mouse and rat) of plague. These data correlated with greatly reduced survivability of the aforementioned mutants in murine macrophages. Furthermore, the Δlpp ΔmsbB double mutant was grossly compromised in its ability to disseminate to distal organs in mice and in evoking cytokines/chemokines in infected animal tissues. Importantly, mice that survived challenge with the Δlpp ΔmsbB double mutant, but not the Δlpp or ΔmsbB single mutant, in a pneumonic plague model were significantly protected against a subsequent lethal WT CO92 rechallenge. These data were substantiated by the fact that the Δlpp ΔmsbB double mutant maintained an immunogenicity comparable to that of the WT strain and induced long-lasting T-cell responses against heat-killed WT CO92 antigens. Taken together, the data indicate that deletion of the msbB gene augmented the attenuation of the Δlpp mutant by crippling the spread of the double mutant to the peripheral organs of animals and by inducing cytokine/chemokine responses. Thus, the Δlpp ΔmsbB double mutant could provide a new live-attenuated background vaccine candidate strain, and this should be explored in the future. PMID:23275092

Stabilization of VEGFR2 signaling by cerebral cavernous malformation 3 is critical for vascular development.

PubMed

He, Yun; Zhang, Haifeng; Yu, Luyang; Gunel, Murat; Boggon, Titus J; Chen, Hong; Min, Wang

2010-04-06

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an early embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.

A 76-bp deletion in the Mip gene causes autosomal dominant cataract in Hfi mice.

PubMed

Sidjanin, D J; Parker-Wilson, D M; Neuhäuser-Klaus, A; Pretsch, W; Favor, J; Deen, P M; Ohtaka-Maruyama, C; Lu, Y; Bragin, A; Skach, W R; Chepelinsky, A B; Grimes, P A; Stambolian, D E

2001-06-15

Hfi is a dominant cataract mutation where heterozygotes show hydropic lens fibers and homozygotes show total lens opacity. The Hfi locus was mapped to the distal part of mouse chromosome 10 close to the major intrinsic protein (Mip), which is expressed only in cell membranes of lens fibers. Molecular analysis of Mip revealed a 76-bp deletion that resulted in exon 2 skipping in Mip mRNA. In Hfi/Hfi this deletion resulted in a complete absence of the wildtype Mip. In contrast, Hfi/+ animals had the same amount of wildtype Mip as +/+. Results from pulse-chase expression studies excluded hetero-oligomerization of wildtype and mutant Mip as a possible mechanism for cataract formation in the Hfi/+. We propose that the cataract phenotype in the Hfi heterozygote mutant is due to a detrimental gain of function by the mutant Mip resulting in either cytotoxicity or disruption in processing of other proteins important for the lens. Cataract formation in the Hfi/Hfi mouse is probably a combined result of both the complete loss of wildtype Mip and a gain of function of the mutant Mip. Copyright 2001 Academic Press.

A mitochondrial locus is necessary for the synthesis of mitochondrial tRNA in the yeast Saccharomyces cerevisiae.

PubMed Central

Martin, N C; Underbrink-Lyon, K

1981-01-01

We have used a cloned yeast mitochondrial tRNAUCNSer gene as a probe to detect RNA species that are transcripts from this gene in wild-type Saccharomyces cerevisiae and in petite deletion mutants. In RNA from wild-type cells, the tRNA is the most prominent transcript of the gene. In RNA from deletion mutants that retain this gene but have lost other regions of mtDNA, high molecular weight transcripts containing the tRNAUCNSer sequences accumulate but tRNAUCNSer is not made. tRNAUCNSer synthesis can be restored in these mutants when they are mated to other deletion mutants that retain a different portion of the mitochondrial genome. Protein synthesis is not necessary for the restoration, and the restoration is not due to a nuclear effect or to an effect of mating alone, because strains without mtDNA are not able to restore tRNA synthesis. These results definitively demonstrate the existence of a yeast mitochondrial locus that is necessary for tRNA synthesis and, because the restoration of tRNAUCNSer synthesis appears to result from intergenic complementation, not recombination, indicate that this locus acts in trans. Images PMID:6795621

Coat protein deletion mutants elicit more severe symptoms than wild-type virus in multiple cereal hosts

USDA-ARS?s Scientific Manuscript database

The coat protein (CP) of Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) tolerates deletion of amino acids 36 to 84 for efficient systemic infection of wheat. This study demonstrates that deletion of CP amino acids 58 to 84, but not 36 to 57, from WSMV genome induced severe ...

Divergent Roles of RPA Homologs of the Model Archaeon Halobacterium salinarum in Survival of DNA Damage.

PubMed

Evans, Jessica J; Gygli, Patrick E; McCaskill, Julienne; DeVeaux, Linda C

2018-04-20

The haloarchaea are unusual in possessing genes for multiple homologs to the ubiquitous single-stranded DNA binding protein (SSB or replication protein A, RPA) found in all three domains of life. Halobacterium salinarum contains five homologs: two are eukaryotic in organization, two are prokaryotic and are encoded on the minichromosomes, and one is uniquely euryarchaeal. Radiation-resistant mutants previously isolated show upregulation of one of the eukaryotic-type RPA genes. Here, we have created deletions in the five RPA operons. These deletion mutants were exposed to DNA-damaging conditions: ionizing radiation, UV radiation, and mitomycin C. Deletion of the euryarchaeal homolog, although not lethal as in Haloferax volcanii , causes severe sensitivity to all of these agents. Deletion of the other RPA/SSB homologs imparts a variable sensitivity to these DNA-damaging agents, suggesting that the different RPA homologs have specialized roles depending on the type of genomic insult encountered.

Deletion of the α-(1,3)-Glucan Synthase Genes Induces a Restructuring of the Conidial Cell Wall Responsible for the Avirulence of Aspergillus fumigatus

PubMed Central

Beauvais, Anne; Bozza, Silvia; Kniemeyer, Olaf; Formosa, Céline; Balloy, Viviane; Henry, Christine; Roberson, Robert W.; Dague, Etienne; Chignard, Michel; Brakhage, Axel A.; Romani, Luigina; Latgé, Jean-Paul

2013-01-01

α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant. PMID:24244155

Screening of polymorphisms for MTHFR and DHFR genes in spina bifida children and their mothers

NASA Astrophysics Data System (ADS)

Husna, M. Z.; Endom, I.; Ibrahim, S.; Selvi, N. Amaramalar; Fakhrurazi, H.; Htwe, R. Ohnmar; Kanehaswari, Y.; Halim, A. R. Abdul; Wong, S. W.; Subashini, K.; Syahira, O. Nur; Aishah, S.

2013-11-01

Mechanism underlying the beneficial effect of folic acid supplementation in reducing the risk of neural tube defect is still not well understood. Current evidences show the involvement of folic acid metabolic gene's polymorphism as contributing factors that regulate this pathway. Therefore, the objective of this research was to determine the presence of C677T polymorphism for methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase (DHFR-19 bp deletion) genes between mother-children pairs of case and control. With the approval of UKMMC ethic committee, genomic DNA was extracted from one hundred and forty consented bloods. Polymerase chain reaction (PCR), PCR-RFLP (Restriction Fragment Length Polymorphism) and sequencing were employed to verify each nucleotide change. Our result shows that mutant MTHFR and DHFR alleles are present in all Malaysian sub-ethnic groups, case and control. Even though mutant MTHFR are found to be slightly higher in the case groups, 75% of the affected child is a non carrier for this allele and 62.5% of the mothers with an affected child are genotypically normal. For DHFR, almost all (87.5-100%) investigated samples are a carrier or having a double DHFR deletion be it a case or control pairs. However, strong maternal inheritance shown by the deleted allele might be due to a cascade effect of lacks of folate consumption or maternal uniparental disomy. In conclusion, the use of MTHFR and DHFR as markers in determining the risk of having spina bifida baby is uninformative and plays a small indirect role as the genetic causes of spina bifida. Therefore, spina bifida remains etiologically unknown polygenic and quantitative developmental trait whereby the searches for positive genetic marker need to be continued.

Vital role for cyclophilin B (CypB) in asexual development, dimorphic transition and virulence of Beauveria bassiana.

PubMed

Chu, Zhen-Jian; Sun, Huan-Huan; Ying, Sheng-Hua; Feng, Ming-Guang

2017-08-01

Cyclophilin B (CypB) was previously revealed as one of many putative secretory proteins in the transcriptome of Beauveria bassiana infection to a lepidopteran pest. Here we show a main localization of CypB in hyphal cell walls and septa and its essential role in the in vitro and in vivo asexual cycles of the fungal insect pathogen. Deletion of cypB reduced colony growth by 16-42% on two rich media and 30 scant media with different carbon or nitrogen sources. The deletion mutant suffered a delayed conidiation on a standard medium and a final 47% reduction in conidial yield, accompanied with drastic transcript depression of several key genes required for conidiation and conidial maturation. The mutant conidia required 10h longer to germinate 50% at optimal 25°C than wild-type conidia. Intriguingly, cultivation of the mutant conidia in a trehalose-peptone broth mimic to insect hemolymph resulted in 83% reduction in blastospore yield but only slight decrease in biomass level, indicating severe defects in transition of hyphae to blastospores. LT 50 for the deletion mutant against Galleria mellonella larvae through normal cuticle infection was prolonged to 7.4d from a wild-type estimate of 4.7d. During colony growth, additionally, the deletion mutant displayed hypersensitivity to Congo red, menadione, H 2 O 2 and heat shock but increased tolerance to cyclosporine A and rapamycin. All of changes were restored by targeted gene complementation. Altogether, CypB takes part in sustaining normal growth, aerial conidiation, conidial germination, dimorphic transition, stress tolerance and pathogenicity in B. bassiana. Copyright © 2017 Elsevier Inc. All rights reserved.

Dystrophin Hot-Spot Mutants Leading to Becker Muscular Dystrophy Insert More Deeply into Membrane Models than the Native Protein.

PubMed

Ameziane-Le Hir, Sarah; Paboeuf, Gilles; Tascon, Christophe; Hubert, Jean-François; Le Rumeur, Elisabeth; Vié, Véronique; Raguénès-Nicol, Céline

2016-07-26

Dystrophin (DYS) is a membrane skeleton protein whose mutations lead to lethal Duchenne muscular dystrophy or to the milder Becker muscular dystrophy (BMD). One third of BMD "in-frame" exon deletions are located in the region that codes for spectrin-like repeats R16 to R21. We focused on four prevalent mutated proteins deleted in this area (called RΔ45-47, RΔ45-48, RΔ45-49, and RΔ45-51 according to the deleted exon numbers), analyzing protein/membrane interactions. Two of the mutants, RΔ45-48 and RΔ45-51, led to mild pathologies and displayed a similar triple coiled-coil structure as the full-length DYS R16-21, whereas the two others, RΔ45-47 and RΔ45-49, induced more severe pathologies and showed "fractional" structures unrelated to the normal one. To explore lipid packing, small unilamellar liposomes (SUVs) and planar monolayers were used at various initial surface pressures. The dissociation constants determined by microscale thermophoresis (MST) were much higher for the full-length DYS R161-21 than for the mutants; thus the wild type protein has weaker SUV binding. Comparing surface pressures after protein adsorption and analysis of atomic force microscopy images of mixed protein/lipid monolayers revealed that the mutants insert more into the lipid monolayer than the wild type does. In fact, in both models every deletion mutant showed more interactions with membranes than the full-length protein did. This means that mutations in the R16-21 part of dystrophin disturb the protein's molecular behavior as it relates to membranes, regardless of whether the accompanying pathology is mild or severe.

Chemical Genomic Screening of a Saccharomyces cerevisiae Genomewide Mutant Collection Reveals Genes Required for Defense against Four Antimicrobial Peptides Derived from Proteins Found in Human Saliva

PubMed Central

Bhatt, Sanjay; Schoenly, Nathan E.; Lee, Anna Y.; Nislow, Corey; Bobek, Libuse A.

2013-01-01

To compare the effects of four antimicrobial peptides (MUC7 12-mer, histatin 12-mer, cathelicidin KR20, and a peptide containing lactoferricin amino acids 1 to 11) on the yeast Saccharomyces cerevisiae, we employed a genomewide fitness screen of combined collections of mutants with homozygous deletions of nonessential genes and heterozygous deletions of essential genes. When an arbitrary fitness score cutoffs of 1 (indicating a fitness defect, or hypersensitivity) and −1 (indicating a fitness gain, or resistance) was used, 425 of the 5,902 mutants tested exhibited altered fitness when treated with at least one peptide. Functional analysis of the 425 strains revealed enrichment among the identified deletions in gene groups associated with the Gene Ontology (GO) terms “ribosomal subunit,” “ribosome biogenesis,” “protein glycosylation,” “vacuolar transport,” “Golgi vesicle transport,” “negative regulation of transcription,” and others. Fitness profiles of all four tested peptides were highly similar, particularly among mutant strains exhibiting the greatest fitness defects. The latter group included deletions in several genes involved in induction of the RIM101 signaling pathway, including several components of the ESCRT sorting machinery. The RIM101 signaling regulates response of yeasts to alkaline and neutral pH and high salts, and our data indicate that this pathway also plays a prominent role in regulating protective measures against all four tested peptides. In summary, the results of the chemical genomic screens of S. cerevisiae mutant collection suggest that the four antimicrobial peptides, despite their differences in structure and physical properties, share many interactions with S. cerevisiae cells and consequently a high degree of similarity between their modes of action. PMID:23208710

Molecular mapping within the mouse albino-deletion complex.

PubMed Central

Johnson, D K; Hand, R E; Rinchik, E M

1989-01-01

Induced germ-line deletion mutations in the mouse provide a malleable experimental system for in-depth molecular and functional analysis of large segments of the mammalian genome. To obtain an initial bank of molecular probes for the region of mouse chromosome 7 associated with the albino-deletion complex, random anonymous DNA clones, derived from a library constructed from flow-sorted chromosomes, were screened on DNAs from Mus musculus-Mus spretus F1 hybrids carrying large, multilocus, lethal albino deletions. Clones falling within a given deletion interval can easily be recognized because hybridization bands that represent restriction fragment length polymorphisms specific for the mutant (deleted) chromosome inherited from the M. musculus parent will be absent. Among 72 informative clones used as probes, one, which defines the locus D7OR1, mapped within two deletions that are 6-11 centimorgans in length. Submapping of this anonymous clone across a panel of 27 smaller deletions localized D7OR1 distal to a chromosomal subregion important for survival of the preimplantation embryo, proximal to globin [beta-chain (Hbb)], and near the shaker-1 (sh-1) locus. The results of these deletion-mapping experiments were also confirmed by standard three-point linkage analysis. This strategy for selection and rapid mapping of anonymous DNA probes to chromosomal segments corresponding to germ-line deletion mutations should contribute to the generation of more detailed physical and functional maps of genomic regions associated with mutant developmental phenotypes. Images PMID:2813427

HU participates in expression of a specific set of genes required for growth and survival at acidic pH in Escherichia coli.

PubMed

Bi, Hongkai; Sun, Lianle; Fukamachi, Toshihiko; Saito, Hiromi; Kobayashi, Hiroshi

2009-05-01

The major histone-like Escherichia coli protein, HU, is composed of alpha and beta subunits respectively encoded by hupA and hupB in Escherichia coli. A mutant deficient in both hupA and hupB grew at a slightly slower rate than the wild type at pH 7.5. Growth of the mutant diminished with a decrease in pH, and no growth was observed at pH 4.6. Mutants of either hupA or hupB grew at all pH levels tested. The arginine-dependent survival at pH 2.5 was diminished approximately 60-fold by the deletion of both hupA and hupB, whereas the survival was slightly affected by the deletion of either hupA or hupB. The mRNA levels of adiA and adiC, which respectively encode arginine decarboxylase and arginine/agmatine antiporter, were low in the mutant deficient in both hupA and hupB. The deletion of both hupA and hupB had little effect on survival at pH 2.5 in the presence of glutamate or lysine, and expression of the genes for glutamate and lysine decarboxylases was not impaired by the deletion of the HU genes. These results suggest that HU regulates expression of the specific set of genes required for growth and survival in acidic environments.

A putative APSES transcription factor is necessary for normal growth and development of Aspergillus nidulans.

PubMed

Lee, Ji-Yeon; Kim, Lee-Han; Kim, Ha-Eun; Park, Jae-Sin; Han, Kap-Hoon; Han, Dong-Min

2013-12-01

The nsdD gene encoding a GATA type transcription factor positively controls sexual development in Aspergillus nidulans. According to microarray data, 20 genes that were upregulated by deleting nsdD during various life cycle stages were randomly selected and deleted for functional analysis. None of the mutants showed apparent changes in growth or development compared with those of the wild-type except the AN3154 gene that encodes a putative APSES transcription factor and is an ortholog of Saccharomyces cerevisiae swi4. Deleting AN3154 resulted in retarded growth and development, and the gene was named rgdA (retared growth and development). The rgdA deletion mutant developed a reduced number of conidia even under favorable conditions for asexual development. The retarded growth and development was partially suppressed by the veA1 mutation. The conidial heads of the mutant aborted, showing reduced and irregular shaped phialides. Fruiting body development was delayed compared with that in the wild-type. The mutant did not respond to various nutritional or environmental factors that affected the development patterns. The rgdA gene was expressed at low levels throughout the life cycle and was not significantly affected by several regulators of sexual and asexual development such as nsdD, veA, stuA, or brlA. However, the rgdA gene affected brlA and abaA expression, which function as key regulators of asexual sporulation, suggesting that rgdA functions upstream of those genes.

Internally deleted WNV genomes isolated from exotic birds in New Mexico: function in cells, mosquitoes, and mice.

PubMed

Pesko, Kendra N; Fitzpatrick, Kelly A; Ryan, Elizabeth M; Shi, Pei-Yong; Zhang, Bo; Lennon, Niall J; Newman, Ruchi M; Henn, Matthew R; Ebel, Gregory D

2012-05-25

Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21 days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts. Copyright © 2012 Elsevier Inc. All rights reserved.

The mir-279/996 cluster represses receptor tyrosine kinase signaling to determine cell fates in the Drosophila eye.

PubMed

Duan, Hong; de Navas, Luis F; Hu, Fuqu; Sun, Kailiang; Mavromatakis, Yannis E; Viets, Kayla; Zhou, Cyrus; Kavaler, Joshua; Johnston, Robert J; Tomlinson, Andrew; Lai, Eric C

2018-04-09

Photoreceptors in the crystalline Drosophila eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the mir-279/996 locus, along with a panel of modified genomic rescue transgenes, show that Drosophila eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mir-279/996 mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since mir-279/996 nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of mir-279/996 sufficiently derepresses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants boss and sev , which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that involves substantial miRNA control. © 2018. Published by The Company of Biologists Ltd.

Identification of the methylation preference region in heterogeneous nuclear ribonucleoprotein K by protein arginine methyltransferase 1 and its implication in regulating nuclear/cytoplasmic distribution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Yuan-I; Hsu, Sheng-Chieh; Chau, Gar-Yang

2011-01-21

Research highlights: {yields} Verifying by direct methylation assay the substrate sites of PRMT1 in the hnRNP K protein. {yields} Identifying the preferred PMRT1 methylation regions in hnRNP K by kinetic analysis. {yields} Linking methylation in regulating nuclear localization of hnRNP K. -- Abstract: Protein arginine methylation plays crucial roles in numerous cellular processes. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multi-functional protein participating in a variety of cellular functions including transcription and RNA processing. HnRNP K is methylated at multiple sites in the glycine- and arginine-rich (RGG) motif. Using various RGG domain deletion mutants of hnRNP K as substrates,more » here we show by direct methylation assay that protein arginine methyltransferase 1 (PRMT1) methylated preferentially in a.a. 280-307 of the RGG motif. Kinetic analysis revealed that deletion of a.a. 280-307, but not a.a. 308-327, significantly inhibited rate of methylation. Importantly, nuclear localization of hnRNP K was significantly impaired in mutant hnRNP K lacking the PRMT1 methylation region or upon pharmacological inhibition of methylation. Together our results identify preferred PRMT1 methylation sequences of hnRNP K by direct methylation assay and implicate a role of arginine methylation in regulating intracellular distribution of hnRNP K.« less

Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms.

PubMed

Liu, Qun; Liu, Wei; Zeng, Baosheng; Wang, Guirong; Hao, Dejun; Huang, Yongping

2017-07-01

Olfaction plays an essential role in many important insect behaviors such as feeding and reproduction. To detect olfactory stimuli, an odorant receptor co-receptor (Orco) is required. In this study, we deleted the Orco gene in the Lepidopteran model insect, Bombyx mori, using a binary transgene-based clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. We initially generated somatic mutations in two targeted sites, from which we obtained homozygous mutants with deletion of a 866 base pair sequence. Because of the flight inability of B. mori, we developed a novel method to examine the adult mating behavior. Considering the specialization in larval feeding, we examined food selection behavior in Orco somatic mutants by the walking trail analysis of silkworm position over time. Single sensillum recordings indicated that the antenna of the homozygous mutant was unable to respond to either of the two sex pheromones, bombykol or bombykal. An adult mating behavior assay revealed that the Orco mutant displayed a significantly impaired mating selection behavior in response to natural pheromone released by a wild-type female moth as well as an 11:1 mixture of bombykol/bombykal. The mutants also exhibited a decreased response to bombykol and, similar to wild-type moths, they displayed no response to bombykal. A larval feeding behavior assay revealed that the Orco mutant displayed defective selection for mulberry leaves and different concentrations of the volatile compound cis-jasmone found in mulberry leaves. Deletion of BmOrco severely disrupts the olfactory system, suggesting that BmOrco is indispensable in the olfactory pathway. The approach used for generating somatic and homozygous mutations also highlights a novel method for mutagenesis. This study on BmOrco function provides insights into the insect olfactory system and also provides a paradigm for agroforestry pest control. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme.

PubMed Central

Fu, C; Javedan, S; Moshiri, F; Maier, R J

1994-01-01

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product. PMID:8197192

Efflux-Mediated Resistance to Tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1

PubMed Central

Dean, Charles R.; Visalli, Melissa A.; Projan, Steven J.; Sum, Phaik-Eng; Bradford, Patricia A.

2003-01-01

Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 μg/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline, P. aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 μg/ml) was specifically associated with loss of MexXY. Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 μg/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from P. aeruginosa, are generally inferior substrates for P. aeruginosa efflux pumps than are narrower-spectrum tetracyclines. PMID:12604529

A Pseudomonas aeruginosa EF-Hand Protein, EfhP (PA4107), Modulates Stress Responses and Virulence at High Calcium Concentration

PubMed Central

Sarkisova, Svetlana A.; Lotlikar, Shalaka R.; Guragain, Manita; Kubat, Ryan; Cloud, John

2014-01-01

Pseudomonas aeruginosa is a facultative human pathogen, and a major cause of nosocomial infections and severe chronic infections in endocarditis and in cystic fibrosis (CF) patients. Calcium (Ca2+) accumulates in pulmonary fluids of CF patients, and plays a role in the hyperinflamatory response to bacterial infection. Earlier we showed that P. aeruginosa responds to increased Ca2+ levels, primarily through the increased production of secreted virulence factors. Here we describe the role of putative Ca2+-binding protein, with an EF-hand domain, PA4107 (EfhP), in this response. Deletion mutations of efhP were generated in P. aeruginosa strain PAO1 and CF pulmonary isolate, strain FRD1. The lack of EfhP abolished the ability of P. aeruginosa PAO1 to maintain intracellular Ca2+ homeostasis. Quantitative high-resolution 2D-PAGE showed that the efhP deletion also affected the proteomes of both strains during growth with added Ca2+. The greatest proteome effects occurred when the pulmonary isolate was cultured in biofilms. Among the proteins that were significantly less abundant or absent in the mutant strains were proteins involved in iron acquisition, biosynthesis of pyocyanin, proteases, and stress response proteins. In support, the phenotypic responses of FRD1 ΔefhP showed that the mutant strain lost its ability to produce pyocyanin, developed less biofilm, and had decreased resistance to oxidative stress (H2O2) when cultured at high [Ca2+]. Furthermore, the mutant strain was unable to produce alginate when grown at high [Ca2+] and no iron. The effect of the ΔefhP mutations on virulence was determined in a lettuce model of infection. Growth of wild-type P. aeruginosa strains at high [Ca2+] causes an increased area of disease. In contrast, the lack of efhP prevented this Ca2+-induced increase in the diseased zone. The results indicate that EfhP is important for Ca2+ homeostasis and virulence of P. aeruginosa when it encounters host environments with high [Ca2+]. PMID:24918783

The Homolog of the Gene bstA of the BTP1 Phage from Salmonella enterica Serovar Typhimurium ST313 Is an Antivirulence Gene in Salmonella enterica Serovar Dublin.

PubMed

Herrero-Fresno, Ana; Espinel, Irene Cartas; Spiegelhauer, Malene Roed; Guerra, Priscila Regina; Andersen, Karsten Wiber; Olsen, John Elmerdahl

2018-01-01

In a previous study, a novel virulence gene, bstA , identified in a Salmonella enterica serovar Typhimurium sequence type 313 (ST313) strain was found to be conserved in all published Salmonella enterica serovar Dublin genomes. In order to analyze the role of this gene in the host-pathogen interaction in S Dublin, a mutant where this gene was deleted ( S Dublin Δ bstA ) and a mutant which was further genetically complemented with bstA ( S Dublin 3246-C) were constructed and tested in models of in vitro and in vivo infection as well as during growth competition assays in M9 medium, Luria-Bertani broth, and cattle blood. In contrast to the results obtained for a strain of S Typhimurium ST313, the lack of bstA was found to be associated with increased virulence in S Dublin. Thus, S Dublin Δ bstA showed higher levels of uptake than the wild-type strain during infection of mouse and cattle macrophages and higher net replication within human THP-1 cells. Furthermore, during mouse infections, S Dublin Δ bstA was more virulent than the wild type following a single intraperitoneal infection and showed an increased competitive index during competitive infection assays. Deletion of bstA did not affect either the amount of cytokines released by THP-1 macrophages or the cytotoxicity toward these cells. The histology of the livers and spleens of mice infected with the wild-type strain and the S Dublin Δ bstA mutant revealed similar levels of inflammation between the two groups. The gene was not important for adherence to or invasion of human epithelial cells and did not influence bacterial growth in rich medium, minimal medium, or cattle blood. In conclusion, a lack of bstA affects the pathogenicity of S Dublin by decreasing its virulence. Therefore, it might be regarded as an antivirulence gene in this serovar. Copyright © 2017 American Society for Microbiology.

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae

PubMed Central

Tong, Kevin; Skibbens, Robert V.

2015-01-01

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures—processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated. PMID:25986377

Pds5 regulators segregate cohesion and condensation pathways in Saccharomyces cerevisiae.

PubMed

Tong, Kevin; Skibbens, Robert V

2015-06-02

Cohesins are required both for the tethering together of sister chromatids (termed cohesion) and subsequent condensation into discrete structures-processes fundamental for faithful chromosome segregation into daughter cells. Differentiating between cohesin roles in cohesion and condensation would provide an important advance in studying chromatin metabolism. Pds5 is a cohesin-associated factor that is essential for both cohesion maintenance and condensation. Recent studies revealed that ELG1 deletion suppresses the temperature sensitivity of pds5 mutant cells. However, the mechanisms through which Elg1 may regulate cohesion and condensation remain unknown. Here, we report that ELG1 deletion from pds5-1 mutant cells results in a significant rescue of cohesion, but not condensation, defects. Based on evidence that Elg1 unloads the DNA replication clamp PCNA from DNA, we tested whether PCNA overexpression would similarly rescue pds5-1 mutant cell cohesion defects. The results indeed reveal that elevated levels of PCNA rescue pds5-1 temperature sensitivity and cohesion defects, but do not rescue pds5-1 mutant cell condensation defects. In contrast, RAD61 deletion rescues the condensation defect, but importantly, neither the temperature sensitivity nor cohesion defects exhibited by pds5-1 mutant cells. In combination, these findings reveal that cohesion and condensation are separable pathways and regulated in nonredundant mechanisms. These results are discussed in terms of a new model through which cohesion and condensation are spatially regulated.

Deletion of Ku80 causes early aging independent of chronic inflammation and Rag-1-induced DSBs.

PubMed

Holcomb, Valerie B; Vogel, Hannes; Hasty, Paul

2007-01-01

Animal models of premature aging are often defective for DNA repair. Ku80-mutant mice are disabled for nonhomologous end joining; a pathway that repairs both spontaneous DNA double-strand breaks (DSBs) and induced DNA DSBs generated by the action of a complex composed of Rag-1 and Rag-2 (Rag). Rag is essential for inducing DSBs important for assembling V(D)J segments of antigen receptor genes that are required for lymphocyte development. Thus, deletion of either Rag-1 or Ku80 causes severe combined immunodeficiency (scid) leading to chronic inflammation. In addition, Rag-1 induces breaks at non-B DNA structures. Previously we reported Ku80-mutant mice undergo premature aging, yet we do not know the root cause of this phenotype. Early aging may be caused by either defective repair of spontaneous DNA damage, defective repair of Rag-1-induced breaks or chronic inflammation caused by scid. To address this issue, we analyzed aging in control and Ku80-mutant mice deleted for Rag-1 such that both cohorts are scid and suffer from chronic inflammation. We make two observations: (1) chronic inflammation does not cause premature aging in these mice and (2) Ku80-mutant mice exhibit early aging independent of Rag-1. Therefore, this study supports defective repair of spontaneous DNA damage as the root cause of early aging in Ku80-mutant mice.

Heavy ion mutagenesis: linear energy transfer effects and genetic linkage

NASA Technical Reports Server (NTRS)

Kronenberg, A.; Gauny, S.; Criddle, K.; Vannais, D.; Ueno, A.; Kraemer, S.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

1995-01-01

We have characterized a series of 69 independent mutants at the endogenous hprt locus of human TK6 lymphoblasts and over 200 independent S1-deficient mutants of the human x hamster hybrid cell line AL arising spontaneously or following low-fluence exposures to densely ionizing Fe ions (600 MeV/amu, linear energy transfer = 190 keV/microns). We find that large deletions are common. The entire hprt gene (> 44 kb) was missing in 19/39 Fe-induced mutants, while only 2/30 spontaneous mutants lost the entire hprt coding sequence. When the gene of interest (S1 locus = M1C1 gene) is located on a nonessential human chromosome 11, multilocus deletions of several million base pairs are observed frequently. The S1 mutation frequency is more than 50-fold greater than the frequency of hprt mutants in the same cells. Taken together, these results suggest that low-fluence exposures to Fe ions are often cytotoxic due to their ability to create multilocus deletions that may often include the loss of essential genes. In addition, the tumorigenic potential of these HZE heavy ions may be due to the high potential for loss of tumor suppressor genes. The relative insensitivity of the hprt locus to mutation is likely due to tight linkage to a gene that is required for viability.

Phenotypic Restoration by Molybdate of Nitrate Reductase Activity in chlD Mutants of Escherichia coli

PubMed Central

Glaser, J. H.; DeMoss, J. A.

1971-01-01

ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10−4m molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chlD gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems. PMID:4942767

Domain-Specific Activation of Death-Associated Intracellular Signalling Cascades by the Cellular Prion Protein in Neuroblastoma Cells.

PubMed

Vilches, Silvia; Vergara, Cristina; Nicolás, Oriol; Mata, Ágata; Del Río, José A; Gavín, Rosalina

2016-09-01

The biological functions of the cellular prion protein remain poorly understood. In fact, numerous studies have aimed to determine specific functions for the different protein domains. Studies of cellular prion protein (PrP(C)) domains through in vivo expression of molecules carrying internal deletions in a mouse Prnp null background have provided helpful data on the implication of the protein in signalling cascades in affected neurons. Nevertheless, understanding of the mechanisms underlying the neurotoxicity induced by these PrP(C) deleted forms is far from complete. To better define the neurotoxic or neuroprotective potential of PrP(C) N-terminal domains, and to overcome the heterogeneity of results due to the lack of a standardized model, we used neuroblastoma cells to analyse the effects of overexpressing PrP(C) deleted forms. Results indicate that PrP(C) N-terminal deleted forms were properly processed through the secretory pathway. However, PrPΔF35 and PrPΔCD mutants led to death by different mechanisms sharing loss of alpha-cleavage and activation of caspase-3. Our data suggest that both gain-of-function and loss-of-function pathogenic mechanisms may be associated with N-terminal domains and may therefore contribute to neurotoxicity in prion disease. Dissecting the molecular response induced by PrPΔF35 may be the key to unravelling the physiological and pathological functions of the prion protein.

Gene replacement in Penicillium roqueforti.

PubMed

Goarin, Anne; Silar, Philippe; Malagnac, Fabienne

2015-05-01

Most cheese-making filamentous fungi lack suitable molecular tools to improve their biotechnology potential. Penicillium roqueforti, a species of high industrial importance, would benefit from functional data yielded by molecular genetic approaches. This work provides the first example of gene replacement by homologous recombination in P. roqueforti, demonstrating that knockout experiments can be performed in this fungus. To do so, we improved the existing transformation method to integrate transgenes into P. roqueforti genome. In the meantime, we cloned the PrNiaD gene, which encodes a NADPH-dependent nitrate reductase that reduces nitrate to nitrite. Then, we performed a deletion of the PrNiaD gene from P. roqueforti strain AGO. The ΔPrNiaD mutant strain is more resistant to chlorate-containing medium than the wild-type strain, but did not grow on nitrate-containing medium. Because genomic data are now available, we believe that generating selective deletions of candidate genes will be a key step to open the way for a comprehensive exploration of gene function in P. roqueforti.

Selective silencing of full-length CD80 but not IgV-CD80 leads to impaired clonal deletion of self-reactive T cells and altered regulation of immune responses.

PubMed

Bugeon, L; Hargreaves, R E; Crompton, T; Outram, S; Rahemtulla, A; Porter, A C; Dallman, M J

2001-01-01

Co-stimulation provided by the B7 family of proteins underpins the development of protective immunity. There are three identified members of this family: CD80, its splice variant IgV-CD80 and CD86. It has hitherto been difficult to analyze the expression and function of IgV-CD80 since there are no appropriate reagents capable of distinguishing it from CD80. We have generated mice, by gene targeting, the lack CD80 whilst maintaining expression of IgV-CD80. Mutant animals did not delete T cells bearing mammary tumor virus-reactive TCR as efficiently as wild-type animals. We also demonstrate the importance of IgV-CD80 in the responses of recently activated cells and reveal a role for CD80 in sustaining T cell responses. CD86, whilst critical to primary T cell activation, made only a minor contribution to re-activation of normal cells.

FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa.

PubMed

Shen, Jiangsheng; Meldrum, Allison; Poole, Keith

2002-06-01

Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P. putida. Deletion of fpvA from the chromosome of P. aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P. aeruginosa.

CCCTC-Binding Factor Acts as a Heterochromatin Barrier on Herpes Simplex Viral Latent Chromatin and Contributes to Poised Latent Infection

PubMed Central

2018-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) establishes latent infection in neurons via a variety of epigenetic mechanisms that silence its genome. The cellular CCCTC-binding factor (CTCF) functions as a mediator of transcriptional control and chromatin organization and has binding sites in the HSV-1 genome. We constructed an HSV-1 deletion mutant that lacked a pair of CTCF-binding sites (CTRL2) within the latency-associated transcript (LAT) coding sequences and found that loss of these CTCF-binding sites did not alter lytic replication or levels of establishment of latent infection, but their deletion reduced the ability of the virus to reactivate from latent infection. We also observed increased heterochromatin modifications on viral chromatin over the LAT promoter and intron. We therefore propose that CTCF binding at the CTRL2 sites acts as a chromatin insulator to keep viral chromatin in a form that is poised for reactivation, a state which we call poised latency. PMID:29437926

Cotargeting the lncRNA-PIP3 Interaction and AKT/PI3K Signaling Axis: A Novel Paradigm for Treating Triple-Negative Breast Cancer

DTIC Science & Technology

2017-10-01

Model Core Facility at MD Anderson has generated a genomic deletion of LINK-A using CRISPR /Cas9 technology based on collaboration. The single...Generation of LINK-A deletion mutant using CRISPR /Cas9 technology. A, Illustration of genomic deletion region of LINK-A (Chr1: 238644075-238644134

A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions

PubMed Central

Shimizu, Kiminori; Paul, Sanjoy; Ohba, Ayumi; Gonoi, Tohru; Watanabe, Akira; Gomi, Katsuya

2017-01-01

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B). PMID:28052140

Adenylate Cyclase and the Cyclic AMP Receptor Protein Modulate Stress Resistance and Virulence Capacity of Uropathogenic Escherichia coli

PubMed Central

Donovan, Grant T.; Norton, J. Paul; Bower, Jean M.

2013-01-01

In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, the cyaA and crp deletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H2O2) and acid stress. In the mutant strains, H2O2 resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract. PMID:23115037

Association of Constitutive Hyperphosphorylation of Hsf1p with a Defective Ethanol Stress Response in Saccharomyces cerevisiae Sake Yeast Strains

PubMed Central

Noguchi, Chiemi; Watanabe, Daisuke; Zhou, Yan; Akao, Takeshi

2012-01-01

Modern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p in Saccharomyces cerevisiae sake yeast. The HSE-lacZ activity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. Since HSF1 allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a Δppt1 mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entire PPT1 gene locus. We confirmed that the expression of laboratory yeast-derived functional PPT1 recovered the HSE-mediated stress response of sake yeast. In addition, deletion of PPT1 in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of the PPT1 gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains. PMID:22057870

Host Hydrogen Rather than That Produced by the Pathogen Is Important for Salmonella enterica Serovar Typhimurium Virulence

PubMed Central

Lamichhane-Khadka, Reena; Benoit, Stéphane L.; Miller-Parks, Erica F.

2014-01-01

Salmonella enterica serovar Typhimurium utilizes molecular hydrogen as a substrate in various respiratory pathways, via H2-uptake enzymes termed Hya, Hyb, and Hyd. A different hydrogenase, the hydrogen-evolving Hyc enzyme, removes excess reductant during fermentative growth. Virulence phenotypes conferred by mutations in hyc genes, either alone or in combination with mutations in the H2-uptake enzyme genes, are addressed. Anaerobically grown ΔhycB or ΔhycC single-deletion strains were more sensitive to acid than the wild-type strain, but the Δhyc strains were like the virulent parent strain with respect to both mouse morbidity and mortality and in organ burden numbers. Even fecal-recovery numbers for both mutant strains at several time points prior to the animals succumbing to salmonellosis were like those seen with the parent. Neither hydrogen uptake nor evolution of the gas was detected in a hydrogenase quadruple-mutant strain containing deletions in the hya, hyb, hyd, and hyc genes. As previously described, a strain lacking all H2-uptake ability was severely attenuated in its virulence characteristics, and the quadruple-mutant strain had the same (greatly attenuated) phenotype. While H2 levels were greatly reduced in ceca of mice treated with antibiotics, both the ΔhycB and ΔhycC strains were still like the parent in their ability to cause typhoid salmonellosis. It seems that the level of H2 produced by the pathogen (through formate hydrogen lyase [FHL] and Hyc) is insignificant in terms of providing respiratory reductant to facilitate either organ colonization or contributions to gut growth leading to pathogenesis. PMID:25368112

Candida albicans ISW2 Regulates Chlamydospore Suspensor Cell Formation and Virulence In Vivo in a Mouse Model of Disseminated Candidiasis

PubMed Central

Lionakis, Michail S.; Nickerson, Kenneth W.

2016-01-01

Formation of chlamydospores by Candida albicans was an established medical diagnostic test to confirm candidiasis before the molecular era. However, the functional role and pathological relevance of this in vitro morphological transition to pathogenesis in vivo remain unclear. We compared the physical properties of in vitro-induced chlamydospores with those of large C. albicans cells purified by density gradient centrifugation from Candida-infected mouse kidneys. The morphological and physical properties of these cells in kidneys of mice infected intravenously with wild type C. albicans confirmed that chlamydospores can form in infected kidneys. A previously reported chlamydospore-null Δisw2/Δisw2 mutant was used to investigate its role in virulence and chlamydospore induction. Virulence of the Δisw2/Δisw2 mutant strain was reduced 3.4-fold compared to wild type C. albicans or the ISW2 reconstituted strain. Altered host inflammatory reactions to the null mutant further indicate that ISW2 is a virulence factor in C. albicans. ISW2 deletion abolished chlamydospore formation within infected mouse kidneys, whereas the reconstituted strain restored chlamydospore formation in kidneys. Under chlamydospore inducing conditions in vitro, deletion of ISW2 significantly delayed chlamydospore formation, and those late induced chlamydospores lacked associated suspensor cells while attaching laterally to hyphae via novel spore-hypha septa. Our findings establish the induction of chlamydospores by C. albicans during mouse kidney colonization. Our results indicate that ISW2 is not strictly required for chlamydospores formation but is necessary for suspensor cell formation. The importance of ISW2 in chlamydospore morphogenesis and virulence may lead to additional insights into morphological differentiation and pathogenesis of C. albicans in the host microenvironment. PMID:27727302

Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum

DOE PAGES

Biswas, Ranjita; Zheng, Tianyong; Olson, Daniel G.; ...

2015-02-12

The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl-CoA reduction to ethanol. C. thermocellum encodes four hydrogenases and rather than delete each individually, we targeted a hydrogenase maturase gene (hydG), involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe]more » hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in hydG ech was undetectable and ethanol yield increased nearly 2-fold compared to wild type. Interestingly, mutant growth improved upon the addition of acetate, which led to increased expression of genes related to sulfate metabolism, suggesting these mutants may use sulfate as a terminal electron acceptor to balance redox reactions. Genomic analysis of hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation is found in ethanol tolerant C. thermocellum strain E50C, hydG and hydG ech are not more ethanol tolerant than wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. The dramatic increase in ethanol production here suggests that targeting protein post-translational modification is a promising new approach for inactivation of multiple enzymes simultaneously for metabolic engineering.« less

Trigger factor of Streptococcus suis is involved in stress tolerance and virulence.

PubMed

Wu, Tao; Zhao, Zhanqin; Zhang, Lin; Ma, Hongwei; Lu, Ka; Ren, Wen; Liu, Zhengya; Chang, Haitao; Bei, Weicheng; Qiu, Yinsheng; Chen, Huanchun

2011-01-01

Streptococcus suis serotype 2 is an important zoonotic pathogen that causes serious diseases such as meningitis, septicemia, endocarditis, arthritis and septic shock in pigs and humans. Little is known about the regulation of virulence gene expression in S. suis serotype 2. In this study, we cloned and deleted the entire tig gene from the chromosome of S. suis serotype 2 SC21 strain, and constructed a mutant strain (Δtig) and a complementation strain (CΔtig). The results demonstrated that the tig gene, encoding trigger factor from S. suis serotype 2 SC21, affects the stress tolerance and the expression of a few virulence genes of S. suis serotype 2. Deletion of the tig gene of S. suis serotype 2 resulted in mutant strain, ΔTig, which exhibited a significant decrease in adherence to cell line HEp-2, and lacked hemolytic activity. Tig deficiency diminishes stresses tolerance of S. suis serotype 2 such as survive thermal, oxidative and acid stresses. Quantification of expression levels of known S. suis serotype 2 SC21 virulence genes by real-time polymerase chain reaction in vitro revealed that trigger factor influences the expression of epf, cps, adh, rpob, fbps, hyl, sly, mrp and hrcA virulence-associated genes. ΔTig was shown to be attenuated in a LD50 assay and bacteriology, indicating that trigger factor plays an important part in the pathogenesis and stress tolerance of. S. suis serotype 2 infection. Mutant ΔTig was 100% defective in virulence in CD1 mice at up to 107 CFU, and provided 100% protection when challenged with 107 CFU of the SC21 strain. Copyright © 2010. Published by Elsevier India Pvt Ltd.

Association of constitutive hyperphosphorylation of Hsf1p with a defective ethanol stress response in Saccharomyces cerevisiae sake yeast strains.

PubMed

Noguchi, Chiemi; Watanabe, Daisuke; Zhou, Yan; Akao, Takeshi; Shimoi, Hitoshi

2012-01-01

Modern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p in Saccharomyces cerevisiae sake yeast. The HSE-lacZ activity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. Since HSF1 allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a Δppt1 mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entire PPT1 gene locus. We confirmed that the expression of laboratory yeast-derived functional PPT1 recovered the HSE-mediated stress response of sake yeast. In addition, deletion of PPT1 in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of the PPT1 gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains.

Role of Interleukin-1 Receptor Signaling in the Behavioral Effects of Ethanol and Benzodiazepines

PubMed Central

Blednov, Yuri A.; Benavidez, Jillian M.; Black, Mendy; Mayfield, Jody; Harris, R. Adron

2015-01-01

Gene expression studies identified the interleukin-1 receptor type I (IL-1R1) as part of a pathway associated with a genetic predisposition to high alcohol consumption, and lack of the endogenous IL-1 receptor antagonist (IL-1ra) strongly reduced ethanol intake in mice. Here, we compared ethanol-mediated behaviors in mice lacking Il1rn or Il1r1. Deletion of Il1rn (the gene encoding IL-1ra) increases sensitivity to the sedative/hypnotic effects of ethanol and flurazepam and reduces severity of acute ethanol withdrawal. Conversely, deletion of Il1r1 (the gene encoding the IL-1 receptor type I, IL-1R1) reduces sensitivity to the sedative effects of ethanol and flurazepam and increases the severity of acute ethanol withdrawal. The sedative effects of ketamine and pentobarbital were not altered in the knockout (KO) strains. Ethanol intake and preference were not changed in mice lacking Il1r1 in three different tests of ethanol consumption. Recovery from ethanol-induced motor incoordination was only altered in female mice lacking Il1r1. Mice lacking Il1rn (but not Il1r1) showed increased ethanol clearance and decreased ethanol-induced conditioned taste aversion. The increased ethanol- and flurazepam-induced sedation in Il1rn KO mice was decreased by administration of IL-1ra (Kineret), and pre-treatment with Kineret also restored the severity of acute ethanol withdrawal. Ethanol-induced sedation and withdrawal severity were changed in opposite directions in the null mutants, indicating that these responses are likely regulated by IL-1R1 signaling, whereas ethanol intake and preference do not appear to be solely regulated by this pathway. PMID:25839897

Role of interleukin-1 receptor signaling in the behavioral effects of ethanol and benzodiazepines.

PubMed

Blednov, Yuri A; Benavidez, Jillian M; Black, Mendy; Mayfield, Jody; Harris, R Adron

2015-08-01

Gene expression studies identified the interleukin-1 receptor type I (IL-1R1) as part of a pathway associated with a genetic predisposition to high alcohol consumption, and lack of the endogenous IL-1 receptor antagonist (IL-1ra) strongly reduced ethanol intake in mice. Here, we compared ethanol-mediated behaviors in mice lacking Il1rn or Il1r1. Deletion of Il1rn (the gene encoding IL-1ra) increases sensitivity to the sedative/hypnotic effects of ethanol and flurazepam and reduces severity of acute ethanol withdrawal. Conversely, deletion of Il1r1 (the gene encoding the IL-1 receptor type I, IL-1R1) reduces sensitivity to the sedative effects of ethanol and flurazepam and increases the severity of acute ethanol withdrawal. The sedative effects of ketamine and pentobarbital were not altered in the knockout (KO) strains. Ethanol intake and preference were not changed in mice lacking Il1r1 in three different tests of ethanol consumption. Recovery from ethanol-induced motor incoordination was only altered in female mice lacking Il1r1. Mice lacking Il1rn (but not Il1r1) showed increased ethanol clearance and decreased ethanol-induced conditioned taste aversion. The increased ethanol- and flurazepam-induced sedation in Il1rn KO mice was decreased by administration of IL-1ra (Kineret), and pre-treatment with Kineret also restored the severity of acute ethanol withdrawal. Ethanol-induced sedation and withdrawal severity were changed in opposite directions in the null mutants, indicating that these responses are likely regulated by IL-1R1 signaling, whereas ethanol intake and preference do not appear to be solely regulated by this pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cj1386 is an ankyrin-containing protein involved in heme trafficking to catalase in Campylobacter jejuni.

PubMed

Flint, Annika; Sun, Yi-Qian; Stintzi, Alain

2012-01-01

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis.

Cj1386 Is an Ankyrin-Containing Protein Involved in Heme Trafficking to Catalase in Campylobacter jejuni

PubMed Central

Flint, Annika; Sun, Yi-Qian

2012-01-01

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ΔCj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ΔCj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ΔkatAΔ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ΔCj1386 and ΔkatA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ΔCj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ΔCj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ΔCj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis. PMID:22081390

Recombinant Escherichia coli K5 strain with the deletion of waaR gene decreases the molecular weight of the heparosan capsular polysaccharide.

PubMed

Huang, Haichan; Liu, Xiaobo; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

2016-09-01

Heparosan, the capsular polysaccharide of Escherichia coli K5 having a carbohydrate backbone similar to that of heparin, has become a potential precursor for bioengineering heparin. In the heparosan biosynthesis pathway, the gene waaR encoding α-1-, 2- glycosyltransferase catalyze s the third glucosyl residues linking to the oligosaccharide chain. In the present study, a waaR deletion mutant of E. coli K5 was constructed. The mutant showed improvement of capsule polysaccharide yield. It is interesting that the heparosan molecular weight of the mutant is reduced and may become more suitable as a precursor for the production of low molecular weight heparin derived from the wild-type K5 capsular polysaccharide.

High-Throughput, Signature-Tagged Mutagenic Approach To Identify Novel Virulence Factors of Yersinia pestis CO92 in a Mouse Model of Infection

PubMed Central

Ponnusamy, Duraisamy; Fitts, Eric C.; Erova, Tatiana E.; Kozlova, Elena V.; Kirtley, Michelle L.; Tiner, Bethany L.; Andersson, Jourdan A.

2015-01-01

The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD50. The mice infected with the Δlpp ΔmsbB ΔrbsA triple mutant at 50 LD50 were 90% protected upon subsequent challenge with 12 LD50 of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s). PMID:25754198

High-throughput, signature-tagged mutagenic approach to identify novel virulence factors of Yersinia pestis CO92 in a mouse model of infection.

PubMed

Ponnusamy, Duraisamy; Fitts, Eric C; Sha, Jian; Erova, Tatiana E; Kozlova, Elena V; Kirtley, Michelle L; Tiner, Bethany L; Andersson, Jourdan A; Chopra, Ashok K

2015-05-01

The identification of new virulence factors in Yersinia pestis and understanding their molecular mechanisms during an infection process are necessary in designing a better vaccine or to formulate an appropriate therapeutic intervention. By using a high-throughput, signature-tagged mutagenic approach, we created 5,088 mutants of Y. pestis strain CO92 and screened them in a mouse model of pneumonic plague at a dose equivalent to 5 50% lethal doses (LD50) of wild-type (WT) CO92. From this screen, we obtained 118 clones showing impairment in disseminating to the spleen, based on hybridization of input versus output DNA from mutant pools with 53 unique signature tags. In the subsequent screen, 20/118 mutants exhibited attenuation at 8 LD50 when tested in a mouse model of bubonic plague, with infection by 10/20 of the aforementioned mutants resulting in 40% or higher survival rates at an infectious dose of 40 LD50. Upon sequencing, six of the attenuated mutants were found to carry interruptions in genes encoding hypothetical proteins or proteins with putative functions. Mutants with in-frame deletion mutations of two of the genes identified from the screen, namely, rbsA, which codes for a putative sugar transport system ATP-binding protein, and vasK, a component of the type VI secretion system, were also found to exhibit some attenuation at 11 or 12 LD50 in a mouse model of pneumonic plague. Likewise, among the remaining 18 signature-tagged mutants, 9 were also attenuated (40 to 100%) at 12 LD50 in a pneumonic plague mouse model. Previously, we found that deleting genes encoding Braun lipoprotein (Lpp) and acyltransferase (MsbB), the latter of which modifies lipopolysaccharide function, reduced the virulence of Y. pestis CO92 in mouse models of bubonic and pneumonic plague. Deletion of rbsA and vasK genes from either the Δlpp single or the Δlpp ΔmsbB double mutant augmented the attenuation to provide 90 to 100% survivability to mice in a pneumonic plague model at 20 to 50 LD50. The mice infected with the Δlpp ΔmsbB ΔrbsA triple mutant at 50 LD50 were 90% protected upon subsequent challenge with 12 LD50 of WT CO92, suggesting that this mutant or others carrying combinational deletions of genes identified through our screen could potentially be further tested and developed into a live attenuated plague vaccine(s). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The C Terminus of the Saccharomyces cerevisiae α-Factor Receptor Contributes to the Formation of Preactivation Complexes with Its Cognate G Protein

PubMed Central

Dosil, Mercedes; Schandel, Kimberly A.; Gupta, Ekta; Jenness, Duane D.; Konopka, James B.

2000-01-01

Binding of the α-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Gα subunits in an α-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Gα subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the α-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for α-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the α-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor–G-protein preactivation complexes. PMID:10866688

Delayed pubertal onset and prepubertal Kiss1 expression in female mice lacking central oestrogen receptor beta.

PubMed

Naulé, Lydie; Robert, Vincent; Parmentier, Caroline; Martini, Mariangela; Keller, Matthieu; Cohen-Solal, Martine; Hardin-Pouzet, Hélène; Grange-Messent, Valérie; Franceschini, Isabelle; Mhaouty-Kodja, Sakina

2015-12-20

Ovarian oestradiol is essential for pubertal maturation and adult physiology of the female reproductive axis. It acts at central and peripheral sites through two main oestrogen receptors (ER) α and β. Here we investigate the role of ERβ on central effects of oestradiol, by generating a mouse line specifically lacking the ERβ gene in neuronal and glial cells. Central ERβ deletion delays the age at vaginal opening and first oestrous and reduces uterine weight without affecting body growth. Analysis of factors necessary for pubertal progression shows reduced levels of Kiss1 transcripts at postnatal (P) day 25 in the preoptic area, but not in the mediobasal hypothalamus (MBH) of mutant females. In agreement with these data, the number of kisspeptin-immunoreactive neurons was decreased by 57-72% in the three subdivisions of the rostral periventricular area of the third ventricle (RP3V), whereas the density of kisspeptin-immunoreactive fibres was unchanged in the arcuate nucleus of mutant mice. These alterations do not involve changes in ERα mRNAs in the preoptic area and protein levels in the RP3V. The number and distribution of GnRH-immunoreactive cells were unaffected, but gonadotropin-releasing hormone (GnRH) transcript levels were higher in the P25 preoptic area of mutants. At adulthood, mutant females have normal oestrous cyclicity, kisspeptin system and exhibit unaltered sexual behaviour. They display, however, reduced ovary weight and increased anxiety-related behaviour during the follicular phase. This argues for the specific involvement of central ERβ in the regulation of pubertal onset in female reproduction, possibly through prepubertal induction of kisspeptin expression in the RP3V. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Role of an archaeal PitA transporter in the copper and arsenic resistance of Metallosphaera sedula, an extreme thermoacidophile.

PubMed

McCarthy, Samuel; Ai, Chenbing; Wheaton, Garrett; Tevatia, Rahul; Eckrich, Valerie; Kelly, Robert; Blum, Paul

2014-10-01

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Role of an Archaeal PitA Transporter in the Copper and Arsenic Resistance of Metallosphaera sedula, an Extreme Thermoacidophile

PubMed Central

McCarthy, Samuel; Ai, Chenbing; Wheaton, Garrett; Tevatia, Rahul; Eckrich, Valerie; Kelly, Robert

2014-01-01

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching. PMID:25092032

Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans.

PubMed

Lermann, Ulrich; Morschhäuser, Joachim

2008-11-01

A well-known virulence attribute of the human-pathogenic yeast Candida albicans is the secretion of aspartic proteases (Saps), which may contribute to colonization and infection of different host niches by degrading tissue barriers, destroying host defence molecules, or digesting proteins for nutrient supply. The role of individual Sap isoenzymes, which are encoded by a large gene family, for the pathogenicity of C. albicans has been investigated by assessing the virulence of mutants lacking specific SAP genes and by studying the expression pattern of the SAP genes in various models of superficial and systemic infections. We used a recombination-based genetic reporter system to detect the induction of the SAP1-SAP6 genes during infection of reconstituted human vaginal epithelium. Only SAP5, but none of the other tested SAP genes, was detectably activated in this in vitro infection model. To directly address the importance of the SAP1-SAP6 genes for invasion of reconstituted human epithelia (RHE), we constructed a set of mutants of the wild-type C. albicans model strain SC5314 in which either single or multiple SAP genes were specifically deleted. Even mutants lacking all of the SAP1-SAP3 or the SAP4-SAP6 genes displayed the same capacity to invade and damage both oral and vaginal RHE as their wild-type parental strain, in contrast to a nonfilamentous efg1Delta mutant that was avirulent under these conditions. We therefore conclude from these results that the secreted aspartic proteases Sap1p-Sap6p are not required for invasion of RHE by C. albicans.

Analysis of Pax6 contiguous gene deletions in the mouse, Mus musculus, identifies regions distinct from Pax6 responsible for extreme small-eye and belly-spotting phenotypes.

PubMed

Favor, Jack; Bradley, Alan; Conte, Nathalie; Janik, Dirk; Pretsch, Walter; Reitmeir, Peter; Rosemann, Michael; Schmahl, Wolfgang; Wienberg, Johannes; Zaus, Irmgard

2009-08-01

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca(+2)-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.

Lack of Methylated Hopanoids Renders the Cyanobacterium Nostoc punctiforme Sensitive to Osmotic and pH Stress

PubMed Central

Garby, Tamsyn J.; Matys, Emily D.; Ongley, Sarah E.; Salih, Anya; Larkum, Anthony W. D.; Walter, Malcolm R.

2017-01-01

ABSTRACT To investigate the function of 2-methylhopanoids in modern cyanobacteria, the hpnP gene coding for the radical S-adenosyl methionine (SAM) methylase protein that acts on the C-2 position of hopanoids was deleted from the filamentous cyanobacterium Nostoc punctiforme ATCC 29133S. The resulting ΔhpnP mutant lacked all 2-methylhopanoids but was found to produce much higher levels of two bacteriohopanepentol isomers than the wild type. Growth rates of the ΔhpnP mutant cultures were not significantly different from those of the wild type under standard growth conditions. Akinete formation was also not impeded by the absence of 2-methylhopanoids. The relative abundances of the different hopanoid structures in akinete-dominated cultures of the wild-type and ΔhpnP mutant strains were similar to those of vegetative cell-dominated cultures. However, the ΔhpnP mutant was found to have decreased growth rates under both pH and osmotic stress, confirming a role for 2-methylhopanoids in stress tolerance. Evidence of elevated photosystem II yield and NAD(P)H-dependent oxidoreductase activity in the ΔhpnP mutant under stress conditions, compared to the wild type, suggested that the absence of 2-methylhopanoids increases cellular metabolic rates under stress conditions. IMPORTANCE As the first group of organisms to develop oxygenic photosynthesis, Cyanobacteria are central to the evolutionary history of life on Earth and the subsequent oxygenation of the atmosphere. To investigate the origin of cyanobacteria and the emergence of oxygenic photosynthesis, geobiologists use biomarkers, the remnants of lipids produced by different organisms that are found in geologic sediments. 2-Methylhopanes have been considered indicative of cyanobacteria in some environmental settings, with the parent lipids 2-methylhopanoids being present in many contemporary cyanobacteria. We have created a Nostoc punctiforme ΔhpnP mutant strain that does not produce 2-methylhopanoids to assess the influence of 2-methylhopanoids on stress tolerance. Increased metabolic activity in the mutant under stress indicates compensatory alterations in metabolism in the absence of 2-methylhopanoids. PMID:28455341

Lack of Methylated Hopanoids Renders the Cyanobacterium Nostoc punctiforme Sensitive to Osmotic and pH Stress.

PubMed

Garby, Tamsyn J; Matys, Emily D; Ongley, Sarah E; Salih, Anya; Larkum, Anthony W D; Walter, Malcolm R; Summons, Roger E; Neilan, Brett A

2017-07-01

To investigate the function of 2-methylhopanoids in modern cyanobacteria, the hpnP gene coding for the radical S -adenosyl methionine (SAM) methylase protein that acts on the C-2 position of hopanoids was deleted from the filamentous cyanobacterium Nostoc punctiforme ATCC 29133S. The resulting Δ hpnP mutant lacked all 2-methylhopanoids but was found to produce much higher levels of two bacteriohopanepentol isomers than the wild type. Growth rates of the Δ hpnP mutant cultures were not significantly different from those of the wild type under standard growth conditions. Akinete formation was also not impeded by the absence of 2-methylhopanoids. The relative abundances of the different hopanoid structures in akinete-dominated cultures of the wild-type and Δ hpnP mutant strains were similar to those of vegetative cell-dominated cultures. However, the Δ hpnP mutant was found to have decreased growth rates under both pH and osmotic stress, confirming a role for 2-methylhopanoids in stress tolerance. Evidence of elevated photosystem II yield and NAD(P)H-dependent oxidoreductase activity in the Δ hpnP mutant under stress conditions, compared to the wild type, suggested that the absence of 2-methylhopanoids increases cellular metabolic rates under stress conditions. IMPORTANCE As the first group of organisms to develop oxygenic photosynthesis, Cyanobacteria are central to the evolutionary history of life on Earth and the subsequent oxygenation of the atmosphere. To investigate the origin of cyanobacteria and the emergence of oxygenic photosynthesis, geobiologists use biomarkers, the remnants of lipids produced by different organisms that are found in geologic sediments. 2-Methylhopanes have been considered indicative of cyanobacteria in some environmental settings, with the parent lipids 2-methylhopanoids being present in many contemporary cyanobacteria. We have created a Nostoc punctiforme Δ hpnP mutant strain that does not produce 2-methylhopanoids to assess the influence of 2-methylhopanoids on stress tolerance. Increased metabolic activity in the mutant under stress indicates compensatory alterations in metabolism in the absence of 2-methylhopanoids. Copyright © 2017 American Society for Microbiology.

Large-mutation spectra induced at hemizygous loci by low-LET radiation: evidence for intrachromosomal proximity effects

NASA Technical Reports Server (NTRS)

Costes, S.; Sachs, R.; Hlatky, L.; Vannais, D.; Waldren, C.; Fouladi, B.; Chatterjee, A. (Principal Investigator)

2001-01-01

A mathematical model is used to analyze mutant spectra for large mutations induced by low-LET radiation. The model equations are based mainly on two-break misrejoining that leads to deletions or translocations. It is assumed, as a working hypothesis, that the initial damage induced by low-LET radiation is located randomly in the genome. Specifically, we analyzed data for two hemizygous loci: CD59- mutants, mainly very large-scale deletions (>3 Mbp), in human-hamster hybrid cells, and data from the literature on those HPRT- mutants which involve at least deletion of the whole gene, and often of additional flanking markers (approximately 50-kbp to approximately 4.4-Mbp deletions). For five data sets, we estimated f, the probability that two given breaks on the same chromosome will misrejoin to make a deletion, as a function of the separation between the breaks. We found that f is larger for nearby breaks than for breaks that are more widely separated; i.e., there is a "proximity effect". For acute irradiation, the values of f determined from the data are consistent with the corresponding break misrejoining parameters found previously in quantitative modeling of chromosome aberrations. The value of f was somewhat smaller for protracted irradiation than for acute irradiation at a given total dose; i.e., the mutation data show a decrease that was smaller than expected for dose protraction by fractionation or low dose rate.

Evaluation of protective efficacy of live attenuated Salmonella enterica serovar Gallinarum vaccine strains against fowl typhoid in chickens.

PubMed

Laniewski, Paweł; Mitra, Arindam; Karaca, Kemal; Khan, Ayub; Prasad, Rajeev; Curtiss, Roy; Roland, Kenneth L

2014-09-01

Salmonella enterica serovar Gallinarum is the etiological agent of fowl typhoid, which constitutes a considerable economic problem for poultry growers in developing countries. The vaccination of chickens seems to be the most effective strategy to control the disease in those areas. We constructed S. Gallinarum strains with a deletion of the global regulatory gene fur and evaluated their virulence and protective efficacy in Rhode Island Red chicks and Brown Leghorn layers. The fur deletion mutant was avirulent and, when delivered orally to chicks, elicited excellent protection against lethal S. Gallinarum challenge. It was not as effective when given orally to older birds, although it was highly immunogenic when delivered by intramuscular injection. We also examined the effect of a pmi mutant and a combination of fur deletions with mutations in the pmi and rfaH genes, which affect O-antigen synthesis, and ansB, whose product inhibits host T-cell responses. The S. Gallinarum Δpmi mutant was only partially attenuated, and the ΔansB mutant was fully virulent. The Δfur Δpmi and Δfur ΔansB double mutants were attenuated but not protective when delivered orally to the chicks. However, a Δpmi Δfur strain was highly immunogenic when administered intramuscularly. All together, our results show that the fur gene is essential for the virulence of S. Gallinarum, and the fur mutant is effective as a live recombinant vaccine against fowl typhoid. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity

PubMed Central

Chittaranjan, Suganthi; Chan, Susanna; Yang, Cindy; Yang, Kevin C.; Chen, Vincent; Moradian, Annie; Firme, Marlo; Song, Jungeun; Go, Nancy E.; Blough, Michael D.; Chan, Jennifer A.; Cairncross, J. Gregory; Gorski, Sharon M.; Morin, Gregg B.; Yip, Stephen; Marra, Marco A.

2014-01-01

The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism of reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples. PMID:25277207

Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

PubMed

Quarterman, Josh; Skerker, Jeffrey M; Feng, Xueyang; Liu, Ian Y; Zhao, Huimin; Arkin, Adam P; Jin, Yong-Su

2016-07-10

In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without respiration. We concluded that the metabolic "death valley" (i.e. no galactose utilization by the Δcox9 mutant) is a necessary intermediate phenotype to facilitate galactose utilization without respiration in yeast. The results in this study demonstrate a promising approach for directing adaptive evolution toward fermentative metabolism and for generating evolved yeast strains with improved phenotypes under anaerobic conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of BIM (BCL-2 like 11 gene) deletion polymorphism in Chinese non-small cell lung cancer patients

PubMed Central

Zhong, Jia; Li, Zheng-Xiang; Zhao, Jun; Duan, Jian-Chun; Bai, Hua; An, Tong-Tong; Yang, Xiao-Dan; Wang, Jie

2014-01-01

Background Drug resistance significantly weakens the efficacy of cancer treatment, and the BIM (also known as the BCL2L11 gene) deletion polymorphism has been identified as a potential biomarker for drug resistance. In this retrospective study, we included a total of 290 patients with advanced non-small cell lung cancer (NSCLC) who received treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and chemotherapy. Methods The BIM deletion polymorphism of each patient was detected by polymerase chain reaction. EGFR mutations were detected by denaturing high-performance liquid chromatography methods and the amplification refractory mutation system. Results The BIM deletion polymorphism was detected in 45/290 (15.5%) Chinese NSCLC patients. No associations were observed between the BIM deletion and clinic-pathologic characteristics of patients. The BIM deletion polymorphism was predictive of shorter progression-free survival in Chinese patients with EGFR-mutant adenocarcinoma and who were treated with EGFR-TKIs (7.30 vs. 9.53 months, P = 0.034). Additionally, we found that the BIM deletion polymorphism was an effective predictor of short progression-free survival in individuals with EGFR-mutant NSCLC and treated with chemotherapy containing pemetrexed (3.32 vs. 5.30, P = 0.012) or second-/beyond-line chemotherapy containing taxanes (1.53 vs. 2.61 months, P = 0.025). The BIM deletion was not correlated with overall survival. Conclusion The BIM deletion polymorphism occurs in 15.5% of Chinese NSCLC patients, and is a biomarker for resistance to TKIs and chemotherapy. However, BIM deletion was not a decisive factor in overall survival. PMID:26767045

Analysis of BIM (BCL-2 like 11 gene) deletion polymorphism in Chinese non-small cell lung cancer patients.

PubMed

Zhong, Jia; Li, Zheng-Xiang; Zhao, Jun; Duan, Jian-Chun; Bai, Hua; An, Tong-Tong; Yang, Xiao-Dan; Wang, Jie

2014-11-01

Drug resistance significantly weakens the efficacy of cancer treatment, and the BIM (also known as the BCL2L11 gene) deletion polymorphism has been identified as a potential biomarker for drug resistance. In this retrospective study, we included a total of 290 patients with advanced non-small cell lung cancer (NSCLC) who received treatment with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and chemotherapy. The BIM deletion polymorphism of each patient was detected by polymerase chain reaction. EGFR mutations were detected by denaturing high-performance liquid chromatography methods and the amplification refractory mutation system. The BIM deletion polymorphism was detected in 45/290 (15.5%) Chinese NSCLC patients. No associations were observed between the BIM deletion and clinic-pathologic characteristics of patients. The BIM deletion polymorphism was predictive of shorter progression-free survival in Chinese patients with EGFR-mutant adenocarcinoma and who were treated with EGFR-TKIs (7.30 vs. 9.53 months, P = 0.034). Additionally, we found that the BIM deletion polymorphism was an effective predictor of short progression-free survival in individuals with EGFR-mutant NSCLC and treated with chemotherapy containing pemetrexed (3.32 vs. 5.30, P = 0.012) or second-/beyond-line chemotherapy containing taxanes (1.53 vs. 2.61 months, P = 0.025). The BIM deletion was not correlated with overall survival. The BIM deletion polymorphism occurs in 15.5% of Chinese NSCLC patients, and is a biomarker for resistance to TKIs and chemotherapy. However, BIM deletion was not a decisive factor in overall survival.

Deletion Mapping of zwf, the Gene for a Constitutive Enzyme, Glucose 6-Phosphate Dehydrogenase in ESCHERICHIA COLI

PubMed Central

Fraenkel, D. G.; Banerjee, Santimoy

1972-01-01

Genes for three enzymes of intermediary sugar metabolism in E. coli, zwf (glucose 6-phosphate dehydrogenase, constitutive), edd (gluconate 6-phosphate dehydrase, inducible), and eda (2-keto-3-deoxygluconate 6-phosphate aldolase, differently inducible) are closely linked on the E. coli genetic map, the overall gene order being man... old... eda. edd. zwf... cheB... uvrC... his. One class of apparent revertants of an eda mutant strain contains a secondary mutation in edd, and some of these mutations are deletions extending into zwf. We have used a series of spontaneous edd-zwf deletions to map a series of point mutants in zwf and thus report the first fine structure map of a gene for a constitutive enzyme (zwf). PMID:4560065

Impact of Branched-Chain Amino Acid Catabolism on Fatty Acid and Alkene Biosynthesis in Micrococcus luteus.

PubMed

Surger, Maximilian J; Angelov, Angel; Stier, Philipp; Übelacker, Maria; Liebl, Wolfgang

2018-01-01

Micrococcus luteus naturally produces alkenes, unsaturated aliphatic hydrocarbons, and represents a promising host to produce hydrocarbons as constituents of biofuels and lubricants. In this work, we identify the genes for key enzymes of the branched-chain amino acid catabolism in M. luteus , whose first metabolic steps lead also to the formation of primer molecules for branched-chain fatty acid and olefin biosynthesis, and demonstrate how these genes can be used to manipulate the production of specific olefins in this organism. We constructed mutants of several gene candidates involved in the branched-chain amino acid metabolism or its regulation and investigated the resulting changes in the cellular fatty acid and olefin profiles by GC/MS. The gene cluster encoding the components of the branched-chain α-keto acid dehydrogenase (BCKD) complex was identified by deletion and promoter exchange mutagenesis. Overexpression of the BCKD gene cluster resulted in about threefold increased olefin production whereas deletion of the cluster led to a drastic reduction in branched-chain fatty acid content and a complete loss of olefin production. The specificities of the acyl-CoA dehydrogenases of the branched amino acid degradation pathways were deduced from the fatty acid and olefin profiles of the respective deletion mutant strains. In addition, growth experiments with branched amino acids as the only nitrogen source were carried out with the mutants in order to confirm our annotations. Both the deletion mutant of the BCKD complex, responsible for the further degradation of all three branched-chain amino acids, as well as the deletion mutant of the proposed isovaleryl-CoA dehydrogenase (specific for leucine degradation) were not able to grow on leucine in contrast to the parental strain. In conclusion, our experiments allow the unambigous assignment of specific functions to the genes for key enzymes of the branched-chain amino acid metabolism of M. luteus . We also show how this knowledge can be used to engineer the isomeric composition and the chain lengths of the olefins produced by this organism.

Deletion of a Cys-His motif from the Alpharetrovirus nucleocapsid domain reveals late domain mutant-like budding defects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Eun-Gyung; Linial, Maxine L.

2006-03-30

The Rous sarcoma virus (RSV) Gag polyprotein is the only protein required for virus assembly and release. We previously found that deletion of either one of the two Cys-His (CH) motifs in the RSV nucleocapsid (NC) protein did not abrogate Gag-Gag interactions, RNA binding, or packaging but greatly reduced virus production (E-G. Lee, A. Alidina et al., J. Virol. 77: 2010-2020, 2003). In this report, we have further investigated the effects of mutations in the CH motifs on virus assembly and release. Precise deletion of either CH motif, without affecting surrounding basic residues, reduced virus production by approximately 10-fold, similarmore » to levels seen for late (L) domain mutants. Strikingly, transmission electron microscopy revealed that virions of both {delta}CH1 and {delta}CH2 mutants were assembled normally at the plasma membrane but were arrested in budding. Virus particles remained tethered to the membrane or to each other, reminiscent of L domain mutants, although the release defect appears to be independent of the L domain functions. Therefore, two CH motifs are likely to be required for budding independent of a requirement for either Gag-Gag interactions or RNA packaging.« less

ClpC regulates the fate of a sporulation initiation sigma factor, sigmaH protein, in Bacillus subtilis at elevated temperatures.

PubMed

Nanamiya, H; Ohashi, Y; Asai, K; Moriya, S; Ogasawara, N; Fujita, M; Sadaie, Y; Kawamura, F

1998-07-01

Using a strain carrying a clpC-bgaB transcriptional fusion at the amyE locus, we found that the expression of a clpC operon was induced at the end of exponential growth in a sigmaB-independent manner and ceased around T3.5 in the wild type but not in a spo0H mutant. This suggests that some gene product(s) whose expression is dependent on sigmaH function is required for the turn-off of clpC transcription during an early stage of sporulation. A clpC deletion mutant showed a temperature-sensitive sporulation phenotype and exhibited an abnormally large accumulation of sigmaH in the cell at 45 degrees C after T2, at which time the sigmaH level in the wild type had begun to decrease. These results, together with the fact that spo0H transcription in the clpC deletion mutant was similar to that of the wild type, suggested that ClpC may be responsible for the degradation of sigmaH after the accomplishment of its role in sporulation. Moreover, as expected from these results, overproduction of Spo0A was also observed after the initiation of sporulation in the clpC deletion mutant at 45 degrees C.

Identification and classification of genes required for tolerance to high-sucrose stress revealed by genome-wide screening of Saccharomyces cerevisiae.

PubMed

Ando, Akira; Tanaka, Fumiko; Murata, Yoshinori; Takagi, Hiroshi; Shima, Jun

2006-03-01

Yeasts used in bread making are exposed to high concentrations of sucrose during sweet dough fermentation. Despite its importance, tolerance to high-sucrose stress is poorly understood at the gene level. To clarify the genes required for tolerance to high-sucrose stress, genome-wide screening was undertaken using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 273 deletions that yielded high sucrose sensitivity, approximately 20 of which were previously uncharacterized. These 273 deleted genes were classified based on their cellular function and localization of their gene products. Cross-sensitivity of the high-sucrose-sensitive mutants to high concentrations of NaCl and sorbitol was studied. Among the 273 sucrose-sensitive deletion mutants, 269 showed cross-sensitivities to sorbitol or NaCl, and four (i.e. ade5,7, ade6, ade8, and pde2) were specifically sensitive to high sucrose. The general stress response pathways via high-osmolarity glycerol and stress response element pathways and the function of the invertase in the ade mutants were similar to those in the wild-type strain. In the presence of high-sucrose stress, intracellular contents of ATP in ade mutants were at least twofold lower than that of the wild-type cells, suggesting that depletion of ATP is a factor in sensitivity to high-sucrose stress. The genes identified in this study might be important for tolerance to high-sucrose stress, and therefore should be target genes in future research into molecular modification for breeding of yeast tolerant to high-sucrose stress.

Construction of upp deletion mutant strains of Lactobacillus casei and Lactococcus lactis based on counterselective system using temperature-sensitive plasmid.

PubMed

Song, Li; Cui, Hongyu; Tang, Lijie; Qiao, Xinyuan; Liu, Min; Jiang, Yanping; Cui, Wen; Li, Yijing

2014-07-01

Integration plasmids are often used in constructing chromosomal mutations, as it enables the alternation of genes at any location by integration or replacement. Food-grade integration vectors can integrate into the host genome without introducing any selectable markers or residual bases, and the recombination often happens in non-coding region. In this study we used the temperature-sensitive pWV01 replicon to construct 2 chloramphenicol-resistant integration plasmids (pGBHC32-upp) containing the uracil phosphoribosyl transferase (upp) gene as a counterselective marker for Lactobacillus casei (L. casei) ATCC393 and Lactococcus lactis (L. lactis) MG1363. We then ligated the designed homologous arms to the pGBHC32-upp plasmids to allow their integration to the bacterial chromosome, and selected upp deletion mutants of L. casei ATCC393 and L. lactis MG1363 in the presence of 5-fluorouracil (5-FU). Analysis of genetic stability, growth curve, carbon utilization and scanning electronic microscopy showed that, except for 5-FU resistance, there were no significant differences between the wild type and mutant lactic acid bacteria. The integration system and the upp deletion strains could be used in the insertion or deletion of genes at any location of the chromosome of both L. casei ATCC 393 and L. lactis MG1363, and the homologous recombination would not introduce any selectable markers or residual bases. These mutant strains can be further investigated for heterologous protein expression and construction of a live mucosal vaccine carrier. Copyright © 2014 Elsevier B.V. All rights reserved.

The HEX1 Gene of Fusarium graminearum Is Required for Fungal Asexual Reproduction and Pathogenesis and for Efficient Viral RNA Accumulation of Fusarium graminearum Virus 1

PubMed Central

Son, Moonil; Lee, Kyung-Mi; Yu, Jisuk; Kang, Minji; Park, Jin Man; Kwon, Sun-Jung

2013-01-01

The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus. PMID:23864619

Targeted mutagenesis in a human-parasitic nematode

PubMed Central

Gang, Spencer S.; Castelletto, Michelle L.

2017-01-01

Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites. PMID:29016680

The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic.

PubMed

Haid, A; Schweyen, R J; Bechmann, H; Kaudewitz, F; Solioz, M; Schatz, G

1979-03-01

Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome. The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho- deletion mapping and recombination analysis. The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence. 1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2 leads to OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type. 2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red. 3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown. 4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).

Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant.

PubMed

Mizutani, Osamu; Arazoe, Takayuki; Toshida, Kenji; Hayashi, Risa; Ohsato, Shuichi; Sakuma, Tetsushi; Yamamoto, Takashi; Kuwata, Shigeru; Yamada, Osamu

2017-03-01

Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALENs (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (ΔligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

PubMed

Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

2013-10-01

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

Patchwork structure-function analysis of the Sendai virus matrix protein.

PubMed

Mottet-Osman, Geneviève; Miazza, Vincent; Vidalain, Pierre-Olivier; Roux, Laurent

2014-09-01

Paramyxoviruses contain a bi-lipidic envelope decorated by two transmembrane glycoproteins and carpeted on the inner surface with a layer of matrix proteins (M), thought to bridge the glycoproteins with the viral nucleocapsids. To characterize M structure-function features, a set of M domains were mutated or deleted. The genes encoding these modified M were incorporated into recombinant Sendai viruses and expressed as supplemental proteins. Using a method of integrated suppression complementation system (ISCS), the functions of these M mutants were analyzed in the context of the infection. Cellular membrane association, localization at the cell periphery, nucleocapsid binding, cellular protein interactions and promotion of viral particle formation were characterized in relation with the mutations. At the end, lack of nucleocapsid binding go together with lack of cell surface localization and both features definitely correlate with loss of M global function estimated by viral particle production. Copyright © 2014 Elsevier Inc. All rights reserved.

Brown adipose tissue macrophages control tissue innervation and homeostatic energy expenditure

PubMed Central

Cortese, Nina; Haimon, Zhana; Sar Shalom, Hadas; Kuperman, Yael; Kalchenko, Vyacheslav; Brandis, Alexander; David, Eyal; Segal-Hayoun, Yifat; Chappell-Maor, Louise; Yaron, Avraham; Jung, Steffen

2017-01-01

Tissue macrophages provide immune defense and contribute to establishment and maintenance of tissue homeostasis. Here we used constitutive and inducible mutagenesis to delete the nuclear transcription regulator methyl-CpG binding protein 2 (Mecp2) in defined tissue macrophages. Animals lacking the Rett syndrome-associated gene in macrophages did not show signs of neurodevelopmental disorder, but displayed spontaneous obesity, which could be linked to impaired brown adipose tissue (BAT) function. Specifically, mutagenesis of a BAT-resident CX3CR1+ macrophage subpopulation compromised homeostatic, though not acute cold-induced thermogenesis. Mechanistically, BAT malfunction of pre-obese mice harboring mutant macrophages was associated with decreased sympathetic innervation and local norepinephrine titers, resulting in reduced adipocyte expression of thermogenic factors. Mutant macrophages over-expressed PlexinA4, which might contribute to the phenotype by repulsion of Sema6A-expressing sympathetic axons. Collectively, we report a previously unappreciated homeostatic role of macrophages in the control of tissue innervation, disruption of which in BAT results in metabolic imbalance. PMID:28459435

A family of membrane-shaping proteins at ER subdomains regulates pre-peroxisomal vesicle biogenesis.

PubMed

Joshi, Amit S; Huang, Xiaofang; Choudhary, Vineet; Levine, Tim P; Hu, Junjie; Prinz, William A

2016-11-21

Saccharomyces cerevisiae contains three conserved reticulon and reticulon-like proteins that help maintain ER structure by stabilizing high membrane curvature in ER tubules and the edges of ER sheets. A mutant lacking all three proteins has dramatically altered ER morphology. We found that ER shape is restored in this mutant when Pex30p or its homologue Pex31p is overexpressed. Pex30p can tubulate membranes both in cells and when reconstituted into proteoliposomes, indicating that Pex30p is a novel ER-shaping protein. In contrast to the reticulons, Pex30p is low abundance, and we found that it localizes to subdomains in the ER. We show that these ER subdomains are the sites where most preperoxisomal vesicles (PPVs) are generated. In addition, overproduction or deletion of Pex30p or Pex31p alters the size, shape, and number of PPVs. Our findings suggest that Pex30p and Pex31p help shape and generate regions of the ER where PPV biogenesis occurs.

Independent signalling cues underpin arbuscular mycorrhizal symbiosis and large lateral root induction in rice.

PubMed

Chiu, Chai Hao; Choi, Jeongmin; Paszkowski, Uta

2018-01-01

Perception of arbuscular mycorrhizal fungi (AMF) triggers distinct plant signalling responses for parallel establishment of symbiosis and induction of lateral root formation. Rice receptor kinase CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) and α/β-fold hydrolase DWARF14-LIKE (D14L) are involved in pre-symbiotic fungal perception. After 6 wk post-inoculation with Rhizophagus irregularis, root developmental responses, fungal colonization and transcriptional responses were monitored in two independent cerk1 null mutants; a deletion mutant lacking D14L, and with D14L complemented as well as their respective wild-type cultivars (cv Nipponbare and Nihonmasari). Here we show that although essential for symbiosis, D14L is dispensable for AMF-induced root architectural modulation, which conversely relies on CERK1. Our results demonstrate uncoupling of symbiosis and the symbiotic root developmental signalling during pre-symbiosis with CERK1 required for AMF-induced root architectural changes. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Apollo contributes to G overhang maintenance and protects leading-end telomeres.

PubMed

Wu, Peng; van Overbeek, Megan; Rooney, Sean; de Lange, Titia

2010-08-27

Mammalian telomeres contain a single-stranded 3' overhang that is thought to mediate telomere protection. Here we identify the TRF2-interacting factor Apollo as a nuclease that contributes to the generation/maintenance of this overhang. The function of mouse Apollo was determined using Cre-mediated gene deletion, complementation with Apollo mutants, and the TRF2-F120A mutant that cannot bind Apollo. Cells lacking Apollo activated the ATM kinase at their telomeres in S phase and showed leading-end telomere fusions. These telomere dysfunction phenotypes were accompanied by a reduction in the telomeric overhang signal. The telomeric functions of Apollo required its TRF2-interaction and nuclease motifs. Thus, TRF2 recruits the Apollo nuclease to process telomere ends synthesized by leading-strand DNA synthesis, thereby creating a terminal structure that avoids ATM activation and resists end-joining. These data establish that the telomeric overhang is required for the protection of telomeres from the DNA damage response. Copyright (c) 2010 Elsevier Inc. All rights reserved.

First report of a deletion encompassing an entire exon in the homogentisate 1,2-dioxygenase gene causing alkaptonuria.

PubMed

Zouheir Habbal, Mohammad; Bou-Assi, Tarek; Zhu, Jun; Owen, Renius; Chehab, Farid F

2014-01-01

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5-16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband's phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin.

First Report of a Deletion Encompassing an Entire Exon in the Homogentisate 1,2-Dioxygenase Gene Causing Alkaptonuria

PubMed Central

Habbal, Mohammad Zouheir; Bou-Assi, Tarek; Zhu, Jun; Owen, Renius; Chehab, Farid F.

2014-01-01

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5–16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband’s phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin. PMID:25233259

Genomic and transcriptomic analyses reveal distinct biological functions for cold shock proteins (VpaCspA and VpaCspD) in Vibrio parahaemolyticus CHN25 during low-temperature survival.

PubMed

Zhu, Chunhua; Sun, Boyi; Liu, Taigang; Zheng, Huajun; Gu, Wenyi; He, Wei; Sun, Fengjiao; Wang, Yaping; Yang, Meicheng; Bei, Weicheng; Peng, Xu; She, Qunxin; Xie, Lu; Chen, Lanming

2017-06-05

Vibrio parahaemolyticus causes serious seafood-borne gastroenteritis and death in humans. Raw seafood is often subjected to post-harvest processing and low-temperature storage. To date, very little information is available regarding the biological functions of cold shock proteins (CSPs) in the low-temperature survival of the bacterium. In this study, we determined the complete genome sequence of V. parahaemolyticus CHN25 (serotype: O5:KUT). The two main CSP-encoding genes (VpacspA and VpacspD) were deleted from the bacterial genome, and comparative transcriptomic analysis between the mutant and wild-type strains was performed to dissect the possible molecular mechanisms that underlie low-temperature adaptation by V. parahaemolyticus. The 5,443,401-bp V. parahaemolyticus CHN25 genome (45.2% G + C) consisted of two circular chromosomes and three plasmids with 4,724 predicted protein-encoding genes. One dual-gene and two single-gene deletion mutants were generated for VpacspA and VpacspD by homologous recombination. The growth of the ΔVpacspA mutant was strongly inhibited at 10 °C, whereas the VpacspD gene deletion strongly stimulated bacterial growth at this low temperature compared with the wild-type strain. The complementary phenotypes were observed in the reverse mutants (ΔVpacspA-com, and ΔVpacspD-com). The transcriptome data revealed that 12.4% of the expressed genes in V. parahaemolyticus CHN25 were significantly altered in the ΔVpacspA mutant when it was grown at 10 °C. These included genes that were involved in amino acid degradation, secretion systems, sulphur metabolism and glycerophospholipid metabolism along with ATP-binding cassette transporters. However, a low temperature elicited significant expression changes for 10.0% of the genes in the ΔVpacspD mutant, including those involved in the phosphotransferase system and in the metabolism of nitrogen and amino acids. The major metabolic pathways that were altered by the dual-gene deletion mutant (ΔVpacspAD) radically differed from those that were altered by single-gene mutants. Comparison of the transcriptome profiles further revealed numerous differentially expressed genes that were shared among the three mutants and regulators that were specifically, coordinately or antagonistically modulated by VpaCspA and VpaCspD. Our data also revealed several possible molecular coping strategies for low-temperature adaptation by the bacterium. This study is the first to describe the complete genome sequence of V. parahaemolyticus (serotype: O5:KUT). The gene deletions, complementary insertions, and comparative transcriptomics demonstrate that VpaCspA is a primary CSP in the bacterium, while VpaCspD functions as a growth inhibitor at 10 °C. These results have improved our understanding of the genetic basis for low-temperature survival by the most common seafood-borne pathogen worldwide.

Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1.

PubMed

Atago, Yuki; Shimodaira, Jun; Araki, Naoto; Bin Othman, Nor'azizi; Zakaria, Zuriati; Fukuda, Masao; Futami, Junichiro; Hara, Hirofumi

2016-05-01

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

Light Regulation of Swarming Motility in Pseudomonas syringae Integrates Signaling Pathways Mediated by a Bacteriophytochrome and a LOV Protein

PubMed Central

Wu, Liang; McGrane, Regina S.; Beattie, Gwyn A.

2013-01-01

ABSTRACT The biological and regulatory roles of photosensory proteins are poorly understood for nonphotosynthetic bacteria. The foliar bacterial pathogen Pseudomonas syringae has three photosensory protein-encoding genes that are predicted to encode the blue-light-sensing LOV (light, oxygen, or voltage) histidine kinase (LOV-HK) and two red/far-red-light-sensing bacteriophytochromes, BphP1 and BphP2. We provide evidence that LOV-HK and BphP1 form an integrated network that regulates swarming motility in response to multiple light wavelengths. The swarming motility of P. syringae B728a deletion mutants indicated that LOV-HK positively regulates swarming motility in response to blue light and BphP1 negatively regulates swarming motility in response to red and far-red light. BphP2 does not detectably regulate swarming motility. The histidine kinase activity of each LOV-HK and BphP1 is required for this regulation based on the loss of complementation upon mutation of residues key to their kinase activity. Surprisingly, mutants lacking both lov and bphP1 were similar in motility to a bphP1 single mutant in blue light, indicating that the loss of bphP1 is epistatic to the loss of lov and also that BphP1 unexpectedly responds to blue light. Moreover, whereas expression of bphP1 did not alter motility under blue light in a bphP1 mutant, it reduced motility in a mutant lacking lov and bphP1, demonstrating that LOV-HK positively regulates motility by suppressing negative regulation by BphP1. These results are the first to show cross talk between the LOV protein and phytochrome signaling pathways in bacteria, and the similarity of this regulatory network to that of photoreceptors in plants suggests a possible common ancestry. PMID:23760465

Whole-Genome Sequences of Variants of Bacillus anthracis Sterne and Their Toxin Gene Deletion Mutants

PubMed Central

Staab, A.; Plaut, R. D.; Pratt, C.; Lovett, S. P.; Wiley, M. R.; Biggs, T. D.; Bernhards, R. C.; Beck, L. C.; Palacios, G. F.; Stibitz, S.; Jones, K. L.; Goodwin, B. G.; Smith, M. A.

2017-01-01

ABSTRACT Here, we report the draft genome sequences of three laboratory variants of Bacillus anthracis Sterne and their double (Δlef Δcya) and triple (Δpag Δlef Δcya) toxin gene deletion derivatives. PMID:29122874

Effects of ion beam irradiation on size of mutant sector and genetic damage in Arabidopsis

NASA Astrophysics Data System (ADS)

Hase, Yoshihiro; Nozawa, Shigeki; Narumi, Issay; Oono, Yutaka

2017-01-01

Size of mutant sector and genetic damage were evaluated in Arabidopsis to further our understanding of effective ion beam use in plant mutation breeding. Arabidopsis seeds, heterozygous for the GLABRA1 (GL1) gene (GL1/gl1-1), were irradiated with 15.8 MeV/u neon ions (mean linear energy transfer (LET): 352 keV/μm), 17.3 MeV/u carbon ions (113 keV/μm), or 60Co gamma rays. The frequency and size of glabrous sectors generated because of inactivation of the GL1 allele were examined. The frequency and overall size of large deletions were evaluated based on the loss of heterozygosity of DNA markers using DNA isolated from glabrous tissue. Irrespective of the radiation properties, plants with mutant sectors were obtained at similar frequencies at the same effective dosage necessary for survival reduction. Ion beams tended to induce larger mutant sectors than gamma rays. The frequency of large deletions (>several kbp) increased as the LET value increased, with chromosome regions larger than 100 kbp lost in most large deletions. The distorted segregation ratio of glabrous plants in the progenies of irradiated GL1/gl1-1 plants suggested frequent occurrence of chromosome rearrangement, especially those subjected to neon ions. Exposure to ion beams with moderate LET values (30-110 keV/μm) is thought effective for inducing mutant sectors without causing extensive genetic damage.

Oxidoreductases that Act as Conditional Virulence Suppressors in Salmonella enterica Serovar Typhimurium

PubMed Central

Anwar, Naeem; Sem, Xiao Hui; Rhen, Mikael

2013-01-01

In Salmonella enterica serovar Typhimurium, oxidoreductases of the thioredoxin superfamily contribute to bacterial invasiveness, intracellular replication and to the virulence in BALB/c mice as well as in the soil nematode Caenorhabditis elegans. The scsABCD gene cluster, present in many but not all enteric bacteria, codes for four putative oxidoreductases of the thioredoxin superfamily. Here we have analyzed the potential role of the scs genes in oxidative stress tolerance and virulence in S. Typhimurium. An scsABCD deletion mutant showed moderate sensitization to the redox-active transition metal ion copper and increased protein carbonylation upon exposure to hydrogen peroxide. Still, the scsABCD mutant was not significantly affected for invasiveness or intracellular replication in respectively cultured epithelial or macrophage-like cells. However, we noted a significant copper chloride sensitivity of SPI1 T3SS mediated invasiveness that strongly depended on the presence of the scs genes. The scsABCD deletion mutant was not attenuated in animal infection models. In contrast, the mutant showed a moderate increase in its competitive index upon intraperitoneal challenge and enhanced invasiveness in small intestinal ileal loops of BALB/c mice. Moreover, deletion of the scsABCD genes restored the invasiveness of a trxA mutant in epithelial cells and its virulence in C. elegans. Our findings thus demonstrate that the scs gene cluster conditionally affects virulence and underscore the complex interactions between oxidoreductases of the thioredoxin superfamily in maintaining host adaptation of S. Typhimurium. PMID:23750221

Involvement of multiple stressors induced by non-thermal plasma-charged aerosols during inactivation of airborne bacteria

PubMed Central

Vaze, Nachiket D.; Park, Sin; Brooks, Ari D.; Fridman, Alexander; Joshi, Suresh G.

2017-01-01

A lab-scale, tunable, single-filament, point-to-point nonthermal dieletric-barrier discharge (DBD) plasma device was built to study the mechanisms of inactivation of aerosolized bacterial pathogens. The system inactivates airborne antibiotic-resistant pathogens efficiently. Nebulization mediated pre-optimized (4 log and 7 log) bacterial loads were challenged to plasma-charged aerosols, and lethal and sublethal doses determined using colony assay, and cell viability assay; and the loss of membrane potential and cellular respiration were determined using cell membrane potential assay and XTT assay. Using the strategies of Escherichia coli wildtype, over-expression mutant, deletion mutants, and peroxide and heat stress scavenging, we analyzed activation of intracellular reactive oxygen species (ROS) and heat shock protein (hsp) chaperons. Superoxide dismutase deletion mutants (ΔsodA, ΔsodB, ΔsodAΔsodB) and catalase mutants ΔkatG and ΔkatEΔkatG did not show significant difference from wildtype strain, and ΔkatE and ΔahpC was found significantly more susceptible to cell death than wildtype. The oxyR regulon was found to mediate plasma-charged aerosol-induced oxidative stress in bacteria. Hsp deficient E. coli (ΔhtpG, ΔgroEL, ΔclpX, ΔgrpE) showed complete inactivation of cells at ambient temperature, and the treatment at cold temperature (4°C) significantly protected hsp deletion mutants and wildtype cells, and indicate a direct involvement of hsp in plasma-charged aerosol mediated E. coli cell death. PMID:28166240

Gα proteins Gvm2 and Gvm3 regulate vegetative growth, asexual development, and pathogenicityon apple in Valsa mali.

PubMed

Song, Na; Dai, Qingqing; Zhu, Baitao; Wu, Yuxing; Xu, Ming; Voegele, Ralf Thomas; Gao, Xiaoning; Kang, Zhensheng; Huang, Lili

2017-01-01

In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. 'Fuji' leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes-important virulence factors in V. mali-are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.

Initial characterization of 17 viruses harboring mutant forms of the immediate-early gene of equine herpesvirus 1.

PubMed

Buczynski, Kimberly A; Kim, Seong K; O'Callaghan, Dennis J

2005-10-01

The sole immediate-early (IE) gene of equine herpesvirus 1 (EHV-1) encodes a major regulatory protein of 1487 amino acids (aa) capable of modulating gene expression from both early and late promoters and also of trans-repressing its own promoter. Using a specially designed recombination system and a library of IE linker-insertion, deletion, point, and nonsense mutant constructs that encode forms of the IE protein (IEP) harboring mutations within all five regions, 17 mutant viruses were generated and characterized. Ribonuclease protection analyses revealed that all 17 mutants synthesize the IE mRNA in RK-13 cells, whereas those that failed to replicate on non-complementing RK-13 cells displayed a defect in the transcription of either an important early gene (EICP0) and/or an essential late gene (glycoprotein D). Western blot analyses showed that the IEP was synthesized and detectable in cells infected with each mutant virus, including those mutants that failed to replicate on non-complementing RK-13 cells. Eleven of the 17 mutants were capable of growth on non-complementing RK-13 cells, whereas mutant viruses with deletions within the serine-rich tract (SRT), nucleus localization signal (NLS), or DNA-binding domain (DBD) were capable of growth only on the IEP-producing cell line (IE13.1). Lastly, temperature shift experiments revealed that mutant viruses containing deletions within the C-terminus (KyAn1029 and KyAn1411) or within the SRT (KyADeltaSRT2) of the IEP exhibited a temperature-sensitive phenotype in that these viruses, in contrast to the parent KyA, failed to replicate at 39 degrees C. Overall, these results indicate that the C-terminus of the IEP is not essential for IEP function in cell culture, but this region contains elements that enhance the function(s) of the IEP. The initial characterization of these 17 EHV-1 mutants has shown that sequences totaling at least 43% of the IEP are not essential for virus replication in cell culture.

An assessment of heavy ion irradiation mutagenesis for reverse genetics in wheat (Triticum aestivum L.).

PubMed

Fitzgerald, Timothy L; Powell, Jonathan J; Stiller, Jiri; Weese, Terri L; Abe, Tomoko; Zhao, Guangyao; Jia, Jizeng; McIntyre, C Lynne; Li, Zhongyi; Manners, John M; Kazan, Kemal

2015-01-01

Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed.

An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)

PubMed Central

Fitzgerald, Timothy L.; Powell, Jonathan J.; Stiller, Jiri; Weese, Terri L.; Abe, Tomoko; Zhao, Guangyao; Jia, Jizeng; McIntyre, C. Lynne; Li, Zhongyi; Manners, John M.; Kazan, Kemal

2015-01-01

Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed. PMID:25719507

Deletion of a target gene in Indica rice via CRISPR/Cas9.

PubMed

Wang, Ying; Geng, Lizhao; Yuan, Menglong; Wei, Juan; Jin, Chen; Li, Min; Yu, Kun; Zhang, Ya; Jin, Huaibing; Wang, Eric; Chai, Zhijian; Fu, Xiangdong; Li, Xianggan

2017-08-01

Using CRISPR/Cas9, we successfully deleted large fragments of the yield-related gene DENSE AND ERECT PANICLE1 in Indica rice at relatively high frequency and generated gain-of-function dep1 mutants. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a rapidly developing technology used to produce gene-specific modifications in both mammalian and plant systems. Most CRISPR-induced modifications in plants reported to date have been small insertions or deletions. Few large target gene deletions have thus far been reported, especially for Indica rice. In this study, we designed multiple CRISPR sgRNAs and successfully deleted DNA fragments in the gene DENSE AND ERECT PANICLE1 (DEP1) in the elite Indica rice line IR58025B. We achieved deletion frequencies of up to 21% for a 430 bp target and 9% for a 10 kb target among T0 events. Constructs with four sgRNAs did not generate higher full-length deletion frequencies than constructs with two sgRNAs. The multiple mutagenesis frequency reached 93% for four targets, and the homozygous mutation frequency reached 21% at the T0 stage. Important yield-related trait characteristics, such as dense and erect panicles and reduced plant height, were observed in dep1 homozygous T0 mutant plants produced by CRISPR/Cas9. Therefore, we successfully obtained deletions in DEP1 in the Indica background using the CRISPR/Cas9 editing tool at relatively high frequency.

Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella enterica Serovar Typhimurium

PubMed Central

Wang-Kan, Xuan; Chirullo, Barbara; Betts, Jonathan; La Ragione, Roberto M.; Ivens, Alasdair; Ricci, Vito; Opperman, Timothy J.

2017-01-01

ABSTRACT AcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq) revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ. Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps. PMID:28720734

Genetic Dissection of Midbrain Dopamine Neuron Development in vivo

PubMed Central

Ellisor, Debra; Rieser, Caroline; Voelcker, Bettina; Machan, Jason T.; Zervas, Mark

2012-01-01

Midbrain dopamine (MbDA) neurons are partitioned into medial and lateral cohorts that control complex functions. However, the genetic underpinnings of MbDA neuron heterogeneity are unclear. While it is known that Wnt1-expressing progenitors contribute to MbDA neurons, the role of Wnt1 in MbDA neuron development in vivo is unresolved. We show that mice with a spontaneous point mutation in Wnt1 have a unique phenotype characterized by the loss of medial MbDA neurons concomitant with a severe depletion of Wnt1-expressing progenitors and diminished LMX1a-expressing progenitors. Wnt1 mutant embryos also have alterations in a hierarchical gene regulatory loop suggesting multiple gene involvement in the Wnt1 mutant MbDA neuron phenotype. To investigate this possibility, we conditionally deleted Gbx2, Fgf8, and En1/2 after their early role in patterning and asked whether these genetic manipulations phenocopied the depletion of MbDA neurons in Wnt1 mutants. The conditional deletion of Gbx2 did not result in re-positioning or distribution of MbDA neurons. The temporal deletion of Fgf8 did not result in the loss of either LMX1a-expressing progenitors nor the initial population of differentiated MbDA neurons, but did result in a complete loss of MbDA neurons at later stages. The temporal deletion and species specific manipulation of En1/2 demonstrated a continued and species specific role of Engrailed genes in MbDA neuron development. Notably, our conditional deletion experiments revealed phenotypes dissimilar to Wnt1 mutants indicating the unique role of Wnt1 in MbDA neuron development. By placing Wnt1, Fgf8, and En1/2 in the context of their temporal requirement for MbDA neuron development, we further deciphered the developmental program underpinning MbDA neuron progenitors. PMID:23041116

The Social Amoeba Polysphondylium pallidum Loses Encystation and Sporulation, but Can Still Erect Fruiting Bodies in the Absence of Cellulose

PubMed Central

Du, Qingyou; Schaap, Pauline

2014-01-01

Amoebas and other freely moving protists differentiate into walled cysts when exposed to stress. As cysts, amoeba pathogens are resistant to biocides, preventing treatment and eradication. Lack of gene modification procedures has left the mechanisms of encystation largely unexplored. Genetically tractable Dictyostelium discoideum amoebas require cellulose synthase for formation of multicellular fructifications with cellulose-rich stalk and spore cells. Amoebas of its distant relative Polysphondylium pallidum (Ppal), can additionally encyst individually in response to stress. Ppal has two cellulose synthase genes, DcsA and DcsB, which we deleted individually and in combination. Dcsa- mutants formed fruiting bodies with normal stalks, but their spore and cyst walls lacked cellulose, which obliterated stress-resistance of spores and rendered cysts entirely non-viable. A dcsa-/dcsb- mutant made no walled spores, stalk cells or cysts, although simple fruiting structures were formed with a droplet of amoeboid cells resting on an sheathed column of decaying cells. DcsB is expressed in prestalk and stalk cells, while DcsA is additionally expressed in spores and cysts. We conclude that cellulose is essential for encystation and that cellulose synthase may be a suitable target for drugs to prevent encystation and render amoeba pathogens susceptible to conventional antibiotics. PMID:25113829

Cross-linking measurements of the Potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus-plant interactions.

PubMed

Chavez, Juan D; Cilia, Michelle; Weisbrod, Chad R; Ju, Ho-Jong; Eng, Jimmy K; Gray, Stewart M; Bruce, James E

2012-05-04

Protein interactions are critical determinants of insect transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus.

Cross-linking measurements of the Potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus-plant interactions

PubMed Central

Chavez, Juan D.; Cilia, Michelle; Weisbrod, Chad R.; Ju, Ho-Jong; Eng, Jimmy K.; Gray, Stewart M.; Bruce, James E.

2012-01-01

Protein interactions are critical determinants of insect-transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and that are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus. PMID:22390342

Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri

PubMed Central

Vaccaro, Brian J.; Lancaster, W. Andrew; Thorgersen, Michael P.; Zane, Grant M.; Younkin, Adam D.; Kazakov, Alexey E.; Wetmore, Kelly M.; Deutschbauer, Adam; Arkin, Adam P.; Novichkov, Pavel S.; Wall, Judy D.

2016-01-01

ABSTRACT Metal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu2+ and Zn2+ resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu2+ and Zn2+ resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu2+ and Zn2+ resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu2+ resistance systems do not show decreased fitness relative to the wild type when exposed to Cu2+. In addition, new genes are identified that have no known connection to Zn2+ (corB, corC, Psest_3226, Psest_3322, and Psest_0618) or Cu2+ resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lacking corB, corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu2+- and Zn2+-based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu2+ and Zn2+ efflux and resistance and their effects on denitrifying metabolism. IMPORTANCE In this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca2+/H+ antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg2+ transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322, corB, corC, or czcI verified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions. PMID:27474723

Stabiliztin of VEGFR2 Signaling by Cerebral Cavernous Malformation 3 is Critical for Vascular Development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Y He; H Zhang; L Yu

2011-12-31

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: CCM1, CCM2, and CCM3. CCM3, also known as PDCD10 (programmed cell death 10), was initially identified as a messenger RNA whose abundance was induced by apoptotic stimuli in vitro. However, the in vivo function of CCM3 has not been determined. Here, we describe mice with a deletion of the CCM3 gene either ubiquitously or specifically in the vascular endothelium, smooth muscle cells, or neurons. Mice with global or endothelial cell-specific deletion of CCM3 exhibited defects in embryonic angiogenesis and died at an earlymore » embryonic stage. CCM3 deletion reduced vascular endothelial growth factor receptor 2 (VEGFR2) signaling in embryos and endothelial cells. In response to VEGF stimulation, CCM3 was recruited to and stabilized VEGFR2, and the carboxyl-terminal domain of CCM3 was required for the stabilization of VEGFR2. Indeed, the CCM3 mutants found in human patients lacking the carboxyl-terminal domain were labile and were unable to stabilize and activate VEGFR2. These results demonstrate that CCM3 promotes VEGFR2 signaling during vascular development.« less

A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs

NASA Technical Reports Server (NTRS)

Pitulle, C.; Hedenstierna, K. O.; Fox, G. E.

1995-01-01

Further improvements in technology for efficient monitoring of genetically engineered microorganisms (GEMs) in the environment are needed. Technology for monitoring rRNA is well established but has not generally been applicable to GEMs because of the lack of unique rRNA target sequences. In the work described herein, it is demonstrated that a deletion mutant of a plasmid-borne Vibrio proteolyticus 5S rRNA gene continues to accumulate to high levels in Escherichia coli although it is no longer incorporated into 70S ribosomes. This deletion construct was subsequently modified by mutagenesis to create a unique recognition site for the restriction endonuclease BstEII, into which new sequences could be readily inserted. Finally, a novel 17-nucleotide identifier sequence from Pennisetum purpureum was embedded into the construct to create an RNA identification cassette. The artificial identifier RNA, expressed from this cassette in vivo, accumulated in E. coli to levels comparable to those of wild-type 5S rRNA without being seriously detrimental to cell survival in laboratory experiments and without entering the ribosomes. These results demonstrate that artificial, stable RNAs containing sequence segments remarkably different from those present in any known rRNA can be designed and that neither the deleted sequence segment nor ribosome incorporation is essential for accumulation of an RNA product.

Autoinducer 2 controls biofilm formation in Escherichia coli through a novel motility quorum-sensing regulator (MqsR, B3022).

PubMed

González Barrios, Andrés F; Zuo, Rongjun; Hashimoto, Yoshifumi; Yang, Li; Bentley, William E; Wood, Thomas K

2006-01-01

The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S-adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-mum) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC, flhD, fliA, and motA, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8- to 27-fold). Adding AI-2 (6.4 muM) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC, fliA, and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene).

Filamentous invasive growth of mutants of the genes encoding ammonia-metabolizing enzymes in the fission yeast Schizosaccharomyces pombe.

PubMed

Sasaki, Yoshie; Kojima, Ayumi; Shibata, Yuriko; Mitsuzawa, Hiroshi

2017-01-01

The fission yeast Schizosaccharomyces pombe undergoes a switch from yeast to filamentous invasive growth in response to certain environmental stimuli. Among them is ammonium limitation. Amt1, one of the three ammonium transporters in this yeast, is required for the ammonium limitation-induced morphological transition; however, the underlying molecular mechanism remains to be understood. Cells lacking Amt1 became capable of invasive growth upon increasing concentrations of ammonium in the medium, suggesting that the ammonium taken up into the cell or a metabolic intermediate in ammonium assimilation might serve as a signal for the ammonium limitation-induced morphological transition. To investigate the possible role of ammonium-metabolizing enzymes in the signaling process, deletion mutants were constructed for the gdh1, gdh2, gln1, and glt1 genes, which were demonstrated by enzyme assays to encode NADP-specific glutamate dehydrogenase, NAD-specific glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, respectively. Growth tests on various nitrogen sources revealed that a gln1Δ mutant was a glutamine auxotroph and that a gdh1Δ mutant had a defect in growth on ammonium, particularly at high concentrations. The latter observation indicates that the NADP-specific glutamate dehydrogenase of S. pombe plays a major role in ammonium assimilation under high ammonium concentrations. Invasive growth assays showed that gdh1Δ and glt1Δ mutants underwent invasive growth to a lesser extent than did wild-type strains. Increasing the ammonium concentration in the medium suppressed the invasive growth defect of the glt1Δ mutant, but not the gdh1Δ mutant. These results suggest that the nitrogen status of the cell is important in the induction of filamentous invasive growth in S. pombe.

The bZIP Transcription Factor Fgap1 Mediates Oxidative Stress Response and Trichothecene Biosynthesis But Not Virulence in Fusarium graminearum

PubMed Central

Montibus, Mathilde; Ducos, Christine; Bonnin-Verdal, Marie-Noelle; Bormann, Jorg; Ponts, Nadia; Richard-Forget, Florence; Barreau, Christian

2013-01-01

Redox sensing is of primary importance for fungi to cope with oxidant compounds found in their environment. Plant pathogens are particularly subject to the oxidative burst during the primary steps of infection. In the budding yeast Saccharomyces cerevisiae, it is the transcription factor Yap1 that mediates the response to oxidative stress via activation of genes coding for detoxification enzymes. In the cereal pathogen Fusarium graminearum, Fgap1 a homologue of Yap1 was identified and its role was investigated. During infection, this pathogen produces mycotoxins belonging to the trichothecenes family that accumulate in the grains. The global regulation of toxin biosynthesis is not completely understood. However, it is now clearly established that an oxidative stress activates the production of toxins by F. graminearum. The involvement of Fgap1 in this activation was investigated. A deleted mutant and a strain expressing a truncated constitutive form of Fgap1 were constructed. None of the mutants was affected in pathogenicity. The deleted mutant showed higher level of trichothecenes production associated with overexpression of Tri genes. Moreover activation of toxin accumulation in response to oxidative stress was no longer observed. Regarding the mutant with the truncated constitutive form of Fgap1, toxin production was strongly reduced. Expression of oxidative stress response genes was not activated in the deleted mutant and expression of the gene encoding the mitochondrial superoxide dismutase MnSOD1 was up-regulated in the mutant with the truncated constitutive form of Fgap1. Our results demonstrate that Fgap1 plays a key role in the link between oxidative stress response and F. graminearum secondary metabolism. PMID:24349499

The contribution of the S-phase checkpoint genes MEC1 and SGS1 to genome stability maintenance in Candida albicans

PubMed Central

Legrand, Melanie; Chan, Christine L.; Jauert, Peter A.; Kirkpatrick, David T.

2011-01-01

Genome rearrangements, a common feature of Candida albicans isolates, are often associated with the acquisition of antifungal drug resistance. In Saccharomyces cerevisiae, perturbations in the S-phase checkpoints result in the same sort of Gross Chromosomal Rearrangements (GCRs) observed in C. albicans. Several proteins are involved in the S. cerevisiae cell cycle checkpoints, including Mec1p, a protein kinase of the PIKK (phosphatidyl inositol 3-kinase-like kinase) family and the central player in the DNA damage checkpoint. Sgs1p, the ortholog of BLM, the Bloom’s syndrome gene, is a RecQ-related DNA helicase; cells from BLM patients are characterized by an increase in genome instability. Yeast strains bearing deletions in MEC1 or SGS1 are viable (in contrast to the inviability seen with loss of MEC1 in S. cerevisiae) but the different deletion mutants have significantly different phenotypes. The mec1Δ/Δ colonies have a wild-type colony morphology, while the sgs1Δ/Δ mutants are slow-growing, producing wrinkled colonies with pseudohyphal-like cells. The mec1Δ/Δ mutants are only sensitive to ethylmethane sulfonate (EMS), methylmethane sulfonate (MMS), and hydroxyurea (HU) but the sgs1Δ/Δ mutants exhibit a high sensitivity to all DNA-damaging agents tested. In an assay for chromosome 1 integrity, the mec1Δ/Δ mutants exhibit an increase in genome instability; no change was observed in the sgs1Δ/Δ mutants. Finally, loss of MEC1 does not affect sensitivity to the antifungal drug fluconazole, while loss of SGS1 leads to an increased susceptibility to fluconazole. Neither deletion elevated the level of antifungal drug resistance acquisition. PMID:21511048

Genetic analysis of the role of yfiR in the ability of Escherichia coli CFT073 to control cellular cyclic dimeric GMP levels and to persist in the urinary tract.

PubMed

Raterman, Erica L; Shapiro, Daniel D; Stevens, Daniel J; Schwartz, Kevin J; Welch, Rodney A

2013-09-01

During urinary tract infections (UTIs), uropathogenic Escherichia coli must maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states in E. coli. The yfiRNB locus in E. coli CFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion of yfiR yielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A double yfiRN mutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in the yfiR mutant. Expression of yhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiR suppressed the overproduction of curli fimbriae and cellulose and further verified that deletion of yfiR results in c-di-GMP accumulation. Additional deletion of csgD and bcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of the yfiR deletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between the yfiR mutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disable E. coli in the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenic E. coli in vivo.

Pyruvate kinase deletion as an effective phenotype to enhance lysine production in Corynebacterium glutamicum ATCC13032: Redirecting the carbon flow to a precursor metabolite.

PubMed

Yanase, Masaki; Aikoh, Tohru; Sawada, Kazunori; Ogura, Kotaro; Hagiwara, Takuya; Imai, Keita; Wada, Masaru; Yokota, Atsushi

2016-08-01

Various attempts have been made to enhance lysine production in Corynebacterium glutamicum. Pyruvate kinase (PYK) defect is one of the strategies used to enhance the supply of oxaloacetic acid (OAA), a precursor metabolite for lysine biosynthesis. However, inconsistent effects of this mutation have been reported: positive effects of PYK defect in mutants having phosphoenolpyruvate carboxylase (PEPC) desensitized to feedback inhibition by aspartic acid, while negative effects in simple PYK gene (pyk) knockout mutants. To address these discrepancies, the effects of pyk deletion on lysine yield were investigated with or without the D299N mutation in ppc rendering PEPC desensitization. C. glutamicum ATCC13032 mutant strain P with a feedback inhibition-desensitized aspartokinase was used as the parent strain, producing 9.36 g/L lysine from 100 g/L glucose in a jar fermentor culture. Under these conditions, while the simple mutant D2 with pyk deletion or R2 with the PEPC-desensitization mutation showed marginally increased lysine yield (∼1.1-fold, not significant), the mutant DR2 strain having both mutations showed synergistically increased lysine productivity (1.38-fold, 12.9 g/L). Therefore, the pyk deletion is effective under a PEPC-desensitized background, which ensures enhanced supply of OAA, thus clarifying the discrepancies. A citrate synthase defective mutation (S252C in gltA) further increased the lysine yield in strain DR2 (1.68-fold, 15.7 g/L). Thus, these three mutations coordinately enhanced the lysine yield. Both the malate:quinone oxidoreductase activity and respiration rate were significantly reduced in strains D2 and DR2. Overall, these results provide valuable knowledge for engineering the anaplerotic reaction to increase lysine yield in C. glutamicum. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains

PubMed Central

Herrgård, Markus J.

2014-01-01

High-cell-density fermentation for industrial production of chemicals can impose numerous stresses on cells due to high substrate, product, and by-product concentrations; high osmolarity; reactive oxygen species; and elevated temperatures. There is a need to develop platform strains of industrial microorganisms that are more tolerant toward these typical processing conditions. In this study, the growth of six industrially relevant strains of Escherichia coli was characterized under eight stress conditions representative of fed-batch fermentation, and strains W and BL21(DE3) were selected as platforms for transposon (Tn) mutagenesis due to favorable resistance characteristics. Selection experiments, followed by either targeted or genome-wide next-generation-sequencing-based Tn insertion site determination, were performed to identify mutants with improved growth properties under a subset of three stress conditions and two combinations of individual stresses. A subset of the identified loss-of-function mutants were selected for a combinatorial approach, where strains with combinations of two and three gene deletions were systematically constructed and tested for single and multistress resistance. These approaches allowed identification of (i) strain-background-specific stress resistance phenotypes, (ii) novel gene deletion mutants in E. coli that confer single and multistress resistance in a strain-background-dependent manner, and (iii) synergistic effects of multiple gene deletions that confer improved resistance over single deletions. The results of this study underscore the suboptimality and strain-specific variability of the genetic network regulating growth under stressful conditions and suggest that further exploration of the combinatorial gene deletion space in multiple strain backgrounds is needed for optimizing strains for microbial bioprocessing applications. PMID:25085490

Impact of epidermal growth factor receptor gene expression level on clinical outcomes in epidermal growth factor receptor mutant lung adenocarcinoma patients taking first-line epidermal growth factor receptor-tyrosine kinase inhibitors.

PubMed

Chang, Huang-Chih; Chen, Yu-Mu; Tseng, Chia-Cheng; Huang, Kuo-Tung; Wang, Chin-Chou; Chen, Yung-Che; Lai, Chien-Hao; Fang, Wen-Feng; Kao, Hsu-Ching; Lin, Meng-Chih

2017-03-01

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are first-choice treatments for advanced non-small-cell lung cancer patients harboring EGFR mutations. Although EGFR mutations are strongly predictive of patients' outcomes and their response to treatment with EGFR-TKIs, early failure of first-line therapy with EGFR-TKIs in patients with EGFR mutations is not rare. Besides several clinical factors influencing EGFR-TKI efficacies studied earlier such as the Eastern Cooperative Oncology Group performance status or uncommon mutation, we would like to see whether semi-quantify EGFR mutation gene expression calculated by 2 -ΔΔct was a prognostic factor in EGFR-mutant non-small cell lung cancer patients receiving first-line EGFR-TKIs. This retrospective study reviews 926 lung cancer patients diagnosed from January 2011 to October 2013 at the Kaohsiung Chang Gung Memorial Hospital in Taiwan. Of 224 EGFR-mutant adenocarcinoma patients, 148 patients who had 2 -ΔΔct data were included. The best cutoff values of 2 -ΔΔct for in-frame deletions in exon 19 (19 deletion) and a position 858 substituted from leucine (L) to an arginine (R) in exon 21 (L858R) were determined using receiver operating characteristic curves. Patients were divided into high and low 2 -ΔΔct expression based on the above cutoff level. The best cutoff point of 2 -ΔΔct value of 19 deletion and L858R was 31.1 and 104.7, respectively. In all, 92 (62.1%) patients showed high 2 -ΔΔct expression and 56 patients (37.9%) low 2 -ΔΔct expression. The mean age was 65.6 years. Progression-free survival of 19 deletion mutant patients with low versus high expression level was 17.07 versus 12.04 months (P = 0.004), respectively. Progression-free survival of L858 mutant patients was 13.75 and 7.96 months (P = 0.008), respectively. EGFR-mutant lung adenocarcinoma patients with lower EGFR gene expression had longer progression-free survival duration without interfering overall survival.

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

PubMed Central

Belfield, Eric J.; Gan, Xiangchao; Mithani, Aziz; Brown, Carly; Jiang, Caifu; Franklin, Keara; Alvey, Elizabeth; Wibowo, Anjar; Jung, Marko; Bailey, Kit; Kalwani, Sharan; Ragoussis, Jiannis; Mott, Richard; Harberd, Nicholas P.

2012-01-01

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)–induced single base substitutions differed substantially from those of “background” mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations. PMID:22499668

Yeast MRX deletions have short chronological life span and more triacylglycerols.

PubMed

Kanagavijayan, Dhanabalan; Rajasekharan, Ram; Srinivasan, Malathi

2016-02-01

Saccharomyces cerevisiae is an excellent model organism for lipid research. Here, we have used yeast haploid RAdiation Damage (RAD) deletion strains to study life span and lipid storage patterns. RAD genes are mainly involved in DNA repair mechanism and hence, their deletions have resulted in shorter life span. Viable RAD mutants were screened for non-polar lipid content, and some of the mutants showed significantly high amounts of triacylglycerol (TAG) and steryl ester, besides short chronological life span. Among these, RAD50, MRE11 and XRS2 form a complex, MRX that is involved in homologous recombination that showed an increase in the amount of TAG. Microarray data of single MRX deletions revealed that besides DNA damage signature genes, lipid metabolism genes are also differentially expressed. Lipid biosynthetic genes (LPP1, SLC1) were upregulated and lipid hydrolytic gene (TGL3) was downregulated. We observed that rad50Δ, mre11Δ, xrs2Δ and mrxΔ strains have high number of lipid droplets (LDs) with fragmented mitochondria. These mutants have a short chronological life span compared to wild type. Aged wild-type cells also accumulated TAG with LDs of ∼2.0 μm in diameter. These results suggest that TAG accumulation and big size LDs could be possible markers for premature or normal aging. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Prevalence and Clinical Relevance of Exon 2 Deletion of COMMD1 in Bedlington Terriers in Korea.

PubMed

Kim, Y G; Kim, S Y; Kim, J H; Lee, K K; Yun, Y M

2016-11-01

Deletion of exon 2 of copper metabolism domain containing 1 (COMMD1) results in copper toxicosis in Bedlington terriers (CT-BT). This study was conducted to identify the prevalence and clinical relevance of the COMMD1 mutation in Bedlington terriers in Korea. A total of 105 purebred Bedlington terriers (50 males, 55 females) from the kennels and pet dog clubs in Korea were examined during the period 2008-2013. A multiplex PCR was carried out to detect exon 2 deletion of COMMD1. Clinical analysis was performed on each genetic group, and clinical status of the dogs was followed up to estimate survival probability. Of the 105 samples, 52 (49%) were wild-type homozygote, 47 (45%) were heterozygote, and 6 (6%) were mutant-type homozygote. Plasma alanine aminotransferase (ALT) activity was increased in the mutant-type homozygous group >2 years of age (P < .0001). The survival probability of 6 mutant-type homozygotes surviving 2.5 years was 0.67, and 4 years was 0.5. Results show the prevalence and clinical relevance of exon 2 deletion of COMMD1 and could help establish a structured selective breeding program to prevent CT-BT in Korea. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Deletion of Smgpi1 encoding a GPI-anchored protein suppresses sterility of the STRIPAK mutant ΔSmmob3 in the filamentous ascomycete Sordaria macrospora.

PubMed

Frey, Stefan; Lahmann, Yasmine; Hartmann, Thomas; Seiler, Stephan; Pöggeler, Stefanie

2015-08-01

The striatin interacting phosphatase and kinase (STRIPAK) complex, which is composed of striatin, protein phosphatase PP2A and kinases, is required for fruiting-body development and cell fusion in the filamentous ascomycete Sordaria macrospora. Here, we report on the interplay of the glycosylphosphatidylinositol (GPI)-anchored protein SmGPI1 with the kinase activator SmMOB3, a core component of human and fungal STRIPAK complexes. SmGPI1 is conserved among filamentous ascomycetes and was first identified in a yeast two-hybrid screen using SmMOB3 as bait. The physical interaction of SmMOB3 and SmGPI1 was verified by co-immunoprecipitation. In vivo localization and differential centrifugation revealed that SmGPI1 is predominantly secreted and attached to the cell wall but is also associated with mitochondria and appears to be a dual-targeted protein. Deletion of Smgpi1 led to an increased number of fruiting bodies that were normally shaped but reduced in size. In addition, Smmob3 and Smgpi1 genetically interact. In the sterile ΔSmmob3 background deletion of Smgpi1 restores fertility, vegetative growth as well as hyphal-fusion defects. The suppression effect was specific for the ΔSmmob3 mutant as deletion of Smgpi1 in other STRIPAK mutants does not restore fertility. © 2015 John Wiley & Sons Ltd.

Acetohydroxyacid synthase FgIlv2 and FgIlv6 are involved in BCAA biosynthesis, mycelial and conidial morphogenesis, and full virulence in Fusarium graminearum.

PubMed

Liu, Xin; Han, Qi; Xu, Jianhong; Wang, Jian; Shi, Jianrong

2015-11-10

In this study, we characterized FgIlv2 and FgIlv6, the catalytic and regulatory subunits of acetohydroxyacid synthase (AHAS) from the important wheat head scab fungus Fusarium graminearum. AHAS catalyzes the first common step in the parallel pathways toward branched-chain amino acids (BCAAs: isoleucine, leucine, valine) and is the inhibitory target of several commercialized herbicides. Both FgILV2 and FgILV6 deletion mutants were BCAA-auxotrophic and showed reduced aerial hyphal growth and red pigmentation when cultured on PDA plates. Conidial formation was completely blocked in the FgILV2 deletion mutant ΔFgIlv2-4 and significantly reduced in the FgILV6 deletion mutant ΔFgIlv6-12. The auxotrophs of ΔFgIlv2-4 and ΔFgIlv6-12 could be restored by exogenous addition of BCAAs but relied on the designated nitrogen source the medium contained. Deletion of FgILV2 or FgILV6 also leads to hypersensitivity to various cellular stresses and reduced deoxynivalenol production. ΔFgIlv2-4 lost virulence completely on flowering wheat heads, whereas ΔFgIlv6-12 could cause scab symptoms in the inoculated spikelet but lost its aggressiveness. Taken together, our study implies the potential value of antifungals targeting both FgIlv2 and FgIlv6 in F. graminearum.

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans▿

PubMed Central

Xie, Zhoujie; Okinaga, Toshinori; Qi, Fengxia; Zhang, Zhijun; Merritt, Justin

2011-01-01

Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches. PMID:21948849

Topoisomerase 3β is the major topoisomerase for mRNAs and linked to neurodevelopment and mental dysfunction.

PubMed

Ahmad, Muzammil; Shen, Weiping; Li, Wen; Xue, Yutong; Zou, Sige; Xu, Dongyi; Wang, Weidong

2017-03-17

Human cells contain five topoisomerases in the nucleus and cytoplasm, but which one is the major topoisomerase for mRNAs is unclear. To date, Top3β is the only known topoisomerase that possesses RNA topoisomerase activity, binds mRNA translation machinery and interacts with an RNA-binding protein, FMRP, to promote synapse formation; and Top3β gene deletion has been linked to schizophrenia. Here, we show that Top3β is also the most abundant mRNA-binding topoisomerase in cells. Top3β, but not other topoisomerases, contains a distinctive RNA-binding domain; and deletion of this domain diminishes the amount of Top3β that associates with mRNAs, indicating that Top3β is specifically targeted to mRNAs by its RNA binding domain. Moreover, Top3β mutants lacking either its RNA-binding domain or catalytic residue fail to promote synapse formation, suggesting that Top3β requires both its mRNA-binding and catalytic activity to facilitate neurodevelopment. Notably, Top3β proteins bearing point mutations from schizophrenia and autism individuals are defective in association with FMRP; whereas one of the mutants is also deficient in binding mRNAs, catalyzing RNA topoisomerase reaction, and promoting synapse formation. Our data suggest that Top3β is the major topoisomerase for mRNAs, and requires both RNA binding and catalytic activity to promote neurodevelopment and prevent mental dysfunction. Published by Oxford University Press on behalf of Nucleic Acids Research 2016.

Nonmuscle Myosin II Regulates the Morphogenesis of Metanephric Mesenchyme–Derived Immature Nephrons

PubMed Central

Recuenco, Mariam C.; Ohmori, Tomoko; Tanigawa, Shunsuke; Taguchi, Atsuhiro; Fujimura, Sayoko; Conti, Mary Anne; Wei, Qize; Kiyonari, Hiroshi; Abe, Takaya; Adelstein, Robert S.

2015-01-01

The kidney develops from reciprocal interactions between the metanephric mesenchyme and ureteric bud. The mesenchyme transforms into epithelia and forms complicated nephron structures, whereas the ureteric bud extends its pre-existing epithelial ducts. Although the roles are well established for extracellular stimuli, such as Wnt and Notch, it is unclear how the intracellular cytoskeleton regulates these morphogenetic processes. Myh9 and Myh10 encode nonmuscle myosin II heavy chains, and Myh9 mutations in humans are implicated in congenital kidney diseases and focal segmental glomerulosclerosis in adults. Here, we analyzed the roles of Myh9 and Myh10 in the developing kidney. Ureteric bud-specific depletion of Myh9 resulted in no apparent phenotypes, whereas mesenchyme-specific Myh9 deletion caused proximal tubule dilations and renal failure. Mesenchyme-specific Myh9/Myh10 mutant mice died shortly after birth and showed a severe defect in nephron formation. The nascent mutant nephrons failed to form a continuous lumen, which likely resulted from impaired apical constriction of the elongating tubules. In addition, nephron progenitors lacking Myh9/Myh10 or the possible interactor Kif26b were less condensed at midgestation and reduced at birth. Taken together, nonmuscle myosin II regulates the morphogenesis of immature nephrons derived from the metanephric mesenchyme and the maintenance of nephron progenitors. Our data also suggest that Myh9 deletion in mice results in failure to maintain renal tubules but not in glomerulosclerosis. PMID:25168025

N-terminal deletions in Rous sarcoma virus p60src: effects on tyrosine kinase and biological activities and on recombination in tissue culture with the cellular src gene.

PubMed Central

Cross, F R; Garber, E A; Hanafusa, H

1985-01-01

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein. Images PMID:2426576

Severe Acute Respiratory Syndrome Coronaviruses with Mutations in the E Protein Are Attenuated and Promising Vaccine Candidates

PubMed Central

Regla-Nava, Jose A.; Nieto-Torres, Jose L.; Jimenez-Guardeño, Jose M.; Fernandez-Delgado, Raul; Fett, Craig; Castaño-Rodríguez, Carlos; Perlman, Stanley; DeDiego, Marta L.

2015-01-01

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouse-adapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo. To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4+ and CD8+ T cell counts in the lungs of BALB/c mice, compared to mice infected with the wild-type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates. IMPORTANCE Human coronaviruses are important zoonotic pathogens. SARS-CoV caused a worldwide epidemic infecting more than 8,000 people with a mortality of around 10%. Therefore, understanding the virulence mechanisms of this pathogen and developing efficacious vaccines are of high importance to prevent epidemics from this and other human coronaviruses. Previously, we demonstrated that a SARS-CoV lacking the E protein was attenuated in vivo. Here, we show that small deletions and modifications within the E protein led to virus attenuation, manifested by minimal lung injury, limited neutrophil influx to the lungs, reduced expression of proinflammatory cytokines, increased anti-inflammatory cytokine levels, and enhanced CD4+ and CD8+ T cell counts in vivo, suggesting that these phenomena contribute to virus attenuation. The attenuated mutants fully protected mice from challenge with virulent virus. These studies show that mutations in the E protein are not well tolerated and indicate that this protein is an excellent target for vaccine development. PMID:25609816

Roles of HAUSP-mediated p53 regulation in central nervous system development.

PubMed

Kon, N; Zhong, J; Kobayashi, Y; Li, M; Szabolcs, M; Ludwig, T; Canoll, P D; Gu, W

2011-08-01

The deubiquitinase HAUSP (herpesvirus-associated ubiquitin-specific protease; also called USP7) has a critical role in regulating the p53-Mdm2 (murine double minute 2) pathway. By using the conventional knockout approach, we previously showed that hausp inactivation leads to early embryonic lethality. To fully understand the physiological functions of hausp, we have generated mice lacking hausp specifically in the brain and examined the impacts of this manipulation on brain development. We found that deletion of hausp in neural cells resulted in neonatal lethality. The brains from these mice displayed hypoplasia and deficiencies in development, which were mainly caused by p53-mediated apoptosis. Detailed analysis also showed an increase of both p53 levels and p53-dependent transcriptional activation in hausp knockout brains. Notably, neural cell survival and brain development of hausp-mutant mice can largely be restored in the p53-null background. Nevertheless, in contrast to the case of mdm2- and mdm4 (murine double minute 4)-mutant mice, inactivation of p53 failed to completely rescue the neonatal lethality of these hausp-mutant mice. These results indicate that HAUSP-mediated p53 regulation is crucial for brain development, and also suggest that both the p53-dependent and the p53-independent functions of HAUSP contribute to the neonatal lethality of hausp-mutant mice.

Role of urease in megasome formation and Helicobacter pylori survival in macrophages

PubMed Central

Schwartz, Justin T.; Allen, Lee-Ann H.

2007-01-01

Previous studies have demonstrated that Helicobacter pylori (Hp) delays its entry into macrophages and persists inside megasomes, which are poorly acidified and accumulate early endosome autoantigen 1. Herein, we explored the role of Hp urease in bacterial survival in murine peritoneal macrophages and J774 cells. Plasmid-free mutagenesis was used to replace ureA and ureB with cat in Hp Strains 11637 and 11916. ureAB null Hp lacked detectable urease activity and did not express UreA or UreB as judged by immunoblotting. Deletion of ureAB had no effect on Hp binding to macrophages or the rate or extent of phagocytosis. However, intracellular survival of mutant organisms was impaired significantly. Immunofluorescence microscopy demonstrated that (in contrast to parental organisms) mutant Hp resided in single phagosomes, which were acidic and accumulated the lysosome marker lysosome-associated membrane protein-1 but not early endosome autoantigen 1. A similar phenotype was observed for spontaneous urease mutants derived from Hp Strain 60190. Treatment of macrophages with bafilomycin A1, NH4Cl, or chloroquine prevented acidification of phagosomes containing mutant Hp. However, only ammonium chloride enhanced bacterial viability significantly. Rescue of ureAB null organisms was also achieved by surface adsorption of active urease. Altogether, our data indicate a role for urease and urease-derived ammonia in megasome formation and Hp survival. PMID:16543403

Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

USDA-ARS?s Scientific Manuscript database

Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. ...

Differential involvement of the related DNA helicases Pif1p and Rrm3p in mtDNA point mutagenesis and stability.

PubMed

O'Rourke, Thomas W; Doudican, Nicole A; Zhang, Hong; Eaton, Jana S; Doetsch, Paul W; Shadel, Gerald S

2005-07-18

With the exception of base excision repair, conserved pathways and mechanisms that maintain mitochondrial genome stability have remained largely undelineated. In the budding yeast, Saccharomyces cerevisiae, Pif1p is a unique DNA helicase that is localized both to the nucleus and mitochondria, where it is involved in maintaining DNA integrity. We previously elucidated a role for Pif1p in oxidative mtDNA damage resistance that appears to be distinct from its postulated function in mtDNA recombination. Strains lacking Pif1p (pif1Delta) exhibit an increased rate of formation of petite mutants (an indicator of mtDNA instability) and elevated mtDNA point mutagenesis. Here we show that deletion of the RRM3 gene, which encodes a DNA helicase closely related to Pif1p, significantly rescues the petite-induction phenotype of a pif1Delta strain. However, suppression of this phenotype was not accompanied by a corresponding decrease in mtDNA point mutagenesis. Instead, deletion of RRM3 alone resulted in an increase in mtDNA point mutagenesis that was synergistic with that caused by a pif1Delta mutation. In addition, we found that over-expression of RNR1, encoding a large subunit of ribonucleotide reductase (RNR), rescued the petite-induction phenotype of a pif1Delta mutation to a similar extent as deletion of RRM3. This, coupled to our finding that the Rad53p protein kinase is phosphorylated in the rrm3Delta pif1Delta double-mutant strain, leads us to conclude that one mechanism whereby deletion of RRM3 influences mtDNA stability is by modulating mitochondrial deoxynucleoside triphosphate pools. We propose that this is accomplished by signaling through the conserved Mec1/Rad53, S-phase checkpoint pathway to induce the expression and activity of RNR. Altogether, our results define a novel role for Rrm3p in mitochondrial function and indicate that Pif1p and Rrm3p influence a common process (or processes) involved in mtDNA replication, repair, or stability.

Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function

PubMed Central

Haziza, Sitvanit; Magnani, Roberta; Lan, Dima; Keinan, Omer; Saada, Ann; Hershkovitz, Eli; Yanay, Nurit; Cohen, Yoram; Nevo, Yoram; Houtz, Robert L.; Sheffield, Val C.; Golan, Hava; Parvari, Ruti

2015-01-01

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the CaM KMT gene. PMID:26247364

Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function.

PubMed

Haziza, Sitvanit; Magnani, Roberta; Lan, Dima; Keinan, Omer; Saada, Ann; Hershkovitz, Eli; Yanay, Nurit; Cohen, Yoram; Nevo, Yoram; Houtz, Robert L; Sheffield, Val C; Golan, Hava; Parvari, Ruti

2015-08-01

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the CaM KMT gene.

Mitochondrial and Cytoplasmic ROS Have Opposing Effects on Lifespan

PubMed Central

Schaar, Claire E.; Dues, Dylan J.; Spielbauer, Katie K.; Machiela, Emily; Cooper, Jason F.; Senchuk, Megan; Hekimi, Siegfried; Van Raamsdonk, Jeremy M.

2015-01-01

Reactive oxygen species (ROS) are highly reactive, oxygen-containing molecules that can cause molecular damage within the cell. While the accumulation of ROS-mediated damage is widely believed to be one of the main causes of aging, ROS also act in signaling pathways. Recent work has demonstrated that increasing levels of superoxide, one form of ROS, through treatment with paraquat, results in increased lifespan. Interestingly, treatment with paraquat robustly increases the already long lifespan of the clk-1 mitochondrial mutant, but not other long-lived mitochondrial mutants such as isp-1 or nuo-6. To genetically dissect the subcellular compartment in which elevated ROS act to increase lifespan, we deleted individual superoxide dismutase (sod) genes in clk-1 mutants, which are sensitized to ROS. We find that only deletion of the primary mitochondrial sod gene, sod-2 results in increased lifespan in clk-1 worms. In contrast, deletion of either of the two cytoplasmic sod genes, sod-1 or sod-5, significantly decreases the lifespan of clk-1 worms. Further, we show that increasing mitochondrial superoxide levels through deletion of sod-2 or treatment with paraquat can still increase lifespan in clk-1;sod-1 double mutants, which live shorter than clk-1 worms. The fact that mitochondrial superoxide can increase lifespan in worms with a detrimental level of cytoplasmic superoxide demonstrates that ROS have a compartment specific effect on lifespan – elevated ROS in the mitochondria acts to increase lifespan, while elevated ROS in the cytoplasm decreases lifespan. This work also suggests that both ROS-dependent and ROS-independent mechanisms contribute to the longevity of clk-1 worms. PMID:25671321

The Role of the Regulator Fur in Gene Regulation and Virulence of Riemerella anatipestifer Assessed Using an Unmarked Gene Deletion System

PubMed Central

Guo, Yunqing; Hu, Di; Guo, Jie; Li, Xiaowen; Guo, Jinyue; Wang, Xiliang; Xiao, Yuncai; Jin, Hui; Liu, Mei; Li, Zili; Bi, Dingren; Zhou, Zutao

2017-01-01

Riemerella anatipestifer, an avian pathogen, has resulted in enormous economic losses to the duck industry globally. Notwithstanding, little is known regarding the physiological, pathogenic and virulence mechanisms of Riemerella anatipestifer (RA) infection. However, the role of Ferric uptake regulator (Fur) in the virulence of R. anatipestifer has not, to date, been demonstrated. Using a genetic approach, unmarked gene deletion system, we evaluated the function of fur gene in the virulence of R. anatipestifer. For this purpose, we constructed a suicide vector containing pheS as a counter selectable marker for unmarked deletion of fur gene to investigate its role in the virulence. After successful transformation of the newly constructed vector, a mutant strain was characterized for genes regulated by iron and Fur using RNA-sequencing and a comparison was made between wild type and mutant strains in both iron restricted and enriched conditions. RNA-seq analysis of the mutant strain in a restricted iron environment showed the downregulation and upregulation of genes which were involved in either important metabolic pathways, transport processes, growth or cell membrane synthesis. Electrophoretic mobility shift assay was performed to identify the putative sequences recognized by Fur. The putative Fur-box sequence was 5′-GATAATGATAATCATTATC-3′. Lastly, the median lethal dose and histopathological investigations of animal tissues also illustrated mild pathological lesions produced by the mutant strain as compared to the wild type RA strain, hence showing declined virulence. Conclusively, an unmarked gene deletion system was successfully developed for RA and the role of the fur gene in virulence was explored comprehensively. PMID:28971067

Role of Tellurite Resistance Operon in Filamentous Growth of Yersinia pestis in Macrophages.

PubMed

Ponnusamy, Duraisamy; Clinkenbeard, Kenneth D

2015-01-01

Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections. In vitro treatment of Y. pestis culture with sodium tellurite (Na2TeO3) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype. These findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a bacterial adaptive strategy to macrophage associated stresses.

Role of the XIAP-Cooper Axis in Prostate Cancer

DTIC Science & Technology

2011-04-01

growing yeast transformed with a plasmid encoding human XIAP in Cu-free selective medium. Supplemental Cu was added to the medium 1-2 hours before...human XIAP into yeast deletion strains. We selected 16 deletion strains from the same background as our wild-type control (BY4741) for analysis. These...transformed with the XIAP expression plasmid. This objective is complete. Assess yeast deletion mutants for delivery of copper to XIAP. After

The Yeast Saccharomyces cerevisiae Contains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

PubMed Central

Luikenhuis, Sandra; Perrone, Gabriel; Dawes, Ian W.; Grant, Chris M.

1998-01-01

Glutaredoxins are small heat-stable proteins that act as glutathione-dependent disulfide oxidoreductases. Two genes, designated GRX1 and GRX2, which share 40–52% identity and 61–76% similarity with glutaredoxins from bacterial and mammalian species, were identified in the yeast Saccharomyces cerevisiae. Strains deleted for both GRX1 and GRX2 were viable but lacked heat-stable oxidoreductase activity using β-hydroxyethylene disulfide as a substrate. Surprisingly, despite the high degree of homology between Grx1 and Grx2 (64% identity), the grx1 mutant was unaffected in oxidoreductase activity, whereas the grx2 mutant displayed only 20% of the wild-type activity, indicating that Grx2 accounted for the majority of this activity in vivo. Expression analysis indicated that this difference in activity did not arise as a result of differential expression of GRX1 and GRX2. In addition, a grx1 mutant was sensitive to oxidative stress induced by the superoxide anion, whereas a strain that lacked GRX2 was sensitive to hydrogen peroxide. Sensitivity to oxidative stress was not attributable to altered glutathione metabolism or cellular redox state, which did not vary between these strains. The expression of both genes was similarly elevated under various stress conditions, including oxidative, osmotic, heat, and stationary phase growth. Thus, Grx1 and Grx2 function differently in the cell, and we suggest that glutaredoxins may act as one of the primary defenses against mixed disulfides formed following oxidative damage to proteins. PMID:9571241

Altered Actions of Memantine and NMDA-Induced Currents in a New Grid2-Deleted Mouse Line

PubMed Central

Kumagai, Ayako; Fujita, Akira; Yokoyama, Tomoki; Nonobe, Yuki; Hasaba, Yasuhiro; Sasaki, Tsutomu; Itoh, Yumi; Koura, Minako; Suzuki, Osamu; Adachi, Shigeki; Ryo, Haruko; Kohara, Arihiro; Tripathi, Lokesh P.; Sanosaka, Masato; Fukushima, Toshiki; Takahashi, Hiroyuki; Kitagawa, Kazuo; Nagaoka, Yasuo; Kawahara, Hidehisa; Mizuguchi, Kenji; Nomura, Taisei; Matsuda, Junichiro; Tabata, Toshihide; Takemori, Hiroshi

2014-01-01

Memantine is a non-competitive antagonist of the N-methyl-d-aspartate (NMDA) receptor, and is an approved drug for the treatment of moderate-to-severe Alzheimer’s disease. We identified a mouse strain with a naturally occurring mutation and an ataxic phenotype that presents with severe leg cramps. To investigate the phenotypes of these mutant mice, we screened several phenotype-modulating drugs and found that memantine (10 mg/kg) disrupted the sense of balance in the mutants. Moreover, the mutant mice showed an attenuated optokinetic response (OKR) and impaired OKR learning, which was also observed in wild-type mice treated with memantine. Microsatellite analyses indicated that the Grid2 gene-deletion is responsible for these phenotypes. Patch-clamp analysis showed a relatively small change in NMDA-dependent current in cultured granule cells from Grid2 gene-deleted mice, suggesting that GRID2 is important for correct NMDA receptor function. In general, NMDA receptors are activated after the activation of non-NMDA receptors, such as AMPA receptors, and AMPA receptor dysregulation also occurs in Grid2 mutant mice. Indeed, the AMPA treatment enhanced memantine susceptibility in wild-type mice, which was indicated by balance sense and OKR impairments. The present study explores a new role for GRID2 and highlights the adverse effects of memantine in different genetic backgrounds. PMID:25513882

Mutant quantity and quality in mammalian cells (AL) exposed to cesium-137 gamma radiation: effect of caffeine

NASA Technical Reports Server (NTRS)

McGuinness, S. M.; Shibuya, M. L.; Ueno, A. M.; Vannais, D. B.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

1995-01-01

We examined the effect of caffeine (1,3,7-trimethylxanthine) on the quantity and quality of mutations in cultured mammalian AL human-hamster hybrid cells exposed to 137Cs gamma radiation. At a dose (1.5 mg/ml for 16 h) that reduced the plating efficiency (PE) by 20%, caffeine was not itself a significant mutagen, but it increased by approximately twofold the slope of the dose-response curve for induction of S1- mutants by 137Cs gamma radiation. Molecular analysis of 235 S1- mutants using a series of DNA probes mapped to the human chromosome 11 in the AL hybrid cells revealed that 73 to 85% of the mutations in unexposed cells and in cells treated with caffeine alone, 137Cs gamma rays alone or 137Cs gamma rays plus caffeine were large deletions involving millions of base pairs of DNA. Most of these deletions were contiguous with the region of the MIC1 gene at 11p13 that encodes the S1 cell surface antigen. In other mutants that had suffered multiple marker loss, the deletions were intermittent along chromosome 11. These "complex" mutations were rare for 137Cs gamma irradiation (1/63 = 1.5%) but relatively prevalent (23-50%) for other exposure conditions. Thus caffeine appears to alter both the quantity and quality of mutations induced by 137Cs gamma irradiation.

Secisbp2 Is Essential for Embryonic Development and Enhances Selenoprotein Expression

PubMed Central

Seeher, Sandra; Atassi, Tarik; Mahdi, Yassin; Carlson, Bradley A.; Braun, Doreen; Wirth, Eva K.; Klein, Marc O.; Reix, Nathalie; Miniard, Angela C.; Schomburg, Lutz; Hatfield, Dolph L.; Driscoll, Donna M.

2014-01-01

Abstract Aims: The selenocysteine insertion sequence (SECIS)-binding protein 2 (Secisbp2) binds to SECIS elements located in the 3′-untranslated region of eukaryotic selenoprotein mRNAs. Selenoproteins contain the rare amino acid selenocysteine (Sec). Mutations in SECISBP2 in humans lead to reduced selenoprotein expression thereby affecting thyroid hormone-dependent growth and differentiation processes. The most severe cases also display myopathy, hearing impairment, male infertility, increased photosensitivity, mental retardation, and ataxia. Mouse models are needed to understand selenoprotein-dependent processes underlying the patients' pleiotropic phenotypes. Results: Unlike tRNA[Ser]Sec-deficient embryos, homozygous Secisbp2-deleted embryos implant, but fail before gastrulation. Heterozygous inactivation of Secisbp2 reduced the amount of selenoprotein expressed, but did not affect the thyroid hormone axis or growth. Conditional deletion of Secisbp2 in hepatocytes significantly decreased selenoprotein expression. Unexpectedly, the loss of Secisbp2 reduced the abundance of many, but not all, selenoprotein mRNAs. Transcript-specific and gender-selective effects on selenoprotein mRNA abundance were greater in Secisbp2-deficient hepatocytes than in tRNA[Ser]Sec-deficient cells. Despite the massive reduction of Dio1 and Sepp1 mRNAs, significantly more corresponding protein was detected in primary hepatocytes lacking Secisbp2 than in cells lacking tRNA[Ser]Sec. Regarding selenoprotein expression, compensatory nuclear factor, erythroid-derived, like 2 (Nrf2)-dependent gene expression, or embryonic development, phenotypes were always milder in Secisbp2-deficient than in tRNA[Ser]Sec-deficient mice. Innovation: We report the first Secisbp2 mutant mouse models. The conditional mutants provide a model for analyzing Secisbp2 function in organs not accessible in patients. Conclusion: In hepatocyte-specific conditional mouse models, Secisbp2 gene inactivation is less detrimental than tRNA[Ser]Sec inactivation. A role of Secisbp2 in stabilizing selenoprotein mRNAs in vivo was uncovered. Antioxid. Redox Signal. 21, 835–849. PMID:24274065

Regulation of synapse development by Vgat deletion from ErbB4-positive interneurons.

PubMed

Lin, Thiri W; Tan, Zhibing; Barik, Arnab; Yin, Dong-Min; Brudvik, Egil; Wang, Hongsheng; Xiong, Wen-Cheng; Mei, Lin

2018-02-05

GABA signaling has been implicated in neural development; however, in vivo genetic evidence is missing because mutant mice lacking GABA activity die prematurely. Here, we studied synapse development by ablating vesicular GABA transporter Vgat in in ErbB4-positive (ErbB4+) interneurons. We show that inhibitory axo-somatic synapses onto pyramidal neurons vary from one cortical layer to another; however, inhibitory synapses on axon initial segments (AISs) were similar across layers. On the other hand, PV-positive (PV+)/ErbB4+ interneurons and PV-only interneurons receive a higher number of inhibitory synapses from PV+ErbB4+ interneurons, compared with ErbB4-only interneurons. Notably, Vgat deletion from ErbB4+ interneurons reduced axo-somatic or axo-axonic synapses from PV+ErbB4+ interneurons onto excitatory neurons. This effect was associated with corresponding changes in neurotransmission. However, the Vgat mutation seemed to have little effect on inhibitory synapses onto PV+ and/or ErbB4+ interneurons. Interestingly, perineuronal nets (PNNs), extracellular matrix structures implicated in maturation, survival, protection and plasticity of PV+ interneurons, were increased in the cortex of ErbB4-Vgat-/- mice. No apparent difference was observed between males and females. These results demonstrate that Vgat of ErbB4+ interneurons is essential for the development of inhibitory synapses onto excitatory neurons and suggest a role of GABA in circuit assembly. SIGNIFICANCE STATEMENT GABA has been implicated in neural development; however, in vivo genetic evidence is missing because mutant mice lacking GABA die prematurely. To this end, we ablated Vgat in ErbB4+ interneurons in an inducible manner. We provide evidence that the formation of inhibitory as well as excitatory synapses onto excitatory neurons requires Vgat in interneurons. In particular, inhibitory axo-somatic and axo-axonic synapses are more vulnerable. Our results suggest a role of GABA in circuit assembly. Copyright © 2018 the authors.

Synthesis of hemoglobin Gun Hill: increased synthesis of the heme-free βGH globin chain and subunit exchange with a free α-chain pool

PubMed Central

Rieder, Ronald F.

1971-01-01

Hemoglobin Gun Hill is an unstable mutant hemoglobin associated with mild compensated hemolysis. This abnormal protein has a deletion of five amino acids in the β-chains. The deletion includes the heme-binding proximal histidine at position 92. The β-chains of hemoglobin Gun Hill lack heme groups. Approximately 32% of the circulating hemoglobin in heterozygous subjects consists of the mutant hemoglobin. When reticulocytes were incubated with radioactive amino acid the specific activity of hemoglobin Gun Hill was three to six times that of hemoglobin A. Total incorporation of radioactivity into hemoglobin Gun Hill was two to three times that into hemoglobin A. There were 20-50% more total counts in β-Gun Hill (βGH) than in βA. These results indicate that in reticulocytes there was greater synthesis of the abnormal β-chains than βA-chains. The ratio of the specific activities of the α-chains of hemoglobin Gun Hill to the α-chains of hemoglobin A was 20: 1. There was evidence of a free pool of α-chains in the reticulocytes containing hemoglobin Gun Hill. After 10 min of incubation approximately 40% of the total α-chain radioactivity was in the free pool. When protein synthesis was blocked by incubation of reticulocytes with puromycin, the specific activity of the α-chains of hemoglobin Gun Hill continued to increase due to direct exchange of α-subunits between the free pool and preformed hemoglobin Gun Hill. Studies of the assembly of βA and βGH revealed that the rates of translation of the two polypeptide chains were equal and uniform. No evidence was obtained for the existence of “slow points” in the process of globin chain assembly. The studies also suggest that lack of strong heme-globin binding does not hinder the synthesis of globin chains. PMID:5540175

Genome-wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato.

PubMed

Shirasawa, Kenta; Hirakawa, Hideki; Nunome, Tsukasa; Tabata, Satoshi; Isobe, Sachiko

2016-01-01

Genome-wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro-Tom genome were identified by a whole-genome shotgun sequencing analysis to estimate the spectrum and distribution of whole-genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired-end reads for four EMS-induced mutants and three gamma-ray-irradiated lines as well as a wild-type line were obtained by next-generation sequencing technology. Using bioinformatics analyses, we identified 5920 induced single nucleotide variations and insertion/deletion (indel) mutations. The predominant mutations in the EMS mutants were C/G to T/A transitions, while in the gamma-ray mutants, C/G to T/A transitions, A/T to T/A transversions, A/T to G/C transitions and deletion mutations were equally common. Biases in the base composition flanking mutations differed between the mutagenesis types. Regarding the effects of the mutations on gene function, >90% of the mutations were located in intergenic regions, and only 0.2% were deleterious. In addition, we detected 1,140,687 spontaneous single nucleotide polymorphisms and indel polymorphisms in wild-type Micro-Tom lines. We also found copy number variation, deletions and insertions of chromosomal segments in both the mutant and wild-type lines. The results provide helpful information not only for mutation research, but also for mutant screening methodology with reverse-genetic approaches. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae.

PubMed

Galli, Alvaro; Cervelli, Tiziana; Schiestl, Robert H

2003-05-01

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.

Deletion of Braun lipoprotein and plasminogen-activating protease-encoding genes attenuates Yersinia pestis in mouse models of bubonic and pneumonic plague.

PubMed

van Lier, Christina J; Sha, Jian; Kirtley, Michelle L; Cao, Anthony; Tiner, Bethany L; Erova, Tatiana E; Cong, Yingzi; Kozlova, Elena V; Popov, Vsevolod L; Baze, Wallace B; Chopra, Ashok K

2014-06-01

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4(+) and CD8(+) T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection.

Deletion of Braun Lipoprotein and Plasminogen-Activating Protease-Encoding Genes Attenuates Yersinia pestis in Mouse Models of Bubonic and Pneumonic Plague

PubMed Central

van Lier, Christina J.; Sha, Jian; Kirtley, Michelle L.; Cao, Anthony; Tiner, Bethany L.; Erova, Tatiana E.; Cong, Yingzi; Kozlova, Elena V.; Popov, Vsevolod L.; Baze, Wallace B.

2014-01-01

Currently, there is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral immunity and cell-mediated immunity are essential in providing the host with protection against plague, we developed a live-attenuated vaccine strain by deleting the Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The Δlpp Δpla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous route were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of proinflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and livers of mice infected with the Δlpp Δpla double mutant compared to the level of damage in wild-type (WT) CO92-challenged animals was observed. The Δlpp Δpla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from mutant-immunized animals exhibited significantly higher proliferative responses, when stimulated ex vivo with heat-killed WT CO92 antigens, than mice immunized with the same sublethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more gamma interferon (IFN-γ) and interleukin-4. These animals had an increasing number of tumor necrosis factor alpha (TNF-α)-producing CD4+ and CD8+ T cells than WT CO92-infected mice. These data emphasize the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our studies provide evidence that deletion of the lpp and pla genes acts synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection. PMID:24686064

Gain-of-function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K-ras

PubMed Central

Acin, Sergio; Li, Zhongyou; Mejia, Olga; Roop, Dennis R; El-Naggar, Adel K; Caulin, Carlos

2015-01-01

Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53R172H, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53R172H. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53R172H. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53. PMID:21952947

Deletion of the Dynein Heavy-Chain Gene DYN1 Leads to Aberrant Nuclear Positioning and Defective Hyphal Development in Candida albicans

PubMed Central

Martin, R.; Walther, A.; Wendland, J.

2004-01-01

Cytoplasmic dynein is a microtubule-associated minus-end-directed motor protein. CaDYN1 encodes the single dynein heavy-chain gene of Candida albicans. The open reading frames of both alleles of CaDYN1 were completely deleted via a PCR-based approach. Cadyn1 mutants are viable but grow more slowly than the wild type. In vivo time-lapse microscopy was used to compare growth of wild-type (SC5314) and dyn1 mutant strains during yeast growth and after hyphal induction. During yeast-like growth, Cadyn1 strains formed chains of cells. Chromosomal TUB1-GFP and HHF1-GFP alleles were used both in wild-type and mutant strains to monitor the orientation of mitotic spindles and nuclear positioning in C. albicans. In vivo fluorescence time-lapse analyses with HHF1-GFP over several generations indicated defects in dyn1 cells in the realignment of spindles with the mother-daughter axis of yeast cells compared to that of the wild type. Mitosis in the dyn1 mutant, in contrast to that of wild-type yeast cells, was very frequently completed in the mother cells. Nevertheless, daughter nuclei were faithfully transported into the daughter cells, resulting in only a small number of multinucleate cells. Cadyn1 mutant strains responded to hypha-inducing media containing l-proline or serum with initial germ tube formation. Elongation of the hyphal tubes eventually came to a halt, and these tubes showed a defect in the tipward localization of nuclei. Using a heterozygous DYN1/dyn1 strain in which the remaining copy was controlled by the regulatable MAL2 promoter, we could switch between wild-type and mutant phenotypes depending on the carbon source, indicating that the observed mutant phenotypes were solely due to deletion of DYN1. PMID:15590831

Proteomic and Functional Analyses of the Virion Transmembrane Proteome of Cyprinid Herpesvirus 3.

PubMed

Vancsok, Catherine; Peñaranda, M Michelle D; Raj, V Stalin; Leroy, Baptiste; Jazowiecka-Rakus, Joanna; Boutier, Maxime; Gao, Yuan; Wilkie, Gavin S; Suárez, Nicolás M; Wattiez, Ruddy; Gillet, Laurent; Davison, Andrew J; Vanderplasschen, Alain F C

2017-11-01

Virion transmembrane proteins (VTPs) mediate key functions in the herpesvirus infectious cycle. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses. The present study was devoted to CyHV-3 VTPs. Using mass spectrometry approaches, we identified 16 VTPs of the CyHV-3 FL strain. Mutagenesis experiments demonstrated that eight of these proteins are essential for viral growth in vitro (open reading frame 32 [ORF32], ORF59, ORF81, ORF83, ORF99, ORF106, ORF115, and ORF131), and eight are nonessential (ORF25, ORF64, ORF65, ORF108, ORF132, ORF136, ORF148, and ORF149). Among the nonessential proteins, deletion of ORF25, ORF132, ORF136, ORF148, or ORF149 affects viral replication in vitro , and deletion of ORF25, ORF64, ORF108, ORF132, or ORF149 impacts plaque size. Lack of ORF148 or ORF25 causes attenuation in vivo to a minor or major extent, respectively. The safety and efficacy of a virus lacking ORF25 were compared to those of a previously described vaccine candidate deleted for ORF56 and ORF57 (Δ56-57). Using quantitative PCR, we demonstrated that the ORF25 deleted virus infects fish through skin infection and then spreads to internal organs as reported previously for the wild-type parental virus and the Δ56-57 virus. However, compared to the parental wild-type virus, the replication of the ORF25-deleted virus was reduced in intensity and duration to levels similar to those observed for the Δ56-57 virus. Vaccination of fish with a virus lacking ORF25 was safe but had low efficacy at the doses tested. This characterization of the virion transmembrane proteome of CyHV-3 provides a firm basis for further research on alloherpesvirus VTPs. IMPORTANCE Virion transmembrane proteins play key roles in the biology of herpesviruses. Cyprinid herpesvirus 3 (CyHV-3) is the archetype of fish alloherpesviruses and the causative agent of major economic losses in common and koi carp worldwide. In this study of the virion transmembrane proteome of CyHV-3, the major findings were: (i) the FL strain encodes 16 virion transmembrane proteins; (ii) eight of these proteins are essential for viral growth in vitro ; (iii) seven of the nonessential proteins affect viral growth in vitro , and two affect virulence in vivo ; and (iv) a mutant lacking ORF25 is highly attenuated but induces moderate immune protection. This study represents a major breakthrough in understanding the biology of CyHV-3 and will contribute to the development of prophylactic methods. It also provides a firm basis for the further research on alloherpesvirus virion transmembrane proteins. Copyright © 2017 Vancsok et al.

The effect of amino acid deletions and substitutions in the longest loop of GFP

PubMed Central

Flores-Ramírez, Gabriela; Rivera, Manuel; Morales-Pablos, Alfredo; Osuna, Joel; Soberón, Xavier; Gaytán, Paul

2007-01-01

Background The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region. Results In this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Δ I129 and sgGFP-Δ D130. Interestingly, both mutants were thermosensitive and at 30°C sgGFP-Δ D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein. Conclusion The behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure. PMID:17594481

Apolipoprotein B-52 mutation associated with hypobetalipoproteinemia is compatible with a misaligned pairing deletion mechanism.

PubMed

Groenewegen, W A; Krul, E S; Schonfeld, G

1993-06-01

We have identified a new truncation of apoB in a large kindred with hypobetalipoproteinemia that arose by an ambiguous deletion of one of four different groups of base-pairs. Eleven affected members of the kindred had total cholesterols (C) of 114 +/- 28, LDL-Cs of 46 +/- 21, and apoBs of 47 +/- 25 (all in mg/dl, mean +/- SD). These levels were lower (P < 0.0001) than in 15 unaffected relatives. On Western blotting, apoB-100 and a second major band corresponding to apoB-52 were seen in the affected individuals. The majority of the plasma apoB-52 was associated with a smaller than normal low density lipoprotein (LDL) particle. The molecular basis for this apoB-52 truncation is a 5-bp deletion, converting the sequence between cDNA nucleotide 7276 and 7283 from 5'-AAGTTAAG-3' into the mutant sequence 5'-AAG-3'. This results in a frameshift starting at amino acid residue 2357 and a termination codon at amino acid residue 2362. Deletion of one of four different groups of five consecutive bases, i.e., AAGTT, AGTTA, GTTAA, and TTAAG, all result in the same mutant sequence. Thus, the precise deletion is ambiguous. We propose that a misaligned pairing mechanism involving repeat sequences is compatible with this deletion mutation. We have noted similar ambiguous deletions associated with apoB-37, apoB-40, and a number of single base deletions and some may also be explained by a misaligned pairing mechanism. Small ambiguous deletions appear to constitute a major proportion of the apoB gene mutation spectrum suggesting that it may be a suitable model for studying the mechanisms of such mutations.

Restoring de novo Coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous Coenzyme Q supplementation

PubMed Central

Gomez, Fernando; Saiki, Ryoichi; Chin, Randall; Srinivasan, Chandra; Clarke, Catherine F.

2012-01-01

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 E. coli, but their second generation homozygous progeny do not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5′ and 3′, respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q10. Here we show that the Q9 content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q9 content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis. PMID:22735617

Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation.

PubMed

Gomez, Fernando; Saiki, Ryoichi; Chin, Randall; Srinivasan, Chandra; Clarke, Catherine F

2012-09-10

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5' and 3', respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q(10). Here we show that the Q(9) content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q(9) content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of C-terminal adaptors, UFD-2 and UFD-3, of CDC-48 on the polyglutamine aggregation in C. elegans.

PubMed

Murayama, Yuki; Ogura, Teru; Yamanaka, Kunitoshi

2015-03-27

CDC-48 (also called VCP or p97 in mammals and Cdc48p in yeast) is a AAA (ATPases associated with diverse cellular activities) chaperone and participates in a wide range of cellular activities including modulation of protein complexes and protein aggregates. UFD-2 and UFD-3, C-terminal adaptors for CDC-48, reportedly bind to CDC-48 in a mutually exclusive manner and they may modulate the fate of substrates for CDC-48. However, their cellular functions have not yet been elucidated. In this study, we found that CDC-48 preferentially interacts with UFD-3 in Caenorhabditis elegans. We also found that the number of polyglutamine (polyQ) aggregates was reduced in the ufd-3 deletion mutant but not in the ufd-2 deletion mutant. Furthermore, the lifespan and motility of the ufd-3 deletion mutant, where polyQ40::GFP was expressed, were greatly decreased. Taken together, we propose that UFD-3 may promote the formation of polyQ aggregates to reduce the polyQ toxicity in C. elegans. Copyright © 2015 Elsevier Inc. All rights reserved.

Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice

PubMed Central

Kraft, Andrew W.; Hu, Xiaoyan; Yoon, Hyejin; Yan, Ping; Xiao, Qingli; Wang, Yan; Gil, So Chon; Brown, Jennifer; Wilhelmsson, Ulrika; Restivo, Jessica L.; Cirrito, John R.; Holtzman, David M.; Kim, Jungsu; Pekny, Milos; Lee, Jin-Moo

2013-01-01

The accumulation of aggregated amyloid-β (Aβ) in amyloid plaques is a neuropathological hallmark of Alzheimer's disease (AD). Reactive astrocytes are intimately associated with amyloid plaques; however, their role in AD pathogenesis is unclear. We deleted the genes encoding two intermediate filament proteins required for astrocyte activation—glial fibrillary acid protein (Gfap) and vimentin (Vim)—in transgenic mice expressing mutant human amyloid precursor protein and presenilin-1 (APP/PS1). The gene deletions increased amyloid plaque load: APP/PS1 Gfap−/−Vim−/− mice had twice the plaque load of APP/PS1 Gfap+/+Vim+/+ mice at 8 and 12 mo of age. APP expression and soluble and interstitial fluid Aβ levels were unchanged, suggesting that the deletions had no effect on APP processing or Aβ generation. Astrocyte morphology was markedly altered by the deletions: wild-type astrocytes had hypertrophied processes that surrounded and infiltrated plaques, whereas Gfap−/−Vim−/− astrocytes had little process hypertrophy and lacked contact with adjacent plaques. Moreover, Gfap and Vim gene deletion resulted in a marked increase in dystrophic neurites (2- to 3-fold higher than APP/PS1 Gfap+/+Vim+/+ mice), even after normalization for amyloid load. These results suggest that astrocyte activation limits plaque growth and attenuates plaque-related dystrophic neurites. These activities may require intimate contact between astrocyte and plaque.—Kraft, A. W., Hu, X., Yoon, H., Yan, P., Xiao, Q., Wang, Y., Gil, S. C., Brown, J., Wilhelmsson, U., Restivo, J. L., Cirrito, J. R., Holtzman, D. M., Kim, J., Pekny, M., Lee, J.-M. Attenuating astrocyte activation accelerates plaque pathogenesis in APP/PS1 mice. PMID:23038755

Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiotherapy response prediction

PubMed Central

Jeong, Youngtae; Hoang, Ngoc T.; Lovejoy, Alexander; Stehr, Henning; Newman, Aaron M.; Gentles, Andrew J.; Kong, William; Truong, Diana; Martin, Shanique; Chaudhuri, Aadel; Heiser, Diane; Zhou, Li; Say, Carmen; Carter, Justin N.; Hiniker, Susan M.; Loo, Billy W.; West, Robert B.; Beachy, Philip; Alizadeh, Ash A.; Diehn, Maximilian

2016-01-01

Lung squamous cell carcinomas (LSCC) pathogenesis remains incompletely understood and biomarkers predicting treatment response remain lacking. Here we describe novel murine LSCC models driven by loss of Trp53 and Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal, suggesting that mutations in these genes lead to expansion of mutant stem cell clones. Deletion of Trp53 and Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histological and molecular features of human LSCCs, indicating that they represent the likely cell of origin in this model. Deletion of Keap1 promoted tumor aggressiveness, metastasis, and resistance to oxidative stress and radiotherapy (RT). KEAP1/NRF2 mutation status predicted risk of local recurrence after RT in non-small lung cancer (NSCLC) patients and could be non-invasively identified in circulating tumor DNA. Thus, KEAP1/NRF2 mutations could serve as predictive biomarkers for personalization of therapeutic strategies for NSCLCs. PMID:27663899

Interactions of neuropathogenic Escherichia coli K1 (RS218) and its derivatives lacking genomic islands with phagocytic Acanthamoeba castellanii and nonphagocytic brain endothelial cells.

PubMed

Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

2014-01-01

Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α -hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity.

Interactions of Neuropathogenic Escherichia coli K1 (RS218) and Its Derivatives Lacking Genomic Islands with Phagocytic Acanthamoeba castellanii and Nonphagocytic Brain Endothelial Cells

PubMed Central

Yousuf, Farzana Abubakar; Yousuf, Zuhair; Iqbal, Junaid; Siddiqui, Ruqaiyyah; Khan, Hafsa; Khan, Naveed Ahmed

2014-01-01

Here we determined the role of various genomic islands in E. coli K1 interactions with phagocytic A. castellanii and nonphagocytic brain microvascular endothelial cells. The findings revealed that the genomic islands deletion mutants of RS218 related to toxins (peptide toxin, α-hemolysin), adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (IbeA, CNF1), metabolism (D-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism) showed reduced interactions with both A. castellanii and brain microvascular endothelial cells. Interestingly, the deletion of RS218-derived genomic island 21 containing adhesins (P fimbriae, F17-like fimbriae, nonfimbrial adhesins, Hek, and hemagglutinin), protein secretion system (T1SS for hemolysin), invasins (CNF1), metabolism (D-serine catabolism) abolished E. coli K1-mediated HBMEC cytotoxicity in a CNF1-independent manner. Therefore, the characterization of these genomic islands should reveal mechanisms of evolutionary gain for E. coli K1 pathogenicity. PMID:24818136

Deletion mutagenesis identifies a haploinsufficient role for gamma-zein in opaque-2 endosperm modification

USDA-ARS?s Scientific Manuscript database

Quality Protein Maize (QPM) is a hard kernel variant of the high-lysine mutant, opaque-2. Using gamma irradiation, we created opaque QPM variants to identify opaque-2 modifier genes and to investigate deletion mutagenesis combined with Illumina sequencing as a maize functional genomics tool. A K0326...

Careful with That Axe, Gene, Genome Perturbation after a PEG-Mediated Protoplast Transformation in Fusarium verticillioides.

PubMed

Scala, Valeria; Grottoli, Alessandro; Aiese Cigliano, Riccardo; Anzar, Irantzu; Beccaccioli, Marzia; Fanelli, Corrado; Dall'Asta, Chiara; Battilani, Paola; Reverberi, Massimo; Sanseverino, Walter

2017-05-31

Fusarium verticillioides causes ear rot disease in maize and its contamination with fumonisins, mycotoxins harmful for humans and livestock. Lipids, and their oxidized forms, may drive the fate of this disease. In a previous study, we have explored the role of oxylipins in this interaction by deleting by standard transformation procedures a linoleate diol synthase-coding gene, lds1 , in F. verticillioides . A profound phenotypic diversity in the mutants generated has prompted us to investigate more deeply the whole genome of two lds1 -deleted strains. Bioinformatics analyses pinpoint significant differences in the genome sequences emerged between the wild type and the lds1 -mutants further than those trivially attributable to the deletion of the lds1 locus, such as single nucleotide polymorphisms, small deletion/insertion polymorphisms and structural variations. Results suggest that the effect of a (theoretically) punctual transformation event might have enhanced the natural mechanisms of genomic variability and that transformation practices, commonly used in the reverse genetics of fungi, may potentially be responsible for unexpected, stochastic and henceforth off-target rearrangements throughout the genome.

Careful with That Axe, Gene, Genome Perturbation after a PEG-Mediated Protoplast Transformation in Fusarium verticillioides

PubMed Central

Scala, Valeria; Grottoli, Alessandro; Aiese Cigliano, Riccardo; Anzar, Irantzu; Beccaccioli, Marzia; Fanelli, Corrado; Dall’Asta, Chiara; Battilani, Paola; Reverberi, Massimo; Sanseverino, Walter

2017-01-01

Fusarium verticillioides causes ear rot disease in maize and its contamination with fumonisins, mycotoxins harmful for humans and livestock. Lipids, and their oxidized forms, may drive the fate of this disease. In a previous study, we have explored the role of oxylipins in this interaction by deleting by standard transformation procedures a linoleate diol synthase-coding gene, lds1, in F. verticillioides. A profound phenotypic diversity in the mutants generated has prompted us to investigate more deeply the whole genome of two lds1-deleted strains. Bioinformatics analyses pinpoint significant differences in the genome sequences emerged between the wild type and the lds1-mutants further than those trivially attributable to the deletion of the lds1 locus, such as single nucleotide polymorphisms, small deletion/insertion polymorphisms and structural variations. Results suggest that the effect of a (theoretically) punctual transformation event might have enhanced the natural mechanisms of genomic variability and that transformation practices, commonly used in the reverse genetics of fungi, may potentially be responsible for unexpected, stochastic and henceforth off-target rearrangements throughout the genome. PMID:28561789

Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine

USDA-ARS?s Scientific Manuscript database

Genomes from fifteen porcine reproductive and respiratory syndrome virus (PRRSV) isolates were derived simultaneously using 454 pyrosequencing technology. The viral isolates sequenced were from a recent swine study, in which engineered Type 2 prototype PRRSV strain VR-2332 mutants, with 87, 184, 200...

A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation.

PubMed

Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Do, Loc Thi Binh Xuan; Mai, Linh Thi Dam; Pham, Duc-Ngoc; Tran, Huyen Thi Thanh; Le, Diep Hong; Nguyen, Huy Quang; Tran, Van-Tuan

2017-06-01

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 10 6 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

Carbon storage regulator A contributes to the virulence of Haemophilus ducreyi in humans by multiple mechanisms.

PubMed

Gangaiah, Dharanesh; Li, Wei; Fortney, Kate R; Janowicz, Diane M; Ellinger, Sheila; Zwickl, Beth; Katz, Barry P; Spinola, Stanley M

2013-02-01

The carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence. Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog of csrA. Here, we generated an unmarked, in-frame deletion mutant of csrA to assess its contribution to H. ducreyi pathogenesis. In human inoculation experiments, the csrA mutant was partially attenuated for pustule formation compared to its parent. Deletion of csrA resulted in decreased adherence of H. ducreyi to human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants of H. ducreyi adherence to HFF cells, were downregulated in the csrA mutant. Compared to its parent, the csrA mutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. The csrA mutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation in trans partially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

A yeast-based genetic screening to identify human proteins that increase homologous recombination.

PubMed

Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

2008-05-01

To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

IDH mutant and 1p/19q co-deleted oligodendrogliomas: tumor grade stratification using diffusion-, susceptibility-, and perfusion-weighted MRI.

PubMed

Lin, Yu; Xing, Zhen; She, Dejun; Yang, Xiefeng; Zheng, Yingyan; Xiao, Zebin; Wang, Xingfu; Cao, Dairong

2017-06-01

Currently, isocitrate dehydrogenase (IDH) mutation and 1p/19q co-deletion are proven diagnostic biomarkers for both grade II and III oligodendrogliomas (ODs). Non-invasive diffusion-weighted imaging (DWI), susceptibility-weighted imaging (SWI), and dynamic susceptibility contrast perfusion-weighted imaging (DSC-PWI) are widely used to provide physiological information (cellularity, hemorrhage, calcifications, and angiogenesis) of neoplastic histology and tumor grade. However, it is unclear whether DWI, SWI, and DSC-PWI are able to stratify grades of IDH-mutant and 1p/19q co-deleted ODs. We retrospectively reviewed the conventional MRI (cMRI), DWI, SWI, and DSC-PWI obtained on 33 patients with IDH-mutated and 1p/19q co-deleted ODs. Features of cMRI, normalized ADC (nADC), intratumoral susceptibility signals (ITSSs), normalized maxim CBV (nCBV), and normalized maximum CBF (nCBF) were compared between low-grade ODs (LGOs) and high-grade ODs (HGOs). Receiver operating characteristic curve and logistic regression were applied to determine diagnostic performances. HGOs tended to present with prominent edema and enhancement. nADC, ITSSs, nCBV, and nCBF were significantly different between groups (all P < 0.05). The combination of SWI and DSC-PWI for grading resulted in sensitivity and specificity of 100.00 and 93.33%, respectively. IDH-mutant and 1p/19q co-deleted ODs can be stratified by grades using cMRI and advanced magnetic resonance imaging techniques including DWI, SWI, and DSC-PWI. Combined ITSSs with nCBV appear to be a promising option for grading molecularly defined ODs in clinical practice.

The Staphylococcus aureus Global Regulator MgrA Modulates Clumping and Virulence by Controlling Surface Protein Expression

PubMed Central

Crosby, Heidi A.; Schlievert, Patrick M.; Merriman, Joseph A.; King, Jessica M.; Salgado-Pabón, Wilmara; Horswill, Alexander R.

2016-01-01

Staphylococcus aureus is a human commensal and opportunistic pathogen that causes devastating infections in a wide range of locations within the body. One of the defining characteristics of S. aureus is its ability to form clumps in the presence of soluble fibrinogen, which likely has a protective benefit and facilitates adhesion to host tissue. We have previously shown that the ArlRS two-component regulatory system controls clumping, in part by repressing production of the large surface protein Ebh. In this work we show that ArlRS does not directly regulate Ebh, but instead ArlRS activates expression of the global regulator MgrA. Strains lacking mgrA fail to clump in the presence of fibrinogen, and clumping can be restored to an arlRS mutant by overexpressing either arlRS or mgrA, indicating that ArlRS and MgrA constitute a regulatory pathway. We used RNA-seq to show that MgrA represses ebh, as well as seven cell wall-associated proteins (SraP, Spa, FnbB, SasG, SasC, FmtB, and SdrD). EMSA analysis showed that MgrA directly represses expression of ebh and sraP. Clumping can be restored to an mgrA mutant by deleting the genes for Ebh, SraP and SasG, suggesting that increased expression of these proteins blocks clumping by steric hindrance. We show that mgrA mutants are less virulent in a rabbit model of endocarditis, and virulence can be partially restored by deleting the genes for the surface proteins ebh, sraP, and sasG. While mgrA mutants are unable to clump, they are known to have enhanced biofilm capacity. We demonstrate that this increase in biofilm formation is partially due to up-regulation of SasG, a surface protein known to promote intercellular interactions. These results confirm that ArlRS and MgrA constitute a regulatory cascade, and that they control expression of a number of genes important for virulence, including those for eight large surface proteins. PMID:27144398

Acute Multiple Organ Failure in Adult Mice Deleted for the Developmental Regulator Wt1

PubMed Central

Chau, You-Ying; Brownstein, David; Mjoseng, Heidi; Lee, Wen-Chin; Buza-Vidas, Natalija; Nerlov, Claus; Jacobsen, Sten Eirik; Perry, Paul; Berry, Rachel; Thornburn, Anna; Sexton, David; Morton, Nik; Hohenstein, Peter; Freyer, Elisabeth; Samuel, Kay; van't Hof, Rob; Hastie, Nicholas

2011-01-01

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal–epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover. PMID:22216009

Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

PubMed Central

Qiu, Z; Hobman, T C; McDonald, H L; Seto, N O; Gillam, S

1992-01-01

The role of N-linked glycosylation in processing and intracellular transport of rubella virus glycoprotein E2 has been studied by expressing glycosylation mutants of E2 in COS cells. A panel of E2 glycosylation mutants were generated by oligonucleotide-directed mutagenesis. Each of the three potential N-linked glycosylation sites was eliminated separately as well as in combination with the other two sites. Expression of the E2 mutant proteins in COS cells indicated that in rubella virus M33 strain, all three sites are used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability, and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number of deleted carbohydrate sites. The mutant proteins were transported to the endoplasmic reticulum and Golgi complex but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site. Images PMID:1583721

A genome-wide screen of bacterial mutants that enhance dauer formation in C. elegans.

PubMed

Khanna, Amit; Kumar, Jitendra; Vargas, Misha A; Barrett, LaKisha; Katewa, Subhash; Li, Patrick; McCloskey, Tom; Sharma, Amit; Naudé, Nicole; Nelson, Christopher; Brem, Rachel; Killilea, David W; Mooney, Sean D; Gill, Matthew; Kapahi, Pankaj

2016-12-13

Molecular pathways involved in dauer formation, an alternate larval stage that allows Caenorhabditis elegans to survive adverse environmental conditions during development, also modulate longevity and metabolism. The decision to proceed with reproductive development or undergo diapause depends on food abundance, population density, and temperature. In recent years, the chemical identities of pheromone signals that modulate dauer entry have been characterized. However, signals derived from bacteria, the major source of nutrients for C. elegans, remain poorly characterized. To systematically identify bacterial components that influence dauer formation and aging in C. elegans, we utilized the individual gene deletion mutants in E. coli (K12). We identified 56 diverse E. coli deletion mutants that enhance dauer formation in an insulin-like receptor mutant (daf-2) background. We describe the mechanism of action of a bacterial mutant cyaA, that is defective in the production of cyclic AMP, which extends lifespan and enhances dauer formation through the modulation of TGF-β (daf-7) signaling in C. elegans. Our results demonstrate the importance of bacterial components in influencing developmental decisions and lifespan in C. elegans. Furthermore, we demonstrate that C. elegans is a useful model to study bacterial-host interactions.

Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion.

PubMed

Luo, Jing; Lu, Liaoxun; Gu, Yanrong; Huang, Rong; Gui, Lin; Li, Saichao; Qi, Xinhui; Zheng, Wenping; Chao, Tianzhu; Zheng, Qianqian; Liang, Yinming; Zhang, Lichen

2018-06-07

Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells. Because of ultra-high efficiency of the CRISPR/Cas9 system with dual reporters and large DNA fragment deletion resulting from independent loci cleavage, monoclonal mutant cells could be easily identified by conventional PCR. In the speed genome editing method presented here, sophisticated genotyping methods are not necessary to identify loss of function mutations after CRISPR/Cas9 genome editing, and desirable loss of function mutant clones could be obtained in less than one month following transfection. Copyright © 2018 Elsevier B.V. All rights reserved.

A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites

PubMed Central

Sajid, Mohammed; Chevalley-Maurel, Séverine; Ramesar, Jai; Klop, Onny; Franke-Fayard, Blandine M. D.; Janse, Chris J.; Khan, Shahid M.

2011-01-01

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research. PMID:22216235

Identification and characterization of Escherichia coli RS218-derived islands in the pathogenesis of E. coli meningitis.

PubMed

Xie, Yi; Kolisnychenko, Vitaliy; Paul-Satyaseela, Maneesh; Elliott, Simon; Parthasarathy, Geetha; Yao, Yufeng; Plunkett, Guy; Blattner, Frederick R; Kim, Kwang Sik

2006-08-01

Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis, but the mechanisms by which E. coli K1 causes meningitis are not clear. We identified 22 E. coli RS218-derived genomic islands (RDIs), using a comparative genome analysis of meningitis-causing E. coli K1 strain RS218 (O18:K1:H7) and laboratory K-12 strain MG1655. Series of RDI deletion mutants were constructed and examined for phenotypes relevant to E. coli K1 meningitis. We identified 9 RDI deletion mutants (RDI 1, 4, 7, 12, 13, 16, 20, 21, and 22) that exhibited defects in meningitis development. RDI 16 and 21 mutants had profound defects in the induction of a high level of bacteremia in neonatal rats, and RDI 4 mutants exhibited a moderate defect in the induction of bacteremia. RDI 1 and 22 mutants showed defects in the ability to invade human brain microvascular endothelial cells (HBMECs), and RDI 12 mutants were defective in the ability to bind to HBMECs. RDI 13 and 20 mutants were defective in the ability to both bind to and invade HBMECs. RDI 7 mutants were defective in the induction of bacteremia and in the ability to both bind to and invade HBMECs. These results provide a framework for the future discovery and analysis of bacteremia and meningitis caused by E. coli K1 strain RS218.

An interactional network of genes involved in chitin synthesis in Saccharomyces cerevisiae.

PubMed

Lesage, Guillaume; Shapiro, Jesse; Specht, Charles A; Sdicu, Anne-Marie; Ménard, Patrice; Hussein, Shamiza; Tong, Amy Hin Yan; Boone, Charles; Bussey, Howard

2005-02-16

In S. cerevisiae the beta-1,4-linked N-acetylglucosamine polymer, chitin, is synthesized by a family of 3 specialized but interacting chitin synthases encoded by CHS1, CHS2 and CHS3. Chs2p makes chitin in the primary septum, while Chs3p makes chitin in the lateral cell wall and in the bud neck, and can partially compensate for the lack of Chs2p. Chs3p requires a pathway of Bni4p, Chs4p, Chs5p, Chs6p and Chs7p for its localization and activity. Chs1p is thought to have a septum repair function after cell separation. To further explore interactions in the chitin synthase family and to find processes buffering chitin synthesis, we compiled a genetic interaction network of genes showing synthetic interactions with CHS1, CHS3 and genes involved in Chs3p localization and function and made a phenotypic analysis of their mutants. Using deletion mutants in CHS1, CHS3, CHS4, CHS5, CHS6, CHS7 and BNI4 in a synthetic genetic array analysis we assembled a network of 316 interactions among 163 genes. The interaction network with CHS3, CHS4, CHS5, CHS6, CHS7 or BNI4 forms a dense neighborhood, with many genes functioning in cell wall assembly or polarized secretion. Chitin levels were altered in 54 of the mutants in individually deleted genes, indicating a functional relationship between them and chitin synthesis. 32 of these mutants triggered the chitin stress response, with elevated chitin levels and a dependence on CHS3. A large fraction of the CHS1-interaction set was distinct from that of the CHS3 network, indicating broad roles for Chs1p in buffering both Chs2p function and more global cell wall robustness. Based on their interaction patterns and chitin levels we group interacting mutants into functional categories. Genes interacting with CHS3 are involved in the amelioration of cell wall defects and in septum or bud neck chitin synthesis, and we newly assign a number of genes to these functions. Our genetic analysis of genes not interacting with CHS3 indicate expanded roles for Chs4p, Chs5p and Chs6p in secretory protein trafficking and of Bni4p in bud neck organization.

GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling

PubMed Central

Marroquin-Guzman, Margarita; Wilson, Richard A.

2015-01-01

Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development. PMID:25901357

Multi-level evaluation of Escherichia coli polyphosphate related mutants using global transcriptomic, proteomic and phenomic analyses.

PubMed

Varas, Macarena; Valdivieso, Camilo; Mauriaca, Cecilia; Ortíz-Severín, Javiera; Paradela, Alberto; Poblete-Castro, Ignacio; Cabrera, Ricardo; Chávez, Francisco P

2017-04-01

Polyphosphate (polyP) is a linear biopolymer found in all living cells. In bacteria, mutants lacking polyphosphate kinase 1 (PPK1), the enzyme responsible for synthesis of most polyP, have many structural and functional defects. However, little is known about the causes of these pleiotropic alterations. The link between ppk1 deletion and those numerous phenotypes observed can be the result of complex molecular interactions that can be elucidated via a systems biology approach. By integrating different omics levels (transcriptome, proteome and phenome), we described the functioning of various metabolic pathways among Escherichia coli polyphosphate mutant strains (Δppk1, Δppx, and ΔpolyP). Bioinformatic analyses reveal the complex metabolic and regulatory bases of the phenotypes unique to polyP mutants. Our results suggest that during polyP deficiency (Δppk1 mutant), metabolic pathways needed for energy supply are up-regulated, including fermentation, aerobic and anaerobic respiration. Transcriptomic and q-proteomic contrasting changes between Δppk1 and Δppx mutant strains were observed in those central metabolic pathways and confirmed by using Phenotypic microarrays. In addition, our results suggest a regulatory connection between polyP, second messenger metabolism, alternative Sigma/Anti-Sigma factors and type-II toxin-antitoxin (TA) systems. We suggest a broader role for polyP via regulation of ATP-dependent proteolysis of type II toxin-antitoxin system and alternative Sigma/Anti-Sigma factors, that could explain the multiple structural and functional deficiencies described due to alteration of polyP metabolism. Understanding the interplay of polyP in bacterial metabolism using a systems biology approach can help to improve design of novel antimicrobials toward pathogens. Copyright © 2017 Elsevier B.V. All rights reserved.

A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus.

PubMed

Coleman, Stewart; Choi, K Yeon; Root, Matthew; McGregor, Alistair

2016-07-01

In human cytomegalovirus (HCMV), tropism to epithelial and endothelial cells is dependent upon a pentameric complex (PC). Given the structure of the placenta, the PC is potentially an important neutralizing antibody target antigen against congenital infection. The guinea pig is the only small animal model for congenital CMV. Guinea pig cytomegalovirus (GPCMV) potentially encodes a UL128-131 HCMV PC homolog locus (GP128-GP133). In transient expression studies, GPCMV gH and gL glycoproteins interacted with UL128, UL130 and UL131 homolog proteins (designated GP129 and GP131 and GP133 respectively) to form PC or subcomplexes which were determined by immunoprecipitation reactions directed to gH or gL. A natural GP129 C-terminal deletion mutant (aa 107-179) and a chimeric HCMV UL128 C-terminal domain swap GP129 mutant failed to form PC with other components. GPCMV infection of a newly established guinea pig epithelial cell line required a complete PC and a GP129 mutant virus lacked epithelial tropism and was attenuated in the guinea pig for pathogenicity and had a low congenital transmission rate. Individual knockout of GP131 or 133 genes resulted in loss of viral epithelial tropism. A GP128 mutant virus retained epithelial tropism and GP128 was determined not to be a PC component. A series of GPCMV mutants demonstrated that gO was not strictly essential for epithelial infection whereas gB and the PC were essential. Ectopic expression of a GP129 cDNA in a GP129 mutant virus restored epithelial tropism, pathogenicity and congenital infection. Overall, GPCMV forms a PC similar to HCMV which enables evaluation of PC based vaccine strategies in the guinea pig model.

A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus

PubMed Central

McGregor, Alistair

2016-01-01

In human cytomegalovirus (HCMV), tropism to epithelial and endothelial cells is dependent upon a pentameric complex (PC). Given the structure of the placenta, the PC is potentially an important neutralizing antibody target antigen against congenital infection. The guinea pig is the only small animal model for congenital CMV. Guinea pig cytomegalovirus (GPCMV) potentially encodes a UL128-131 HCMV PC homolog locus (GP128-GP133). In transient expression studies, GPCMV gH and gL glycoproteins interacted with UL128, UL130 and UL131 homolog proteins (designated GP129 and GP131 and GP133 respectively) to form PC or subcomplexes which were determined by immunoprecipitation reactions directed to gH or gL. A natural GP129 C-terminal deletion mutant (aa 107–179) and a chimeric HCMV UL128 C-terminal domain swap GP129 mutant failed to form PC with other components. GPCMV infection of a newly established guinea pig epithelial cell line required a complete PC and a GP129 mutant virus lacked epithelial tropism and was attenuated in the guinea pig for pathogenicity and had a low congenital transmission rate. Individual knockout of GP131 or 133 genes resulted in loss of viral epithelial tropism. A GP128 mutant virus retained epithelial tropism and GP128 was determined not to be a PC component. A series of GPCMV mutants demonstrated that gO was not strictly essential for epithelial infection whereas gB and the PC were essential. Ectopic expression of a GP129 cDNA in a GP129 mutant virus restored epithelial tropism, pathogenicity and congenital infection. Overall, GPCMV forms a PC similar to HCMV which enables evaluation of PC based vaccine strategies in the guinea pig model. PMID:27387220

Genetic characterization of moaB mutants of Escherichia coli

PubMed Central

Kozmin, Stanislav G.; Schaaper, Roel M.

2013-01-01

The moaABCDE operon of Escherichia coli encodes enzymes essential for the biosynthesis of the molybdenum cofactor (Moco). However, the role of the moaB gene within this operon has remained enigmatic. Here, we have investigated the effect of moaB defects on two phenotypes diagnostic for Moco-deficiency: chlorate-resistance and sensitivity to the base analog 6-N-hydroxylaminopurine (HAP). We found that transposon insertions in moaB caused partial Moco-deficiency associated with chlorate-resistance, but not for HAP-sensitivity. On the other hand, in-frame deletions of moaB, or moaB overexpression, had no effect on either phenotype. Our combined data are consistent with the lack of any role for MoaB in Moco biosynthesis in E. coli. PMID:23680484

The transcription factor Ace2 and its paralog Swi5 regulate ethanol production during static fermentation through their targets Cts1 and Rps4a in Saccharomyces cerevisiae.

PubMed

Wu, Yao; Du, Jie; Xu, Guoqiang; Jiang, Linghuo

2016-05-01

Saccharomyces cerevisiae is the most widely used fermentation organism for ethanol production. However, the gene expression regulatory networks behind the ethanol fermentation are still not fully understood. Using a static fermentation model, we examined the ethanol yields on biomass of deletion mutants for 77 yeast genes encoding nonessential transcription factors, and found that deletion mutants for ACE2 and SWI5 showed dramatically increased ethanol yields. Overexpression of ACE2 or SWI5 in wild type cells reduced their ethanol yields. Furthermore, among the 34 target genes regulated by Ace2 and Swi5, deletion of CTS1,RPS4a,SIC1,EGT2,DSE2, or SCP160 led to increased ethanol yields, with the former two showing higher effects. Overexpression of CTS1 or RPS4a in both ace2/ace2 and swi5/swi5 mutants reduced their ethanol yields. In contrast, deletion of MCR1 or HO significantly decreased ethanol yields, with the former one showing the highest effect. Therefore, Ace2 and Swi5 are two negative regulators of ethanol yield during static fermentation of yeast cells, and both CTS1 and RPS4a are major effectors mediating these two transcription factors in regulating ethanol production. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Transcription Factor RFX1 Is Crucial for Maintenance of Genome Integrity in Fusarium graminearum

PubMed Central

Min, Kyunghun; Son, Hokyoung; Lim, Jae Yun; Choi, Gyung Ja; Kim, Jin-Cheol; Harris, Steven D.

2014-01-01

The survival of cellular organisms depends on the faithful replication and transmission of DNA. Regulatory factor X (RFX) transcription factors are well conserved in animals and fungi, but their functions are diverse, ranging from the DNA damage response to ciliary gene regulation. We investigated the role of the sole RFX transcription factor, RFX1, in the plant-pathogenic fungus Fusarium graminearum. Deletion of rfx1 resulted in multiple defects in hyphal growth, conidiation, virulence, and sexual development. Deletion mutants of rfx1 were more sensitive to various types of DNA damage than the wild-type strain. Septum formation was inhibited and micronuclei were produced in the rfx1 deletion mutants. The results of the neutral comet assay demonstrated that disruption of rfx1 function caused spontaneous DNA double-strand breaks (DSBs). The transcript levels of genes involved in DNA DSB repair were upregulated in the rfx1 deletion mutants. DNA DSBs produced micronuclei and delayed septum formation in F. graminearum. Green fluorescent protein (GFP)-tagged RFX1 localized in nuclei and exhibited high expression levels in growing hyphae and conidiophores, where nuclear division was actively occurring. RNA-sequencing-based transcriptomic analysis revealed that RFX1 suppressed the expression of many genes, including those required for the repair of DNA damage. Taken together, these findings indicate that the transcriptional repressor rfx1 performs crucial roles during normal cell growth by maintaining genome integrity. PMID:24465002

Effects of ompA deletion on expression of type 1 fimbriae in Escherichia coli K1 strain RS218 and on the association of E. coli with human brain microvascular endothelial cells.

PubMed

Teng, Ching-Hao; Xie, Yi; Shin, Sooan; Di Cello, Francescopaolo; Paul-Satyaseela, Maneesh; Cai, Mian; Kim, Kwang Sik

2006-10-01

We have previously shown that outer membrane protein A (OmpA) and type 1 fimbriae are the bacterial determinants involved in Escherichia coli K1 binding to human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In investigating the role of OmpA in E. coli K1 binding to HBMEC, we showed for the first time that ompA deletion decreased the expression of type 1 fimbriae in E. coli K1. Decreased expression of type 1 fimbriae in the ompA deletion mutant was largely the result of driving the fim promoter toward the type 1 fimbrial phase-OFF orientation. mRNA levels of fimB and fimE were found to be decreased with the OmpA mutant compared to the parent strain. Of interest, the ompA deletion further decreased the abilities of E. coli K1 to bind to and invade HBMEC under the conditions of fixing type 1 fimbria expression in the phase-ON or phase-OFF status. These findings suggest that the decreased ability of the OmpA mutant to interact with HBMEC is not entirely due to its decreased type 1 fimbrial expression and that OmpA and type 1 fimbriae facilitate the interaction of E. coli K1 with HBMEC at least in an additive manner.

Flagellin and F4 fimbriae have opposite effects on biofilm formation and quorum sensing in F4ac+ enterotoxigenic Escherichia coli.

PubMed

Zhou, Mingxu; Guo, Zhiyan; Yang, Yang; Duan, Qiangde; Zhang, Qi; Yao, Fenghua; Zhu, Jun; Zhang, Xinjun; Hardwidge, Philip R; Zhu, Guoqiang

2014-01-10

Bacteria that form biofilms are often highly resistant to antibiotics and are capable of evading the host immune system. To evaluate the role of flagellin and F4 fimbriae on biofilm formation by enterotoxigenic Escherichia coli (ETEC), we deleted the fliC (encoding the major flagellin protein) and/or the faeG (encoding the major subunit of F4 fimbriae) genes from ETEC C83902. Biofilm formation was reduced in the fliC mutant but increased in the faeG mutant, as compared with the wild-type strain. The expression of AI-2 quorum sensing associated genes was regulated in the fliC and faeG mutants, consistent with the biofilm formation of these strains. But, deleting fliC and/or faeG also inhibited AI-2 quorum sensing activity. Copyright © 2013 Elsevier B.V. All rights reserved.

The Deletion of the Succinate Dehydrogenase Gene KlSDH1 in Kluyveromyces lactis Does Not Lead to Respiratory Deficiency

PubMed Central

Saliola, Michele; Bartoccioni, Paola Chiara; De Maria, Ilaria; Lodi, Tiziana; Falcone, Claudio

2004-01-01

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose. PMID:15189981

Shp2 Acts Downstream of SDF-1α/CXCR4 in Guiding Granule Cell Migration During Cerebellar Development

PubMed Central

Hagihara, Kazuki; Zhang, Eric E.; Ke, Yue-Hai; Liu, Guofa; Liu, Jan-Jan; Rao, Yi; Feng, Gen-Sheng

2009-01-01

Shp2 is a non-receptor protein tyrosine phosphatase containing two Src homology 2 (SH2) domains that is implicated in intracellular signaling events controlling cell proliferation, differentiation and migration. To examine the role of Shp2 in brain development, we created mice with Shp2 selectively deleted in neural stem/progenitor cells. Homozygous mutant mice exhibited early postnatal lethality with defects in neural stem cell self-renewal and neuronal/glial cell fate specification. Here we report a critical role of Shp2 in guiding neuronal cell migration in the cerebellum. In homozygous mutants, we observed reduced and less foliated cerebellum, ectopic presence of external granule cells and mispositioned Purkinje cells, a phenotype very similar to that of mutant mice lacking either SDF-1α or CXCR4. Consistently, Shp2-deficient granule cells failed to migrate toward SDF-1α in an in vitro cell migration assay, and SDF-1α treatment triggered a robust induction of tyrosyl phosphorylation on Shp2. Together, these results suggest that although Shp2 is involved in multiple signaling events during brain development, a prominent role of the phosphatase is to mediate SDF-1α/CXCR4 signal in guiding cerebellar granule cell migration. PMID:19635473

A masked NES in INI1/hSNF5 mediates hCRM1-dependent nuclear export: implications for tumorigenesis

PubMed Central

Craig, Errol; Zhang, Zhi-Kai; Davies, Kelvin P.; Kalpana, Ganjam V.

2002-01-01

INI1 (integrase interactor 1)/hSNF5 is a component of the mammalian SWI/SNF complex and a tumor suppressor mutated in malignant rhabdoid tumors (MRT). We have identified a nuclear export signal (NES) in the highly conserved repeat 2 domain of INI1 that is unmasked upon deletion of a downstream sequence. Mutation of conserved hydrophobic residues within the NES, as well as leptomycin B treatment abrogated the nuclear export. Full-length INI1 specifically associated with hCRM1/exportin1 in vivo and in vitro. A mutant INI1 [INI1(1–319) delG950] found in MRT lacking the 66 C-terminal amino acids mislocalized to the cytoplasm. Full-length INI1 but not the INI1(1–319 delG950) mutant caused flat cell formation and cell cycle arrest in cell lines derived from MRT. Disruption of the NES in the delG950 mutant caused nuclear localization of the protein and restored its ability to cause cell cycle arrest. These observations demonstrate that INI1 has a masked NES that mediates regulated hCRM1/exportin1-dependent nuclear export and we propose that mutations that cause deregulated nuclear export of the protein could lead to tumorigenesis. PMID:11782423

β-1,6-glucan synthesis-associated genes are required for proper spore wall formation in Saccharomyces cerevisiae.

PubMed

Pan, Hua-Ping; Wang, Ning; Tachikawa, Hiroyuki; Nakanishi, Hideki; Gao, Xiao-Dong

2017-11-01

The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in β-1,6-glucan synthesis, including kre1∆, kre6∆, kre9∆ and big1∆, were sporulated to analyse the effect of β-1,6-glucan defects on the spore wall. Except for kre6∆, these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild-type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1∆ spores. The majority of the big1∆chs3∆ mutants failed to form visible spores at a higher temperature. Given that the big1∆ mutation caused a failure to attach a GPI-anchored reporter, Cwp2-GFP, to the spore wall, β-1,6-glucan is involved in tethering of GPI-anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, β-1,6-glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Deletion of exons 9 and 10 of the Presenilin 1 gene in a patient with Early-onset Alzheimer Disease generates longer amyloid seeds.

PubMed

Le Guennec, Kilan; Veugelen, Sarah; Quenez, Olivier; Szaruga, Maria; Rousseau, Stéphane; Nicolas, Gaël; Wallon, David; Fluchere, Frédérique; Frébourg, Thierry; De Strooper, Bart; Campion, Dominique; Chávez-Gutiérrez, Lucía; Rovelet-Lecrux, Anne

2017-08-01

Presenilin 1 (PSEN1) mutations are the main cause of autosomal dominant Early-onset Alzheimer Disease (EOAD). Among them, deletions of exon 9 have been reported to be associated with a phenotype of spastic paraparesis. Using exome data from a large sample of 522 EOAD cases and 584 controls to search for genomic copy-number variations (CNVs), we report here a novel partial, in-frame deletion of PSEN1, removing both exons 9 and 10. The patient presented with memory impairment associated with spastic paraparesis, both starting from the age of 56years. He presented a positive family history of EOAD. We performed functional analysis to elucidate the impact of this novel deletion on PSEN1 activity as part of the γ-secretase complex. The deletion does not affect the assembly of a mature protease complex but has an extreme impact on its global endopeptidase activity. The mutant carboxypeptidase-like activity is also strongly impaired and the deleterious mutant effect leads to an incomplete digestion of long Aβ peptides and enhances the production of Aβ43, which has been shown to be potently amyloidogenic and neurotoxic in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

PubMed

van Heemst, D; Swart, K; Holub, E F; van Dijk, R; Offenberg, H H; Goosen, T; van den Broek, H W; Heyting, C

1997-05-01

We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

A new regulatory mechanism controlling carotenogenesis in the fungus Mucor circinelloides as a target to generate β-carotene over-producing strains by genetic engineering.

PubMed

Zhang, Yingtong; Navarro, Eusebio; Cánovas-Márquez, José T; Almagro, Lorena; Chen, Haiqin; Chen, Yong Q; Zhang, Hao; Torres-Martínez, Santiago; Chen, Wei; Garre, Victoriano

2016-06-07

Carotenoids are natural pigments with antioxidant properties that have important functions in human physiology and must be supplied through the diet. They also have important industrial applications as food colourants, animal feed additives and nutraceuticals. Some of them, such as β-carotene, are produced on an industrial scale with the use of microorganisms, including fungi. The mucoral Blakeslea trispora is used by the industry to produce β-carotene, although optimisation of production by molecular genetic engineering is unfeasible. However, the phylogenetically closely related Mucor circinelloides, which is also able to accumulate β-carotene, possesses a vast collection of genetic tools with which to manipulate its genome. This work combines classical forward and modern reverse genetic techniques to deepen the regulation of carotenoid synthesis and generate candidate strains for biotechnological production of β-carotene. Mutagenesis followed by screening for mutants with altered colour in the dark and/or in light led to the isolation of 26 mutants that, together with eight previously isolated mutants, have been analysed in this work. Although most of the mutants harboured mutations in known structural and regulatory carotenogenic genes, eight of them lacked mutations in those genes. Whole-genome sequencing of six of these strains revealed the presence of many mutations throughout their genomes, which makes identification of the mutation that produced the phenotype difficult. However, deletion of the crgA gene, a well-known repressor of carotenoid biosynthesis in M. circinelloides, in two mutants (MU206 and MU218) with high levels of β-carotene resulted in a further increase in β-carotene content to differing extents with respect to the crgA single-null strain; in particular, one strain derived from MU218 was able to accumulate up to 4 mg/g of β-carotene. The additive effect of crgA deletion and the mutations present in MU218 suggests the existence of a previously unknown regulatory mechanism that represses carotenoid biosynthesis independently and in parallel to crgA. The use of a mucoral model such as M. circinelloides can allow the identification of the regulatory mechanisms that control carotenoid biosynthesis, which can then be manipulated to generate tailored strains of biotechnological interest. Mutants in the repressor crgA and in the newly identified regulatory mechanism generated in this work accumulate high levels of β-carotene and are candidates for further improvements in biotechnological β-carotene production.

Cch1 and Mid1 Are Functionally Required for Vegetative Growth under Low-Calcium Conditions in the Phytopathogenic Ascomycete Botrytis cinerea

PubMed Central

Harren, Karin

2013-01-01

In the filamentous phytopathogen Botrytis cinerea, the Ca2+/calcineurin signaling cascade has been shown to play an important role in fungal growth, differentiation, and virulence. This study deals with the functional characterization of two components of this pathway, the putative calcium channel proteins Cch1 and Mid1. The cch1 and mid1 genes were deleted, and single and double knockout mutants were analyzed during different stages of the fungal life cycle. Our data indicate that Cch1 and Mid1 are functionally required for vegetative growth under conditions of low extracellular calcium, since the growth of both deletion mutants is strongly impaired when they are exposed to the Ca2+-chelating agents EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). The impact of external Ca2+ was investigated by supplementing with CaCl2 and the ionophore A23187, both of which resulted in elevated growth for all mutants. However, deletion of either gene had no impact on germination, sporulation, hyphal morphology, or virulence. By use of the aequorin reporter system to measure intracellular calcium levels, no differences between the mutant strains and the wild type were obtained. Localization studies revealed a subcellular distribution of the Mid1–green fluorescent protein (GFP) fusion protein in network-like filaments, probably the endoplasmic reticulum (ER) membranes, indicating that Mid1 is not a plasma membrane-located calcium channel in B. cinerea. PMID:23475703

Sequence analysis of laci mutations obtained from lung cells of radon-exposed big blue{trademark} transgenic mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Layton, A.D.; Cross, F.T.; Steigler, G.L.

1994-12-31

We have exposed Big Blue{trademark} transgenic mice by inhalation to 320, 640 and 960 Working Level Months (WLM) of radon progeny. Mice were sacrificed after 3, 6 and 9 days; the time periods required to obtain the exposures. Control mice were also sacrificed at each time interval. In each case all tissues were excised, flash frozen in liquid nitrogen, and stored at -80{degrees}C for further analysis. Twelve lacI mutations have been isolated from the lung tissue of a mouse from the 960-WLM exposure group; the lacI genes from these mutants have been sequenced. Sequence data indicate that three of themore » mutants have a C;G deletion at BP 978 and are possibly clonal in origin. Two mutants have multiple events within the gene: one has a an A:T to C:G transversion and a C:G insertion separated by 291 BPs; the second has a G:C to A:T transition as well as an A:T deletion followed by 6 base pairs downstream by a T:A insertion. Other mutations include a single G:C to A:T transition, a two base pair deletion, and a C:G to T:A transition. Mutant plaques are being evaluated from individual mice at other dose levels. Time course experiments are also planned. These studies will help define the molecular fine structure of mutations induced by high-LET radiation exposure.« less

A Deubiquitinating Enzyme Ubp14 Is Required for Development, Stress Response, Nutrient Utilization, and Pathogenesis of Magnaporthe oryzae.

PubMed

Wang, Zhao; Zhang, Hong; Liu, Caiyun; Xing, Junjie; Chen, Xiao-Lin

2018-01-01

Ubiquitination is an essential protein modification in eukaryotic cells, which is reversible. Deubiquitinating enzymes (DUBs) catalyze deubiquitination process to reverse ubiquitination, maintain ubiquitin homeostasis or promote protein degradation by recycling ubiquitins. In order to investigate effects of deubiquitination process in plant pathogenic fungus Magnaporthe oryzae , we generated deletion mutants of MoUBP14 . Ortholog of MoUbp14 was reported to play general roles in ubiquitin-mediated protein degradation in Saccharomyces cerevisiae . The Δ Moubp14 mutant lost its pathogenicity and was severely reduced in mycelial growth, sporulation, carbon source utilization, and increased in sensitivity to distinct stresses. The mutant was blocked in penetration, which could due to defect in turgor generation. It is also blocked in invasive growth, which could due to reduction in stress tolerance and nutrient utilization. Deletion of UBP14 also led to accumulation of free polyubiquitin chains. Pulldown assay identified some proteins related to carbohydrate metabolism and stress response may putatively interact with MoUbp14, including two key rate-limiting enzymes of gluconeogenesis, MoFbp1 and MoPck1. These two proteins were degraded when the glucose was supplied to M. oryzae grown in low glucose media for a short period of time (∼12 h), and this process required MoUbp14. In summary, pleiotropic phenotypes of the deletion mutants indicated that MoUbp14 is required for different developments and pathogenicity of M. oryzae .

A defective mutant of Salmonella enterica Serovar Gallinarum in cobalamin biosynthesis is avirulent in chickens

PubMed Central

de Paiva, Jacqueline Boldrin; Penha Filho, Rafael Antonio Casarin; Arguello, Yuli Melisa Sierra; Berchieri Junior, Ângelo; Lemos, Manuel Victor Franco; Barrow, Paul A.

2009-01-01

Salmonella enterica serovar Gallinarum (SG) is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80%. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out, the Salmonella Gallinarum mutant was unable to provoke mortality in susceptible chickens. This work showed that B12 biosynthesis is a very important step in the metabolism of Salmonella Gallinarum during the infection of the chickens. Further research on bacterium physiology should be carried out to elucidate the events described in this research and to assess the mutant as a vaccine strain. PMID:24031393

Chromosomal mutations and chromosome loss measured in a new human-hamster hybrid cell line, ALC: studies with colcemid, ultraviolet irradiation, and 137Cs gamma-rays

NASA Technical Reports Server (NTRS)

Kraemer, S. M.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

1997-01-01

Small mutations, megabase deletions, and aneuploidy are involved in carcinogenesis and genetic defects, so it is important to be able to quantify these mutations and understand mechanisms of their creation. We have previously quantified a spectrum of mutations, including megabase deletions, in human chromosome 11, the sole human chromosome in a hamster-human hybrid cell line AL. S1- mutants have lost expression of a human cell surface antigen, S1, which is encoded by the M1C1 gene at 11p13 so that mutants can be detected via a complement-mediated cytotoxicity assay in which S1+ cells are killed and S1- cells survive. But loss of genes located on the tip of the short arm of 11 (11p15.5) is lethal to the AL hybrid, so that mutants that have lost the entire chromosome 11 die and escape detection. To circumvent this, we fused AL with Chinese hamster ovary (CHO) cells to produce a new hybrid, ALC, in which the requirement for maintaining 11p15.5 is relieved, allowing us to detect mutations events involving loss of 11p15.5. We evaluated the usefulness of this hybrid by conducting mutagenesis studies with colcemid, 137Cs gamma-radiation and UV 254 nm light. Colcemid induced 1000 more S1- mutants per unit dose in ALC than in AL; the increase for UV 254 nm light was only two-fold; and the increase for 137Cs gamma-rays was 12-fold. The increase in S1- mutant fraction in ALC cells treated with colcemid and 137Cs gamma-rays were largely due to chromosome loss and 11p deletions often containing a breakpoint within the centromeric region.

Induction of endoplasmic reticulum stress by deletion of Grp78 depletes Apc mutant intestinal epithelial stem cells.

PubMed

van Lidth de Jeude, J F; Meijer, B J; Wielenga, M C B; Spaan, C N; Baan, B; Rosekrans, S L; Meisner, S; Shen, Y H; Lee, A S; Paton, J C; Paton, A W; Muncan, V; van den Brink, G R; Heijmans, J

2017-06-15

Intestinal epithelial stem cells are highly sensitive to differentiation induced by endoplasmic reticulum (ER) stress. Colorectal cancer develops from mutated intestinal epithelial stem cells. The most frequent initiating mutation occurs in Apc, which results in hyperactivated Wnt signalling. This causes hyperproliferation and reduced sensitivity to chemotherapy, but whether these mutated stem cells are sensitive to ER stress induced differentiation remains unknown. Here we examined this by generating mice in which both Apc and ER stress repressor chaperone Grp78 can be conditionally deleted from the intestinal epithelium. For molecular studies, we used intestinal organoids derived from these mice. Homozygous loss of Apc alone resulted in crypt elongation, activation of the Wnt signature and accumulation of intestinal epithelial stem cells, as expected. This phenotype was however completely rescued on activation of ER stress by additional deletion of Grp78. In these Apc-Grp78 double mutant animals, stem cells were rapidly lost and repopulation occurred by non-mutant cells that had escaped recombination, suggesting that Apc-Grp78 double mutant stem cells had lost self-renewal capacity. Although in Apc-Grp78 double mutant mice the Wnt signature was lost, these intestines exhibited ubiquitous epithelial presence of nuclear β-catenin. This suggests that ER stress interferes with Wnt signalling downstream of nuclear β-catenin. In conclusion, our findings indicate that ER stress signalling results in loss of Apc mutated intestinal epithelial stem cells by interference with the Wnt signature. In contrast to many known inhibitors of Wnt signalling, ER stress acts downstream of β-catenin. Therefore, ER stress poses a promising target in colorectal cancers, which develop as a result of Wnt activating mutations.

Global Proteome Response to Deletion of Genes Related to Mercury Methylation and Dissimilatory Metal Reduction Reveals Changes in Respiratory Metabolism in Geobacter sulfurreducens PCA

DOE PAGES

Qian, Chen; Johs, Alexander; Chen, Hongmei; ...

2016-07-27

Geobacter sulfurreducens PCA can reduce, sorb, and methylate mercury (Hg); however, the underlying biochemical mechanisms of these processes and interdependent metabolic pathways remain unknown. In this study, shotgun proteomics was used to compare global proteome profiles between wild-type G. sulfurreducens PCA and two mutant strains: a ΔhgcAB mutant, which is deficient in two genes known to be essential for Hg methylation and a ΔomcBESTZ mutant, which is deficient in five outer membrane c-type cytochromes and thus impaired in its ability for dissimilatory metal ion reduction. We were able to delineate the global response of G. sulfurreducens PCA in both mutantsmore » and identify cellular networks and metabolic pathways that were affected by the loss of these genes. Deletion of hgcAB increased the relative abundances of proteins implicated in extracellular electron transfer, including most of the c-type cytochromes, PilA-C, and OmpB, and is consistent with a previously observed increase in Hg reduction in the hgcAB mutant. Deletion of omcBESTZ was found to significantly increase relative abundances of various methyltransferases, suggesting that a loss of dissimilatory reduction capacity results in elevated activity among one-carbon metabolic pathways and thus increased methylation. We show that G. sulfurreducens PCA encodes only the folate branch of the Wood Ljungdahl pathway, and proteins associated with the folate branch were found at lower abundance in the ΔhgcAB mutant strain than the wild type. In conclusion, this observation supports the hypothesis that the function of HgcA and HgcB may be linked to one carbon metabolism through the folate branch of the Wood-Ljungdahl pathway by providing methyl groups required for Hg methylation.« less

Influence of minor displacements in loops of the porcine parvovirus VP2 capsid on virus-like particles assembly and the induction of antibody responses.

PubMed

Pan, Qunxing; He, Kongwang; Wang, Yongshan; Wang, Xiaoli; Ouyang, Wei

2013-06-01

An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine parvovirus (PPV) and expressing foreign peptides offers an alternative method for vaccination. In this study, the three-dimensional structure of the PPV capsid protein and surface loops deletion mutants were analyzed to define essential domains in PPV VP2 for the assembly of VLPs. Electron microscopic analysis and SDS-PAGE analysis confirmed the presence of abundant VLPs in a loop2 deletion mutant of expected size and appropriate morphology. Loop4 and loop2-loop4 deletion mutants, however, resulted in a lower number of particles and the morphology of the particles was not well preserved. Furthermore, the green fluorescent protein (gfp) gene was used as a model. GFP was observed at the same level in displacements mutants. However, GFP displacement mutants in loop2 construct allowed better adaptation for the fusion GFP to be further displayed on the surface of the capsid-like structure. Immunogenicity study showed that there is no obvious difference in mice inoculated with rAd-VP2(Δloop2), rAd-VP2(Δloop4), rAd-VP2(Δloop2-Δloop4), and PPV inactivated vaccine. The results suggested the possibility of inserting simultaneously B and T cell epitopes in the surface loop2 and the N-terminus. The combination of different types of epitopes (B, CD4+, and CD8+) in different positions of the PPV particles opens the way to the development of highly efficient vaccines, able to stimulate at the same time the different branches of the immune system.

The Arthroderma benhamiae hydrophobin HypA mediates hydrophobicity and influences recognition by human immune effector cells.

PubMed

Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf; Brakhage, Axel A

2012-05-01

Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypA(C)). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited "easily wettable" mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA.

The Arthroderma benhamiae Hydrophobin HypA Mediates Hydrophobicity and Influences Recognition by Human Immune Effector Cells

PubMed Central

Heddergott, Christoph; Bruns, Sandra; Nietzsche, Sandor; Leonhardt, Ines; Kurzai, Oliver; Kniemeyer, Olaf

2012-01-01

Dermatophytes are the most common cause of superficial mycoses in humans and animals. They can coexist with their hosts for many years without causing significant symptoms but also cause highly inflammatory diseases. To identify mechanisms involved in the modulation of the host response during infection caused by the zoophilic dermatophyte Arthroderma benhamiae, cell wall-associated surface proteins were studied. By two-dimensional gel electrophoresis, we found that a hydrophobin protein designated HypA was the dominant cell surface protein. HypA was also detected in the supernatant during the growth and conidiation of the fungus. The A. benhamiae genome harbors only a single hydrophobin gene, designated hypA. A hypA deletion mutant was generated, as was a complemented hypA mutant strain (hypAC). In contrast to the wild type and the complemented strain, the hypA deletion mutant exhibited “easily wettable” mycelia and conidia, indicating the loss of surface hydrophobicity of both morphotypes. Compared with the wild type, the hypA deletion mutant triggered an increased activation of human neutrophil granulocytes and dendritic cells, characterized by an increased release of the immune mediators interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha (TNF-α). For the first time, we observed the formation of neutrophil extracellular traps against dermatophytes, whose level of formation was increased by the ΔhypA mutant compared with the wild type. Furthermore, conidia of the ΔhypA strain were killed more effectively by neutrophils. Our data suggest that the recognition of A. benhamiae by the cellular immune defense system is notably influenced by the presence of the surface rodlet layer formed by the hydrophobin HypA. PMID:22408226

Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri

The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells.more » vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.« less

Centrosome Linker-induced Tetraploid Segregation Errors Link Rhabdoid Phenotypes and Lethal Colorectal Cancers.

PubMed

Remo, Andrea; Manfrin, Erminia; Parcesepe, Pietro; Ferrarini, Alberto; Han, Hye Seung; Ugnius, Mickys; Laudanna, Carmelo; Simbolo, Michele; Malanga, Donatella; Mendes Oliveira, Duarte; Baritono, Elisabetta; Colangelo, Tommaso; Sabatino, Lina; Giuliani, Jacopo; Molinari, Enrico; Garonzi, Marianna; Xumerle, Luciano; Delledonne, Massimo; Giordano, Guido; Ghimenton, Claudio; Lonardo, Fortunato; D'angelo, Fulvio; Grillo, Federica; Mastracci, Luca; Viglietto, Giuseppe; Ceccarelli, Michele; Colantuoni, Vittorio; Scarpa, Aldo; Pancione, Massimo

2018-05-21

Centrosome anomalies contribute to tumorigenesis but it remains unclear how they are generated in lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and BRAF(V600E) mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of rootletin (CROCC), a centrosomal-linker component required for centrosome cohesion and separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent in a subgroup of BRAF(V600E) mutant and microsatellite stable (MSS) rhabdoid colorectal cancers but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC expression in near-diploid BRAF(V600E) mutant/MSI colon cancer cells disrupts bipolar mitotic spindle architecture, promotes tetraploid segregation errors resulting in a highly aggressive rhabdoid-like phenotype in vitro. Restoring near-wild-type levels of CROCC in a metastatic model harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to centrosome spindle pole stabilizing agents in vitro. These data shed light on a previously unknown link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into pathways underlying genome instability. Mis-segregation of chromosomes is a prominent feature of chromosome instability and intra-tumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. The present study provides insight into the mechanism by which defects in rootletin, a centrosome linker component causes tetraploid segregation errors and phenotypic transition to a clinically devastating form of malignant rhabdoid tumor. Copyright ©2018, American Association for Cancer Research.

Functional analysis reveals the possible role of the C-terminal sequences and PI motif in the function of lily (Lilium longiflorum) PISTILLATA (PI) orthologues

PubMed Central

Chen, Ming-Kun; Hsieh, Wen-Ping; Yang, Chang-Hsien

2012-01-01

Two lily (Lilium longiflorum) PISTILLATA (PI) genes, Lily MADS Box Gene 8 and 9 (LMADS8/9), were characterized. LMADS9 lacked 29 C-terminal amino acids including the PI motif that was present in LMADS8. Both LMADS8/9 mRNAs were prevalent in the first and second whorl tepals during all stages of development and were expressed in the stamen only in young flower buds. LMADS8/9 could both form homodimers, but the ability of LMADS8 homodimers to bind to CArG1 was relatively stronger than that of LMADS9 homodimers. 35S:LMADS8 completely, and 35S:LMADS9 only partially, rescued the second whorl petal formation and partially converted the first whorl sepal into a petal-like structure in Arabidopsis pi-1 mutants. Ectopic expression of LMADS8-C (with deletion of the 29 amino acids of the C-terminal sequence) or LMADS8-PI (with only the PI motif deleted) only partially rescued petal formation in pi mutants, which was similar to what was observed in 35S:LMADS9/pi plants. In contrast, 35:LMADS9+L8C (with the addition of the 29 amino acids of the LMADS8 C-terminal sequence) or 35S:LMADS9+L8PI (with the addition of the LMADS8 PI motif) demonstrated an increased ability to rescue petal formation in pi mutants, which was similar to what was observed in 35S:LMADS8/pi plants. Furthermore, ectopic expression of LMADS8-M (with the MADS domain truncated) generated more severe dominant negative phenotypes than those seen in 35S:LMADS9-M flowers. These results revealed that the 29 amino acids including the PI motif in the C-terminal region of the lily PI orthologue are valuable for its function in regulating perianth organ formation. PMID:22068145

Contributions of EspA Filaments and Curli Fimbriae in Cellular Adherence and Biofilm Formation of Enterohemorrhagic Escherichia coli O157:H7

PubMed Central

Sharma, Vijay K.; Kudva, Indira T.; Bearson, Bradley L.; Stasko, Judith A.

2016-01-01

In Escherichia coli O157:H7 (O157), the filamentous structure of the type III secretion system is produced from the polymerization of the EspA protein. EspA filaments are essential for O157 adherence to epithelial cells. In previous studies, we demonstrated that O157 hha deletion mutants showed increased adherence to HEp-2 cells and produced abundant biofilms. Transcriptional analysis revealed increased expression of espA as well as the csgA gene, which encodes curli fimbriae that are essential for biofilm formation. In the present study, we constructed hha espA, hha csgA, and hha csgA espA deletion mutants to determine the relative importance of EspA and CsgA in O157 adherence to HEp-2 cells and biofilm formation. In vitro adherence assays, conducted at 37°C in a tissue culture medium containing 0.1% glucose, showed that HEp-2 cell adherence required EspA because hha espA and hha csgA espA mutants adhered to HEp-2 cells at higher levels only when complemented with an espA-expressing plasmid. Biofilm assays performed at 28°C in a medium lacking glucose showed dependency of biofilm formation on CsgA; however EspA was not produced under these conditions. Despite production of detectable levels of EspA at 37°C in media supplemented with 0.1% glucose, the biofilm formation occurred independent of EspA. These results indicate dependency of O157 adherence to epithelial cells on EspA filaments, while CsgA promoted biofilm formation under conditions mimicking those found in the environment (low temperature with nutrient limitations) and in the digestive tract of an host animal (higher temperature and low levels of glucose). PMID:26900701

Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is incorporated into HIV-1 virions through interactions with viral and nonviral RNAs.

PubMed

Svarovskaia, Evguenia S; Xu, Hongzhan; Mbisa, Jean L; Barr, Rebekah; Gorelick, Robert J; Ono, Akira; Freed, Eric O; Hu, Wei-Shau; Pathak, Vinay K

2004-08-20

Apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is a host cytidine deaminase that is packaged into virions and confers resistance to retroviral infection. APOBEC3G deaminates deoxycytidines in minus strand DNA to deoxyuridines, resulting in G to A hypermutation and viral inactivation. Human immunodeficiency virus type 1 (HIV-1) virion infectivity factor counteracts the antiviral activity of APOBEC3G by inducing its proteosomal degradation and preventing virion incorporation. To elucidate the mechanism of viral suppression by APOBEC3G, we developed a sensitive cytidine deamination assay and analyzed APOBEC3G virion incorporation in a series of HIV-1 deletion mutants. Virus-like particles derived from constructs in which pol, env, and most of gag were deleted still contained high levels of cytidine deaminase activity; in addition, coimmunoprecipitation of APOBEC3G and HIV-1 Gag in the presence and absence of RNase A indicated that the two proteins do not interact directly but form an RNase-sensitive complex. Viral particles lacking HIV-1 genomic RNA which were generated from the gag-pol expression constructs pC-Help and pSYNGP packaged APOBEC3G at 30-40% of the wild-type level, indicating that interactions with viral RNA are not necessary for incorporation. In addition, viral particles produced from an nucleocapsid zinc finger mutant contained approximately 1% of the viral genomic RNA but approximately 30% of the cytidine deaminase activity. The reduction in APOBEC3G incorporation was equivalent to the reduction in the total RNA present in the nucleocapsid mutant virions. These results indicate that interactions with viral proteins or viral genomic RNA are not essential for APOBEC3G incorporation and suggest that APOBEC3G interactions with viral and nonviral RNAs that are packaged into viral particles are sufficient for APOBEC3G virion incorporation.

A genome-wide nanotoxicology screen of Saccharomyces cerevisiae mutants reveals the basis for cadmium sulphide quantum dot tolerance and sensitivity.

PubMed

Marmiroli, M; Pagano, L; Pasquali, F; Zappettini, A; Tosato, V; Bruschi, C V; Marmiroli, N

2016-01-01

The use of cadmium sulphide quantum dots (CdS QDs) is increasing, particularly in the electronics industry. Their size (1-10 nm in diameter) is, however, such that they can be taken up by living cells. Here, a bakers' yeast (Saccharomyces cerevisiae) deletion mutant collection has been exploited to provide a high-throughput means of revealing the genetic basis for tolerance/susceptibility to CdS QD exposure. The deletion of 112 genes, some associated with the abiotic stress response, some with various metabolic processes, some with mitochondrial organization, some with transport and some with DNA repair, reduced the level of tolerance to CdS QDs. A gene ontology analysis highlighted the role of oxidative stress in determining the cellular response. The transformation of sensitive mutants with centromeric plasmids harbouring DNA from a wild type strain restored the wild type growth phenotype when the complemented genes encoded either HSC82, DSK2 or ALD3. The use of these simple eukaryote knock-out mutants for functional toxicogenomic analysis will inform studies focusing on higher organisms.

Increased resistance to ionizing and ultraviolet radiation in Escherichia coli JM83 is associated with a chromosomal rearrangement.

PubMed

McLean, K M; Gutman, P D; Minton, K W; Clark, E P

1992-06-01

Cells cope with radiation damage through several mechanisms: (1) increased DNA repair activity, (2) scavenging and inactivation of radiation-induced radical molecules, and (3) entry into a G0-like quiescent state. We have investigated a chromosomal rearrangement to elucidate further the molecular and genetic mechanisms underlying these phenomena. A mutant of Escherichia coli JM83 (phi 80dlacZ delta M15) was isolated that demonstrated significantly increased resistance to both ionizing and ultraviolet radiation. Surviving fractions of mutant and wild-type cells were measured following exposure to standardized doses of radiation. Increased radioresistance was directly related to a chromosomal alteration near the bacteriophage phi 80 attachment site (attB), as initially detected by the LacZ- phenotype of the isolate. Southern hybridization of chromosomal DNA from the mutant and wild-type E. coli JM83 strains indicated that a deletion had occurred. We propose that the deletion near the attB locus produces the radioresistant phenotype of the E. coli JM83 LacZ- mutant, perhaps through the alteration or inactivation of a gene or its controlling element(s).

Next generation sequencing analysis reveals that the ribonucleases RNase II, RNase R and PNPase affect bacterial motility and biofilm formation in E. coli.

PubMed

Pobre, Vânia; Arraiano, Cecília M

2015-02-14

The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome. In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms. In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color.

PubMed

Takahashi, Ryoji; Githiri, Stephen M; Hatayama, Kouta; Dubouzet, Emilyn G; Shimada, Norimoto; Aoki, Toshio; Ayabe, Shin-ichi; Iwashina, Tsukasa; Toda, Kyoko; Matsumura, Hisakazu

2007-01-01

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5' RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59-72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F(2) population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

Dominant negative mutant of ionotropic glutamate receptor subunit GluR3: implications for the role of a cysteine residue for its channel activity and pharmacological properties.

PubMed Central

Watase, K; Sekiguchi, M; Matsui, T A; Tagawa, Y; Wada, K

1997-01-01

We reported that a 33-amino-acid deletion (from tyrosine-715 to glycine-747) in a putative extracellular loop of GluR3 produced a mutant that exhibited dominant negative effects upon the functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [Sekiguchi et al. (1994) J. Biol. Chem. 269, 14559-14565]. In this study, we searched for a key residue in the dominant negative effects to explore the mechanism and examined the role of the residue in the function of the AMPA receptor. We prepared 20 GluR3 mutants with amino acid substitutions within the 33-amino-acid-region, and dominant negative effects were tested electrophysiologically in Xenopus oocytes co-expressing the mutant and normal subunits. Among the mutants, only a GluR3 mutant in which an original cysteine (Cys)-722 was replaced by alanine exhibited a dominant negative effect comparable with that of the original mutant in which the entire 33-amino-acid segment is deleted. The co-expression of the Cys-722 mutant did not inhibit the translation of normal subunits in oocytes. The Cys-722 mutant formed a functional homomeric receptor with significantly higher affinity for glutamate or kainate than a homomeric GluR3 receptor. The Cys-722 mutation greatly enhanced the sensitivity of GluR3 for aniracetam, which alters kinetic properties of AMPA receptors. The kainate-induced currents in oocytes expressing the Cys-722 mutant alone showed strong inward rectification. These results suggest that the Cys-722 in GluR3 is important for dominant negative effects and plays a crucial role in the determination of pharmacological properties in AMPA receptor function. PMID:9065754

Targeted proteome analysis of single-gene deletion strains of Saccharomyces cerevisiae lacking enzymes in the central carbon metabolism.

PubMed

Matsuda, Fumio; Kinoshita, Syohei; Nishino, Shunsuke; Tomita, Atsumi; Shimizu, Hiroshi

2017-01-01

Central carbon metabolism is controlled by modulating the protein abundance profiles of enzymes that maintain the essential systems in living organisms. In this study, metabolic adaptation mechanisms in the model organism Saccharomyces cerevisiae were investigated by direct determination of enzyme abundance levels in 30 wild type and mutant strains. We performed a targeted proteome analysis using S. cerevisiae strains that lack genes encoding the enzymes responsible for central carbon metabolism. Our analysis revealed that at least 30% of the observed variations in enzyme abundance levels could be explained by global regulatory mechanisms. A enzyme-enzyme co-abundance analysis revealed that the abundances of enzyme proteins involved in the trehalose metabolism and glycolysis changed in a coordinated manner under the control of the transcription factors for global regulation. The remaining variations were derived from local mechanisms such as a mutant-specific increase in the abundances of remote enzymes. The proteome data also suggested that, although the functional compensation of the deficient enzyme was attained by using more resources for protein biosynthesis, available resources for the biosynthesis of the enzymes responsible for central metabolism were not abundant in S. cerevisiae cells. These results showed that global and local regulation of enzyme abundance levels shape central carbon metabolism in S. cerevisiae by using a limited resource for protein biosynthesis.

Defective prohormone processing and altered pancreatic islet morphology in mice lacking active SPC2

PubMed Central

Furuta, Machi; Yano, Hideki; Zhou, An; Rouillé, Yves; Holst, Jens J.; Carroll, Raymond; Ravazzola, Mariella; Orci, Lelio; Furuta, Hiroto; Steiner, Donald F.

1997-01-01

The prohormone convertase SPC2 (PC2) participates in the processing of proinsulin, proglucagon, and a variety of other neuroendocrine precursors, acting either alone or in conjunction with the structurally related dense-core granule convertase SPC3 (PC3/PC1). We have generated a strain of mice lacking active SPC2 by introducing the neomycin resistance gene (Neor) into the third exon of the mSPC2 gene. This gene insertion results in the synthesis of an exon 3-deleted form of SPC2 that does not undergo autoactivation and is not secreted. The homozygous mutant mice appear to be normal at birth. However, they exhibit a small decrease in rate of growth. They also have chronic fasting hypoglycemia and a reduced rise in blood glucose levels during an intraperitoneal glucose tolerance test, which is consistent with a deficiency of circulating glucagon. The processing of proglucagon, prosomatostatin, and proinsulin in the alpha, delta, and beta cells, respectively, of the pancreatic islets is severely impaired. The islets in mutant mice at 3 months of age show marked hyperplasia of alpha and delta cells and a relative diminution of beta cells. SPC2-defective mice offer many possibilities for further delineating neuroendocrine precursor processing mechanisms and for exploring more fully the physiological roles of many neuropeptides and peptide hormones. PMID:9192619

Construction of an Escherichia coli strain unable to synthesize putrescine, spermidine, or cadaverine: characterization of two genes controlling lysine decarboxylase.

PubMed

Tabor, H; Hafner, E W; Tabor, C W

1980-12-01

We have previously described a polyamine-deficient strain of Escherichia coli that contained deletions in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). Although this strain completely lacked putrescine and spermidine, it was still able to grow at a slow rate indefinitely on amine-deficient media. However, these cells contained some cadaverine (1,5-diaminopentane). To rule out the possibility that the presence of cadaverine permitted the growth of this strain, we isolated a mutant (cadA) that is deficient in cadaverine biosynthesis, namely, a mutant lacking lysine decarboxylase, and transduced this cadA gene into the delta (speA-speB) delta speC delta D strain. The resultant strain had essentially no cadaverine but showed the same phenotypic characteristics as the parent. Thus, these results confirm our previous findings that the polyamines are not essential for the growth of E. coli or for the replication of bacteriophages T4 and T7. We have mapped the cadA gene at 92 min; the gene order is mel cadA groE ampA purA. A regulatory gene for lysine decarboxylase (cadR) was also obtained and mapped at 46 min; the gene order is his cdd cadR fpk gyrA.

Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms

PubMed Central

Antony, N.; McDougall, A. R.; Mantamadiotis, T.; Cole, T. J.; Bird, A. D.

2016-01-01

During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1−/− mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1−/− mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1−/− mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development. PMID:27150575

Streptococcal heme binding protein (Shp) promotes virulence and contributes to the pathogenesis of group A Streptococcus infection.

PubMed

Zhang, Xiaolan; Lu, Chunmei; Zhang, Fengmin; Song, Yingli; Cai, Minghui; Zhu, Hui

2017-09-29

Streptococcal heme binding protein (Shp) is involved in the process of heme acquisition in group A Streptococcus (GAS). However, no research thus far has examined the contribution of Shp to the virulence of GAS. To this end, we generated an isogenic strain lacking the shp gene (Δshp) and its complemented strain (Δshp-c) using the parent strain MGAS5005 (WT). Deletion of shp increased survival rates and neutrophil recruitment and reduced skin lesion sizes and GAS loads in the blood and the liver, lung, kidney and spleen in subcutaneous infections of mice. These results indicate that Shp significantly contributes to the skin and systemic invasion of GAS. The growth of the Δshp mutant was significantly slower than MGAS5005 and Δshp-c than in non-immune human blood and in incubation with isolated rat neutrophils. Microarray transcriptional analyses found no alteration in expression of virulence genes, indicating that the phenotype of the Δshp mutant was directly linked to the lack of Shp. The findings indicate that Shp significantly contributes to GAS skin invasion, systemic infection and virulence and that these contributions of Shp are mediated by the effects of Shp on systemic GAS growth and neutrophil responses. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Inappropriate Expression of an NLP Effector in Colletotrichum orbiculare Impairs Infection on Cucurbitaceae Cultivars via Plant Recognition of the C-Terminal Region.

PubMed

Azmi, Nur Sabrina Ahmad; Singkaravanit-Ogawa, Suthitar; Ikeda, Kyoko; Kitakura, Saeko; Inoue, Yoshihiro; Narusaka, Yoshihiro; Shirasu, Ken; Kaido, Masanori; Mise, Kazuyuki; Takano, Yoshitaka

2018-01-01

The hemibiotrophic pathogen Colletotrichum orbiculare preferentially expresses a necrosis and ethylene-inducing peptide 1 (Nep1)-like protein named NLP1 during the switch to necrotrophy. Here, we report that the constitutive expression of NLP1 in C. orbiculare blocks pathogen infection in multiple Cucurbitaceae cultivars via their enhanced defense responses. NLP1 has a cytotoxic activity that induces cell death in Nicotiana benthamiana. However, C. orbiculare transgenic lines constitutively expressing a mutant NLP1 lacking the cytotoxic activity still failed to infect cucumber, indicating no clear relationship between cytotoxic activity and the NLP1-dependent enhanced defense. NLP1 also possesses the microbe-associated molecular pattern (MAMP) sequence called nlp24, recognized by Arabidopsis thaliana at its central region, similar to NLPs of other pathogens. Surprisingly, inappropriate expression of a mutant NLP1 lacking the MAMP signature is also effective for blocking pathogen infection, uncoupling the infection block from the corresponding MAMP. Notably, the deletion analyses of NLP1 suggested that the C-terminal region of NLP1 is critical to enhance defense in cucumber. The expression of mCherry fused with the C-terminal 32 amino acids of NLP1 was enough to trigger the defense of cucurbits, revealing that the C-terminal region of the NLP1 protein is recognized by cucurbits and, then, terminates C. orbiculare infection.

Immune Escape Variants of H9N2 Influenza Viruses Containing Deletions at the Hemagglutinin Receptor Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic Characteristics.

PubMed

Peacock, Thomas P; Benton, Donald J; James, Joe; Sadeyen, Jean-Remy; Chang, Pengxiang; Sealy, Joshua E; Bryant, Juliet E; Martin, Stephen R; Shelton, Holly; Barclay, Wendy S; Iqbal, Munir

2017-07-15

H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence. IMPORTANCE Avian influenza viruses, such as H9N2, cause disease in poultry as well as occasionally infecting humans and are therefore considered viruses with pandemic potential. Many countries have introduced vaccination of poultry to try to control the disease burden; however, influenza viruses are able to rapidly evolve to escape immune pressure in a process known as "antigenic drift." Previously, we experimentally generated antigenic-drift variants in the laboratory, and here, we test our "drifted" viruses to assess their zoonotic infection characteristics and transmissibility in chickens. We found that the drifted viruses were able to infect and be transmitted between chickens and showed increased binding to human-like receptors. However, the drift mutant viruses displayed reduced stability, and we predict that they are unlikely to be transmitted from human to human and cause an influenza pandemic. These results demonstrate the complex relationship between antigenic drift and the potential of avian influenza viruses to infect humans. Copyright © 2017 Peacock et al.

Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

PubMed

Honda, Ryo; Kuwata, Kazuo

2018-02-01

Amyloid fibrils are filamentous protein aggregates associated with the pathogenesis of a wide variety of human diseases. The formation of such aggregates typically follows nucleation-dependent kinetics, wherein the assembly and structural conversion of amyloidogenic proteins into oligomeric aggregates (nuclei) is the rate-limiting step of the overall reaction. In this study, we sought to gain structural insights into the oligomeric nuclei of the human prion protein (PrP) by preparing a series of deletion mutants lacking 14-44 of the C-terminal 107 residues of PrP and examined the kinetics and thermodynamics of these mutants in amyloid formation. An analysis of the experimental data using the concepts of the Φ-value analysis indicated that the helix 2 region (residues 168-196) acquires an amyloid-like β-sheet during nucleation, whereas the other regions preserves a relatively disordered structure in the nuclei. This finding suggests that the helix 2 region serves as the nucleation site for the assembly of amyloid fibrils.-Honda, R., Kuwata, K. Evidence for a central role of PrP helix 2 in the nucleation of amyloid fibrils.

Phenotypic and Genetic Analysis of Clock, a New Circadian Rhythm Mutant in Drosophila Melanogaster

PubMed Central

Dushay, M. S.; Konopka, R. J.; Orr, D.; Greenacre, M. L.; Kyriacou, C. P.; Rosbash, M.; Hall, J. C.

1990-01-01

Clock is a semidominant X-linked mutation that results in shortening the period of Drosophila melanogaster's free-running locomotor activity rhythm from ca. 24.0 to ca. 22.5 hr. This mutation similarly shortened the phase response curve, determined by resetting activity rhythms with light pulses. Eclosion peaks for Clk cultures were separated by only 22.5 hr instead of the normal 24 hr. Clk was mapped close to, but separable from, another rhythm mutation--period(01)--by recombination. The estimated distance between these two mutations was short enough to suggest that Clk could be a per allele. If this is the case, the new mutant is unique in that it, unlike other per variants, is associated with essentially normal 1-min courtship song rhythms when Clk is expressed in males. Also, the new rhythm variant could not, in contrast to a short-period per mutation, have its effects on free-running activity rhythms uncovered by deletions. This result, and the lack of coverage of Clk's effects by duplications, suggest that it is not a simple hypomorphic or amorphic mutation. PMID:2116357

Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.

PubMed

Feyerabend, Thorsten B; Weiser, Anne; Tietz, Annette; Stassen, Michael; Harris, Nicola; Kopf, Manfred; Radermacher, Peter; Möller, Peter; Benoist, Christophe; Mathis, Diane; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2011-11-23

Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy. Copyright © 2011 Elsevier Inc. All rights reserved.

Chimeric Saccharomyces cerevisiae Msh6 protein with an Msh3 mispair-binding domain combines properties of both proteins.

PubMed

Shell, Scarlet S; Putnam, Christopher D; Kolodner, Richard D

2007-06-26

Msh2-Msh3 and Msh2-Msh6 are two partially redundant mispair-recognition complexes that initiate mismatch repair in eukaryotes. Crystal structures of the prokaryotic homolog MutS suggest the mechanism by which Msh6 interacts with mispairs because key mispair-contacting residues are conserved in these two proteins. Because Msh3 lacks these conserved residues, we constructed a series of mutants to investigate the requirements for mispair interaction by Msh3. We found that a chimeric protein in which the mispair-binding domain (MBD) of Msh6 was replaced by the equivalent domain of Msh3 was functional for mismatch repair. This chimera possessed the mispair-binding specificity of Msh3 and revealed that communication between the MBD and the ATPase domain is conserved between Msh2-Msh3 and Msh2-Msh6. Further, the chimeric protein retained Msh6-like properties with respect to genetic interactions with the MutL homologs and an Msh2 MBD deletion mutant, indicating that Msh3-like behaviors beyond mispair specificity are not features controlled by the MBD.

Zernike phase contrast cryo-electron tomography of sodium-driven flagellar hook-basal bodies from Vibrio alginolyticus.

PubMed

Hosogi, Naoki; Shigematsu, Hideki; Terashima, Hiroyuki; Homma, Michio; Nagayama, Kuniaki

2011-01-01

Vibrio alginolyticus use flagella to swim. A flagellum consists of a filament, hook and basal body. The basal body is made up of a rod and several ring structures. This study investigates the structure of the T ring which is a unique component of the V. alginolyticus sodium ion-driven flagellar basal body. Using Zernike phase contrast (ZPC) cryo-electron tomography, we compared the 3D structures of purified hook-basal bodies (HBB) from a wild-type strain (KK148) and a deletion mutant lacking MotX and MotY (TH3), which are thought to form the T ring. ZPC images of HBBs had highly improved signal-to-noise ratio compared to conventional phase contrast images. We observed the outline of the HBBs from strains KK148 and TH3, and the TH3 mutant was missing its T ring. In the wild-type strain, the T ring was beneath the LP ring and seemed to form a ring shape with diameter of 32 nm. Copyright © 2010 Elsevier Inc. All rights reserved.

Mycobacterium tuberculosis PhoY Proteins Promote Persister Formation by Mediating Pst/SenX3-RegX3 Phosphate Sensing.

PubMed

Namugenyi, Sarah B; Aagesen, Alisha M; Elliott, Sarah R; Tischler, Anna D

2017-07-11

The Mycobacterium tuberculosis phosphate-specific transport (Pst) system controls gene expression in response to phosphate availability by inhibiting the activation of the SenX3-RegX3 two-component system under phosphate-rich conditions, but the mechanism of communication between these systems is unknown. In Escherichia coli , inhibition of the two-component system PhoR-PhoB under phosphate-rich conditions requires both the Pst system and PhoU, a putative adaptor protein. E. coli PhoU is also involved in the formation of persisters, a subpopulation of phenotypically antibiotic-tolerant bacteria. M. tuberculosis encodes two PhoU orthologs, PhoY1 and PhoY2. We generated phoY single- and double-deletion mutants and examined the expression of RegX3-regulated genes by quantitative reverse transcription-PCR (qRT-PCR). Gene expression was increased only in the Δ phoY1 Δ phoY2 double mutant and could be restored to the wild-type level by complementation with either phoY1 or phoY2 or by deletion of regX3 These data suggest that the PhoY proteins function redundantly to inhibit SenX3-RegX3 activation. We analyzed the frequencies of antibiotic-tolerant persister variants in the phoY mutants using several antibiotic combinations. Persister frequency was decreased at least 40-fold in the Δ phoY1 Δ phoY2 mutant compared to the frequency in the wild type, and this phenotype was RegX3 dependent. A Δ pstA1 mutant lacking a Pst system transmembrane component exhibited a similar RegX3-dependent decrease in persister frequency. In aerosol-infected mice, the Δ phoY1 Δ phoY2 and Δ pstA1 mutants were more susceptible to treatment with rifampin but not isoniazid. Our data demonstrate that disrupting phosphate sensing mediated by the PhoY proteins and the Pst system enhances the susceptibility of M. tuberculosis to antibiotics both in vitro and during infection. IMPORTANCE Persister variants, subpopulations of bacteria that are phenotypically antibiotic tolerant, contribute to the lengthy treatment times required to cure Mycobacterium tuberculosis infection, but the molecular mechanisms governing their formation and maintenance are poorly characterized. Here, we demonstrate that a phosphate-sensing signal transduction system, comprising the Pst phosphate transporter, the two-component system SenX3-RegX3, and functionally redundant PhoY proteins that mediate signaling between Pst and SenX3-RegX3, influences persister formation. Activation of RegX3 by deletion of the phoY genes or a Pst system component resulted in decreased persister formation in vitro Activated RegX3 also limited persister formation during growth under phosphate-limiting conditions. Importantly, increased susceptibility to the front-line drug rifampin was also observed in a mouse infection model. Thus, the M. tuberculosis phosphate-sensing signal transduction system contributes to antibiotic tolerance and is a potential target for the development of novel therapeutics that may shorten the duration of tuberculosis treatment. Copyright © 2017 Namugenyi et al.

Enamel-free teeth: Tbx1 deletion affects amelogenesis in rodent incisors.

PubMed

Catón, Javier; Luder, Hans-Ulrich; Zoupa, Maria; Bradman, Matthew; Bluteau, Gilles; Tucker, Abigail S; Klein, Ophir; Mitsiadis, Thimios A

2009-04-15

TBX1 is a principal candidate gene for DiGeorge syndrome, a developmental anomaly that affects the heart, thymus, parathyroid, face, and teeth. A mouse model carrying a deletion in a functional region of the Tbx1 gene has been extensively used to study anomalies related to this syndrome. We have used the Tbx1 null mouse to understand the tooth phenotype reported in patients afflicted by DiGeorge syndrome. Because of the early lethality of the Tbx1-/- mice, we used long-term culture techniques that allow the unharmed growth of incisors until their full maturity. All cultured incisors of Tbx1-/- mice were hypoplastic and lacked enamel, while thorough histological examinations demonstrated the complete absence of ameloblasts. The absence of enamel is preceded by a decrease in proliferation of the ameloblast precursor cells and a reduction in amelogenin gene expression. The cervical loop area of the incisor, which contains the niche for the epithelial stem cells, was either severely reduced or completely missing in mutant incisors. In contrast, ectopic expression of Tbx1 was observed in incisors from mice with upregulated Fibroblast Growth Factor signalling and was closely linked to ectopic enamel formation and deposition in these incisors. These results demonstrate that Tbx1 is essential for the maintenance of ameloblast progenitor cells in rodent incisors and that its deletion results in the absence of enamel formation.