Science.gov

Sample records for agr mutant strains

  1. Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis.

    PubMed Central

    Cheung, A L; Eberhardt, K J; Chung, E; Yeaman, M R; Sullam, P M; Ramos, M; Bayer, A S

    1994-01-01

    Microbial pathogenicity in Staphylococcus aureus is a complex process involving a number of virulence genes that are regulated by global regulatory systems including sar and agr. To evaluate the roles of these two loci in virulence, we constructed sar-/agr- mutants of strains RN6390 and RN450 and compared their phenotypic profiles to the corresponding single sar- and agr- mutants and parents. The secretion of all hemolysins was absent in the sar-/agr- mutants while residual beta-hemolysin activity remained in single agr- mutants. The fibronectin binding capacity was significantly diminished in both single sar- mutants and double mutants when compared with parents while the reduction in fibrinogen binding capacity in the double mutants was modest. In the rabbit endocarditis model, there was a significant decrease in both infectivity rates and intravegetation bacterial densities with the double mutant as compared to the parent (RN6390) at 10(3)-10(6) CFU inocula despite comparable levels of early bacteremia among various challenge groups. Notably, fewer bacteria in the double mutant group adhered to valvular vegetations at 30 min after challenge (10(6) CFU) than the parent group. These studies suggest that both the sar and agr loci are involved in initial valvular adherence, intravegetation persistence and multiplication of S. aureus in endocarditis. Images PMID:7962526

  2. Staphylococcal superantigen-like genes, ssl5 and ssl8, are positively regulated by Sae and negatively by Agr in the Newman strain.

    PubMed

    Pantrangi, Madhulatha; Singh, Vineet K; Wolz, Christiane; Shukla, Sanjay K

    2010-07-01

    Some of the staphylococcal superantigen-like (SSL) proteins SSL5, SSL7, SSL9, and SSL11 act as immunomodulatory proteins in Staphylococcus aureus. However, little is known about their regulatory mechanisms. We determined the expression levels of ssl5 and ssl8 in seven clinically important S. aureus strains and their regulatory mechanisms in the Newman strain, which had the highest ssl5 and ssl8 expression. Independent comparisons of ssl5 or ssl8 coding and upstream sequences in these strains identified multiple haplotypes that did not correlate with the differential expression of ssl5 and ssl8, suggesting the role of additional regulatory elements. Using knockout mutant strains of known S. aureus global regulators such as Agr, Sae, and SigB in the Newman strain, we showed that both ssl5 and ssl8 were induced by Sae and repressed by Agr, suggesting that Sae and Agr are the positive and the negative regulators, respectively, of these two ssl genes. Moreover, we observed upregulation of sae in the agr mutant and upregulation of agr in the sae mutant compared with the isogenic Newman strain, suggesting that the Agr and Sae may be inhibiting each other. The SigB mutation did not affect ssl5 and ssl8 expression, but they were downregulated in the agr/sigB double mutant, indicating that SigB probably acts synergistically with Agr in their upregulation.

  3. Genome Sequence of Streptomyces auratus Strain AGR0001, a Phoslactomycin-Producing Actinomycete

    PubMed Central

    Han, Xiulin; Li, Minggang; Ding, Zhanggui; Zhao, Jiangyuan; Ji, Kaiyan

    2012-01-01

    Streptomyces auratus strain AGR0001 produces neophoslactomycin A, a novel analog of phoslactomycin that possesses potent activity against some phytopathogenic fungi. Here, the draft genome sequence of S. auratus strain AGR0001 is presented, which would provide insight into the biosynthetic mechanism of neophoslactomycin A. PMID:22965094

  4. agr-Mediated Dispersal of Staphylococcus aureus Biofilms

    PubMed Central

    Boles, Blaise R.; Horswill, Alexander R.

    2008-01-01

    The agr quorum-sensing system of Staphylococcus aureus modulates the expression of virulence factors in response to autoinducing peptides (AIPs). Recent studies have suggested a role for the agr system in S. aureus biofilm development, as agr mutants exhibit a high propensity to form biofilms, and cells dispersing from a biofilm have been observed displaying an active agr system. Here, we report that repression of agr is necessary to form a biofilm and that reactivation of agr in established biofilms through AIP addition or glucose depletion triggers detachment. Inhibitory AIP molecules did not induce detachment and an agr mutant was non-responsive, indicating a dependence on a functional, active agr system for dispersal. Biofilm detachment occurred in multiple S. aureus strains possessing divergent agr systems, suggesting it is a general S. aureus phenomenon. Importantly, detachment also restored sensitivity of the dispersed cells to the antibiotic rifampicin. Proteinase K inhibited biofilm formation and dispersed established biofilms, suggesting agr-mediated detachment occurred in an ica-independent manner. Consistent with a protease-mediated mechanism, increased levels of serine proteases were detected in detaching biofilm effluents, and the serine protease inhibitor PMSF reduced the degree of agr-mediated detachment. Through genetic analysis, a double mutant in the agr-regulated Aur metalloprotease and the SplABCDEF serine proteases displayed minimal extracellular protease activity, improved biofilm formation, and a strongly attenuated detachment phenotype. These findings indicate that induction of the agr system in established S. aureus biofilms detaches cells and demonstrate that the dispersal mechanism requires extracellular protease activity. PMID:18437240

  5. Mutant strain of C. acetobutylicum and process for making butanol

    DOEpatents

    Jain, Mahendra K.; Beacom, Daniel; Datta, Rathin

    1993-01-01

    A biologically pure asporogenic mutant of Clostridium acetobutylicum is produced by growing sporogenic C. acetobutylicum ATCC 4259 and treating the parent strain with ethane methane sulfonate. The mutant which as been designated C. acetobutylicum ATCC 55025 is useful in an improved ABE fermentation process, and produces high concentrations of butanol and total solvents.

  6. Existence of two groups of Staphylococcus aureus strains isolated from bovine mastitis based on biofilm formation, intracellular survival, capsular profile and agr-typing.

    PubMed

    Bardiau, Marjorie; Caplin, Jonathan; Detilleux, Johann; Graber, Hans; Moroni, Paolo; Taminiau, Bernard; Mainil, Jacques G

    2016-03-15

    Staphylococcus (S.) aureus is recognised worldwide as an important pathogen causing contagious acute and chronic bovine mastitis. Chronic mastitis account for a significant part of all bovine cases and represent an important economic problem for dairy producers. Several properties (biofilm formation, intracellular survival, capsular expression and group agr) are thought to be associated with this chronic status. In a previous study, we found the existence of two groups of strains based on the association of these features. The aim of the present work was to confirm on a large international and non-related collection of strains the existence of these clusters and to associate them with case history records. In addition, the genomes of eight strains were sequenced to study the genomic differences between strains of each cluster. The results confirmed the existence of both groups based on capsular typing, intracellular survival and agr-typing: strains cap8-positive, belonging to agr group II, showing a low invasion rate and strains cap5-positive, belonging to agr group I, showing a high invasion rate. None of the two clusters were associated with the chronic status of the cow. When comparing the genomes of strains belonging to both clusters, the genes specific to the group "cap5-agrI" would suggest that these strains are better adapted to live in hostile environment. The existence of these two groups is highly important as they may represent two clusters that are adapted differently to the host and/or the surrounding environment.

  7. The Agr communication system provides a benefit to the populations of Listeria monocytogenes in soil

    PubMed Central

    Vivant, Anne-Laure; Garmyn, Dominique; Gal, Laurent; Piveteau, Pascal

    2014-01-01

    In this study, we investigated whether the Agr communication system of the pathogenic bacterium Listeria monocytogenes was involved in adaptation and competitiveness in soil. Alteration of the ability to communicate, either by deletion of the gene coding the response regulator AgrA (response-negative mutant) or the signal pro-peptide AgrD (signal-negative mutant), did not affect population dynamics in soil that had been sterilized but survival was altered in biotic soil suggesting that the Agr system of L. monocytogenes was involved to face the complex soil biotic environment. This was confirmed by a set of co-incubation experiments. The fitness of the response-negative mutant was lower either in the presence or absence of the parental strain but the fitness of the signal-negative mutant depended on the strain with which it was co-incubated. The survival of the signal-negative mutant was higher when co-cultured with the parental strain than when co-cultured with the response-negative mutant. These results showed that the ability to respond to Agr communication provided a benefit to listerial cells to compete. These results might also indicate that in soil, the Agr system controls private goods rather than public goods. PMID:25414837

  8. Applications of mutant yeast strains with low glycogen storage capability

    NASA Technical Reports Server (NTRS)

    Petersen, G. R.; Schubert, W. W.; Stokes, B. O.

    1981-01-01

    Several strains of Hansenula polymorpha were selected for possible low glycogen storage characteristics based on a selective I2 staining procedure. The levels of storage carbohydrates in the mutant strains were found to be 44-70% of the levels in the parent strain for cultures harvested in stationary phase. Similar differences generally were not found for cells harvested in exponential phase. Yeast strains deficient in glycogen storage capability are valuable in increasing the relative protein value of microbial biomass and also may provide significant cost savings in substrate utilization in fermentative processes.

  9. Identification of the agr Peptide of Listeria monocytogenes.

    PubMed

    Zetzmann, Marion; Sánchez-Kopper, Andrés; Waidmann, Mark S; Blombach, Bastian; Riedel, Christian U

    2016-01-01

    Listeria monocytogenes (Lm) is an important food-borne human pathogen that is able to strive under a wide range of environmental conditions. Its accessory gene regulator (agr) system was shown to impact on biofilm formation and virulence and has been proposed as one of the regulatory mechanisms involved in adaptation to these changing environments. The Lm agr operon is homologous to the Staphylococcus aureus system, which includes an agrD-encoded autoinducing peptide that stimulates expression of the agr genes via the AgrCA two-component system and is required for regulation of target genes. The aim of the present study was to identify the native autoinducing peptide (AIP) of Lm using a luciferase reporter system in wildtype and agrD deficient strains, rational design of synthetic peptides and mass spectrometry. Upon deletion of agrD, luciferase reporter activity driven by the PII promoter of the agr operon was completely abolished and this defect was restored by co-cultivation of the agrD-negative reporter strain with a producer strain. Based on the sequence and structures of known AIPs of other organisms, a set of potential Lm AIPs was designed and tested for PII-activation. This led to the identification of a cyclic pentapeptide that was able to induce PII-driven luciferase reporter activity and restore defective invasion of the agrD deletion mutant into Caco-2 cells. Analysis of supernatants of a recombinant Escherichia coli strain expressing AgrBD identified a peptide identical in mass and charge to the cyclic pentapeptide. The Lm agr system is specific for this pentapeptide since the AIP of Lactobacillus plantarum, which also is a pentapeptide yet with different amino acid sequence, did not induce PII activity. In summary, the presented results provide further evidence for the hypothesis that the agrD gene of Lm encodes a secreted AIP responsible for autoregulation of the agr system of Lm. Additionally, the structure of the native Lm AIP was identified. PMID

  10. Identification of the agr Peptide of Listeria monocytogenes

    PubMed Central

    Zetzmann, Marion; Sánchez-Kopper, Andrés; Waidmann, Mark S.; Blombach, Bastian; Riedel, Christian U.

    2016-01-01

    Listeria monocytogenes (Lm) is an important food-borne human pathogen that is able to strive under a wide range of environmental conditions. Its accessory gene regulator (agr) system was shown to impact on biofilm formation and virulence and has been proposed as one of the regulatory mechanisms involved in adaptation to these changing environments. The Lm agr operon is homologous to the Staphylococcus aureus system, which includes an agrD-encoded autoinducing peptide that stimulates expression of the agr genes via the AgrCA two-component system and is required for regulation of target genes. The aim of the present study was to identify the native autoinducing peptide (AIP) of Lm using a luciferase reporter system in wildtype and agrD deficient strains, rational design of synthetic peptides and mass spectrometry. Upon deletion of agrD, luciferase reporter activity driven by the PII promoter of the agr operon was completely abolished and this defect was restored by co-cultivation of the agrD-negative reporter strain with a producer strain. Based on the sequence and structures of known AIPs of other organisms, a set of potential Lm AIPs was designed and tested for PII-activation. This led to the identification of a cyclic pentapeptide that was able to induce PII-driven luciferase reporter activity and restore defective invasion of the agrD deletion mutant into Caco-2 cells. Analysis of supernatants of a recombinant Escherichia coli strain expressing AgrBD identified a peptide identical in mass and charge to the cyclic pentapeptide. The Lm agr system is specific for this pentapeptide since the AIP of Lactobacillus plantarum, which also is a pentapeptide yet with different amino acid sequence, did not induce PII activity. In summary, the presented results provide further evidence for the hypothesis that the agrD gene of Lm encodes a secreted AIP responsible for autoregulation of the agr system of Lm. Additionally, the structure of the native Lm AIP was identified. PMID

  11. A point mutation in AgrC determines cytotoxic or colonizing properties associated with phenotypic variants of ST22 MRSA strains

    PubMed Central

    Mairpady Shambat, Srikanth; Siemens, Nikolai; Monk, Ian R.; Mohan, Disha B.; Mukundan, Santhosh; Krishnan, Karthickeyan Chella; Prabhakara, Sushma; Snäll, Johanna; Kearns, Angela; Vandenesch, Francois; Svensson, Mattias; Kotb, Malak; Gopal, Balasubramanian; Arakere, Gayathri; Norrby-Teglund, Anna

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of skin and soft tissue infections. One of the highly successful and rapidly disseminating clones is MRSA ST22 commonly associated with skin tropism. Here we show that a naturally occurring single amino acid substitution (tyrosine to cysteine) at position 223 of AgrC determines starkly different ST22 S. aureus virulence phenotypes, e.g. cytotoxic or colonizing, as evident in both in vitro and in vivo skin infections. Y223C amino acid substitution destabilizes AgrC-AgrA interaction leading to a colonizing phenotype characterized by upregulation of bacterial surface proteins. The colonizing phenotype strains cause less severe skin tissue damage, show decreased susceptibility towards the antimicrobial LL-37 and induce autophagy. In contrast, cytotoxic strains with tyrosine at position 223 of AgrC cause infections characterized by inflammasome activation and severe skin tissue pathology. Taken together, the study demonstrates how a single amino acid substitution in the histidine kinase receptor AgrC of ST22 strains determines virulence properties and infection outcome. PMID:27511873

  12. A point mutation in AgrC determines cytotoxic or colonizing properties associated with phenotypic variants of ST22 MRSA strains.

    PubMed

    Mairpady Shambat, Srikanth; Siemens, Nikolai; Monk, Ian R; Mohan, Disha B; Mukundan, Santhosh; Krishnan, Karthickeyan Chella; Prabhakara, Sushma; Snäll, Johanna; Kearns, Angela; Vandenesch, Francois; Svensson, Mattias; Kotb, Malak; Gopal, Balasubramanian; Arakere, Gayathri; Norrby-Teglund, Anna

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of skin and soft tissue infections. One of the highly successful and rapidly disseminating clones is MRSA ST22 commonly associated with skin tropism. Here we show that a naturally occurring single amino acid substitution (tyrosine to cysteine) at position 223 of AgrC determines starkly different ST22 S. aureus virulence phenotypes, e.g. cytotoxic or colonizing, as evident in both in vitro and in vivo skin infections. Y223C amino acid substitution destabilizes AgrC-AgrA interaction leading to a colonizing phenotype characterized by upregulation of bacterial surface proteins. The colonizing phenotype strains cause less severe skin tissue damage, show decreased susceptibility towards the antimicrobial LL-37 and induce autophagy. In contrast, cytotoxic strains with tyrosine at position 223 of AgrC cause infections characterized by inflammasome activation and severe skin tissue pathology. Taken together, the study demonstrates how a single amino acid substitution in the histidine kinase receptor AgrC of ST22 strains determines virulence properties and infection outcome. PMID:27511873

  13. Staphylococcus epidermidis agr quorum-sensing system: signal identification, cross talk, and importance in colonization.

    PubMed

    Olson, Michael E; Todd, Daniel A; Schaeffer, Carolyn R; Paharik, Alexandra E; Van Dyke, Michael J; Büttner, Henning; Dunman, Paul M; Rohde, Holger; Cech, Nadja B; Fey, Paul D; Horswill, Alexander R

    2014-10-01

    Staphylococcus epidermidis is an opportunistic pathogen that is one of the leading causes of medical device infections. Global regulators like the agr quorum-sensing system in this pathogen have received a limited amount of attention, leaving important questions unanswered. There are three agr types in S. epidermidis strains, but only one of the autoinducing peptide (AIP) signals has been identified (AIP-I), and cross talk between agr systems has not been tested. We structurally characterized all three AIP types using mass spectrometry and discovered that the AIP-II and AIP-III signals are 12 residues in length, making them the largest staphylococcal AIPs identified to date. S. epidermidis agr reporter strains were developed for each system, and we determined that cross-inhibitory interactions occur between the agr type I and II systems and between the agr type I and III systems. In contrast, no cross talk was observed between the type II and III systems. To further understand the outputs of the S. epidermidis agr system, an RNAIII mutant was constructed, and microarray studies revealed that exoenzymes (Ecp protease and Geh lipase) and low-molecular-weight toxins were downregulated in the mutant. Follow-up analysis of Ecp confirmed the RNAIII is required to induce protease activity and that agr cross talk modulates Ecp activity in a manner that mirrors the agr reporter results. Finally, we demonstrated that the agr system enhances skin colonization by S. epidermidis using a porcine model. This work expands our knowledge of S. epidermidis agr system function and will aid future studies on cell-cell communication in this important opportunistic pathogen.

  14. Staphylococcus epidermidis agr Quorum-Sensing System: Signal Identification, Cross Talk, and Importance in Colonization

    PubMed Central

    Olson, Michael E.; Todd, Daniel A.; Schaeffer, Carolyn R.; Paharik, Alexandra E.; Van Dyke, Michael J.; Büttner, Henning; Dunman, Paul M.; Rohde, Holger; Cech, Nadja B.; Fey, Paul D.

    2014-01-01

    Staphylococcus epidermidis is an opportunistic pathogen that is one of the leading causes of medical device infections. Global regulators like the agr quorum-sensing system in this pathogen have received a limited amount of attention, leaving important questions unanswered. There are three agr types in S. epidermidis strains, but only one of the autoinducing peptide (AIP) signals has been identified (AIP-I), and cross talk between agr systems has not been tested. We structurally characterized all three AIP types using mass spectrometry and discovered that the AIP-II and AIP-III signals are 12 residues in length, making them the largest staphylococcal AIPs identified to date. S. epidermidis agr reporter strains were developed for each system, and we determined that cross-inhibitory interactions occur between the agr type I and II systems and between the agr type I and III systems. In contrast, no cross talk was observed between the type II and III systems. To further understand the outputs of the S. epidermidis agr system, an RNAIII mutant was constructed, and microarray studies revealed that exoenzymes (Ecp protease and Geh lipase) and low-molecular-weight toxins were downregulated in the mutant. Follow-up analysis of Ecp confirmed the RNAIII is required to induce protease activity and that agr cross talk modulates Ecp activity in a manner that mirrors the agr reporter results. Finally, we demonstrated that the agr system enhances skin colonization by S. epidermidis using a porcine model. This work expands our knowledge of S. epidermidis agr system function and will aid future studies on cell-cell communication in this important opportunistic pathogen. PMID:25070736

  15. CodY-Mediated Regulation of the Staphylococcus aureus Agr System Integrates Nutritional and Population Density Signals

    PubMed Central

    Roux, Agnès; Todd, Daniel A.; Velázquez, Jose V.; Cech, Nadja B.

    2014-01-01

    The Staphylococcus aureus Agr system regulates virulence gene expression by responding to cell population density (quorum sensing). When an extracellular peptide signal (AIP-III in strain UAMS-1, used for these experiments) reaches a concentration threshold, the AgrC-AgrA two-component regulatory system is activated through a cascade of phosphorylation events, leading to induction of the divergently transcribed agrBDCA operon and the RNAIII gene. RNAIII is a posttranscriptional regulator of numerous metabolic and pathogenesis genes. CodY, a global regulatory protein, is known to repress agrBDCA and RNAIII transcription during exponential growth in rich medium, but the mechanism of this regulation has remained elusive. Here we report that phosphorylation of AgrA by the AgrC protein kinase is required for the overexpression of the agrBDCA operon and the RNAIII gene in a codY mutant during the exponential-growth phase, suggesting that the quorum-sensing system, which normally controls AgrC activation, is active even in exponential-phase cells in the absence of CodY. In part, such premature expression of RNAIII was attributable to higher-than-normal accumulation of AIP-III in a codY mutant strain, as determined using ultrahigh-performance liquid chromatography coupled to mass spectrometry. Although CodY is a strong repressor of the agr locus, CodY bound only weakly to the agrBDCA-RNAIII promoter region, suggesting that direct regulation by CodY is unlikely to be the principal mechanism by which CodY regulates agr and RNAIII expression. Taken together, these results strongly suggest that cell population density signals inducing virulence gene expression can be overridden by nutrient availability, a condition monitored by CodY. PMID:24391052

  16. XerC Contributes to Diverse Forms of Staphylococcus aureus Infection via agr-Dependent and agr-Independent Pathways.

    PubMed

    Atwood, Danielle N; Beenken, Karen E; Loughran, Allister J; Meeker, Daniel G; Lantz, Tamara L; Graham, Justin W; Spencer, Horace J; Smeltzer, Mark S

    2016-04-01

    We demonstrate that mutation of xerC, which reportedly encodes a homologue of an Escherichia coli recombinase, limits biofilm formation in the methicillin-resistant Staphylococcus aureus strain LAC and the methicillin-sensitive strain UAMS-1. This was not due to the decreased production of the polysaccharide intracellular adhesin (PIA) in either strain because the amount of PIA was increased in a UAMS-1xerC mutant and undetectable in both LAC and its isogenic xerC mutant. Mutation of xerC also resulted in the increased production of extracellular proteases and nucleases in both LAC and UAMS-1, and limiting the production of either class of enzymes increased biofilm formation in the isogenic xerC mutants. More importantly, the limited capacity to form a biofilm was correlated with increased antibiotic susceptibility in both strains in the context of an established biofilm in vivo. Mutation of xerC also attenuated virulence in a murine bacteremia model, as assessed on the basis of the bacterial loads in internal organs and overall lethality. It also resulted in the decreased accumulation of alpha toxin and the increased accumulation of protein A. These findings suggest that xerC may impact the functional status of agr. This was confirmed by demonstrating the reduced accumulation of RNAIII and AgrA in LAC and UAMS-1xerC mutants. However, this cannot account for the biofilm-deficient phenotype of xerC mutants because mutation of agr did not limit biofilm formation in either strain. These results demonstrate that xerC contributes to biofilm-associated infections and acute bacteremia and that this is likely due to agr-independent and -dependent pathways, respectively.

  17. XerC Contributes to Diverse Forms of Staphylococcus aureus Infection via agr-Dependent and agr-Independent Pathways

    PubMed Central

    Beenken, Karen E.; Loughran, Allister J.; Meeker, Daniel G.; Lantz, Tamara L.; Graham, Justin W.; Spencer, Horace J.

    2016-01-01

    We demonstrate that mutation of xerC, which reportedly encodes a homologue of an Escherichia coli recombinase, limits biofilm formation in the methicillin-resistant Staphylococcus aureus strain LAC and the methicillin-sensitive strain UAMS-1. This was not due to the decreased production of the polysaccharide intracellular adhesin (PIA) in either strain because the amount of PIA was increased in a UAMS-1 xerC mutant and undetectable in both LAC and its isogenic xerC mutant. Mutation of xerC also resulted in the increased production of extracellular proteases and nucleases in both LAC and UAMS-1, and limiting the production of either class of enzymes increased biofilm formation in the isogenic xerC mutants. More importantly, the limited capacity to form a biofilm was correlated with increased antibiotic susceptibility in both strains in the context of an established biofilm in vivo. Mutation of xerC also attenuated virulence in a murine bacteremia model, as assessed on the basis of the bacterial loads in internal organs and overall lethality. It also resulted in the decreased accumulation of alpha toxin and the increased accumulation of protein A. These findings suggest that xerC may impact the functional status of agr. This was confirmed by demonstrating the reduced accumulation of RNAIII and AgrA in LAC and UAMS-1 xerC mutants. However, this cannot account for the biofilm-deficient phenotype of xerC mutants because mutation of agr did not limit biofilm formation in either strain. These results demonstrate that xerC contributes to biofilm-associated infections and acute bacteremia and that this is likely due to agr-independent and -dependent pathways, respectively. PMID:26857575

  18. Revealing Differences in Metabolic Flux Distributions between a Mutant Strain and Its Parent Strain Gluconacetobacter xylinus CGMCC 2955

    PubMed Central

    Liu, Miao; Yang, Xiao-Ning; Zhu, Hui-Xia; Jia, Yuan-Yuan; Jia, Shi-Ru; Piergiovanni, Luciano

    2014-01-01

    A better understanding of metabolic fluxes is important for manipulating microbial metabolism toward desired end products, or away from undesirable by-products. A mutant strain, Gluconacetobacter xylinus AX2-16, was obtained by combined chemical mutation of the parent strain (G. xylinus CGMCC 2955) using DEC (diethyl sulfate) and LiCl. The highest bacterial cellulose production for this mutant was obtained at about 11.75 g/L, which was an increase of 62% compared with that by the parent strain. In contrast, gluconic acid (the main byproduct) concentration was only 5.71 g/L for mutant strain, which was 55.7% lower than that of parent strain. Metabolic flux analysis indicated that 40.1% of the carbon source was transformed to bacterial cellulose in mutant strain, compared with 24.2% for parent strain. Only 32.7% and 4.0% of the carbon source were converted into gluconic acid and acetic acid in mutant strain, compared with 58.5% and 9.5% of that in parent strain. In addition, a higher flux of tricarboxylic acid (TCA) cycle was obtained in mutant strain (57.0%) compared with parent strain (17.0%). It was also indicated from the flux analysis that more ATP was produced in mutant strain from pentose phosphate pathway (PPP) and TCA cycle. The enzymatic activity of succinate dehydrogenase (SDH), which is one of the key enzymes in TCA cycle, was 1.65-fold higher in mutant strain than that in parent strain at the end of culture. It was further validated by the measurement of ATPase that 3.53–6.41 fold higher enzymatic activity was obtained from mutant strain compared with parent strain. PMID:24901455

  19. Rhizobium japonicum mutant strains unable to grow chemoautotrophically with H2.

    PubMed Central

    Maier, R J

    1981-01-01

    Rhizobium japonicum strain SR grows chemoautotrophically on a mineral salts medium when incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow or that grow very poorly chemoautotrophically with H2 have been isolated from strain SR. The mutant isolation procedure involved mutagenesis with ethyl methane sulfonate, penicillin selection under chemoautotrophic growth conditions, and plating of the survivors onto medium containing carbon. The resulting colonies were replica plated onto medium that did not contain carbon, and the plates were incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow under these conditions were chosen. Over 100 mutant strains with defects in chemoautotrophic metabolism were obtained. The phenotypes of the mutants fall into various classes. These include strains unable to oxidize H2 and strains deficient in CO2 uptake. Some of the mutant strains were capable of oxidizing H2 only when artificial electron acceptors were provided. Two mutant strains specifically lack activity of the key CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase. Other mutant strains lack both H2-oxidizing ability and ribulose 1,5-bisphosphate carboxylase activity. PMID:6780521

  20. AgrAbility Project

    MedlinePlus

    ... It’s About Hope AgrAbility on Twitter AgrAbility on Facebook AgrAbility on You Tube AgrAbility… It’s About Hope ... anniversary throughout 2016... AgrAbility Harvest Get a copy Facebook Posts National AgrAbility Project 5 days ago AgrAbility's ...

  1. Survival of Listeria monocytogenes in Soil Requires AgrA-Mediated Regulation

    PubMed Central

    Vivant, Anne-Laure; Garmyn, Dominique; Gal, Laurent; Hartmann, Alain

    2015-01-01

    In a recent paper, we demonstrated that inactivation of the Agr system affects the patterns of survival of Listeria monocytogenes (A.-L. Vivant, D. Garmyn, L. Gal, and P. Piveteau, Front Cell Infect Microbiol 4:160, http://dx.doi.org/10.3389/fcimb.2014.00160). In this study, we investigated whether the Agr-mediated response is triggered during adaptation in soil, and we compared survival patterns in a set of 10 soils. The fate of the parental strain L. monocytogenes L9 (a rifampin-resistant mutant of L. monocytogenes EGD-e) and that of a ΔagrA deletion mutant were compared in a collection of 10 soil microcosms. The ΔagrA mutant displayed significantly reduced survival in these biotic soil microcosms, and differential transcriptome analyses showed large alterations of the transcriptome when AgrA was not functional, while the variations in the transcriptomes between the wild type and the ΔagrA deletion mutant were modest under abiotic conditions. Indeed, in biotic soil environments, 578 protein-coding genes and an extensive repertoire of noncoding RNAs (ncRNAs) were differentially transcribed. The transcription of genes coding for proteins involved in cell envelope and cellular processes, including the phosphotransferase system and ABC transporters, and proteins involved in resistance to antimicrobial peptides was affected. Under sterilized soil conditions, the differences were limited to 86 genes and 29 ncRNAs. These results suggest that the response regulator AgrA of the Agr communication system plays important roles during the saprophytic life of L. monocytogenes in soil. PMID:26002901

  2. Antibacterial Activity of Cold Atmospheric Pressure Argon Plasma against 78 Genetically Different (mecA, luk-P, agr or Capsular Polysaccharide Type) Staphylococcus aureus Strains.

    PubMed

    Matthes, Rutger; Lührman, Anne; Holtfreter, Silva; Kolata, Julia; Radke, Dörte; Hübner, Nils-Olaf; Assadian, Ojan; Kramer, Axel

    2016-01-01

    Previous studies on the antimicrobial activity of cold atmospheric pressure argon plasma showed varying effects against mecA+ or mecA-Staphylococcus aureus strains. This observation may have important clinical and epidemiological implications. Here, the antibacterial activity of argon plasma was investigated against 78 genetically different S. aureus strains, stratified by mecA, luk-P, agr1-4, or the cell wall capsule polysaccharide types 5 and 8. kINPen09® served as the plasma source for all experiments. On agar plates, mecA+luk-P-S. aureus strains showed a decreased susceptibility against plasma compared to other S. aureus strains. This study underlines the high complexity of microbial defence against antimicrobial treatment and confirms a previously reported strain-dependent susceptibility of S. aureus to plasma treatment.

  3. Gastrointestinal Colonization by Candida albicans Mutant Strains in Antibiotic-Treated Mice

    PubMed Central

    Wiesner, Stephen M.; Jechorek, Robert P.; Garni, Robb M.; Bendel, Catherine M.; Wells, Carol L.

    2001-01-01

    Antibiotic-treated mice orally inoculated with one of three Candida albicans strains (including two mutant strains) or indigenous Candida pelliculosa showed levels of candidal gastrointestinal colonization that were strain specific. However, regardless of strain, the numbers of viable candida were intermediate to high in the stomach, were consistently lowest in the upper small intestine, and increased progressively down the intestinal tract. PMID:11139219

  4. Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii.

    PubMed

    Tajima, Satoshi; Itoh, Yoko; Sugimoto, Takashi; Kato, Tatsuya; Park, Enoch Y

    2009-10-01

    The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct. PMID:19716523

  5. Survival of smooth, rough and transposon mutant strains of Brucella abortus in bovine mammary macrophages.

    PubMed

    Price, R E; Templeton, J W; Adams, L G

    1990-12-01

    Transposon mutants offer a unique way to evaluate the role of lipopolysaccharide (LPS) by producing a theoretical single-gene difference between the original strain and the transposon mutant strain. Comparative survival of Brucella abortus smooth strain 2308, rough RB51, smooth strain 19, and two transposon mutant strains (rough strain 2308::Tn5 Lac Z [m106] and rough strain 19::Tn5 Lac Z [m3], was tested in restrictive bovine mammary macrophages that were able to effectively reduce the percentage of intracellular bacterial survival and permissive bovine mammary macrophages that were unable to control the intracellular replication of B. abortus. The theoretical single-gene difference between strain 19 and strain 19::Tn5 lac Z [m3] and between smooth virulent strain 2308 and rough transposon mutant 2308::Tn5 lacZ [m106] is likely related to differences in LPS content or structure. Significant (P less than 0.05) reduction in the survival of rough strain 19::Tn5 Lac Z [m3] with no significant reduction in the rough transposon mutant strain 2308::Tn5 lacZ [m106] indicated that at least one factor other than LPS contributes to the intracellular survival of B. abortus in bovine macrophages.

  6. The agr locus regulates virulence and colonization genes in Clostridium difficile 027.

    PubMed

    Martin, Melissa J; Clare, Simon; Goulding, David; Faulds-Pain, Alexandra; Barquist, Lars; Browne, Hilary P; Pettit, Laura; Dougan, Gordon; Lawley, Trevor D; Wren, Brendan W

    2013-08-01

    The transcriptional regulator AgrA, a member of the LytTR family of proteins, plays a key role in controlling gene expression in some Gram-positive pathogens, including Staphylococcus aureus and Enterococcus faecalis. AgrA is encoded by the agrACDB global regulatory locus, and orthologues are found within the genome of most Clostridium difficile isolates, including the epidemic lineage 027/BI/NAP1. Comparative RNA sequencing of the wild type and otherwise isogenic agrA null mutant derivatives of C. difficile R20291 revealed a network of approximately 75 differentially regulated transcripts at late exponential growth phase, including many genes associated with flagellar assembly and function, such as the major structural subunit, FliC. Other differentially regulated genes include several involved in bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) synthesis and toxin A expression. C. difficile 027 R20291 agrA mutant derivatives were poorly flagellated and exhibited reduced levels of colonization and relapses in the murine infection model. Thus, the agr locus likely plays a contributory role in the fitness and virulence potential of C. difficile strains in the 027/BI/NAP1 lineage. PMID:23772065

  7. Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: Biochemical and genetic characterization

    SciTech Connect

    Cornish, K.V.; Pearlman, R.E.

    1982-08-01

    Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

  8. Properties of Mutants of Synechocystis sp. Strain PCC 6803 Lacking Inorganic Carbon Sequestration Systems

    SciTech Connect

    Xu, Min; Bernat, Gabor; Singh, Abhay K.; Mi, Hualing; Rogner, Matthias; Pakrasi, Himadri B.; Ogawa, Teruo

    2008-09-10

    A mutant ( 5) of Synechocystis sp. strain PCC 6803 constructed by inactivating five inorganic carbon sequestration systems did not take up CO2 or HCO3– and was unable to grow in air with or without glucose. The 4 mutant in which BicA is the only active inorganic carbon sequestration system showed low activity of HCO3– uptake and grew under these conditions but more slowly than the wild-type strain. The 5 mutant required 1.7% CO2 to attain half the maximal growth rate. Electron transport activity of the mutants was strongly inhibited under high light intensities, with the 5 mutant more susceptible to high light than the 4 mutant. The results implicated the significance of carbon sequestration in dissipating excess light energy.

  9. Development of a mutant strain of Escherichia coli for molecular cloning of highly methylated DNA

    SciTech Connect

    Bishr, M.A.

    1991-01-01

    A mutant strain of Escherichia coli designated as GR219 that allows efficient molecular cloning of highly methylated bean DNA has been developed by UV light mutation of the parent LE392 str[sup r] strain. This mutant strain, like the parent, is streptomycin resistant and is biologically contained, because it requires thymidine for growth. Both the wild type and the mutant strain have lambda phage receptors so both can be utilized for construction of genomic libraries using the phase as a vector. The efficiency of transformation of the parent and the mutant strain with a recombinant plasmid containing bean DNA was compared to the efficiency of transformation of the PLK-F[prime] strain, which has a deletion of mcrA and mcrB genes and, therefore, allows transformation with methylated bean DNA. It has been found that the GR219 strain has the highest efficiency of transformation, while the PLK-F[prime] strain shows less, and the parent LE392 str[sup r] strain the least efficiency of transformation. These results indicate that strains of E. coli with mcrA and mcrB genes can recognize and degrade highly methylated DNA. However, other undefined factors affected by the altered gene(s) in the GR219 strain are also involved in the recognition and degradation of any cloned foreign DNA.

  10. [Composition of cell walls of 2 mutant strains of Streptomyces chrysomallus].

    PubMed

    Zaretskaia, M Sh; Nefelova, M V; Baratova, L A; Polin, A N

    1984-12-01

    The cell walls and peptidoglycans of two mutant strains, Streptomyces chrysomallus var. carotenoides and Streptomyces chrysomallus var. macrotetrolidi, were studied. The strains are organisms producing carotenes and antibiotics of the macrotetrolide group. By the qualitative composition of the peptidoglycans the mutants belong to Streptomyces and are similar. Their glycan portion consists of equimolar quantities of N-acetyl glucosamine and muramic acid. The peptide subunit is presented by glutamic acid, L, L-diaminopimelic acid, glycine and alanine. The molar ratio of alanine is 1.2-1.3. The mutant strains differ in the content of carbohydrates, total phosphorus and phosphorus belonging to teichoic acids. Teichoic acids of the cell walls of the both strains are of the ribitolhosphate nature. The cell walls of the mutants contain polysaccharides differing from teichoic acids and consisting of glucose, galactose, arabinose and fucose. The influence of the cell wall composition of the mutant strains on their morphology and metabolism and comparison of the data relative to the mutant strains with those relative to the starting strain are discussed.

  11. Induction, isolation, and characterization of aspergillus niger mutant strains producing elevated levels of beta-galactosidase.

    PubMed Central

    Nevalainen, K M

    1981-01-01

    An Aspergillus niger mutant strain, VTT-D-80144, with an improvement of three- to fourfold in the production of extracellular beta-galactosidase was isolated after mutagenesis. The production of beta-galactosidase by this mutant was unaffected by fermentor size, and the enzyme was also suitable for immobilization. PMID:6784672

  12. Pathogenicity and immunogenicity of a mutant strain of Listeria monocytogenes in the chicken infection model.

    PubMed

    Yin, Yuelan; Tian, Debin; Jiao, Hongmei; Zhang, Chenju; Pan, Zhiming; Zhang, Xiaoming; Wang, Xiaobo; Jiao, Xinan

    2011-03-01

    Listeria monocytogenes has been exploited as a vaccine carrier based upon its ability to induce a strong cell-mediated immune response. At present, the safety of live, attenuated L. monocytogenes vaccines in patients is being studied in clinical trials. L. monocytogenes is also an attractive vaccine vector for use in poultry; however, the pathogenicity and immunogenicity of this organism in poultry remain to be fully elucidated. In this study, we investigated the pathogenicity and immunogenicity of an actA- and plcB-deficient L. monocytogenes strain, yzuLM4ΔactA/plcB, and its wild-type parent strain, yzuLM4, in an avian infection model. The results showed that the wild-type strain could infect ISA brown chickens, causing serious tissue disruptions, including various degrees of degeneration, necrotic lesions, and inflammatory cell infiltration in the liver, spleen, heart, and kidney. However, the mutant strain showed reduced virulence in embryonated eggs compared with that of the parent strain (the 50% lethal dose [LD(50)] was 3 logs higher). The mutant strain also showed low virulence in chickens and was rapidly eliminated by the host. There were no obvious pathological changes in tissue sections, but the mutant strain still retained the ability to stimulate high levels of antibody against the protein listeriolysin O (LLO). Booster immunization with the mutant strain led to rapid bacterial clearance from the livers and spleens of chickens challenged by the intramuscular route or the oral route. Collectively, our data suggest that the wild-type serotype 1/2a L. monocytogenes strain can cause serious disease in chickens but the mutant strain with a deletion of the actA and plcB genes is less virulent but induces a strong immune response. This mutant strain of L. monocytogenes is therefore a promising candidate as a safe and effective vector for the delivery of heterologous antigens to prevent zoonosis and infectious disease in poultry.

  13. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, M.; Millard, C.S.; Stols, L.

    1998-06-23

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria. 2 figs.

  14. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2002-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  15. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2001-09-25

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  16. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1998-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  17. Quorum-sensing agr mediates bacterial oxidation response via an intramolecular disulfide redox switch in the response regulator AgrA

    PubMed Central

    Sun, Fei; Liang, Haihua; Kong, Xiangqian; Xie, Sherrie; Cho, Hoonsik; Deng, Xin; Ji, Quanjiang; Zhang, Haiyan; Alvarez, Sophie; Hicks, Leslie M.; Bae, Taeok; Luo, Cheng; Jiang, Hualiang; He, Chuan

    2012-01-01

    Oxidation sensing and quorum sensing significantly affect bacterial physiology and host–pathogen interactions. However, little attention has been paid to the cross-talk between these two seemingly orthogonal signaling pathways. Here we show that the quorum-sensing agr system has a built-in oxidation-sensing mechanism through an intramolecular disulfide switch possessed by the DNA-binding domain of the response regulator AgrA. Biochemical and mass spectrometric analysis revealed that oxidation induces the intracellular disulfide bond formation between Cys-199 and Cys-228, thus leading to dissociation of AgrA from DNA. Molecular dynamics (MD) simulations suggest that the disulfide bond formation generates a steric clash responsible for the abolished DNA binding of the oxidized AgrA. Mutagenesis studies further established that Cys-199 is crucial for oxidation sensing. The oxidation-sensing role of Cys-199 is further supported by the observation that the mutant Staphylococcus aureus strain expressing AgrAC199S is more susceptible to H2O2 owing to repression of the antioxidant bsaA gene under oxidative stress. Together, our results show that oxidation sensing is a component of the quorum-sensing agr signaling system, which serves as an intrinsic checkpoint to ameliorate the oxidation burden caused by intense metabolic activity and potential host immune response. PMID:22586129

  18. Isolation and characterization of nitrogenase-derepressed mutant strains of cyanobacterium Anabaena variabilis.

    PubMed Central

    Spiller, H; Latorre, C; Hassan, M E; Shanmugam, K T

    1986-01-01

    A positive selection method for isolation of nitrogenase-derepressed mutant strains of a filamentous cyanobacterium, Anabaena variabilis, is described. Mutant strains that are resistant to a glutamate analog, L-methionine-D,L-sulfoximine, were screened for their ability to produce and excrete NH4+ into medium. Mutant strains capable of producing nitrogenase in the presence of NH4+ were selected from a population of NH4+-excreting mutants. One of the mutant strains (SA-1) studied in detail was found to be a conditional glutamine auxotroph requiring glutamine for growth in media containing N2, NO3-, or low concentrations of NH4+ (less than 0.5 mM). This glutamine requirement is a consequence of a block in the assimilation of NH4+ produced by an enzyme system like nitrogenase. Glutamate and aspartate failed to substitute for glutamine because of a defect in the transport and utilization of these amino acids. Strain SA-1 assimilated NH4+ when the concentration in the medium reached about 0.5 mM, and under these conditions the growth rate was similar to that of the parent. Mutant strain SA-1 produced L-methionine-D,L-sulfoximine-resistant glutamine synthetase activity. Kinetic properties of the enzyme from the parent and mutant were similar. Mutant strain SA-1 can potentially serve as a source of fertilizer nitrogen to support growth of crop plants, since the NH4+ produced by nitrogenase, utilizing sunlight and water as sources of energy and reductant, respectively, is excreted into the environment. PMID:2867990

  19. Enhanced cellulase producing mutants developed from heterokaryotic Aspergillus strain.

    PubMed

    Kaur, Baljit; Oberoi, H S; Chadha, B S

    2014-03-01

    A heterokaryon 28, derived through protoplast fusion between Aspergillus nidulans and Aspergillus tubingensis (Dal8), was subjected cyclic mutagenesis followed by selection on increasing levels of 2-deoxy glucose (2-DG) as selection marker. The derived deregulated cellulase hyper producing mutant '64', when compared to fusant 28, produced 9.83, 7.8, 3.2, 4.2 and 19.74 folds higher endoglucanase, β-glucosidase, cellobiohydrolase, FPase and xylanase, respectively, under shake cultures. The sequence analysis of PCR amplified β-glucosidase gene from wild and mutant showed nucleotide deletion/substitution. The mutants showed highly catalytic efficient β-glucosidase as evident from low Km and high Vmax values. The expression profiling through zymogram analysis also indicated towards over-expression of cellulases. The up/down regulated expressed proteins observed through SDS-PAGE were identified by Peptide mass fingerprinting The cellulase produced by mutants in conjunction with cellulase free xylanase derived from Thermomyces lanuginosus was used for efficient utilization of alkali treated rice straw for obtaining xylo-oligosaccharides and ethanol.

  20. Mutant breeding of Serratia marcescens strain for enhancing prodigiosin production and application to textiles.

    PubMed

    Liu, Xiaoxia; Wang, Yujie; Sun, Shiqing; Zhu, Changjun; Xu, Wei; Park, Yongdoo; Zhou, Haimeng

    2013-01-01

    Microwaves have been used as a mutant agent to select mutant strains with high-yield and high-purity pigment. Mass spectrometry and nuclear magnetic resonance spectroscopic techniques were used to elucidate the structures of the pigment. High-performance liquid chromatography was used to measure pigment purity. The analysis of the mutant strain showed that pigment yield increased by 109% and was 98% pure. Prodigiosin in ethanol solution had good stability under ambient temperature and natural indoor light. However, prodigiosin rapidly decomposed under intense sunlight. Prodigiosin is an ecological colorant to dye fabrics, including synthetic and natural fibers. Synthetic fabrics dyed with prodigiosin, such as polyamide and acrylic, have high colorfastness to washing (≥4th grade) and antimicrobial properties (>90%) against Escherichia coli and Staphylococcus aureus. Antimicrobial properties were significantly different between synthetic and natural fabrics. The mutant strain Serratia marcescens jx1-1, with high prodigiosin yield and purity, has promising prospects in food, cosmetic, and textile industries.

  1. Influence of the AgrC-AgrA complex on the response time of Staphylococcus aureus quorum sensing.

    PubMed

    Srivastava, Sandeep K; Rajasree, Kalagiri; Fasim, Aneesa; Arakere, Gayathri; Gopal, Balasubramanian

    2014-08-01

    The Staphylococcus aureus agr quorum-sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type I to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrCCyto) and the response regulator domain of AgrA (AgrARR) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrCCyto provided a basis for a mechanistic model of AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity, and kinetics of the phosphotransfer reaction. This step, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which to validate simulations of the quorum-sensing response and for strategies that employ the agr quorum-sensing system to combat biofilm formation in S. aureus infections. PMID:24858185

  2. Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

    PubMed Central

    Srivastava, Sandeep K.; Rajasree, Kalagiri; Fasim, Aneesa; Arakere, Gayathri

    2014-01-01

    The Staphylococcus aureus agr quorum-sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type I to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrCCyto) and the response regulator domain of AgrA (AgrARR) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrCCyto provided a basis for a mechanistic model of AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity, and kinetics of the phosphotransfer reaction. This step, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which to validate simulations of the quorum-sensing response and for strategies that employ the agr quorum-sensing system to combat biofilm formation in S. aureus infections. PMID:24858185

  3. Bovine and rabbit models for the study of a Staphylococcus aureus avirulent mutant strain, RC122

    PubMed Central

    Reinoso, Elina; Magnano, Gabriel; Giraudo, Jose; Calzolari, Aldo; Bogni, Cristina

    2002-01-01

    Staphylococcus aureus is the main etiological agent of bovine mastitis. Intramammary infections are difficult to cure and vaccination appears to be an alternative to prevent the disease. Research has focused on the development of mutants affected in the synthesis of pathogenicity determinants. We constructed a mutant strain (RC122) after chemical mutagenesis. In a mouse model, the strain was shown to be 1500 times less virulent, showed similar kinetics of disappearance in the kidney as its parental strain, and a good degree of protection against a challenge from homologous and heterologous strains. The objective of the present report was to study the avirulent RC122 S. aureus mutant strain in rabbit and bovine infection models. The results clearly show that RC122 was less virulent than its parental strain in a rabbit skin model, and was also correlated with its avirulence as an udder pathogen. These traits make the RC122 mutant strain interesting as a potential strain for an experimental vaccine trial in dairy herds. PMID:12418786

  4. Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

    PubMed Central

    King, Stephen J.; Dutcher, Susan K.

    1997-01-01

    To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning. PMID:9008712

  5. [Generation of nalidixic acid-resistant strains and signature-tagged mutants of Actinobacillus pleuropneumoniae].

    PubMed

    Shang, Lin; Li, Wei; Li, Liangjun; Li, Lu; Zhang, Sihua; Li, Tingting; Li, Yaokun; Liu, Lei; Guo, Zhiwei; Zhou, Rui; Chen, Huanchun

    2008-01-01

    Actinobacillus pleuropneumoniae is a very important respiratory pathogen for swine and causes great economic losses in pig industry worldwide. Signature-tagged mutagenesis (STM) is an effective method to identify virulence genes in bacteria. In this study, we selected nalidixic acid-resistant strains of APP serotypes 1 and 3 by in vitro cultivation, and used as receipt strains for constructing transposon mutants by mating with E. coli CC 118 lambdapir or S17-1 lambdapir containing mini-Tn10 tag plasmids pLOF/TAG1-48, with or without the help of E. coli DH5alpha (pRK2073). We screened mutant strains by antibiotics selection, PCR and Southern blot identification. Our data revealed that nalidixic acid-resistance of APP strains could easily be induced in vitro and the resistance was due to the mutation in the DNA gyrase A subunit gene gyrA. In the mating experiments, the bi-parental mating was more effective and easier than tri-parental mating. Different APP strains showed a different mating and transposon efficiency in the bi-parental mating, with the strains of serotype 1 much higher than serotype 3 and the reference strain of serotype 3 higher than the field strains. These data were helpful for the construction of STM mutants and pickup of virulence genes of APP. PMID:18338580

  6. Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina.

    PubMed

    Druzhinina, Irina S; Schmoll, Monika; Seiboth, Bernhard; Kubicek, Christian P

    2006-03-01

    The ascomycete Hypocrea jecorina (Trichoderma reesei), an industrial producer of cellulases and hemicellulases, can efficiently degrade plant polysaccharides. However, the catabolic pathways for the resulting monomers and their relationship to enzyme induction are not well known. Here we used the Biolog Phenotype MicroArrays technique to evaluate the growth of H. jecorina on 95 carbon sources. For this purpose, we compared several wild-type isolates, mutants producing different amounts of cellulases, and strains transformed with a heterologous antibiotic resistance marker gene. The wild-type isolates and transformed strains had the highest variation in growth patterns on individual carbon sources. The cellulase mutants were relatively similar to their parental strains. Both in the mutant and in the transformed strains, the most significant changes occurred in utilization of xylitol, erythritol, D-sorbitol, D-ribose, D-galactose, L-arabinose, N-acetyl-D-glucosamine, maltotriose, and beta-methyl-glucoside. Increased production of cellulases was negatively correlated with the ability to grow on gamma-aminobutyrate, adonitol, and 2-ketogluconate; and positively correlated with that on d-sorbitol and saccharic acid. The reproducibility, relative simplicity, and high resolution (+/-10% of increase in mycelial density) of the phenotypic microarrays make them a useful tool for the characterization of mutant and transformed strains and for a global analysis of gene function.

  7. Genetics of Peripheral Vestibular Dysfunction: Lessons from Mutant Mouse Strains

    PubMed Central

    Jones, Sherri M.; Jones, Timothy A.

    2015-01-01

    Background A considerable amount of research has been published about genetic hearing impairment. Fifty to sixty percent of hearing loss is thought to have a genetic cause. Genes may also play a significant role in acquired hearing loss due to aging, noise exposure, or ototoxic medications. Between 1995 and 2012, over 100 causative genes have been identified for syndromic and nonsyndromic forms of hereditary hearing loss (see Hereditary Hearing Loss Homepage http://hereditaryhearingloss.org). Mouse models have been extremely valuable in facilitating the discovery of hearing loss genes, and in understanding inner ear pathology due to genetic mutations or elucidating fundamental mechanisms of inner ear development. Purpose Whereas much is being learned about hereditary hearing loss and the genetics of cochlear disorders, relatively little is known about the role genes may play in peripheral vestibular impairment. Here we review the literature with regard to genetics of vestibular dysfunction and discuss what we have learned from studies using mutant mouse models and direct measures of peripheral vestibular neural function. Results Several genes are considered that when mutated lead to varying degrees of inner ear vestibular dysfunction due to deficits in otoconia, stereocilia, hair cells, or neurons. Behavior often does not reveal the inner ear deficit. Many of the examples presented are also known to cause human disorders. Conclusions Knowledge regarding the roles of particular genes in the operation of the vestibular sensory apparatus is growing and it is clear that gene products co-expressed in the cochlea and vestibule may play different roles in the respective end organs. The discovery of new genes mediating critical inner ear vestibular function carries the promise of new strategies in diagnosing, treating and managing patients as well as predicting the course and level of morbidity in human vestibular disease. PMID:25032973

  8. Frequency-dependent viability in mutant strains of Drosophila melanogaster.

    PubMed

    Curtsinger, J W; Sheen, F M

    1991-01-01

    We investigated the effects of genotypic frequencies on egg-to-adult viabilities in pairwise combinations of four strains of Drosophila melanogaster. The experiments involved mixture of a total of 42,000 eggs in varying proportions under controlled densities and observation of surviving adults. Viabilities were found to depend on frequencies in several genotypic combinations. In the most extreme case, the absolute viability of cn;bw females increased monotonically from 54% when common to 70% when rare. The results illustrate several statistical and methodological problems that might explain why some experiments have failed to detect frequency-dependent viabilities. These problems include heterogeneity between replications, sex differences in susceptibility to competition, and strong dependence of the experimental outcome on the choice of competitor genotypes. PMID:1901577

  9. Selection and Characterization of Phage-Resistant Mutant Strains of Listeria monocytogenes Reveal Host Genes Linked to Phage Adsorption

    PubMed Central

    Denes, Thomas; den Bakker, Henk C.; Tokman, Jeffrey I.; Guldimann, Claudia

    2015-01-01

    Listeria-infecting phages are readily isolated from Listeria-containing environments, yet little is known about the selective forces they exert on their host. Here, we identified that two virulent phages, LP-048 and LP-125, adsorb to the surface of Listeria monocytogenes strain 10403S through different mechanisms. We isolated and sequenced, using whole-genome sequencing, 69 spontaneous mutant strains of 10403S that were resistant to either one or both phages. Mutations from 56 phage-resistant mutant strains with only a single mutation mapped to 10 genes representing five loci on the 10403S chromosome. An additional 12 mutant strains showed two mutations, and one mutant strain showed three mutations. Two of the loci, containing seven of the genes, accumulated the majority (n = 64) of the mutations. A representative mutant strain for each of the 10 genes was shown to resist phage infection through mechanisms of adsorption inhibition. Complementation of mutant strains with the associated wild-type allele was able to rescue phage susceptibility for 6 out of the 10 representative mutant strains. Wheat germ agglutinin, which specifically binds to N-acetylglucosamine, bound to 10403S and mutant strains resistant to LP-048 but did not bind to mutant strains resistant to only LP-125. We conclude that mutant strains resistant to only LP-125 lack terminal N-acetylglucosamine in their wall teichoic acid (WTA), whereas mutant strains resistant to both phages have disruptive mutations in their rhamnose biosynthesis operon but still possess N-acetylglucosamine in their WTA. PMID:25888172

  10. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants

    PubMed Central

    Liberati, Nicole T.; Urbach, Jonathan M.; Miyata, Sachiko; Lee, Daniel G.; Drenkard, Eliana; Wu, Gang; Villanueva, Jacinto; Wei, Tao; Ausubel, Frederick M.

    2006-01-01

    Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes. PMID:16477005

  11. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants.

    PubMed

    Liberati, Nicole T; Urbach, Jonathan M; Miyata, Sachiko; Lee, Daniel G; Drenkard, Eliana; Wu, Gang; Villanueva, Jacinto; Wei, Tao; Ausubel, Frederick M

    2006-02-21

    Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes.

  12. Derepression of colicin E1 synthesis in the constitutive tif mutant strain (spr tif sfi) and in a tif sfi mutant strain of Escherichia coli K-12.

    PubMed Central

    Tessman, E S; Gritzmacher, C A; Peterson, P K

    1978-01-01

    We show here that expression of the colicin gene of the ColE1 plasmid is greatly derepressed in Escherichia coli K-12 strain DM1187 spr tif sfi, which is a constitutive tif mutant, altered in the lexA gene, and which shows constitutive expression of various pathways of the recA-dependent, lexA-blocked (SOS) repair system. In this strain colicin E1 synthesis is at least 100-fold greater than that observed in uninduced control strains (spr+ tif sfi and spr+ tif+ sfi). This result confirms the regulatory role of the lexA product in colicin E1 synthesis. Colicin yields by the uninduced strain DM1187 are as high as the maximum yields from mitomycin-induced control strains and often are several-fold higher. When the nonconstitutive tif sfi strain GC467 is raised to 43 degrees C to induce the SOS system, a low level of colicin synthesis is observed which is less than one-tenth of the yield obtained by induction with mitomycin C. Addition of adenine at the time of shift-up can increase the colicin yield of tif sfi to about one-third of the yield obtained with mitomycin C. We have also found that colicin overproduction can be detected by altered colony appearance in an overlay assay with colicin-sensitive bacteria. In addition, the lethality of the process of colicin synthesis is observed here without the use of bacteriostatic inducing agents. Images PMID:353034

  13. Mechanical properties of elytra from Tribolium castaneum wild-type and body color mutant strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cuticle tanning in insects involves simultaneous cuticular hardening and pigmentation. The dynamic mechanical properties of the highly modified and cuticle-rich forewings (elytra) from Tribolium castaneum (red flour beetle) body color mutant strains were investigated to determine the relationship b...

  14. Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins.

    PubMed

    Larsen, Sasha; Weaver, Jun; de Sa Campos, Katherine; Bulahan, Rhobe; Nguyen, Jackson; Grove, Heather; Huang, Amy; Low, Lauren; Tran, Namphuong; Gomez, Seth; Yau, Jennifer; Ilustrisimo, Thomas; Kawilarang, Jessica; Lau, Jonathan; Tranphung, Maivi; Chen, Irene; Tran, Christina; Fox, Marcia; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2013-11-01

    Although Pichia pastoris is a popular protein expression system, it exhibits limitations in its ability to secrete heterologous proteins. Therefore, a REMI (restriction enzyme mediated insertion) strategy was utilized to select mutant beta-g alactosidase s upersecretion (bgs) strains that secreted increased levels of a β-galactosidase reporter. Many of the twelve BGS genes may have functions in intracellular signaling or vesicle transport. Several of these strains also appeared to contain a more permeable cell wall. Preliminary characterization of four bgs mutants showed that they differed in the ability to enhance the export of other reporter proteins. bgs13, which has a disruption in a gene homologous to Saccharomyces cerevisiae protein kinase C (PKC1), gave enhanced secretion of most recombinant proteins that were tested, raising the possibility that it has the universal super-secreter phenotype needed in an industrial production strain of P. pastoris. PMID:23881328

  15. Construction and characterization of a H19 epitope point mutant of MDV CVI988/Rispens strain.

    PubMed

    Cui, Z; Qin, A; Lee, L F; Wu, P; Kung, H J

    1999-01-01

    A recombinant virus, CVI/rpp38, was developed from the Marek's disease virus (MDV) CVI988/Rispens vaccine strain. This recombinant was obtained by transfection of CVI988/Rispens-infected chick embryo fibroblasts (CEFs) with plasmid pHA25 DNA containing pp38 gene from GA strain of MDV. Monoclonal antibody (MAb) H19 which reacts with pp38 from GA but not with that from CVI988 was used to screen for recombinant viruses in transfected cell culture plates by immunofluorescent assay (IFA). A positive plaque was isolated, propagated, and purified from cell-free virus particles after sonication of infected CEFs. The mutant CVI/rpp38 was not only reactive with MAb H19 in IFA but also in immunoprecipitation. A 38 kDa protein was immunoprecipitated from the CVI/rpp38 mutant virus but not from parental CVI988 virus. DNA sequence of the mutant virus showed a substitution of G at position 320 by a resulting in a change of an amino acid residue from arginine to glutamine. Comparison of nucleotide sequence of pp38 from strains GA, Md5 and Md11/75c/R2 and CVI988 revealed change to glutamine in this position. The result of this study provides a direct evidence for the location of the identified H19 epitope in pp38. This mutant is potentially useful to further explore the biological function of pp38 and its H19 epitope.

  16. Biotransformation of ethanol to acetaldehyde by wild and mutant strains of methylotrophic yeast

    SciTech Connect

    Moroz, O.M.; Sibirnyi, A.A.; Ksheminskaya, G.P. |

    1995-05-01

    The conversion of ethanol to acetaldehyde by intact cells of wild and mutant strains of methylotrophic yeast Hansenula polymorpha was studied. It was established that mutations that lower the activity of aldehyde reductase and acetaldehyde dehydrogenase stimulate acetaldehyde accumulation. The highest accumulation of acetaldehyde was found in a mutant that possessed increased alcohol oxidase activity in growth on a medium with glucose. A decrease in formaldehyde dehydrogenase did not stimulate acetaldehyde accumulation. Bioconversion of ethanol to acetaldehyde was most effective at lowered temperatures due to marked suppression of catabolic alcohol oxidase inactivation, but not to the activity of this enzyme under indicated conditions. 27 refs., 4 figs., 3 tabs.

  17. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

    SciTech Connect

    Shao, Xiongjun; Raman, Babu; Zhu, Mingjun; Mielenz, Jonathan R; Brown, Steven D; Guss, Adam M; Lynd, Lee R

    2011-01-01

    Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

  18. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

    SciTech Connect

    Lynd, Lee R; Shao, Xiongjun; Raman, Babu; Mielenz, Jonathan R; Brown, Steven D; Guss, Adam M; Zhu, Mingjun

    2011-01-01

    Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1 2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

  19. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum.

    PubMed

    Shao, Xiongjun; Raman, Babu; Zhu, Mingjun; Mielenz, Jonathan R; Brown, Steven D; Guss, Adam M; Lynd, Lee R

    2011-11-01

    Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed. PMID:21874277

  20. Development of a Mutant Strain of Bacillus polymyxa Showing Enhanced Production of 2,3-Butanediol

    PubMed Central

    Mallonee, D. H.; Speckman, R. A.

    1988-01-01

    2,3-Butanediol is a feedstock chemical of potential industrial importance. It can serve as a monomer for many polymers of consumer interest that are currently supplied by the fossil fuel industry. Bacillus polymyxa can grow on inexpensive waste products of the food-processing industry and produce this glycol. This paper describes a mutant strain of B. polymyxa which displays constitutive production of catabolic α-acetolactate synthase, an enzyme in the 2,3-butanediol pathway which is normally produced only in the late log or stationary phase of growth. The mutant was obtained by treating the wild type with nitrosoguanidine and subjecting it to a penicillin counterselection procedure. One of the selected mutant strains produced four times as much of the glycol as the wild type and utilized approximately 25% of the energy source, compared with essentially complete utilization of the energy source by the wild type. Studies are under way to optimize the production of the glycol by the mutant. PMID:16347522

  1. Development of a mutant strain of Bacillus polymyxa showing enhanced production of 2,3-butanediol

    SciTech Connect

    Mallonee, D.H.; Speckman, R.A.

    1988-01-01

    2,3-Butanediol is a feedstock chemical of potential industrial importance. It can serve as a monomer for many polymers of consumer interest that are currently supplied by the fossil fuel industry. Bacillus polymyxa can grow on inexpensive waste products of the food-processing industry and produce this glycol. This paper describes a mutant strain of B. polymyxa which displays constitutive production of catabolic ..cap alpha..-acetolactate synthase, an enzyme in the 2,3-butanediol pathway which is normally produced only in the late log or stationary phase of growth. The mutant was obtained by treating the wild type with nitrosoguanidine and subjecting it to a penicillin counterselection procedure. One of the selected mutant strains produced four times as much of the glycol as the wild type and utilized approximately 25% of the energy source, compared with essentially complete utilization of the energy source by the wild type. Studies are under way to optimize the production of the glycol by the mutant.

  2. Live Attenuated Borrelia burgdorferi Targeted Mutants in an Infectious Strain Background Protect Mice from Challenge Infection.

    PubMed

    Hahn, Beth L; Padmore, Lavinia J; Ristow, Laura C; Curtis, Michael W; Coburn, Jenifer

    2016-08-01

    Borrelia burgdorferi, B. garinii, and B. afzelii are all agents of Lyme disease in different geographic locations. If left untreated, Lyme disease can cause significant and long-term morbidity, which may continue after appropriate antibiotic therapy has been administered and live bacteria are no longer detectable. The increasing incidence and geographic spread of Lyme disease are renewing interest in the vaccination of at-risk populations. We took the approach of vaccinating mice with two targeted mutant strains of B. burgdorferi that, unlike the parental strain, are avirulent in mice. Mice vaccinated with both strains were protected against a challenge with the parental strain and a heterologous B. burgdorferi strain by either needle inoculation or tick bite. In ticks, the homologous strain was eliminated but the heterologous strain was not, suggesting that the vaccines generated a response to antigens that are produced by the bacteria both early in mammalian infection and in the tick. Partial protection against B. garinii infection was also conferred. Protection was antibody mediated, and reactivity to a variety of proteins was observed. These experiments suggest that live attenuated B. burgdorferi strains may be informative regarding the identification of protective antigens produced by the bacteria and recognized by the mouse immune system in vivo Further work may illuminate new candidates that are effective and safe for the development of Lyme disease vaccines. PMID:27335385

  3. Pseudomonas mutant strains that accumulate androstane and seco-androstane intermediates from bile acids.

    PubMed Central

    Leppik, R A; Sinden, D J

    1987-01-01

    Transposon mutant strains which were affected in bile acid catabolism were isolated from four Pseudomonas spp. Two of the mutant groups isolated were found to accumulate 12 alpha-hydroxyandrosta-1,4-diene-3,17-dione as the major product from deoxycholic acid. Strains in one of these two groups were able to grow on steroids such as chenodeoxycholic acid, which lacks a 12 alpha-hydroxy function, whereas the one member of the second group could not. With chenodeoxycholic acid, this latter strain accumulated a yellow muconic-like derivative, tentatively identified as 3,7-dihydroxy-5,9,17-trioxo-4(5),9(10)-disecoandrosta-1(10)2 -dien-4-oic acid. Members of two further mutant groups accumulated either 12 beta-hydroxyandrosta-1,4-diene-3,17-dione or 3,12 beta-dihydroxy-9(10)-secoandrosta-1,3,5(10)-triene-9,17-dione as the major product from deoxycholic acid. The relationship between the catabolism of m- and p-cresol, 3-ethylphenol and the bile acids was also examined. PMID:3038076

  4. Characterization of a mutant strain of a filamentous fungus Cladosporium phlei for the mass production of the secondary metabolite phleichrome.

    PubMed

    Yi, Min-Hee; Kim, Jung-Ae; Kim, Jung-Mi; Park, Jin-Ah; Kim, Beom-Tae; Park, Seung-Moon; Yang, Moon-Sik; Hwang, Ki-Jun; Kim, Dae-Hyuk

    2011-08-01

    UV-mutagenesis was performed to obtain mutant strains that demonstrate altered production of phleichrome, a secondary metabolite of Cladosporium phlei. Among fifty mutants selected, based on the increased area and intensity of the purple pigment surrounding the colonies, the strain M0035 showed the highest production of phleichrome, more than seven fold over wild type. Plate cultures of the M0035 strain resulted in a total of 592 mg phleichrome consisting of 146 mg and 446 mg from the mycelia and agar media, respectively. The M0035 strain displayed a growth rate and a mycelial mass comparable to the parental strain but had significantly reduced asexual sporulation.

  5. Isolation and characterization of symbiotic mutants of bradyrhizobium sp. (Arachis) strain NC92: mutants with host-specific defects in nodulation and nitrogen fixation.

    PubMed Central

    Wilson, K J; Anjaiah, V; Nambiar, P T; Ausubel, F M

    1987-01-01

    Random transposon Tn5 mutagenesis of Bradyrhizobium sp. (Arachis) strain NC92, a member of the cowpea cross-inoculation group, was carried out, and kanamycin-resistant transconjugants were tested for their symbiotic phenotype on three host plants: groundnut, siratro, and pigeonpea. Two nodulation (Nod- phenotype) mutants were isolated. One is unable to nodulate all three hosts and appears to contain an insertion in one of the common nodulation genes (nodABCD); the other is a host-specific nodulation mutant that fails to nodulate pigeonpea, elicits uninvaded nodules on siratro, and elicits normal, nitrogen-fixing nodules on groundnut. In addition, nine mutants defective in nitrogen fixation (Fix- phenotype) were isolated. Three fail to supply symbiotically fixed nitrogen to all three host plants. Surprisingly, nodules elicited by one of these mutants exhibit high levels of acetylene reduction activity, demonstrating the presence of the enzyme nitrogenase. Three more mutants have partially effective phenotypes (Fix +/-) in symbiosis with all three host plants. The remaining three mutants fail to supply fixed nitrogen to one of the host plants tested while remaining partially or fully effective on the other two hosts; two of these mutants are Fix- in pigeonpea and Fix +/- on groundnut and on siratro, whereas the other one is Fix- on groundnut but Fix+ on siratro and on pigeonpea. These latter mutants also retain significant nodule acetylene reduction activity, even in the ineffective symbioses. Such bacterial host-specific fixation (Hsf) mutants have not previously been reported. Images PMID:3032910

  6. Draft Genome Sequences for Clostridium thermocellum Wild-Type Strain YS and Derived Cellulose Adhesion-Defective Mutant Strain AD2

    SciTech Connect

    Brown, Steven D; Lamed, Raphael; Morag, Ely; Borovok, Ilya; Shoham, Yuval; Klingeman, Dawn Marie; Johnson, Courtney M; Yang, Zamin; Land, Miriam L; Utturkar, Sagar M; Keller, Martin; Bayer, Edward A

    2012-01-01

    Clostridium thermocellum wild-type strain YS is an anaerobic, thermophilic, cellulolytic bacterium capable of directly converting cellulosic substrates into ethanol. Strain YS and a derived cellulose adhesion-defective mutant strain AD2 played pivotal roles in describing the original cellulosome concept. We present their draft genome sequences.

  7. In vitro characterization of Salmonella typhi mutant strains for live oral vaccines.

    PubMed

    Dragunsky, E M; Rivera, E; Hochstein, H D; Levenbook, I S

    1990-06-01

    Several Salmonella typhi attenuated mutant strains, suggested as candidates for live oral vaccine, were examined for their characteristics in vitro in comparison with parental strains Ty2 and CDC10-80. Three methods were used: interaction of bacteria with the human monocyte-macrophage U937 cell line evaluated by microscopic examination, bacterial growth in the cell culture medium estimated by absorbance and bacterial resistance to human plasma assessed by the viable count technique. The most informative data were obtained in the test with U937 cells. Ty2 penetrated almost 100% of the cells, multiplied rapidly and caused death of the cells. CDC10-80 infected about 30% of the cells, multiplied slightly and did not kill the cells. The Ty2 mutant galE via EX462 behaved like CDC10-80. Bacteria of the galE Ty21a, Vi + Ty21a, 541 Ty and 543 Ty, found in only 3-4% of the cells, did not multiply within the cells and decreased in number with time. These findings correlate with the reported virulence of these strains for humans. With the second method, the rate of bacterial growth in cell culture medium did not differentiate Ty2, CDC10-80 and EX462. They grew at the same rate and faster than the remaining mutants. The plasma resistance test did not discriminate between EX462 and other mutants. These tests did not reveal any difference between Vi + Ty21a and Vi-Ty21a.

  8. Asexual development is increased in Neurospora crassa cat-3-null mutant strains.

    PubMed

    Michán, Shaday; Lledías, Fernando; Hansberg, Wilhelm

    2003-08-01

    We use asexual development of Neurospora crassa as a model system with which to determine the causes of cell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerial hyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the start of these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3 (CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here we analyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was not compensated by other catalases, even under oxidative stress conditions, and cat-3(RIP) colonies were sensitive to H(2)O(2), indicating that wild-type (Wt) resistance to external H(2)O(2) was due to CAT-3. cat-3(RIP) colonies grown in the dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidative stress and further increased carotene synthesis. In the cat-3(RIP) mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3(RIP) mutant strain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longer and more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed more aerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lack of CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

  9. Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

    PubMed

    Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

    2015-11-01

    Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. PMID:26319139

  10. Cutaneous delayed hypersensitivity reactions of cattle vaccinated with mutant strains of Brucella abortus, using brucellins prepared from various brucellar strains.

    PubMed

    Cheville, N F; Jensen, A E; Morfitt, D C; Stabel, T J

    1994-09-01

    Cutaneous reactivity to brucellin was evaluated in 10-month-old heifers vaccinated with low-virulence mutant strains of Brucella abortus and was compared with brucellin reactions in postparturient cows with active brucellosis. In the cows, the cutaneous lesion was characterized microscopically as severe, acute, serofibrinous vasculitis; dermal lesions at 6, 12, 25, and 48 hours after brucellin injection consisted of endothelial activation and perivascular exudation that led to progressive accumulation of fibrin, monocytes, macrophages, and lymphocytes. In vaccinated heifers, cutaneous tests were done, using standard brucellin, brucellin prepared from strain RB51, and the purified brucellar proteins-31K and superoxide dismutase. Negative-control cattle given saline solution, did not have cutaneous reactions. Standard brucellin induced the most marked reactions in vaccinated heifers. Brucellin from rough strain RB51 caused positive reactions in heifers vaccinated with strain 19, but reactions were variable in other groups. Skin lesions induced by purified superoxide dismutase and 31-kd proteins in vaccinated cattle were not acceptable for diagnosis. Marked variability of test responses in vaccinated cattle precludes field use of this test to determine vaccination status.

  11. Regulation of allantoate transport in wild-type and mutant strains of Saccharomyces cerevisiae.

    PubMed Central

    Chisholm, V T; Lea, H Z; Rai, R; Cooper, T G

    1987-01-01

    Accumulation of intracellular allantoin and allantoate is mediated by two distinct active transport systems in Saccharomyces cerevisiae. Allantoin transport (DAL4 gene) is inducible, while allantoate uptake is constitutive (it occurs at full levels in the absence of any allantoate-related compounds from the culture medium). Both systems appear to be sensitive to nitrogen catabolite repression, feedback inhibition, and trans-inhibition. Mutants (dal5) that lack allantoate transport have been isolated. These strains also exhibit a 60% loss of allantoin transport capability. Conversely, dal4 mutants previously described are unable to transport allantoin and exhibit a 50% loss of allantoate transport. We interpret the pleiotropic behavior of the dal4 and dal5 mutations as deriving from a functional interaction between elements of the two transport systems. PMID:3549700

  12. Production and downstream processing of (1→3)-β-D-glucan from mutant strain of Agrobacterium sp. ATCC 31750

    PubMed Central

    2012-01-01

    We isolated a mutant that produced higher levels of curdlan than the wild strain Agrobacterium sp. ATCC 31750 by chemical mutagenesis using N-methyl-N-nitro-nitrosoguanidine. The mutant strain produced 66 g/L of curdlan in 120 h with a yield of (0.88) while, the wild strain produced 41 g/L in 120 h with a yield of (0.62) in a stirred bioreactor. The mutant could not produce curdlan when the pH was shifted from 7.0 to 5.5 after nitrogen depletion as followed for wild strain. In contrast, pH optimum for cell growth and curdlan production for mutant was found to be 7.0. We optimized the downstream processing of curdlan by varying different volumes of NaOH and HCl for extraction and precipitation of curdlan. The molecular weight of the purified curdlan from the wild and mutant strain was 6.6 × 105 Da and 5.8 × 105 Da respectively. The monosaccharide analyses confirm that curdlan from both wild and mutant strain contains only glucose units. From the NMR and FTIR data, it has been confirmed that curdlan was exclusively composed of β (1 → 3)-D-glucan residues. PMID:22681895

  13. Differential Abilities of Capsulated and Noncapsulated Staphylococcus aureus Isolates from Diverse agr Groups To Invade Mammary Epithelial Cells▿

    PubMed Central

    Buzzola, Fernanda R.; Alvarez, Lucía P.; Tuchscherr, Lorena P. N.; Barbagelata, María S.; Lattar, Santiago M.; Calvinho, Luis; Sordelli, Daniel O.

    2007-01-01

    Staphylococcus aureus is the bacterium most frequently isolated from milk of bovines with mastitis. Four allelic groups, which interfere with the regulatory activities among the different groups, have been identified in the accessory gene regulator (agr) system. The aim of this study was to ascertain the prevalence of the different agr groups in capsulated and noncapsulated S. aureus bacteria isolated from mastitic bovines in Argentina and whether a given agr group was associated with MAC-T cell invasion and in vivo persistence. Eighty-eight percent of the bovine S. aureus strains were classified in agr group I. The remainder belonged in agr groups II, III, and IV (2, 8, and 2%, respectively). By restriction fragment length polymorphism analysis after PCR amplification of the agr locus variable region, six agr restriction types were identified. All agr group I strains presented a unique allele (A/1), whereas strains from groups II, III, and IV exhibited more diversity. Bovine S. aureus strains defined as being in agr group I (capsulated or noncapsulated) showed significantly increased abilities to be internalized within MAC-T cells, compared with isolates from agr groups II, III, and IV. agr group II or IV S. aureus strains were cleared more efficiently than agr group I strains from the murine mammary gland. The results suggest that agr group I S. aureus strains are more efficiently internalized within epithelial cells and can persist in higher numbers in mammary gland tissue than S. aureus strains classified in agr group II, III, or IV. PMID:17145949

  14. CHLOROPLAST STRUCTURE AND FUNCTION IN ac-20, A MUTANT STRAIN OF CHLAMYDOMONAS REINHARDI

    PubMed Central

    Goodenough, Ursula W.; Levine, R. P.

    1970-01-01

    The fine structure of the ac-20 strain of Chlamydomonas reinhardi is described. Cells grown mixotrophically in the presence of acetate have a highly disordered chloroplast membrane organization and usually lack pyrenoids. Chloroplast ribosome levels are only 5–10% of wild-type levels. Cells grown phototrophically without acetate possess more chloroplast ribosomes and have more normal membrane and pyrenoid organization. Chloroplast ribosome levels rise rapidly when cells are transferred from acetate to minimal medium, whereas membrane reorganization occurs only after a lag. These results, combined with earlier studies of the photosynthetic properties of the mutant strain, suggest that proper membrane organization, Photosystem II activity, and ribulose-1,5-diphosphate carboxylase formation are dependent on the presence of chloroplast ribosomes. Other chloroplast components tested are unaffected by a 10-fold reduction in levels of chloroplast ribosomes. PMID:5415236

  15. Symbiotic effectiveness of antibiotic-resistant mutants of fast- and slow-growing strains of Rhizobium nodulating Lotus species.

    PubMed

    Pankhurst, C E

    1977-08-01

    Mutants resistant ot 16 individual antibiotics were isolated from two fast-growing and two slow-growing strains of Lotus rhizobia and their symbiotic effectiveness on Lotus pedunculatus evaluated. Resistance to streptomycin, spectinomycin, chloramphenicol, and tetracycline (inhibitors of protein synthesis) was associated with little or no loss of effectiveness with all four strains but resistance to nalidixic acid and rifampicin (inhibitors of nucleic acid synthesis), and to D-cycloserine, novobiocin, and penicillin (inhibitors of cell wall-cell membrane synthesis) was associated with significant loss of effectiveness in 20-100% of the mutants. Resistance to viomycin, neomycin, kanamycin, and vibramycin was associated with loss of effectiveness with mutants of the two fast-growing strains but not with mutants of the two slow-growing strains. When tested on four alternate host legumes individual mutants of a slow-growing strain showed significantly different levels of effectiveness. The results suggest that both the inherent characteristics of the bacterium and of the host plant will influence the symbiotic effectiveness of antibiotic-resistant mutants of Rhizobium. PMID:890601

  16. A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins

    PubMed Central

    Orr, Sally L; Le, Dzung; Long, Jeffrey M; Sobieszczuk, Peter; Ma, Bo; Tian, Hua; Fang, Xiaoqun; Paulson, James C; Marth, Jamey D; Varki, Nissi

    2013-01-01

    The consortium for functional glycomics (CFG) was a large research initiative providing networking and resources for investigators studying the role of glycans and glycan-binding proteins in health and disease. Starting in 2001, six scientific cores were established to generate data, materials and new technologies. By the end of funding in 2011, the mouse phenotype core (MPC) submitted data to a website from the phenotype screen of 36 mutant mouse strains deficient in a gene for either a glycan-binding protein (GBP) or glycosyltransferase (GT). Each mutant strain was allotted three months for analysis and screened by standard phenotype assays used in the fields of immunology, histology, hematology, coagulation, serum chemistry, metabolism and behavior. Twenty of the deficient mouse strains had been studied in other laboratories, and additional tests were performed on these strains to confirm previous observations and discover new data. The CFG constructed 16 new homozygous mutant mouse strains and completed the initial phenotype screen of the majority of these new mutant strains. In total, >300 phenotype changes were observed, but considering the over 100 assays performed on each strain, most of the phenotypes were unchanged. Phenotype differences include abnormal testis morphology in GlcNAcT9- and Siglec-H-deficient mice and lethality in Pomgnt1-deficient mice. The numerous altered phenotypes discovered, along with the consideration of the significant findings of normality, will provide a platform for future characterization to understand the important roles of glycans and GBPs in the mechanisms of health and disease. PMID:23118208

  17. Influence of Sae-regulated and Agr-regulated factors on the escape of Staphylococcus aureus from human macrophages.

    PubMed

    Münzenmayer, Lisa; Geiger, Tobias; Daiber, Ellen; Schulte, Berit; Autenrieth, Stella E; Fraunholz, Martin; Wolz, Christiane

    2016-08-01

    Although Staphylococcus aureus is not a classical intracellular pathogen, it can survive within phagocytes and many other cell types. However, the pathogen is also able to escape from cells by mechanisms that are only partially understood. We analysed a series of isogenic S. aureus mutants of the USA300 derivative JE2 for their capacity to destroy human macrophages from within. Intracellular S. aureus JE2 caused severe cell damage in human macrophages and could efficiently escape from within the cells. To obtain this full escape phenotype including an intermittent residency in the cytoplasm, the combined action of the regulatory systems Sae and Agr is required. Mutants in Sae or mutants deficient in the Sae target genes lukAB and pvl remained in high numbers within the macrophages causing reduced cell damage. Mutants in the regulatory system Agr or in the Agr target gene psmα were largely similar to wild-type bacteria concerning cell damage and escape efficiency. However, these strains were rarely detectable in the cytoplasm, emphasizing the role of phenol-soluble modulins (PSMs) for phagosomal escape. Thus, Sae-regulated toxins largely determine damage and escape from within macrophages, whereas PSMs are mainly responsible for the escape from the phagosome into the cytoplasm. Damage of macrophages induced by intracellular bacteria was linked neither to activation of apoptosis-related caspase 3, 7 or 8 nor to NLRP3-dependent inflammasome activation.

  18. Activation of dormant bacterial genes by Nonomuraea sp. strain ATCC 39727 mutant-type RNA polymerase.

    PubMed

    Talà, Adelfia; Wang, Guojun; Zemanova, Martina; Okamoto, Susumu; Ochi, Kozo; Alifano, Pietro

    2009-02-01

    There is accumulating evidence that the ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to the presence of cryptic gene clusters. The activation of dormant genes is therefore one of the most important areas of experimental research for the discovery of drugs in these organisms. The recent observation that several actinomycetes possess two RNA polymerase beta-chain genes (rpoB) has opened up the possibility, explored in this study, of developing a new strategy to activate dormant gene expression in bacteria. Two rpoB paralogs, rpoB(S) and rpoB(R), provide Nonomuraea sp. strain ATCC 39727 with two functionally distinct and developmentally regulated RNA polymerases. The product of rpoB(R), the expression of which increases after transition to stationary phase, is characterized by five amino acid substitutions located within or close to the so-called rifampin resistance clusters that play a key role in fundamental activities of RNA polymerase. Here, we report that rpoB(R) markedly activated antibiotic biosynthesis in the wild-type Streptomyces lividans strain 1326 and also in strain KO-421, a relaxed (rel) mutant unable to produce ppGpp. Site-directed mutagenesis demonstrated that the rpoB(R)-specific missense H426N mutation was essential for the activation of secondary metabolism. Our observations also indicated that mutant-type or duplicated, rpoB often exists in nature among rare actinomycetes and will thus provide a basis for further basic and applied research.

  19. Recovery of Nonpathogenic Mutant Bacteria from Tumors Caused by Several Agrobacterium tumefaciens Strains: a Frequent Event?▿

    PubMed Central

    Llop, Pablo; Murillo, Jesús; Lastra, Beatriz; López, María M.

    2009-01-01

    We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor. PMID:19700547

  20. Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase.

    PubMed

    McNeely, Kelsey; Xu, Yu; Ananyev, Gennady; Bennette, Nicholas; Bryant, Donald A; Dismukes, G Charles

    2011-04-01

    The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (F(v)/F(m)). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H(2) production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H(2) production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H(2) revealed two temporally resolved phases of H(2) production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD(+) ratio revealed that the reductant generated by PFOR contributing to the first phase of H(2) production was not in equilibrium with bulk NADH/NAD(+) and that the second phase corresponded to the equilibrium NADH-mediated process. PMID:21317262

  1. Biosynthesis of a substituted cellulose from a mutant strain of Xanthomonas campestris.

    PubMed

    Vojnov, Adrián A; Bassi, Daniel E; Daniels, Michael J; Dankert, Marcelo A

    2002-02-18

    In Xanthomonas campestris the genes involved in polysaccharide (xanthan) biosynthesis are located in a gene cluster (gum) of 16 kb. A Tn5 insertion mutant with a reduced slimy phenotype has been characterized. This mutant failed to produce the pentasaccharide repeating-unit of xanthan. Only three sugars were transferred to the prenyl phosphate intermediate. Several lines of evidence suggested that the lipid-associated saccharide was the trisaccharide reducing end of the pentasaccharide from the wild-type strain. This trisaccharide was built up from UDP-Glc and GDP-Man, and a glucose residue was at the reducing end, linked to an allylic prenol through a diphosphate bridge. Results from one- or two-stage reactions showed that the trisaccharide-P-P-polyprenol was the precursor of the polymer. This new polymer, a polytrisaccharide, was detected also in vivo. The transposon responsible for the mutation was located within gumK gene. Therefore, this gene encodes for the glycosyltransferase IV, which catalyses the transfer of glucuronic acid to the lipid-linked beta-D-Manp-(1-->3)-beta-D-Glcp-(1-->4)-beta-D-Glcp trisaccharide. A recombinant plasmid with the whole gum cluster restored the wild type phenotype.

  2. Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6

    SciTech Connect

    Haigler, B.E.; Spain, J.C. )

    1989-02-01

    Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3- dihydroxy-1-methyl benzene (3-chloro-6- methylcatechol), 4-chloro- 2,3-dihydroxy-1-methyl cyclohexa- 4,6-diene (p-CT dihydrodoil), and 2-methyl-4-carboxy methylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.

  3. Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant.

    PubMed

    Kramer, Annemarie; Beck, Hans Christian; Kumar, Abhishek; Kristensen, Lars Peter; Imhoff, Johannes F; Labes, Antje

    2015-01-01

    The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum. PMID:26460745

  4. Biotransformation of L-tyrosine to Dopamine by a Calcium Alginate Immobilized Mutant Strain of Aspergillus oryzae.

    PubMed

    Ali, Sikander; Nawaz, Wajeeha

    2016-08-01

    The present research work is concerned with the biotransformation of L-tyrosine to dopamine (DA) by calcium alginate entrapped conidiospores of a mutant strain of Aspergillus oryzae. Different strains of A. oryzae were isolated from soil. Out of 13 isolated strains, isolate-2 (I-2) was found to be a better DA producer. The wild-type I-2 was chemically improved by treating it with different concentrations of ethyl methyl sulfonate (EMS). Among seven mutant variants, EMS-6 exhibiting maximal DA activity of 43 μg/ml was selected. The strain was further exposed with L-cysteine HCl to make it resistant against diversion and environmental stress. The conidiospores of selected mutant variant A. oryzae EMS-6 strain were entrapped in calcium alginate beads. Different parameters for immobilization were investigated. The activity was further improved from 44 to 62 μg/ml under optimized conditions (1.5 % sodium alginate, 2 ml inoculum, and 2 mm bead size). The best resistant mutant variable exhibited over threefold increase in DA activity (62 μg/ml) than did wild-type I-2 (21 μg/ml) in the reaction mixture. From the results presented in the study, it was observed that high titers of DA activity in vitro could effectively be achieved by the EMS-induced mutagenesis of filamentous fungus culture used.

  5. Induced drought tolerance through wild and mutant bacterial strain Pseudomonas simiae in mung bean (Vigna radiata L.).

    PubMed

    Kumari, Sarita; Vaishnav, Anukool; Jain, Shekhar; Varma, Ajit; Choudhary, Devendra Kumar

    2016-01-01

    The present study focused on the overproducing mutant of a plant growth promoting rhizobacterium (PGPR) Pseudomonas simiae strain AU (MTCC-12057) for significant drought tolerance in mung bean plants. Five mutants namely AU-M1, AU-M2, AU-M3, AU-M4 and AU-M5 were made after treatment of wild type strain with N-methyl-N-nitro-N-nitrosoguanidine. Mutant strain AU-M4 was recorded for enhanced ACC deaminase (ACC-D) activity, indole acetic acid (IAA) production and inorganic phosphate (Pi) solubilization compared to wild strain and other four mutant strains under drought condition. AU-M4 showed higher phosphate solubilization index (8.17) together with higher ACC-D activity (98 nmol/mg/h) and IAA concentration (69.35 µg/ml) compared with the wild type P. simiae strain AU ACC-D activity (79 nmol/mg/h) and IAA concentration (38.98 µg/ml) respectively. In this report, we investigated the effect of both wild and mutant type bacterial strain on mung bean plants under drought stress. Results showed that mutant AU-M4 and wild type strain AU inoculated plants exhibited superior tolerance against drought stress, as shown by their enhanced plant biomass (fresh weight), higher water content, higher proline accumulation and lower osmotic stress injury. Mutant AU-M4 and wild strain AU inoculated plants reduced the ethylene level by 59 and 45% respectively, compared to the control under stress condition. Furthermore, bacterial inoculated plants showed enhanced induced systemic drought tolerance by reducing stomata size and net photosynthesis resulting higher water content in mung bean plants that may help in survival of plants during drought condition. To mitigate the effects of drought stress, use of PGPR will be needed to ensure sufficient production of food from crop plants. Taking current leads available, concerted future research is needed in this area, particularly on field evaluation with application of potential microorganisms.

  6. Induced drought tolerance through wild and mutant bacterial strain Pseudomonas simiae in mung bean (Vigna radiata L.).

    PubMed

    Kumari, Sarita; Vaishnav, Anukool; Jain, Shekhar; Varma, Ajit; Choudhary, Devendra Kumar

    2016-01-01

    The present study focused on the overproducing mutant of a plant growth promoting rhizobacterium (PGPR) Pseudomonas simiae strain AU (MTCC-12057) for significant drought tolerance in mung bean plants. Five mutants namely AU-M1, AU-M2, AU-M3, AU-M4 and AU-M5 were made after treatment of wild type strain with N-methyl-N-nitro-N-nitrosoguanidine. Mutant strain AU-M4 was recorded for enhanced ACC deaminase (ACC-D) activity, indole acetic acid (IAA) production and inorganic phosphate (Pi) solubilization compared to wild strain and other four mutant strains under drought condition. AU-M4 showed higher phosphate solubilization index (8.17) together with higher ACC-D activity (98 nmol/mg/h) and IAA concentration (69.35 µg/ml) compared with the wild type P. simiae strain AU ACC-D activity (79 nmol/mg/h) and IAA concentration (38.98 µg/ml) respectively. In this report, we investigated the effect of both wild and mutant type bacterial strain on mung bean plants under drought stress. Results showed that mutant AU-M4 and wild type strain AU inoculated plants exhibited superior tolerance against drought stress, as shown by their enhanced plant biomass (fresh weight), higher water content, higher proline accumulation and lower osmotic stress injury. Mutant AU-M4 and wild strain AU inoculated plants reduced the ethylene level by 59 and 45% respectively, compared to the control under stress condition. Furthermore, bacterial inoculated plants showed enhanced induced systemic drought tolerance by reducing stomata size and net photosynthesis resulting higher water content in mung bean plants that may help in survival of plants during drought condition. To mitigate the effects of drought stress, use of PGPR will be needed to ensure sufficient production of food from crop plants. Taking current leads available, concerted future research is needed in this area, particularly on field evaluation with application of potential microorganisms. PMID:26712619

  7. The extracellular protein regulator (xpr) affects exoprotein and agr mRNA levels in Staphylococcus aureus.

    PubMed Central

    Hart, M E; Smeltzer, M S; Iandolo, J J

    1993-01-01

    xpr, a regulatory element of exoprotein synthesis in Staphylococcus aureus, defined by an insertion of Tn551 into the chromosome of strain S6C, affects the expression of several exoproteins at the mRNA level. Drastic reduction in transcript levels for staphylococcal enterotoxin B (seb), lipase (geh), alpha-toxin (hla), and delta-toxin (hld) were detected, while mRNA levels for coagulase (coa) and protein A (spa) were elevated. Because the delta-toxin gene resides within the RNAIII transcript of the exoprotein regulator, agr, the reduction in hld message in the mutant strain of S6C is indicative of additional regulatory events in exoprotein gene expression. Northern (RNA) analysis of total cellular RNA hybridized with probes specific for RNAII and RNAIII (the two major transcripts of the agr operon) showed that both transcripts were reduced 16- to 32-fold at 3 h (late exponential phase) and 8- to 16-fold at 12 h (postexponential phase). These data confirm our original findings (M. S. Smeltzer, M. E. Hart, and J. J. Iandolo, Infect. Immun. 61:919-925, 1993) that two regulatory loci, agr and xpr, are interactive at the genotypic level. Images PMID:7504665

  8. Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

    PubMed

    Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

    2016-10-01

    We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression. PMID:27309759

  9. A short-filament mutant of Anabaena sp. strain PCC 7120 that fragments in nitrogen-deficient medium.

    PubMed Central

    Bauer, C C; Buikema, W J; Black, K; Haselkorn, R

    1995-01-01

    Strain 129 is a fragmentation mutant of the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Growing with fixed nitrogen, this mutant forms filaments that are much shorter than wild-type filaments. Following starvation for fixed nitrogen, strain 129 becomes nearly unicellular and forms few heterocysts, although electron microscopy suggests that proheterocysts form while fragmentation occurs. Starvation for sulfate, phosphate, iron, and calcium does not cause this fragmentation. The affected gene in strain 129, fraC, was cloned by complementation and characterized. It encodes a unique 179-amino-acid protein rich in phenylalanine. Insertional inactivation of the chromosomal copy of fraC results in a phenotype identical to that of strain 129, while complementation using a truncated version of FraC results in only partial complementation of the original mutant. Heterocysts could be induced to form in N-replete cultures of strain 129, as in wild-type cells, by supplying extra copies of the hetR gene on a plasmid. Thus, FraC is required for the integrity of cell junctions in general but is apparently not directly involved in normal differentiation and nitrogen fixation. PMID:7883709

  10. Draft Genome Sequence of the Intermediate Rough Vaccine Strain Brucella abortus S19Δper Mutant.

    PubMed

    Chaudhuri, Pallab; Goswami T, Tapas K; Lalsiamthara, Jonathan; Kaur, Gurpreet; Vishnu, Udayakumar S; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2015-11-12

    Here, we report the genome sequence of the intermediate rough vaccine strain mutant, Brucella abortus S19Δper. The length of the draft genome was 3,271,238 bp, with 57.2% G+C content. A total of 3,204 protein-coding genes and 56 RNA genes were predicted.

  11. Draft Genome Sequence of the Intermediate Rough Vaccine Strain Brucella abortus S19Δper Mutant

    PubMed Central

    Chaudhuri, Pallab; Goswami T, Tapas K.; Lalsiamthara, Jonathan; Kaur, Gurpreet; Vishnu, Udayakumar S.; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy

    2015-01-01

    Here, we report the genome sequence of the intermediate rough vaccine strain mutant, Brucella abortus S19Δper. The length of the draft genome was 3,271,238 bp, with 57.2% G+C content. A total of 3,204 protein-coding genes and 56 RNA genes were predicted. PMID:26564050

  12. Altered calmodulin activity in fluphenazine-resistant mutant strains. Pleiotropic effect on development and cellular organization in Volvox carteri.

    PubMed

    Kurn, N; Sela, B A

    1981-12-01

    Genetically altered calmodulin activity in spontaneously derived mutant strains, which were selected for resistance to the toxic effect of a specific inhibitor, the phenothiazine drug fluphenazine, is demonstrated. Partially purified calmodulin preparations from wild-type and fluphenazine-resistant strains of the multicellular alga Volvox carteri, were tested for the ability to activate Ca2+-ATPase of the erythrocyte membranes, and the inhibition of this stimulatory activity by fluphenazine. Unlike the preparation obtained from wild-type cells, mutant calmodulin is shown to be insensitive to fluphenazine inhibition, in one case, and calmodulin from another strain was found to be inactive in vitro, i.e. it did not activate Ca2+-ATPase. The pleiotropic phenotype of the spontaneously derived mutant strains involved aberrant multicellular organization and hormone-independent commitment of the multipotent asexual reproductive cells, gonodia, to sexual development. These results clearly implicate calmodulin in the control of development and morphogenesis in this simple multicellular eukaryote. In addition, intracellular inhibition of calmodulin in wild-type cells is shown to block the morphogenic process of embryo inversion and to arrest motility. The availability of mutant calmodulin will facilitate further investigation of the role of this ubiquitous regulatory protein in the control of development and differentiation in multicellular eukarytes, as well as the fine structure/function relationship with regard to calmodulin modulation of a wide variety of cellular processes. PMID:6459931

  13. Enhanced production of thrombinase by Streptomyces venezuelae: kinetic studies on growth and enzyme production of mutant strain.

    PubMed

    Naveena, Balakrishnan; Gopinath, Kannapan Panchamoorthy; Sakthiselvan, Punniavan; Partha, Nagarajan

    2012-05-01

    This investigation provides the enhanced production of thrombinase, a fibrinolytic enzyme using mutant Streptomyces venezuelae. Initially the mutagenesis of the marine isolate was done by UV and Ethyl methane sulfonate (EMS) and their mutational efficiencies were compared. The mutants were selected based on their high thrombinase activity and used for further studies. The mutant was found to be more halo and thermo tolerant comparing to wild. The effect of Dissolved oxygen level was also determined and the mutant offered the maximum specific growth rate as 0.2404 (h(-1)). The mutant showed high resistance to higher initial lactose concentration and the inhibition concentration was found to be 155.1mg/mL. The effect of S(0)/X(0) ratio on specific substrate consumption and production rate were also investigated. Both mutant and wild showed increase in specific substrate consumption and production rate at higher S(0)/X(0) ratio but the mutant showed better values than the wild strain. PMID:22401714

  14. Enhanced bioethanol production from wheat straw hemicellulose by mutant strains of pentose fermenting organisms Pichia stipitis and Candida shehatae.

    PubMed

    Koti, Sravanthi; Govumoni, Sai Prashanthi; Gentela, Jahnavi; Venkateswar Rao, L

    2016-01-01

    The main aim of the present study was to mutate yeast strains, Pichia stipitis NCIM 3498 and Candida shehatae NCIM 3501 and assess the mutant's ability to utilize, ferment wheat straw hemicellulose with enhanced ethanol yield. The organisms were subjected to random mutagenesis using physical (ultraviolet radiation) and chemical (ethidium bromide) mutagens. The mutant and wild strains were used to ferment the hemicellulosic hydrolysates of wheat straw obtained by 2 % dilute sulphuric acid and enzymatic hydrolysis by crude xylanase separately. Among all the mutant strains, PSUV9 and CSEB7 showed enhanced ethanol production (12.15 ± 0.57, 9.55 ± 0.47 g/L and yield 0.450 ± 0.009, 0.440 ± 0.001 g/g) as compared to the wild strains (8.28 ± 0.54, 7.92 ± 0.89 g/L and yield 0.380 ± 0.006 and 0.370 ± 0.002 g/g) in both the hydrolysates. The mutant strains were also checked for their consistency in ethanol production and found stable for 19 cycles in hemicellulosic hydrolysates of wheat straw. A novel element in the present study was introduction of chemical mutagenesis in wild type as well as UV induced mutants. This combination of treatments i.e., UV followed by chemical mutagenesis was practically successful. PMID:27652118

  15. Assessing benzene-induced toxicity on wild type Euglena gracilis Z and its mutant strain SMZ.

    PubMed

    Peng, Cheng; Arthur, Dionne M; Sichani, Homa Teimouri; Xia, Qing; Ng, Jack C

    2013-11-01

    Benzene is a representative member of volatile organic compounds and has been widely used as an industrial solvent. Groundwater contamination of benzene may pose risks to human health and ecosystems. Detection of benzene in the groundwater using chemical analysis is expensive and time consuming. In addition, biological responses to environmental exposures are uninformative using such analysis. Therefore, the aim of this study was to employ a microorganism, Euglena gracilis (E. gracilis) as a putative model to monitor the contamination of benzene in groundwater. To this end, we examined the wild type of E. gracilis Z and its mutant form, SMZ in their growth rate, morphology, chlorophyll content, formation of reactive oxygen species (ROS) and DNA damage in response to benzene exposure. The results showed that benzene inhibited cell growth in a dose response manner up to 48 h of exposure. SMZ showed a greater sensitivity compared to Z in response to benzene exposure. The difference was more evident at lower concentrations of benzene (0.005-5 μM) where growth inhibition occurred in SMZ but not in Z cells. We found that benzene induced morphological changes, formation of lipofuscin, and decreased chlorophyll content in Z strain in a dose response manner. No significant differences were found between the two strains in ROS formation and DNA damage by benzene at concentrations affecting cell growth. Based on these results, we conclude that E. gracilis cells were sensitive to benzene-induced toxicities for certain endpoints such as cell growth rate, morphological change, depletion of chlorophyll. Therefore, it is a potentially suitable model for monitoring the contamination of benzene and its effects in the groundwater.

  16. Cellulase production and saccharification of rice straw by the mutant strain Hypocrea koningii RSC1.

    PubMed

    Palaniyandi, Sasikumar Arunachalam; Yang, Seung Hwan; Suh, Joo-Won

    2014-01-01

    The production of cellulase using solid-state fermentation of rice straw by the mutant strain Hypocrea koningii RSC1 was studied. Optimization of culture conditions, such as the nitrogen source, pH, and temperature, resulted in a maximum filter paper cellulase activity of 44.15 U g(-1) substrate, a carboxymethylcellulase activity of 324.6 U g(-1) substrate, and a β-glucosidase activity of 7.45 U g(-1) substrate. Saccharification of untreated, 1% H(2)SO(4)-treated, and 2.5% NaOH-treated rice straw using the RSC1 cellulase resulted in 19, 17, and 34 g L(-1) of reducing sugar, respectively. Further studies on the morphological and compositional changes of rice straw upon treatment with the cellulase by scanning electron microscopy analysis and Fourier transform infrared spectroscopy revealed the disruption of the arrangement of fibers and changes in the functional groups that occur in cellulose. X-ray diffraction analysis revealed a reduction in crystallinity of the rice straw upon treatment with the cellulase. Our study shows that H. koningii RSC1 could be a good choice for the production of cellulase and reducing sugars from rice straw.

  17. Pilin regulation in the pilT mutant of Neisseria gonorrhoeae strain MS11

    PubMed Central

    Dietrich, Manuela; Mollenkopf, Hans; So, Magdalene; Friedrich, Alexandra

    2009-01-01

    The ATPase protein PilT mediates retraction of type IV pili (Tfp). Tfp retraction of Neisseria gonorrhoeae causes many signal transduction events and changes in gene expression in infected epithelial cells. To find out whether a pilT mutation and lack of Tfp retraction, respectively, lead also to gene regulation in bacteria we performed microarrays comparing the transcriptional profiles of the N. gonorrhoeae parent strain MS11 and its isogenic pilT mutant during growth in vitro. A loss-of-function-mutation in pilT led to altered transcript levels of 63 open reading frames. Levels of pilE transcripts and its deduced protein the major Tfp subunit pilin, were increased most markedly by a mutation in pilT. Further studies revealed that pilE expression was also controlled by two other genes encoding Tfp biogenesis proteins, pilD and pilF. Our studies strongly suggest that pilE expression is a finely-tuned process. PMID:19486161

  18. Shedding of Brucella abortus rough mutant strain RB51 in milk of water buffalo (Bubalus bubalis).

    PubMed

    Longo, Mariangela; Mallardo, Karina; Montagnaro, Serena; De Martino, Luisa; Gallo, Sergio; Fusco, Giovanna; Galiero, Giorgio; Guarino, Achille; Pagnini, Ugo; Iovane, Giuseppe

    2009-07-01

    The objective of this study was to determine if Brucella abortus rough mutant strain RB51 (SRB51) is eliminated in buffalo milk. Thirty Brucella-free female buffaloes were used in this study: ten 4-5 years old were inoculated with the triple of the recommended calfhood dose of SRB51 by subcutaneous route, ten 2-3 years old at the first lactation were previously vaccinated twice as calves with triple the recommended calf dose of RB51, while five 4-5 years old and five 2-3 years old not vaccinated Brucella-free female buffaloes served as controls. Milk samples were taken aseptically on a daily basis for the first 30 days and weekly for the second and third months. The samples were inoculated on selective media for isolation of SRB51 and incubated for 11 days. Moreover, PCR analysis was also performed directly on milk samples. SRB51 was isolated from milk samples only during the first week post-vaccination while RB51 DNA was detected during the first week till the fourth week post-vaccination only in water buffaloes vaccinated as adults. The identification of Brucella RB51 in milk samples, strongly suggests that this Brucella vaccine could be excreted in milk of buffalo cows vaccinated as adults, while our data demonstrate that the vaccine is safe for use in buffaloes vaccinated as calves in which it was not excreted in milk.

  19. Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen

    SciTech Connect

    Wolk, C.P.; Cai, Y.; Cardemil, L.; Flores, E.; Hohn, B.; Murry, M.; Schmetterer, G.; Schrautemeier, B.; Wilson, R.

    1988-03-01

    Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.

  20. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

    PubMed Central

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; dos Reis, Ana Beatriz de Bragança; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-01-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins. PMID:27223868

  1. Dynamics of Photosynthesis in a Glycogen-Deficient glgC Mutant of Synechococcus sp. Strain PCC 7002

    PubMed Central

    Jackson, Simon A.; Eaton-Rye, Julian J.; Bryant, Donald A.; Posewitz, Matthew C.

    2015-01-01

    Cyanobacterial glycogen-deficient mutants display impaired degradation of light-harvesting phycobilisomes under nitrogen-limiting growth conditions and secrete a suite of organic acids as a putative reductant-spilling mechanism. This genetic background, therefore, represents an important platform to better understand the complex relationships between light harvesting, photosynthetic electron transport, carbon fixation, and carbon/nitrogen metabolisms. In this study, we conducted a comprehensive analysis of the dynamics of photosynthesis as a function of reductant sink manipulation in a glycogen-deficient glgC mutant of Synechococcus sp. strain PCC 7002. The glgC mutant showed increased susceptibility to photoinhibition during the initial phase of nitrogen deprivation. However, after extended periods of nitrogen deprivation, glgC mutant cells maintained higher levels of photosynthetic activity than the wild type, supporting continuous organic acid secretion in the absence of biomass accumulation. In contrast to the wild type, the glgC mutant maintained efficient energy transfer from phycobilisomes to photosystem II (PSII) reaction centers, had an elevated PSII/PSI ratio as a result of reduced PSII degradation, and retained a nitrogen-replete-type ultrastructure, including an extensive thylakoid membrane network, after prolonged nitrogen deprivation. Together, these results suggest that multiple global signals for nitrogen deprivation are not activated in the glgC mutant, allowing the maintenance of active photosynthetic complexes under conditions where photosynthesis would normally be abolished. PMID:26150450

  2. Mutant strains of Spirulina (Arthrospira) platensis to increase the efficiency of micro-ecological life support systems

    NASA Astrophysics Data System (ADS)

    Brown, Igor

    The European Micro-Ecological Life Support System Alternative (MELiSSA) is an advanced idea for organizing a bioregenerative system for long term space flights and extraterrestrial settlements (Hendrickx, De Wever et al., 2005). Despite the hostility of both lunar and Martian environments to unprotected life, it seems possible to cultivate photosynthetic bacteria using closed bioreactors illuminated and heated by solar energy. Such reactors might be employed in critical processes, e.g. air revitalization, foodcaloric and protein source, as well as an immunomodulators production. The MELiSSA team suggested cyanobacterium Spirulina as most appropriate agent to revitalize air and produce a simple "fast" food. This is right suggestion because Spirulina was recently shown to be an oxygenic organism with the highest level of O2 production per unit mass (Ananyev et al., 2005). Chemical composition of Spirulina includes proteins (55Aiming to make Spirulina cultivation in life support systems like MELiSSA more efficient, we selected Spirulina mutant strains with increased fraction of methionine in the biomass of this cyanobacterium and compared the effect of parental wild strain of Spirulina and its mutants on the tendency of such experimental illnesses as radiationinduced lesions and hemolythic anemia. Results: It was found that mutant strains 198B and 27G contain higher quantities of total protein, essential amino acids, c-phycocyanin, allophycocyanin and chlorophyll a than parental wild strain of S. platensis. The strain 198B is also characterized with increased content of carotenoids. Revealed biochemical peculiarities of mutant strains suggest that these strains can serve as an additional source of essential amino acids as well as phycobiliproteins and carotenoids for the astronauts. Feeding animals suffering from radiation-induced lesions, c-phycocyanin, extracted from strain 27G, led to a correction in deficient dehydrogenase activity and energy-rich phosphate levels

  3. Transcriptome Profiling of Wild-Type and pga-Knockout Mutant Strains Reveal the Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans

    PubMed Central

    Shanmugam, Mayilvahanan; El Abbar, Faiha; Ramasubbu, Narayanan

    2015-01-01

    Exopolysaccharides have a diverse set of functions in most bacteria including a mechanistic role in protecting bacteria against environmental stresses. Among the many functions attributed to the exopolysaccharides, biofilm formation, antibiotic resistance, immune evasion and colonization have been studied most extensively. The exopolysaccharide produced by many Gram positive as well as Gram negative bacteria including the oral pathogen Aggregatibacter actinomycetemcomitans is the homopolymer of β(1,6)-linked N-acetylglucosamine. Recently, we reported that the PGA-deficient mutant of A. actinomycetemcomitans failed to colonize or induce bone resorption in a rat model of periodontal disease, and the colonization genes, apiA and aae, were significantly down regulated in the mutant strain. To understand the role of exopolysaccharide and the pga locus in the global expression of A. actinomycetemcomitans, we have used comparative transcriptome profiling to identify differentially expressed genes in the wild-type strain in relation to the PGA-deficient strain. Transcriptome analysis revealed that about 50% of the genes are differently expressed (P < 0.05 and fold change >1.5). Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence. Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth. Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans. PMID:26221956

  4. Immune responses and protection against infection and abortion in cattle experimentally vaccinated with mutant strains of Brucella abortus.

    PubMed

    Cheville, N F; Stevens, M G; Jensen, A E; Tatum, F M; Halling, S M

    1993-10-01

    Twenty-four 10-month-old Polled Hereford heifers were inoculated SC with live cells of one of the following strains of Brucella abortus: S19 delta 31K (n = 4), S19 delta SOD (n = 4), RB51 (n = 4), and strain 19 (n = 6); controls (n = 6) were given saline solution. Heifers given the deletion mutants S19 delta 31K and S19 delta SOD, and those given strain 19 developed antibody responses to B abortus and cutaneous reactions to brucellin. Heifers given strain RB51 did not develop antibodies that reacted in the standard tube agglutination test, but sera reacted in tests, using an antibody dot-blot assay containing RB51 antigen. The S19 delta 31K and S19 delta SOD strains of B abortus isolated from lymph node tissue after vaccination did not differ genetically from the master stock strain. All heifers were bred naturally at 16 to 17 months of age, and were challenge-exposed intraconjunctivally with virulent B abortus strain 2308 during the fifth month of pregnancy. All vaccinated heifers were protected (ie, none aborted and none had B abortus isolated from their tissues after parturition). Calves born from vaccinated dams were free of B abortus. Antibody responses in heifers after challenge exposure were an indicator of immunity. All 5 control heifers (nonvaccinated) developed serum antibodies after challenge exposure; 3 aborted, and 1 delivered a small, weak calf at 8.5 months of gestation. Thus live mutant strains of B abortus can induce protective immunity when given at 10 months of age, and strain RB51 is a strong candidate for further testing.

  5. Anthraquinone dyes decolorization capacity of anamorphic Bjerkandera adusta CCBAS 930 strain and its HRP-like negative mutants.

    PubMed

    Korniłłowicz-Kowalska, Teresa; Rybczyńska, Kamila

    2014-06-01

    Cultures of the anamorphic fungus Bjerkandera adusta CCBAS 930 decolorizing, in stationary cultures, 0.01 % solutions of carminic acid and Poly R-478, were characterised by a strong increase in the activity of the horseradish peroxidase (HRP-like) and manganese-dependent peroxidase (MnP) at a low activity of lignin peroxidase. Genotypically modified mutants of B. adusta CCBAS 930: 930-5 and 930-14, with total or partial loss of decolorization capabilities relative to anthraquinonic dyes, showed inhibition of the activity of HRP-like peroxidase and MnP. Whereas, compared to the parental strain, in the mutant cultures there was an increase in the activity of lignin peroxidase and laccase. The paper presents a discussion of the role of the studied enzymatic activities in the process of decolorization of anthraquinonic dyes by the strain B. adusta CCBAS 930. PMID:24415463

  6. Transcriptional changes in the nuc-2A mutant strain of Neurospora crassa cultivated under conditions of phosphate shortage.

    PubMed

    Gras, Diana E; Silveira, Henrique C S; Peres, Nalu T A; Sanches, Pablo R; Martinez-Rossi, Nilce M; Rossi, Antonio

    2009-01-01

    The molecular mechanism that controls the response to phosphate shortage in Neurospora crassa involves four regulatory genes -nuc-2, preg, pgov, and nuc-1. Phosphate shortage is sensed by the nuc-2 gene, the product of which inhibits the functioning of the PREG-PGOV complex. This allows the translocation of the transcriptional factor NUC-1 into the nucleus, which activates the transcription of phosphate-repressible phosphatases. The nuc-2A mutant strain of N. crassa carries a loss-of-function mutation in the nuc-2 gene, which encodes an ankyrin-like repeat protein. In this study, we identified transcripts that are downregulated in the nuc-2A mutant strain. Functional grouping of these expressed sequence tags allowed the identification of genes that play essential roles in different cellular processes such as transport, transcriptional regulation, signal transduction, metabolism, protein synthesis, protein fate, and development. These results reveal novel aspects of the phosphorus-sensing network in N. crassa.

  7. [Study of the transcriptional and transpositional activities of the Tirant retrotransposon in Drosophila melanogaster strains mutant for the flamenco locus].

    PubMed

    Nefedova, L N; Urusov, F A; Romanova, N I; Shmel'kova, A O; Kim, A I

    2012-11-01

    Transpositions of the gypsy retrotransposon in the Drosophila melanogaster genome are controlled by the flamenco locus, which is represented as an accumulation of defective copies of transposable elements. In the present work, genetic control by the flamenco locus of the transcriptional and transpositional activities of the Tirant retrotransposon from the gypsy group was studied. Tissue-specific expression of Tirant was detected in the tissues of ovaries in a strain mutant for the flamenco locus. Tirant was found to be transpositionally active in isogenic D. melanogaster strains mutant for the flamenco locus. The sites of two new insertions have been localized by the method of subtractive hybridization. It has been concluded from the results obtained that the flamenco locus is involved in the genetic control of Tirant transpositions. PMID:23297482

  8. Constitutive expression of the tzs gene from Agrobacterium tumefaciens virG mutant strains is responsible for improved transgenic plant regeneration in cotton meristem transformation.

    PubMed

    Ye, Xudong; Chen, Yurong; Wan, Yuechun; Hong, Yun-Jeong; Ruebelt, Martin C; Gilbertson, Larry A

    2016-03-01

    KEY MESSAGE : virG mutant strains of a nopaline type of Agrobacterium tumefaciens increase the transformation frequency in cotton meristem transformation. Constitutive cytokinin expression from the tzs gene in the virG mutant strains is responsible for the improvement. Strains of Agrobacterium tumefaciens were tested for their ability to improve cotton meristem transformation frequency. Two disarmed A. tumefaciens nopaline strains with either a virGN54D constitutively active mutation or virGI77V hypersensitive induction mutation significantly increased the transformation frequency in a cotton meristem transformation system. The virG mutant strains resulted in greener explants after three days of co-culture in the presence of light, which could be attributed to a cytokinin effect of the mutants. A tzs knockout strain of virGI77V mutant showed more elongated, less green explants and decreased cotton transformation frequency, as compared to a wild type parental strain, suggesting that expression of the tzs gene is required for transformation frequency improvement in cotton meristem transformation. In vitro cytokinin levels in culture media were tenfold higher in the virGN54D strain, and approximately 30-fold higher in the virGI77V strain, in the absence of acetosyringone induction, compared to the wild type strain. The cytokinin level in the virGN54D strain is further increased upon acetosyringone induction, while the cytokinin level in the virGI77V mutant is decreased by induction, suggesting that different tzs gene expression regulation mechanisms are present in the two virG mutant strains. Based on these data, we suggest that the increased cytokinin levels play a major role in increasing Agrobacterium attachment and stimulating localized division of the attached plant cells. PMID:26650837

  9. Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants.

    PubMed Central

    Drigues, P; Demery-Lafforgue, D; Trigalet, A; Dupin, P; Samain, D; Asselineau, J

    1985-01-01

    The composition of the Pseudomonas solanacearum lipolysaccharide (LPS) was found to be similar to that described for the LPS of enterobacteria. The lipid A contained fatty acids and glucosamine in a molar ratio of 5:2. The LPS fraction contained 2-keto-3-deoxyoctulosonic acid, L-glycero-D-mannoheptose, hexoses (glucose, rhamnose, and glucosamine), and a pentose (xylose). The LPSs from the wild-type strain (GMI1000), from the spontaneous rough mutant (GMI2000), and from their respective acridine orange-resistant (Acrr) mutants (GMI1178 and GMI2179) contained the same component sugars in their polysaccharide moieties, but the relative amounts of each sugar varied greatly. Spontaneous mutation to the rough type was characterized by a decrease in the ratio of rhamnose to glucose, whereas a reverse effect was seen for the acridine orange resistance mutation from the parent strains (GMI1000 and GMI2000) to the respective mutant strains (GMI1178 and GMI2179). The exopolysaccharide (EPS) from GMI1000 was found to be composed of two fractions: a heteropolysaccharide (galactosamine, glucose, and rhamnose) excluded from Sephadex G-50 and an additional glucan with a lower molecular weight. Strains GMI1000 and GMI1178 produced comparable amounts of EPS, GMI2179 synthesized less EPS, and GMI2000 produced no detectable EPS. High-pressure liquid chromatography and 13C nuclear magnetic resonance analyses revealed some differences between these EPSs. The glucan fraction seemed to be the major component of the EPS from GMI2179, whereas GMI1000 and GMI1178 EPSs contained both fractions and appeared to differ in the structures of their heteropolysaccharide fractions. Viscosity measurements confirmed differences between whole EPSs produced by the three strains. PMID:3988700

  10. [Analysis of the structure and expression of the DIP1 gene in Drosophila melanogaster strains mutant for the flamenco gene].

    PubMed

    Nefedova, L N; Potanova, M V; Romanova, N I; Kim, A I

    2009-02-01

    DIP1 gene transcription was analyzed with the use of RT-PCR in three Drosophila melanogaster strains with the flamenco- phenotype (flam(SS), flam(MS), and flam(Ore)) and in one flamenco+ strain at the stages of embryos (0-24 h), third-instar larvae, and adult flies. The mutant strains flam(SS) and flam(Ore) lack an active copy of transposon gypsy. Theflam(MS) strain was obtained by introducing an active copy of gypsy in flies of theflam(SS) strain and is characterized by a high rate of gypsy transpositions. The experiments showed that at least five forms of DIP1 gene transcripts are produced. The form of cDNA corresponding to CDS DIP1-d was discovered only in embryos. It was found that DIP1 gene transcription depends on the age of flies: at the larval stage the level of transcription is significantly reduced. However, no reduction of gene transcription is observed in theflam(Ore) strain. These results suggest that the flamenco- phenotype may be associated with an alteration of DIP1 gene transcription, as in differentflamenco- strains the DIP1 gene expression is changed differently. PMID:19334614

  11. Enhanced desulfurization in a transposon-mutant strain of Rhodococcus erythropolis.

    PubMed

    Watanabe, Kimiko; Noda, Ken-ichi; Maruhashi, Kenji

    2003-08-01

    The dsz desulfurization gene cluster from Rhodococcus erythropolis strain KA2-5-1 was transferred into R. erythropolis strain MC1109, unable to desulfurize light gas oil (LGO), using a transposon-transposase complex. As a result, two recombinant strains, named MC0203 and MC0122, were isolated. Resting cells of strain MC0203 decreased the sulfur concentration of LGO from 120 mg l(-1) to 70 mg l(-1) in 2 h. The LGO-desulfurization activity of strain MC0203 was about twice that of strain MC0122 and KA2-5-1. The 10-methyl fatty acids of strain MC0203 were about 28%-41% that of strain MC1109. It is likely that strain MC0203 had a mutation involving alkylenation or methylation of delta9-unsaturated fatty acids caused by the transposon inserted in the chromosome, which increased the fluidity of cell membranes and enhanced the desulfurization activity.

  12. Increase in Ty1 cDNA Recombination in Yeast sir4 Mutant Strains at High Temperature

    PubMed Central

    Radford, Sarah J.; Boyle, Meredith L.; Sheely, Catherine J.; Graham, Joel; Haeusser, Daniel P.; Zimmerman, Leigh; Keeney, Jill B.

    2004-01-01

    Transposition of the Ty1 element of the yeast Saccharomyces cerevisiae is temperature sensitive. We have identified a null allele of the silent information regulator gene SIR4 as a host mutant that allows for transposition at high temperature. We show that the apparent increase in transposition activity in sir4 mutant strains at high temperature is dependent on the RAD52 gene and is thus likely resulting from an increase in Ty1 cDNA recombination, rather than in IN-mediated integration. General cellular recombination is not increased at high temperature, suggesting that the increase in recombination at high temperature in sir4 mutants is specific for Ty1 cDNA. Additionally, this high-temperature Ty1 recombination was found to be dependent on functional Sir2p and Sir3p. We speculate that the increase in recombination seen in sir4 mutants at high temperature may be due to changes in chromatin structure or Ty1 interactions with chromosomal structures resulting in higher recombination rates. PMID:15454529

  13. Tissue persistence and vaccine efficacy of tricarboxylic acid cycle and one-carbon metabolism mutant strains of Edwardsiella ictaluri.

    PubMed

    Dahal, Neeti; Abdelhamed, Hossam; Karsi, Attila; Lawrence, Mark L

    2014-06-30

    Edwardsiella ictaluri causes enteric septicemia in fish. Recently, we reported construction of E. ictaluri mutants with single and double gene deletions in tricarboxylic acid cycle (TCA) and one-carbon (C-1) metabolism. Here, we report the tissue persistence, virulence, and vaccine efficacy of TCA cycle (EiΔsdhC, EiΔfrdA, and EiΔmdh), C-1 metabolism (EiΔgcvP and EiΔglyA), and combination mutants (EiΔfrdAΔsdhC, EiΔgcvPΔsdhC, EiΔmdhΔsdhC, and EiΔgcvPΔglyA) in channel catfish. The tissue persistence study showed that EiΔsdhC, EiΔfrdA, EiΔfrdAΔsdhC, and EiΔgcvPΔsdhC were able to invade catfish and persist until 11 days post-infection. Vaccination of catfish fingerlings with all nine mutants provided significant (P<0.05) protection against subsequent challenge with the virulent parental strain. Vaccinated catfish fingerlings had 100% survival when subsequently challenged by immersion with wild-type E. ictaluri except for EiΔgcvPΔglyA and EiΔgcvP. Mutant EiΔgcvPΔsdhC was found to be very good at protecting catfish fry, as evidenced by 10-fold higher survival compared to non-vaccinated fish.

  14. Francisella tularensis live vaccine strain folate metabolism and pseudouridine synthase gene mutants modulate macrophage caspase-1 activation.

    PubMed

    Ulland, Tyler K; Janowski, Ann M; Buchan, Blake W; Faron, Matthew; Cassel, Suzanne L; Jones, Bradley D; Sutterwala, Fayyaz S

    2013-01-01

    Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses to F. tularensis. We have identified the 5-formyltetrahydrofolate cycloligase gene (FTL_0724) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL_0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL_0724 mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophages in vitro. In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase (FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL_0699 mutant was not attenuated in vivo. These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo.

  15. Francisella tularensis Live Vaccine Strain Folate Metabolism and Pseudouridine Synthase Gene Mutants Modulate Macrophage Caspase-1 Activation

    PubMed Central

    Ulland, Tyler K.; Janowski, Ann M.; Buchan, Blake W.; Faron, Matthew; Cassel, Suzanne L.; Jones, Bradley D.

    2013-01-01

    Francisella tularensis is a Gram-negative bacterium and the causative agent of the disease tularemia. Escape of F. tularensis from the phagosome into the cytosol of the macrophage triggers the activation of the AIM2 inflammasome through a mechanism that is not well understood. Activation of the AIM2 inflammasome results in autocatalytic cleavage of caspase-1, resulting in the processing and secretion of interleukin-1β (IL-1β) and IL-18, which play a crucial role in innate immune responses to F. tularensis. We have identified the 5-formyltetrahydrofolate cycloligase gene (FTL_0724) as being important for F. tularensis live vaccine strain (LVS) virulence. Infection of mice in vivo with a F. tularensis LVS FTL_0724 mutant resulted in diminished mortality compared to infection of mice with wild-type LVS. The FTL_0724 mutant also induced increased inflammasome-dependent IL-1β and IL-18 secretion and cytotoxicity in macrophages in vitro. In contrast, infection of macrophages with a F. tularensis LVS rluD pseudouridine synthase (FTL_0699) mutant resulted in diminished IL-1β and IL-18 secretion from macrophages in vitro compared to infection of macrophages with wild-type LVS. In addition, the FTL_0699 mutant was not attenuated in vivo. These findings further illustrate that F. tularensis LVS possesses numerous genes that influence its ability to activate the inflammasome, which is a key host strategy to control infection with this pathogen in vivo. PMID:23115038

  16. Strain-Dependent Anterior Segment Dysgenesis and Progression to Glaucoma in Col4a1 Mutant Mice

    PubMed Central

    Mao, Mao; Smith, Richard S.; Alavi, Marcel V.; Marchant, Jeffrey K.; Cosma, Mihai; Libby, Richard T.; John, Simon W. M.; Gould, Douglas B.

    2015-01-01

    Purpose Mutations in the gene encoding collagen type IV alpha 1 (COL4A1) cause multisystem disorders including anterior segment dysgenesis (ASD) and optic nerve hypoplasia. The penetrance and severity of individual phenotypes depends on genetic context. Here, we tested the effects of a Col4a1 mutation in two different genetic backgrounds to compare how genetic context influences ocular dysgenesis, IOP, and progression to glaucoma. Methods Col4a1 mutant mice maintained on a C57BL/6J background were crossed to either 129S6/SvEvTac or CAST/EiJ and the F1 progeny were analyzed by slit-lamp biomicroscopy and optical coherence tomography. We also measured IOPs and compared tissue sections of eyes and optic nerves. Results. We found that the CAST/EiJ inbred strain has a relatively uniform and profound suppression on the effects of Col4a1 mutation and that mutant CASTB6F1 mice were generally only very mildly affected. In contrast, mutant 129B6F1 mice had more variable and severe ASD and IOP dysregulation that were associated with glaucomatous signs including lost or damaged retinal ganglion cell axons and excavation of the optic nerve head. Conclusions. Ocular defects in Col4a1 mutant mice model ASD and glaucoma that are observed in a subset of patients with COL4A1 mutations. We demonstrate that different inbred strains of mice give graded severities of ASD and we detected elevated IOP and glaucomatous damage in 129B6F1, but not CASTB6F1 mice that carried a Col4a1 mutation. These data demonstrate that genetic context differences are one factor that may contribute to the variable penetrance and severity of ASD and glaucoma in patients with COL4A1 mutations. PMID:26567795

  17. In vitro and in vivo characterization of a Bordetella bronchiseptica mutant strain with a deep rough lipopolysaccharide structure.

    PubMed

    Sisti, Federico; Fernández, Julieta; Rodríguez, María Eugenia; Lagares, Antonio; Guiso, Nicole; Hozbor, Daniela Flavia

    2002-04-01

    Bordetella bronchiseptica is closely related to Bordetella pertussis, which produces respiratory disease primarily in mammals other than humans. However, its importance as a human pathogen is being increasingly recognized. Although a large amount of research on Bordetella has been generated regarding protein virulence factors, the participation of the surface lipopolysaccharide (LPS) during B. bronchiseptica infection is less understood. To get a better insight into this matter, we constructed and characterized the behavior of an LPS mutant with the deepest possible rough phenotype. We generated the defective mutant B. bronchiseptica LP39 on the waaC gene, which codes for a heptosyl transferase involved in the biosynthesis of the core region of the LPS molecule. Although in B. bronchiseptica LP39 the production of the principal virulence determinants adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin persisted, the quantity of the two latter factors was diminished, with the levels of pertactin being the most greatly affected. Furthermore, the LPS of B. bronchiseptica LP39 did not react with sera obtained from mice that had been infected with the parental strain, indicating that this defective LPS is immunologically different from the wild-type LPS. In vivo experiments demonstrated that the ability to colonize the respiratory tract is reduced in the mutant, being effectively cleared from lungs within 5 days, whereas the parental strain survived at least for 30 days. In vitro experiments have demonstrated that, although B. bronchiseptica LP39 was impaired for adhesion to human epithelial cells, it is still able to survive within the host cells as efficiently as the parental strain. These results seem to indicate that the deep rough form of B. bronchiseptica LPS cannot represent a dominant phenotype at the first stage of colonization. Since isolates with deep rough LPS phenotype have already been obtained from human B. bronchiseptica chronic

  18. [Separation of the biosynthesis products of a mutant strain of Actinomyces chrysomallus var. carotenoides and the identification of actinomycin antibiotics].

    PubMed

    Sverdlova, A N; Nefelova, M V; Silaev, A B

    1979-01-01

    An orange antibiotically active substance isolated from the mycelium of a mutant strain of Actinomyces chrysomallus var. carotenoides was identified as a mixture of actinomycins according to its light absorption spectra, circular dichroism spectra, IR spectra and chromatographic comparison with the standard samples. A scheme for successive extraction of the biologically active substances from the mycelium resulting in isolation of a fraction enriched with antibiotic substances and a fraction enriched with pigments is presented. A method for separation and purification of 3 groups of biologically active substances from the mycelium enriched extract was developed.

  19. Characterization of the endemic equilibrium and response to mutant injection in a multi-strain disease model.

    PubMed

    Aquino, Tomás; Bolster, Diogo; Nunes, Ana

    2015-03-01

    We explore a model of an antigenically diverse infection whose otherwise identical strains compete through cross-immunity. We assume that individuals may produce upon infection different numbers of antibody types, each of which matches the antigenic configuration of a particular epitope, and that one matching antibody type grants total immunity against a challenging strain. In order to reduce the number of equations involved in the analytic description of the dynamics, we follow the strategy proposed by Kryazhimskiy et al. (2007) and apply a low-order closure reminiscent of a pair approximation. Using this approximation, we go beyond the numerical studies of Kryazhimskiy et al. (2007) and explore the analytic properties of the ensuing model in the absence of mutation. We characterize its endemic equilibrium, comparing with the results of agent based simulations of the full model to assess the performance of the closure assumption. We show that a particular choice of immune response leads to a degenerate endemic equilibrium, where different strain prevalences may exist, breaking the symmetry of the model. Finally we study the behavior of the system under the injection of mutant strains. We find that the build up of diversity from a single founding strain is extremely unlikely for different choices of the population׳s immune response. PMID:25496729

  20. Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multicopy suppressor analysis in ammonium permease mutant strains.

    PubMed Central

    Lorenz, M C; Heitman, J

    1998-01-01

    Nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. Recognition of nitrogen starvation is mediated, at least in part, by the ammonium permease Mep2p and the Galpha subunit Gpa2p. Genetic activation of the pheromone-responsive MAP kinase cascade, which is also required for filamentous growth, only weakly suppresses the filamentation defect of Deltamep2/Deltamep2 and Deltagpa2/Deltagpa2 strain. Surprisingly, deletion of Mep1p, an ammonium permease not previously thought to regulate differentiation, significantly enhances the potency of MAP kinase activation, such that the STE11-4 allele induces filamentation to near wild-type levels in Deltamep1/Deltamep1 Deltamep2/Deltamep2 and Deltamep1/Deltamep1 Deltagpa2/Deltagpa2 strains. To identify additional regulatory components, we isolated high-copy suppressors of the filamentation defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant. Multicopy expression of TEC1, PHD1, PHD2 (MSS10/MSN1/FUP4), MSN5, CDC6, MSS11, MGA1, SKN7, DOT6, HMS1, HMS2, or MEP2 each restored filamentation in a Deltamep1/Deltamep1 Deltamep2/Deltamep2 strain. Overexpression of SRK1 (SSD1), URE2, DAL80, MEP1, or MEP3 suppressed only the growth defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant strain. Characterization of these genes through deletion analysis and epistasis underscores the complexity of this developmental pathway and suggests that stress conditions other than nitrogen deprivation may also promote filamentous growth. PMID:9832522

  1. Industrial Robustness: Understanding the Mechanism of Tolerance for the Populus Hydrolysate-Tolerant Mutant Strain of Clostridium thermocellum

    PubMed Central

    Linville, Jessica L.; Rodriguez, Miguel; Land, Miriam; Syed, Mustafa H.; Engle, Nancy L.; Tschaplinski, Timothy J.; Mielenz, Jonathan R.; Cox, Chris D.

    2013-01-01

    Background An industrially robust microorganism that can efficiently degrade and convert lignocellulosic biomass into ethanol and next-generation fuels is required to economically produce future sustainable liquid transportation fuels. The anaerobic, thermophilic, cellulolytic bacterium Clostridium thermocellum is a candidate microorganism for such conversions but it, like many bacteria, is sensitive to potential toxic inhibitors developed in the liquid hydrolysate produced during biomass processing. Microbial processes leading to tolerance of these inhibitory compounds found in the pretreated biomass hydrolysate are likely complex and involve multiple genes. Methodology/Principal Findings In this study, we developed a 17.5% v/v Populus hydrolysate tolerant mutant strain of C. thermocellum by directed evolution. The genome of the wild type strain, six intermediate population samples and seven single colony isolates were sequenced to elucidate the mechanism of tolerance. Analysis of the 224 putative mutations revealed 73 high confidence mutations. A longitudinal analysis of the intermediate population samples, a pan-genomic analysis of the isolates, and a hotspot analysis revealed 24 core genes common to all seven isolates and 8 hotspots. Genetic mutations were matched with the observed phenotype through comparison of RNA expression levels during fermentation by the wild type strain and mutant isolate 6 in various concentrations of Populus hydrolysate (0%, 10%, and 17.5% v/v). Conclusion/Significance The findings suggest that there are multiple mutations responsible for the Populus hydrolysate tolerant phenotype resulting in several simultaneous mechanisms of action, including increases in cellular repair, and altered energy metabolism. To date, this study provides the most comprehensive elucidation of the mechanism of tolerance to a pretreated biomass hydrolysate by C. thermocellum. These findings make important contributions to the development of industrially

  2. Elevated AIM2-mediated pyroptosis triggered by hypercytotoxic Francisella mutant strains is attributed to increased intracellular bacteriolysis

    PubMed Central

    Peng, Kaitian; Broz, Petr; Jones, Jonathan; Joubert, Lydia-Marie; Monack, Denise

    2011-01-01

    Intracellular bacterial pathogens Francisella novicida and the Live Vaccine Strain (LVS) are recognized in the macrophage cytosol by the AIM2 inflammasome, which leads to the activation of caspase-1 and the processing and secretion of active IL-1β, IL-18 and pyroptosis. Previous studies have reported that F. novicida and LVS mutants in specific genes (e.g., FTT0584, mviN and ripA) induce elevated inflammasome activation and hypercytotoxicity in host cells, leading to the proposal that F. novicida and LVS may have proteins that actively modulate inflammasome activation. However, there has been no direct evidence of such inflammasome evasion mechanisms. Here, we demonstrate for the first time that the above mutants, along with a wide range of F. novicida hypercytotoxic mutants that are deficient for membrane-associated proteins (ΔFTT0584, ΔmviN, ΔripA, ΔfopA and ΔFTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (ΔwbtA and ΔlpxH) lyse more intracellularly, thus activating increased levels of AIM2-dependent pyroptosis and other innate immune signaling pathways. This suggests that an inflammasome-specific evasion mechanism may not be present in F. novicida and LVS. Furthermore, future studies may need to consider increased bacterial lysis as a possible cause of elevated stimulation of multiple innate immune pathways when the protein composition or surface carbohydrates of the bacterial membrane is altered. PMID:21883803

  3. Comparative Proteomic Analysis of Streptomyces lividans Wild-Type and ppk Mutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis

    PubMed Central

    Le Maréchal, Pierre; Decottignies, Paulette; Marchand, Christophe H.; Degrouard, Jeril; Jaillard, Danièle; Dulermo, Thierry; Froissard, Marine; Smirnov, Aleksey; Chapuis, Violaine

    2013-01-01

    Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces. PMID:23872561

  4. Comparative proteomic analysis of Streptomyces lividans Wild-Type and ppk mutant strains reveals the importance of storage lipids for antibiotic biosynthesis.

    PubMed

    Le Maréchal, Pierre; Decottignies, Paulette; Marchand, Christophe H; Degrouard, Jeril; Jaillard, Danièle; Dulermo, Thierry; Froissard, Marine; Smirnov, Aleksey; Chapuis, Violaine; Virolle, Marie-Joelle

    2013-10-01

    Streptomyces lividans TK24 is a strain that naturally produces antibiotics at low levels, but dramatic overproduction of antibiotics occurs upon interruption of the ppk gene. However, the role of the Ppk enzyme in relation to the regulation of antibiotic biosynthesis remains poorly understood. In order to gain a better understanding of the phenotype of the ppk mutant, the proteomes of the wild-type (wt) and ppk mutant strains, grown for 96 h on R2YE medium limited in phosphate, were analyzed. Intracellular proteins were separated on two-dimensional (2D) gels, spots were quantified, and those showing a 3-fold variation or more were identified by mass spectrometry. The expression of 12 proteins increased and that of 29 decreased in the ppk mutant strain. Our results suggested that storage lipid degradation rather than hexose catabolism was taking place in the mutant. In order to validate this hypothesis, the triacylglycerol contents of the wt and ppk mutant strains of S. lividans as well as that of Streptomyces coelicolor M145, a strain that produces antibiotics at high levels and is closely related to S. lividans, were assessed using electron microscopy and thin-layer chromatography. These studies highlighted the large difference in triacylglycerol contents of the three strains and confirmed the hypothetical link between storage lipid metabolism and antibiotic biosynthesis in Streptomyces.

  5. Establishing a Markerless Genetic Exchange System for Methanosarcina mazei Strain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

    PubMed Central

    Ehlers, Claudia; Jäger, Dominik; Schmitz, Ruth A.

    2011-01-01

    A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazeiΔsRNA154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation. PMID:21941461

  6. Pigmentation restored in mutant laboratory strain of the lady beetle Coleomegilla maculata through dietary supplementation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A laboratory colony of Coleomegilla maculata (DeGeer), ye, selected for a pigmentation deficiency, was restored to near wild type cuticle coloration by adding crushed heads and wings of the red colored parental strain to the diet. While the wings and other colored portions of the cuticle regained th...

  7. Modelling Hepatitis B Virus Antiviral Therapy and Drug Resistant Mutant Strains

    NASA Astrophysics Data System (ADS)

    Bernal, Julie; Dix, Trevor; Allison, Lloyd; Bartholomeusz, Angeline; Yuen, Lilly

    Despite the existence of vaccines, the Hepatitis B virus (HBV) is still a serious global health concern. HBV targets liver cells. It has an unusual replication process involving an RNA pre-genome that the reverse transcriptase domain of the viral polymerase protein translates into viral DNA. The reverse transcription process is error prone and together with the high replication rates of the virus, allows the virus to exist as a heterogeneous population of mutants, known as a quasispecies, that can adapt and become resistant to antiviral therapy. This study presents an individual-based model of HBV inside an artificial liver, and associated blood serum, undergoing antiviral therapy. This model aims to provide insights into the evolution of the HBV quasispecies and the individual contribution of HBV mutations in the outcome of therapy.

  8. Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.

    PubMed

    Lin, Xiaorong; Chacko, Nadia; Wang, Linqi; Pavuluri, Yashwant

    2015-04-01

    Cryptococcus neoformans is the etiologic agent of cryptococcal meningitis that causes more than half a million deaths worldwide each year. This capsulated basidiomycetous yeast also serves as a model for micropathogenic studies. The ability to make stable mutants, either via ectopic integration or homologous recombination, has been accomplished using biolistic transformation. This technical advance has greatly facilitated the research on the basic biology and pathogenic mechanisms of this pathogen in the past two decades. However, biolistic transformation is costly, and its reproducibility varies widely. Here we found that stable ectopic integration or targeted gene deletion via homologous replacement could be accomplished through electroporative transformation. The stability of the transformants obtained through electroporation and the frequency of homologous replacement is highly dependent on the selective marker. A frequency of homologous recombination among the stable transformants obtained by electroporation is comparable to those obtained by biolistic transformation (∼10%) when dominant drug selection markers are used, which is much higher than what has been previously reported for electroporation when auxotrophic markers were used (0.001% to 0.1%). Furthermore, disruption of the KU80 gene or generation of gene deletion constructs using the split marker strategy, two approaches known to increase homologous replacement among transformants obtained through biolistic transformation, also increase the frequency of homologous replacement among transformants obtained through electroporation. Therefore, electroporation provides a low cost alternative for mutagenesis in Cryptococcus.

  9. Safety, efficacy and efficiency of laser-assisted IVF in subfertile mutant mouse strains

    PubMed Central

    Li, Ming-Wen; Kinchen, Kristy L; Vallelunga, Jadine M; Young, Diana L; Wright, Kaleb D K; Gorano, Lisa N; Wasson, Katherine; Lloyd, K C Kent

    2013-01-01

    In the present report we studied the safety, efficacy and efficiency of using an infrared laser to facilitate IVF by assessing fertilization, development and birth rates after laser-zona drilling (LZD) in 30 subfertile genetically modified (GM) mouse lines. We determined that LZD increased the fertilization rate four to ten times that of regular IVF, thus facilitating the derivation of 26 of 30 (86.7%) GM mouse lines. Cryopreserved two-cell stage embryos derived by LZD-assisted IVF were recovered and developed to blastocysts in vitro at the same rate as frozen–thawed embryos derived by regular IVF. Surprisingly after surgical transfer to pseudopregnant recipients the birth rate of embryos derived by LZD-assisted IVF was significantly lower than that of embryos derived by regular IVF. However this result could be completely mitigated by the addition of 0.25 M sucrose to the culture medium during LZD which caused the oocyte to shrink in volume relative to the perivitelline space. By increasing the distance from the laser target site on the zona pellucida, we hypothesize that the hyperosmotic effect of sucrose reduced the potential for laser-induced cytotoxic thermal damage to the underlying oocytes. With appropriate preparation and cautious application, our results indicate that LZD-assisted IVF is a safe, efficacious and efficient assisted reproductive technology for deriving mutant mouse lines with male factor infertility and subfertility caused by sperm–zona penetration defects. PMID:23315689

  10. Isolation of a novel mutant strain of Saccharomyces cerevisiae by an ethyl methane sulfonate-induced mutagenesis approach as a high producer of bioethanol.

    PubMed

    Mobini-Dehkordi, Mohsen; Nahvi, Iraj; Zarkesh-Esfahani, Hamid; Ghaedi, Kamran; Tavassoli, Manoochehr; Akada, Rinji

    2008-04-01

    In order to obtain mutant strains showing higher bioethanol production than wild-type strains, a commercial Saccharomyces cerevisiae type was subjected to mutagenesis using ethyl methane sulfonate (EMS). After adding EMS to a shaken yeast suspension, the viability of yeast cells was assessed by diluted sample inoculation to solid yeast-extract peptone glucose (YEPG) medium at 15-min intervals. At 45 min, the viability of yeast cells was estimated to be about 40%. Mutagenized cells were recovered from YEPG broth after incubation at 30 degrees C for 18 h. After this period, EMS-treated yeast cells were grown on solid aerobic low-peptone (ALP) medium containing 2-12% (v/v) ethanol. All plates were incubated at 30 degrees C for 2-6 d in order to form colonies. The mutant strains that tolerated high concentrations of ethanol were selected for bioethanol production in microfuge tubes containing fermentation medium. Formation of bioethanol in small tubes was detected by the distillation-colorimetric method. In addition, trehalose content and invertase activity were determined in each mutant strain. Among many isolated mutant strains, there were six isolated colonies that grew on ALP medium supplemented with 10% (v/v) ethanol and one of them produced bioethanol 17.3% more than the wild type.

  11. Regulation of Staphylococcal Superantigen-Like Gene, ssl8, Expression in Staphylococcus aureus strain, RN6390

    PubMed Central

    Pantrangi, Madhulatha; Singh, Vineet K; Shukla, Sanjay K

    2015-01-01

    Staphylococcal superantigen-like (SSL) proteins, which are encoded by a cluster of eleven ssl genes, contribute to the Staphylococcus aureus virulence. Recently we reported ssl8 expression profiles in seven clinically important strains—MW2, USA300FPR3757, MSSA476, Newman, RN6390, Mu50, and N315—and showed the differential expression of ssl8 in Newman, RN6390, and USA300FPR3757 strains, despite harboring identical allelic forms of ssl8, suggesting the roles for different regulatory elements for this gene in different S. aureus strains. In this communication, using RN6390, a common laboratory S. aureus strain and its isogenic knockout mutant strains of agr, sae, sarA, sigB, rot, and the agr-/sigB− double mutant, we showed that SarA and Rot are inducer and repressor, respectively, for ssl8 expression in RN6390. This is in contrast to the Newman strain, where ssl8 is positively regulated by Sae but negatively by Agr, indicating the variable expression of ssl8 in clinical strains is more likely due to strain-specific regulatory elements. PMID:24899694

  12. Transcriptomic analysis of Clostridium thermocellum Populus hydrolysate-tolerant mutant strain shows increased cellular efficiency in response to Populus hydrolysate compared to the wild type strain

    PubMed Central

    2014-01-01

    Background The thermophilic, anaerobic bacterium, Clostridium thermocellum is a model organism for consolidated processing due to its efficient fermentation of cellulose. Constituents of dilute acid pretreatment hydrolysate are known to inhibit C. thermocellum and other microorganisms. To evaluate the biological impact of this type of hydrolysate, a transcriptomic analysis of growth in hydrolysate-containing medium was conducted on 17.5% v/v Populus hydrolysate-tolerant mutant (PM) and wild type (WT) strains of C. thermocellum. Results In two levels of Populus hydrolysate medium (0% and 10% v/v), the PM showed both gene specific increases and decreases of gene expression compared to the wild-type strain. The PM had increased expression of genes in energy production and conversion, and amino acid transport and metabolism in both standard and 10% v/v Populus hydrolysate media. In particular, expression of the histidine metabolism increased up to 100 fold. In contrast, the PM decreased gene expression in cell division and sporulation (standard medium only), cell defense mechanisms, cell envelope, cell motility, and cellulosome in both media. The PM downregulated inorganic ion transport and metabolism in standard medium but upregulated it in the hydrolysate media when compared to the WT. The WT differentially expressed 1072 genes in response to the hydrolysate medium which included increased transcription of cell defense mechanisms, cell motility, and cellulosome, and decreased expression in cell envelope, amino acid transport and metabolism, inorganic ion transport and metabolism, and lipid metabolism, while the PM only differentially expressed 92 genes. The PM tolerates up to 17.5% v/v Populus hydrolysate and growth in it elicited 489 genes with differential expression, which included increased expression in energy production and conversion, cellulosome production, and inorganic ion transport and metabolism and decreased expression in transcription and cell

  13. Decreased coenzyme A levels in ridA mutant strains of Salmonella enterica result from inactivated serine hydroxymethyltransferase.

    PubMed

    Flynn, Jeffrey M; Christopherson, Melissa R; Downs, Diana M

    2013-08-01

    The RidA/Yer057/UK114 family of proteins is well represented across the domains of life and recent work has defined both an in vitro activity and an in vivo role for RidA. RidA proteins have enamine deaminase activity, and in their absence the reactive 2-aminoacrylate (2-AA) accumulates and inactivates at least some pyridoxal 5'-phosphate (PLP)-containing enzymes in Salmonella enterica. The conservation of RidA suggested that 2-AA was a ubiquitous cellular stressor that was generated in central metabolism. Phenotypically, strains of S. enterica that lack RidA accumulated significantly more pyruvate in the growth medium than wild-type strains. Here we dissected this ridA mutant phenotype and showed it was an indirect consequence of damage to serine hydroxymethyltransferase (GlyA; E.C. 2.1.2.1). The results here identified a fourth PLP enzyme as a target of enamine stress in Salmonella.

  14. AGR-1 Thermocouple Data Analysis

    SciTech Connect

    Jeff Einerson

    2012-05-01

    This report documents an effort to analyze measured and simulated data obtained in the Advanced Gas Reactor (AGR) fuel irradiation test program conducted in the INL's Advanced Test Reactor (ATR) to support the Next Generation Nuclear Plant (NGNP) R&D program. The work follows up on a previous study (Pham and Einerson, 2010), in which statistical analysis methods were applied for AGR-1 thermocouple data qualification. The present work exercises the idea that, while recognizing uncertainties inherent in physics and thermal simulations of the AGR-1 test, results of the numerical simulations can be used in combination with the statistical analysis methods to further improve qualification of measured data. Additionally, the combined analysis of measured and simulation data can generate insights about simulation model uncertainty that can be useful for model improvement. This report also describes an experimental control procedure to maintain fuel target temperature in the future AGR tests using regression relationships that include simulation results. The report is organized into four chapters. Chapter 1 introduces the AGR Fuel Development and Qualification program, AGR-1 test configuration and test procedure, overview of AGR-1 measured data, and overview of physics and thermal simulation, including modeling assumptions and uncertainties. A brief summary of statistical analysis methods developed in (Pham and Einerson 2010) for AGR-1 measured data qualification within NGNP Data Management and Analysis System (NDMAS) is also included for completeness. Chapters 2-3 describe and discuss cases, in which the combined use of experimental and simulation data is realized. A set of issues associated with measurement and modeling uncertainties resulted from the combined analysis are identified. This includes demonstration that such a combined analysis led to important insights for reducing uncertainty in presentation of AGR-1 measured data (Chapter 2) and interpretation of

  15. Constitutive synthesis of enzymes of the protocatechuate pathway and of the beta-ketoadipate uptake system in mutant strains of Pseudomonas putida.

    PubMed Central

    Parke, D; Ornston, L N

    1976-01-01

    Mutant Pseudomonas putida strains that produce constitutive levels of the beta-ketoadipate uptake system are selected by the sequential transfer of cultures between mineral growth media supplemented with the noninducing growth substrate succinate and growth media containing beta-ketoadipate as the sole carbon and energy source. The mutant strains also produce constitutively three catabolic enzymes that give rise to beta-ketoadipate from the metabolic precursor beta-carboxy-cis, cis-muconate, and thus a single regulatory gene appears to govern the expression of the enzymes as well as the uptake system. The three enzymes that convert beta-carboxy-cis, cis-muconate to beta-ketoadipate are induced to higher levels when the orgainisms are grown with p-hydroxybenzoate (a compound that is catabolized via beta-ketoadipate); the beta-ketoadipate uptake system is partially repressed when the cells are grwon at the expense of p-hydroxybenzoate. The transferase that acts upon beta-ketoadipate remains inducible in the constitutive mutant strains. Thus a minimum of three biosynthetic controls must be exerted over the expression of the five genes. Since the regulatory mutation does not alter the expression of the gene for the transferase, the physiological target of the selection procedure appears to be mutant strains that produce the uptake system constitutively. Levels of the uptake system are higher in uninduced constitutive mutant cultures than in induced cultures of the wild type. Hence procedures analogous to the one we employed may be of general use in obtaining mutant strains that produce high levels of uptake systems. PMID:1262305

  16. Characterization of the Burkholderia mallei tonB Mutant and Its Potential as a Backbone Strain for Vaccine Development

    PubMed Central

    Mott, Tiffany M.; Vijayakumar, Sudhamathi; Sbrana, Elena; Endsley, Janice J.; Torres, Alfredo G.

    2015-01-01

    Background In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis. Methodology/Principal Findings Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 104 CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001. Conclusions/Significance Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis. PMID:26114445

  17. Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus.

    PubMed

    Gokce, Abdulmecit; Cakar, Zeynep Petek; Yucel, Meral; Ozcan, Orhan; Sencan, Sevde; Sertdemir, Ibrahim; Erguner, Bekir; Yuceturk, Betul; Sarac, Aydan; Yuksel, Bayram; Ozturk, Yavuz

    2016-01-01

    The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus.

  18. Draft Genome Sequences of Two Heat-Resistant Mutant Strains (A52 and B41) of the Photosynthetic Hydrogen-Producing Bacterium Rhodobacter capsulatus

    PubMed Central

    Gokce, Abdulmecit; Cakar, Zeynep Petek; Yucel, Meral; Ozcan, Orhan; Sencan, Sevde; Sertdemir, Ibrahim; Erguner, Bekir; Yuceturk, Betul; Sarac, Aydan; Yuksel, Bayram

    2016-01-01

    The draft genome sequences of two heat-resistant mutant strains, A52 and B41, derived from Rhodobacter capsulatus DSM 1710, and with different hydrogen production levels, are reported here. These sequences may help understand the molecular basis of heat resistance and hydrogen production in R. capsulatus. PMID:27284151

  19. Complete Closed Genome Sequences of a Mannheimia haemolytica Serotype A1 Leukotoxin Deletion Mutant and Its Wild-Type Parent Strain

    PubMed Central

    Harhay, Gregory P.; Smith, Timothy P. L.; Bono, James L.; Chitko-McKown, Carol G.

    2015-01-01

    Mannheimia haemolytica is a bacterial pathogen that secretes leukotoxin (LktA) which binds to leukocyte membranes via CD18, causing bacterial pneumonia in ruminants. We report the complete closed genome sequences of a leukotoxin mutant and its parent strain that are frequently used in respiratory disease studies. PMID:25953160

  20. luxS mutants of Serratia defective in autoinducer-2-dependent 'quorum sensing' show strain-dependent impacts on virulence and production of carbapenem and prodigiosin.

    PubMed

    Coulthurst, Sarah J; Kurz, C Léopold; Salmond, George P C

    2004-06-01

    The enzyme LuxS is responsible for the production of autoinducer-2 (AI-2), a molecule that has been implicated in quorum sensing in many bacterial species. This study investigated whether there is a luxS-dependent signalling system in the Gram-negative bacteria Serratia spp. Serratia marcescens is a broad-host-range pathogen and an important cause of nosocomial infections. Production of AI-2 activity was detected in S. marcescens ATCC 274 and Serratia ATCC 39006 and their luxS genes were sequenced. luxS mutants were constructed in these strains and were analysed to determine which phenotypes are regulated by luxS and therefore, potentially, by AI-2. The phenotypes of the luxS mutants included decreased carbapenem antibiotic production in Serratia ATCC 39006 and decreased prodigiosin and secreted haemolysin production in S. marcescens ATCC 274. The luxS mutant of S. marcescens ATCC 274 was also found to exhibit modestly reduced virulence in a Caenorhabditis elegans model. Finally, it was shown that the culture supernatant of a wild-type strain contains a signal, presumably AI-2, capable of complementing the prodigiosin defect of the luxS mutant of another strain, even when substantially diluted. It is concluded that luxS modulates virulence and antibiotic production in Serratia, in a strain-dependent manner, and that, for at least one phenotype, this regulation is via extracellular signalling.

  1. The Brucella abortus phosphoglycerate kinase mutant is highly attenuated and induces protection superior to that of vaccine strain 19 in immunocompromised and immunocompetent mice.

    PubMed

    Trant, Cyntia G M C; Lacerda, Thais L S; Carvalho, Natalia B; Azevedo, Vasco; Rosinha, Gracia M S; Salcedo, Suzana P; Gorvel, Jean-Pierre; Oliveira, Sergio C

    2010-05-01

    Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that can function as virulence factors to better understand the host-pathogen interplay. Herein, we identified the gene encoding the phosphoglycerate kinase (PGK) of B. abortus strain 2308. To test the role of PGK in Brucella pathogenesis, a pgk deletion mutant was constructed. Replacement of the wild-type pgk by recombination was demonstrated by Southern and Western blot analyses. The B. abortus Delta pgk mutant strain exhibited extreme attenuation in bone marrow-derived macrophages and in vivo in BALB/c, C57BL/6, 129/Sv, and interferon regulatory factor-1 knockout (IRF-1 KO) mice. Additionally, at 24 h postinfection the Delta pgk mutant was not found within the same endoplasmic reticulum-derived compartment as the wild-type bacteria, but, instead, over 60% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP1. Furthermore, the B. abortus Delta pgk deletion mutant was used as a live vaccine. Challenge experiments revealed that the Delta pgk mutant strain induced protective immunity in 129/Sv or IRF-1 KO mice that was superior to the protection conferred by commercial strain 19 or RB51. Finally, the results shown here demonstrated that Brucella PGK is critical for full bacterial virulence and that a Delta pgk mutant may serve as a potential vaccine candidate in future studies. PMID:20194591

  2. Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Kim, Kiju; Park, Bo-Kyoung; Hahn, Tae-Wook

    2015-11-01

    Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

  3. Construction of a Quadruple Auxotrophic Mutant of an Industrial Polyploid Saccharomyces cerevisiae Strain by Using RNA-Guided Cas9 Nuclease

    PubMed Central

    Zhang, Guo-Chang; Kong, In Iok; Kim, Heejin; Liu, Jing-Jing; Cate, Jamie H. D.

    2014-01-01

    Industrial polyploid yeast strains harbor numerous beneficial traits but suffer from a lack of available auxotrophic markers for genetic manipulation. Here we demonstrated a quick and efficient strategy to generate auxotrophic markers in industrial polyploid yeast strains with the RNA-guided Cas9 nuclease. We successfully constructed a quadruple auxotrophic mutant of a popular industrial polyploid yeast strain, Saccharomyces cerevisiae ATCC 4124, with ura3, trp1, leu2, and his3 auxotrophies through RNA-guided Cas9 nuclease. Even though multiple alleles of auxotrophic marker genes had to be disrupted simultaneously, we observed knockouts in up to 60% of the positive colonies after targeted gene disruption. In addition, growth-based spotting assays and fermentation experiments showed that the auxotrophic mutants inherited the beneficial traits of the parental strain, such as tolerance of major fermentation inhibitors and high temperature. Moreover, the auxotrophic mutants could be transformed with plasmids containing selection marker genes. These results indicate that precise gene disruptions based on the RNA-guided Cas9 nuclease now enable metabolic engineering of polyploid S. cerevisiae strains that have been widely used in the wine, beer, and fermentation industries. PMID:25281382

  4. Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees.

    PubMed

    López, M M; Gorris, M T; Salcedo, C I; Montojo, A M; Miró, M

    1989-03-01

    The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr against the sensitive strain but was similar against the resistant strain.

  5. Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

    PubMed

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2016-12-01

    Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823. PMID:27622208

  6. Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

    PubMed

    Neves, Gabriela Westerlund Peixoto; Curty, Nathália; Kubitschek-Barreira, Paula Helena; Fontaine, Thierry; Souza, Gustavo Henrique Martins Ferreira; Cunha, Marcel Lyra; Goldman, Gustavo H; Beauvais, Anne; Latgé, Jean-Paul; Lopes-Bezerra, Leila M

    2016-12-01

    Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823.

  7. The cbs mutant strain of Rhodococcus erythropolis KA2-5-1 expresses high levels of Dsz enzymes in the presence of sulfate.

    PubMed

    Tanaka, Yasuhiro; Yoshikawa, Osamu; Maruhashi, Kenji; Kurane, Ryuichiro

    2002-11-01

    Two mutants of the dibenzothiophene-desulfurizing Rhodococcus erythropolis KA2-5-1, strains MS51 and MS316, which express a high level of desulfurizing activity in the presence of sulfate, were isolated using the transposome technique. The level of dibenzothiophene-desulfurization by cell-free extracts prepared from mutants MS51 and MS316 grown on sulfate was about five-fold higher than that by cell-free extracts of the wild-type. This result was consistent with results of Western-blot analysis using antisera specific for DszA, DszB and DszC, the enzymes involved in the desulfurization of dibenzothiophene. Gene analysis of the mutants revealed that the same gene was disrupted in mutants MS51 and MS316 and that the transposon-inserted gene in these strains was the gene for cystathionine beta-synthase, cbs. The cbs mutants also expressed high levels of Dsz enzymes when methionine was used as the sole source of sulfur.

  8. Molecular identification of an ABC transporter complex for manganese: analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process.

    PubMed Central

    Bartsevich, V V; Pakrasi, H B

    1995-01-01

    During photosynthesis, the photosystem II (PSII) pigment-protein complex catalyzes oxygen evolution, a reaction in which a four-manganese ensemble plays a crucial role. Using a newly developed selection scheme, we have isolated BP13, a random photosynthesis-deficient mutant strain of the cyanobacterium, Synechocystis 6803. This mutant grew slowly under photoautotrophic conditions, and had a low oxygen evolution activity. Biochemical analysis revealed that the lesion in this mutant strain had specifically affected the Mn ensemble in PSII. Interestingly, incubation of BP13 cells with micromolar levels of added Mn induced rapid recovery of oxygen evolution activity. The mutant could be complemented with a fragment of wild-type chromosomal DNA containing three closely linked genes, mntA, mntB and mntC. These gene products showed significant sequence similarities with polypeptide components of bacterial permeases that are members of the 'ABC (ATP binding cassette) superfamily' of transporter proteins. We determined that in the BP13 strain, a single nucleotide change had resulted in the replacement of an alanine by an aspartic acid residue in MntA, a soluble protein containing ATP binding motifs. These results suggest that the mntCAB gene cluster encodes polypeptide components of a Mn transporter, the first such protein complex identified in any organism. PMID:7743991

  9. The cefazolin inoculum effect in methicillin-susceptible Staphylococcus aureus blood isolates: their association with dysfunctional accessory gene regulator (agr).

    PubMed

    Wi, Yu Mi; Park, Young Kyoung; Moon, Chisook; Ryu, Seong Yeol; Lee, Hyuck; Ki, Hyun Kyun; Cheong, Hae Suk; Son, Jun Seong; Lee, Jin Seo; Kwon, Ki Tae; Kim, June Myong; Ha, Young Eun; Kang, Cheol In; Ko, Kwan Soo; Chung, Doo Ryeon; Peck, Kyong Ran; Song, Jae-Hoon

    2015-11-01

    We evaluated the clinical significance of the cefazolin inoculum effect (CIE) in methicillin-susceptible Staphylococcus aureus (MSSA) isolates. In total, 146 isolates were recovered from patients with MSSA bacteremia at 9 hospitals in Korea. The CIE was observed in 16 MSSA isolates, and while type A was the only detected β-lactamase in MSSA isolates exhibiting the CIE, no strains expressing type B, C, or D β-lactamases exhibited this effect. The CIE was only observed in agr group III and I isolates and was significantly more common in isolates with agr dysfunction than in those with functional agr (P<0.001). Even among isolates producing type A β-lactamase, the CIE was also prevalent in isolates with dysfunctional agr than in isolates with functional agr (P=0.025). This study demonstrates an association between the CIE of MSSA isolates and agr dysfunction, in addition to those between the CIE and type A β-lactamase.

  10. Isolation and characterization of the ndhF gene of Synechococcus sp. strain PCC 7002 and initial characterization of an interposon mutant.

    PubMed Central

    Schluchter, W M; Zhao, J; Bryant, D A

    1993-01-01

    The ndhF gene of the unicellular marine cyanobacterium Synechococcus sp. strain PCC 7002 was cloned and characterized. NdhF is a subunit of the type 1, multisubunit NADH:plastoquinone oxidoreductase (NADH dehydrogenase). The nucleotide sequence of the gene predicts an extremely hydrophobic protein of 664 amino acids with a calculated mass of 72.9 kDa. The ndhF gene was shown to be single copy and transcribed into a monocistronic mRNA of 2,300 nucleotides. An ndhF null mutation was successfully constructed by interposon mutagenesis, demonstrating that NdhF is not required for cell viability under photoautotrophic growth conditions. The mutant strain exhibited a negligible rate of oxygen uptake in the dark, but its photosynthetic properties (oxygen evolution, chlorophyll/P700 ratio, and chlorophyll/P680 ratio) were generally similar to those of the wild type. Although the ndhF mutant strain grew as rapidly as the wild-type strain at high light intensity, the mutant grew more slowly than the wild type at lower light intensities and did not grow at all under photoheterotrophic conditions. The roles of the NADH:plastoquinone oxidoreductase in photosynthetic and respiratory electron transport are discussed. Images PMID:8501038

  11. Rapid, Sensitive, and Accurate Evaluation of Drug Resistant Mutant (NS5A-Y93H) Strain Frequency in Genotype 1b HCV by Invader Assay.

    PubMed

    Yoshimi, Satoshi; Ochi, Hidenori; Murakami, Eisuke; Uchida, Takuro; Kan, Hiromi; Akamatsu, Sakura; Hayes, C Nelson; Abe, Hiromi; Miki, Daiki; Hiraga, Nobuhiko; Imamura, Michio; Aikata, Hiroshi; Chayama, Kazuaki

    2015-01-01

    Daclatasvir and asunaprevir dual oral therapy is expected to achieve high sustained virological response (SVR) rates in patients with HCV genotype 1b infection. However, presence of the NS5A-Y93H substitution at baseline has been shown to be an independent predictor of treatment failure for this regimen. By using the Invader assay, we developed a system to rapidly and accurately detect the presence of mutant strains and evaluate the proportion of patients harboring a pre-treatment Y93H mutation. This assay system, consisting of nested PCR followed by Invader reaction with well-designed primers and probes, attained a high overall assay success rate of 98.9% among a total of 702 Japanese HCV genotype 1b patients. Even in serum samples with low HCV titers, more than half of the samples could be successfully assayed. Our assay system showed a better lower detection limit of Y93H proportion than using direct sequencing, and Y93H frequencies obtained by this method correlated well with those of deep-sequencing analysis (r = 0.85, P <0.001). The proportion of the patients with the mutant strain estimated by this assay was 23.6% (164/694). Interestingly, patients with the Y93H mutant strain showed significantly lower ALT levels (p=8.8 x 10-4), higher serum HCV RNA levels (p=4.3 x 10-7), and lower HCC risk (p=6.9 x 10-3) than those with the wild type strain. Because the method is both sensitive and rapid, the NS5A-Y93H mutant strain detection system established in this study may provide important pre-treatment information valuable not only for treatment decisions but also for prediction of disease progression in HCV genotype 1b patients. PMID:26083687

  12. Oligonucleotide-directed mutagenesis of psbB, the gene encoding CP47, employing a deletion mutant strain of the cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Eaton-Rye, J J; Vermaas, W F

    1991-12-01

    A mutant strain of the cyanobacterium Synechocystis sp. PCC (Pasteur Culture Collection) 6803 has been developed in which psbB, the gene coding for the chlorophyl alpha-binding protein CP47 in Photosystem II (PSII), has been deleted. This deletion mutant can be used for the reintroduction of modified psbB into the cyanobacterium. To study the role of a large hydrophilic region in CP47, presumably located on the lumenal side of the thylakoid membrane between the fifth and sixth membrane-spanning regions, specific deletions have been introduced in psbB coding for regions within this domain. One psbB mutation leads to deletion of Gly-351 to Thr-365 in CP47, another psbB mutation was targeted towards deletion of Arg-384 to Val-392 in this protein. The deletion from Gly-351 to Thr-365 results in a loss of PSII activity and of photoautotrophic growth of the mutant, but the deletion between Arg-384 and Val-392 retains PSII activity and the ability to grow photoautotrophically. The mutant strain with the deletion from Gly-351 to Thr-365 does not assemble a stable PSII reaction center complex in its thylakoid membranes, and exhibits diminished levels of CP47 and of the reaction center proteins D1 and D2. In contrast to the Arg-384 to Val-392 portion of this domain, the region between Gly-351 and Thr-365 appears essential for the normal structure and function of photosystem II. PMID:1932693

  13. Low-proline environments impair growth, proline transport and in vivo survival of Staphylococcus aureus strain-specific putP mutants.

    PubMed

    Schwan, William R; Wetzel, Keith J; Gomez, Timothy S; Stiles, Melissa A; Beitlich, Brian D; Grunwald, Sandra

    2004-04-01

    Staphylococcus aureus is a common cause of disease in humans, particularly in hospitalized patients. This species needs to import several amino acids to survive, including proline. Previously, it was shown that an insertion mutation in the high-affinity proline uptake gene putP in strain RN6390 affected proline uptake by the bacteria as well as reducing their ability to survive in vivo. To further delineate the effect of the putP mutation on growth of S. aureus strain RN6390, a proline uptake assay that spanned less than 1 min was done to measure transport. An eightfold difference in proline levels was observed between the wild-type strain and the high-affinity proline transport mutant strain after 15 s, indicating that the defect was only in proline transport and not a combination of proline transport, metabolism and accumulation that would have been assessed with longer assays. A putP mutant of S. aureus strain RN4220 was then grown in minimal medium with different concentrations of proline. When compared to the wild-type strain, the putP mutant strain was significantly growth impaired when the level of proline was decreased to 1.74 microM. An assessment of proline concentrations in mouse livers and spleens showed proline concentrations of 7.5 micromol per spleen and 88.4 micromol per liver. To verify that the effects on proline transport and bacterial survival were indeed caused solely by a mutation in putP, the putP mutation was complemented by cloning a full-length putP gene on a plasmid that replicates in S. aureus. Complementation of the putP mutant strains restored proline transport, in vitro growth in low-proline medium, and in vivo survival within mice. These results show that the mutation in putP led to attenuated growth in low-proline media and by corollary low-proline murine organ tissues due to less efficient transport of proline into the bacteria.

  14. Development of a fluorescent monoclonal antibody-based assay to measure the allosteric effects of synthetic peptides on self-oligomerization of AGR2 protein.

    PubMed

    Gray, Terry A; Murray, Euan; Nowicki, Matthew W; Remnant, Lucy; Scherl, Alexander; Muller, Petr; Vojtesek, Borek; Hupp, Ted R

    2013-09-01

    Many regulatory proteins are homo-oligomeric and designing assays that measure self-assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer-associated oncoprotein AGR2. A two site-sandwich microtiter assay ((2S) MTA) was designed using a DyLight800-labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self-peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N-terminal region of AGR2 increase in trans oligomer stability as defined using the (2S) MTA assay. A DSS-crosslinking assay that traps the AGR2 dimer through K95-K95 adducts confirmed that Δ45-AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt-AGR2, Δ45-AGR2 (more stable dimer), and monomeric AGR2(E60A) revealed that Δ45-AGR2 was more active in binding to Reptin than either wt-AGR2 or the AGR2(E60A) mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential.

  15. Selective Chemical Inhibition of agr Quorum Sensing in Staphylococcus aureus Promotes Host Defense with Minimal Impact on Resistance

    PubMed Central

    Sully, Erin K.; Malachowa, Natalia; Elmore, Bradley O.; Alexander, Susan M.; Femling, Jon K.; Gray, Brian M.; DeLeo, Frank R.; Otto, Michael; Cheung, Ambrose L.; Edwards, Bruce S.; Sklar, Larry A.; Horswill, Alexander R.; Hall, Pamela R.; Gresham, Hattie D.

    2014-01-01

    Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in

  16. Selective chemical inhibition of agr quorum sensing in Staphylococcus aureus promotes host defense with minimal impact on resistance.

    PubMed

    Sully, Erin K; Malachowa, Natalia; Elmore, Bradley O; Alexander, Susan M; Femling, Jon K; Gray, Brian M; DeLeo, Frank R; Otto, Michael; Cheung, Ambrose L; Edwards, Bruce S; Sklar, Larry A; Horswill, Alexander R; Hall, Pamela R; Gresham, Hattie D

    2014-06-01

    Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in

  17. Adhesion in vitro and in vivo associated with an adhesive antigen (F41) produced by a K99 mutant of the reference strain Escherichia coli B41.

    PubMed Central

    Morris, J A; Thorns, C; Scott, A C; Sojka, W J; Wells, G A

    1982-01-01

    A K99-negative mutant of the K99 reference strain Escherichia coli B41 (O101:K99) was isolated (strain B41M). It did not react with OK antiserum to a K12 K99+ recombinant or with OK antiserum to K99-positive organisms from the O8, O20, or O64 serogroups, but it did react with OK antiserum to K99-positive organisms from the O101 and O9 serogroups. The mutant hemagglutinated sheep erythrocytes and attached in vitro to calf enterocytes when cultured at 37 degrees C but not when grown at 18 degrees C. Attachment was mannose resistant but susceptible to heating and formaldehyde. These properties were associated with the presence of fimbriae. The isolated hemagglutinin migrated to the anode in immunoelectrophoresis experiments, competitively inhibited attachment of strain B41M to calf enterocytes, and could be demonstrated adhering to these cells in vitro by indirect immunofluorescent staining. The anionic hemagglutinin is referred to provisionally as F41. Germfree piglets infected with strain B41M developed diarrhea within 16 h. Scanning electron microscopy showed groups of bacteria adherent to the microvilli of villous enterocytes. Indirect immunofluorescent staining demonstrated the presence of F41 antigen in vivo. Images FIG. 1 FIG. 2 FIG. 4 PMID:6124503

  18. Frequency of specific agr groups and antibiotic resistance in Staphylococcus aureus isolated from bovine mastitis in the northeast of Iran

    PubMed Central

    Mohsenzadeh, Mohammad; Ghazvini, Kiarash; Azimian, Amir

    2015-01-01

    Staphylococcus aureus is generally regarded as a leading cause of mastitis in dairy cattle. The aim of this study was to investigate the pattern of agr groups and any possible relationship between agr groups and antibiotic resistance among S. aureus strains isolated from bovine mastitis in Northeast of Iran. For this purpose, a total of 300 bovine mastitic milk samples were taken from dairy industry farms of Khorasan Razavi Province, Iran. S. aureus were isolated and identified according to the standard methods. Antibiotic susceptibility testing was conducted by disk diffusion method. In this study a total of 31 isolates of S. aureus were evaluated for agrD gene polymorphism by specific primers. Most of the isolates belonged to agr group I (54.8%), followed by agr group III (25.8%) and agr group II (19.4%). There was not any isolates belonging to group IV. Resistance to methicillin in agr group I isolates was more than other groups. Agr groups II and III were quite susceptible to methicillin. Due to high prevalent of S. aureus isolates and high antibiotic resistance rate in bovine mastitic isolates, it is important to verify the characteristics of S. aureus strains in Iran. PMID:26973764

  19. A strategic study using mutant-strain entrapment in calcium alginate for the production of Saccharomyces cerevisiae cells with high invertase activity.

    PubMed

    Rossi-Alva, Juan Carlos; Rocha-Leão, Maria Helena Miguez

    2003-08-01

    Entrapped cells and entrapped cells grown inside of a calcium alginate matrix as well as free cells have been investigated using Saccharomyces cerevisiae mutant strains with regard to their pattern of growth and invertase activity. The repression of invertase by glucose and glucose-consumption ability were considered in the selection process of the mutants. Efficient sucrose bioconversion due to high invertase activity was obtained when entrapped mutant strain Q6R2 cells were grown within calcium alginate gel beads using sucrose plus glucose as the carbon source. Under these conditions, 1 mg (dry weight) of entrapped cells is able to produce 20 micromol of inverted sugar in 3 min (the maximum activity obtained was 20 units x mg(-1)). The experiments were carried out for 6 months without appreciable loss of either bead integrity or invertase activity. The biocatalyst was also stored at 4 degrees C for 6 months without appreciable loss of the invertase activity. This work shows that entrapped yeast cells with a weak ability to consume sugar may be used to produce inverted sugar.

  20. [Construction and function analysis of Bac operon mutants of bio-control strain Bacillus subtilis Bs-916].

    PubMed

    Luo, Chuping; Chen, Zhiyi; Liu, Yongfeng; Zhang, Jie; Liu, Youzhou; Wang, Xiaoyu; Nie, Yafeng

    2009-04-01

    Bacillus subtilis Bs-916 is an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. We identified and analyzed the operon Bac in Bs-916 responsible for synthesis of iturin-like lipopeptides. The research plays an important part in genetic engineered Bs-916 for further improving its bio-control activity. Taking advantage of homologous recombination method, the one mutant obtained by replacement of the Bac original promoter by a constitutive promoter P (repU) and designated BGG104; the other mutant was obtained by disruption of the Bac promoter by insertion and designated BGG105. The biological activities results showed the mutant BGG104 enhanced antagonistic activities against several pathogenic fungi and also clearly increased in hemolytic activities. However, the mutant BGG105 decreased clearly in both antagonistic activities and hemolytic activities. Crude lipopeptides were extracted with methanol from precipitates, which were obtained by adding 6 mol/L HCl to the cell-free culture broth. The results of reversed-phase high-performance liquid chromatography (HPLC) analysis of crude lipopeptides showed lipopeptides produced by Bs-916 have a different retention time compared to iturinA. The mutant BGG104 produced up to 3-fold more lipopeptides than Bs-916. However, the mutant BGG105 has not been detected countpart lipopeptides production. The molecular weights of the lipopeptides synthesized by Bac determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) were 1007.7 Da, 1021.7 Da and 1035.7 Da. They were presumed to belong to homologues differed by a structure of --CH2 and their molecular weights were not the same as the other iturin-like liopeptides', such as iturinA, mycosubtilin and bacillomycin, molecular weights. In conclusion, this paper showed the lipopeptides synthesized by Bac play crucial part in Bs-916 antifungal activities and the lipopeptides overproduction were able to enhance Bs

  1. Genetic analysis of developmental mechanisms in hydra. XVI. Effect of food on budding and developmental gradients in a mutant strain L4.

    PubMed

    Takano, J; Sugiyama, T

    1985-12-01

    Effect of food was examined on the budding rate and the developmental gradients of a mutant hydra strain L4. This mutant strain has very high levels of head-inhibition potential gradient along its body axis (Takano & Sugiyama, 1983). It also has a reduced budding capacity when it is cultured using brine shrimp nauplii as food, but its budding capacity is significantly improved when a small amount of tubifex worm tissue is added to its diet of brine shrimp (Takano, 1984). To test whether or not this change of budding rate is correlated with the change in the levels of the head-activation or head-inhibition potential gradients, L4 animals were cultured on the diet of brine shrimp with or without addition of tubifex worm tissue and the budding rates and the gradient levels were examined in these animals. The results showed that food affected the budding rate in L4 without affecting its gradient levels. This suggests that the gradient levels and the budding rate in L4 are are uncorrelated to each other, and that therefore the high levels of head-inhibition potential are not the cause for the low budding rate in this strain (cf., Takano & Sugiyama, 1983). PMID:3834025

  2. AGR-1 Data Qualification Report

    SciTech Connect

    Michael Abbott

    2010-03-01

    ABSTRACT Projects for the very high temperature reactor (VHTR) Technology Development Office (TDO) program provide data in support of Nuclear Regulatory Commission licensing of the VHTR. Fuel and materials to be used in the reactor are tested and characterized to quantify performance in high temperature and high fluence environments. The VHTR program has established the NGNP Data Management and Analysis System (NDMAS) to ensure that VHTR data are (1) qualified for use, (2) stored in a readily accessible electronic form, and (3) analyzed to extract useful results. This document focuses on the first NDMAS objective. It describes the data streams associated with the first Advanced Gas Reactor experiment (AGR-1), the processing of these data within NDMAS, and reports the qualification status of the data. Data qualification activities within NDMAS for specific types of data are determined by the data qualification category assigned by the data generator. They include: (1) capture testing, to confirm that the data stored within NDMAS are identical to the raw data supplied, (2) accuracy testing, to confirm that the data are an accurate representation of the system or object being measured, and (3) documentation that the data were collected under an NQA-1 or equivalent quality assurance program. The NDMAS database processing and qualification status of the following five data streams is reported in this document: 1. Fuel fabrication data. All data have been processed into the NDMAS database and qualified (1,819 records). 2. Fuel irradiation data. Data from all 13 AGR-1 reactor cycles have been processed into the NDMAS database and tested. Of these, 85% have been qualified and 15% have failed NDMAS accuracy testing. 3. FPMS data. Reprocessed (January 2010) data from all 13 AGR-1 reactor cycles have been processed into the database and capture tested. Final qualification of these data will be recorded after QA approval of an Engineering Calculations and Analysis Report

  3. Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: Isolation of a new ssb allele, ssb-3

    SciTech Connect

    Schmellik-Sandage, C.S.; Tessman, E.S. )

    1990-08-01

    Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found.

  4. Signal strains that can detect certain DNA replication and membrane mutants of Escherichia coli: isolation of a new ssb allele, ssb-3.

    PubMed Central

    Schmellik-Sandage, C S; Tessman, E S

    1990-01-01

    Mutations in several dna genes of Escherichia coli, when introduced into a strain with a lac fusion in the SOS gene sulA, resulted in formation of blue colonies on plates containing 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal). Unexpectedly, several lines of evidence indicated that the blue colony color was not primarily due to induction of the SOS system but rather was due to a membrane defect, along with the replication defect, making the cell X-Gal extrasensitive (phenotypically Xgx), possibly because of enhanced permeability to X-Gal or leakage of beta-galactosidase. (i) In most cases, beta-galactosidase specific activity increased only two- to threefold. (ii) Mutations conferring tolerance to colicin E1 resulted in blue colony color with no increase in beta-galactosidase specific activity. (iii) Mutations in either the dnaA, dnaB, dnaC, dnaE, dnaG, or ssb gene, when introduced into a strain containing a bioA::lac fusion, produced a blue colony color without an increase in beta-galactosidase synthesis. These lac fusion strains can serve as signal strains to detect dna mutations as well as membrane mutations. By localized mutagenesis of the 92-min region of the chromosome of the sulA::lac signal strain and picking blue colonies, we isolated a novel ssb allele that confers the same extreme UV sensitivity as a delta recA allele, which is a considerably greater sensitivity than that conferred by the two well-studied ssb alleles, ssb-1 and ssb-113. The technique also yielded dnaB mutants; fortuitously, uvrA mutants were also found. PMID:2142938

  5. Sulphide Resistance in the Cyanobacterium Microcystis aeruginosa: a Comparative Study of Morphology and Photosynthetic Performance Between the Sulphide-Resistant Mutant and the Wild-Type Strain.

    PubMed

    Bañares-España, Elena; del Mar Fernández-Arjona, María; García-Sánchez, María Jesús; Hernández-López, Miguel; Reul, Andreas; Mariné, Mariona Hernández; Flores-Moya, Antonio

    2016-05-01

    The cyanobacterium Microcystis aeruginosa is a mesophilic freshwater organism, which cannot tolerate sulphide. However, it was possible to isolate a sulphide-resistant (S(r)) mutant strain that was able to survive in a normally lethal medium sulphide. In order to evaluate the cost of the mutation conferring sulphide resistance in the S(r) strain of M. aeruginosa, the morphology and the photosynthetic performance were compared to that found in the wild-type, sulphide-sensitive (S(s)) strain. An increase in size and a disrupted morphology was observed in S(r) cells in comparison to the S(s) counterpart. Phycoerythrin and phycocyanin levels were higher in the S(r) than in the S(s) cells, whereas a higher carotenoid content, per unit volume, was found in the S(s) strain. The irradiance-saturated photosynthetic oxygen-production rate (GPR max) and the photosynthetic efficiency (measured both by oxygen production and fluorescence, α(GPR) and α(ETR)) were lower in the S(r) strain than in the wild-type. These results appear to be the result of package effect. On the other hand, the S(r) strain showed higher quantum yield of non-photochemical quenching, especially those regulated mechanisms (estimated throughout qN and Y(NPQ)) and a significantly lower slope in the maximum quantum yield of light-adapted samples (Fv'/Fm') compared to the S(s) strain. These findings point to a change in the regulation of the quenching of the transition states (qT) in the S(r) strain which may be generated by a change in the distribution of thylakoidal membranes, which somehow could protect metalloenzymes of the electron transport chain from the lethal effect of sulphide. PMID:26677166

  6. Sulphide Resistance in the Cyanobacterium Microcystis aeruginosa: a Comparative Study of Morphology and Photosynthetic Performance Between the Sulphide-Resistant Mutant and the Wild-Type Strain.

    PubMed

    Bañares-España, Elena; del Mar Fernández-Arjona, María; García-Sánchez, María Jesús; Hernández-López, Miguel; Reul, Andreas; Mariné, Mariona Hernández; Flores-Moya, Antonio

    2016-05-01

    The cyanobacterium Microcystis aeruginosa is a mesophilic freshwater organism, which cannot tolerate sulphide. However, it was possible to isolate a sulphide-resistant (S(r)) mutant strain that was able to survive in a normally lethal medium sulphide. In order to evaluate the cost of the mutation conferring sulphide resistance in the S(r) strain of M. aeruginosa, the morphology and the photosynthetic performance were compared to that found in the wild-type, sulphide-sensitive (S(s)) strain. An increase in size and a disrupted morphology was observed in S(r) cells in comparison to the S(s) counterpart. Phycoerythrin and phycocyanin levels were higher in the S(r) than in the S(s) cells, whereas a higher carotenoid content, per unit volume, was found in the S(s) strain. The irradiance-saturated photosynthetic oxygen-production rate (GPR max) and the photosynthetic efficiency (measured both by oxygen production and fluorescence, α(GPR) and α(ETR)) were lower in the S(r) strain than in the wild-type. These results appear to be the result of package effect. On the other hand, the S(r) strain showed higher quantum yield of non-photochemical quenching, especially those regulated mechanisms (estimated throughout qN and Y(NPQ)) and a significantly lower slope in the maximum quantum yield of light-adapted samples (Fv'/Fm') compared to the S(s) strain. These findings point to a change in the regulation of the quenching of the transition states (qT) in the S(r) strain which may be generated by a change in the distribution of thylakoidal membranes, which somehow could protect metalloenzymes of the electron transport chain from the lethal effect of sulphide.

  7. NAD(P)+-Malic Enzyme Mutants of Sinorhizobium sp. Strain NGR234, but Not Azorhizobium caulinodans ORS571, Maintain Symbiotic N2 Fixation Capabilities

    PubMed Central

    Zhang, Ye; Aono, Toshihiro; Poole, Phillip

    2012-01-01

    C4-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N2-fixing bacteria (bacteroids) within legume nodules. In Sinorhizobium meliloti bacteroids from alfalfa, NAD+-malic enzyme (DME) is required for N2 fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiont Rhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report that dme mutants of the broad-host-range Sinorhizobium sp. strain NGR234 formed nodules whose level of N2 fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while single pckA and single dme mutants fixed N2 at reduced rates, a pckA dme double mutant had no N2-fixing activity (Fix−). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix− phenotype of S. meliloti dme mutants may be specific to the alfalfa-S. meliloti symbiosis. We therefore examined the ME-like genes azc3656 and azc0119 from Azorhizobium caulinodans, as azc3656 mutants were previously shown to form Fix− nodules on the tropical legume Sesbania rostrata. We found that purified AZC3656 protein is an NAD(P)+-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N2 fixation in A. caulinodans and S. meliloti, in other rhizobia this activity can be bypassed via another pathway(s). PMID:22307295

  8. Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination.

    PubMed

    Rockx-Brouwer, Dedeke; Chong, Audrey; Wehrly, Tara D; Child, Robert; Crane, Deborah D; Celli, Jean; Bosio, Catharine M

    2012-01-01

    Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.

  9. Open fermentative production of fuel ethanol from food waste by an acid-tolerant mutant strain of Zymomonas mobilis.

    PubMed

    Ma, Kedong; Ruan, Zhiyong; Shui, Zongxia; Wang, Yanwei; Hu, Guoquan; He, Mingxiong

    2016-03-01

    The aim of present study was to develop a process for open ethanol fermentation from food waste using an acid-tolerant mutant of Zymomonas mobilis (ZMA7-2). The mutant showed strong tolerance to acid condition of food waste hydrolysate and high ethanol production performance. By optimizing fermentation parameters, ethanol fermentation with initial glucose concentration of 200 g/L, pH value around 4.0, inoculum size of 10% and without nutrient addition was considered as best conditions. Moreover, the potential of bench scales fermentation and cell reusability was also examined. The fermentation in bench scales (44 h) was faster than flask scale (48 h), and the maximum ethanol concentration and ethanol yield (99.78 g/L, 0.50 g/g) higher than that of flask scale (98.31 g/L, 0.49 g/g). In addition, the stable cell growth and ethanol production profile in five cycles successive fermentation was observed, indicating the mutant was suitable for industrial ethanol production.

  10. Development of a Nuclear Transformation System for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and Genetic Complementation of a Mutant Strain, Deficient in Arachidonic Acid Biosynthesis

    PubMed Central

    Khozin-Goldberg, Inna; Leu, Stefan; Shapira, Michal; Kaye, Yuval; Tourasse, Nicolas; Vallon, Olivier; Boussiba, Sammy

    2014-01-01

    Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2–5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism. PMID:25133787

  11. Metabolic Engineering of Light and Dark Biochemical Pathways in Wild-Type and Mutant Strains of Synechocystis PCC 6803 for Maximal, 24-Hour Production of Hydrogen Gas

    SciTech Connect

    Ely, Roger L.; Chaplen, Frank W.R.

    2014-03-11

    enhanced H2 production profiles using selected culture conditions and inhibitors of specific pathways in WT cells and an NDH-1 mutant; 3. Create Synechocystis PCC 6803 mutant strains with modified hydrogenases exhibiting increased O2 tolerance and greater H2 production; and 4. Integrate enhanced hydrogenase mutants and culture and metabolic factor studies to maximize 24-hour H2 production.

  12. Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

    PubMed

    Peters, S E; Paterson, G K; Bandularatne, E S D; Northen, H C; Pleasance, S; Willers, C; Wang, J; Foote, A K; Constantino-Casas, F; Scase, T J; Blacklaws, B A; Bryant, C E; Mastroeni, P; Charles, I G; Maskell, D J

    2010-01-01

    In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

  13. In vivo-selected mutations in methyl-directed mismatch repair suppress the virulence attenuation of Salmonella dam mutant strains following intraperitoneal, but not oral, infection of naïve mice.

    PubMed

    Heithoff, Douglas M; Badie, Golnaz; Julio, Steven M; Enioutina, Elena Y; Daynes, Raymond A; Sinsheimer, Robert L; Mahan, Michael J

    2007-07-01

    Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety of dam mutant Salmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Since dam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-AP(r)) isolates in systemic tissues of mice infected with dam mutant Salmonella. Such 2-AP(r) derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naïve mice. These 2-AP(r) derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parental dam mutant strain. Additionally, introduction of a mutH null mutation into dam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required for Salmonella systemic survival. These data suggest that restoration of intraperitoneal virulence of dam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.

  14. Protective effects of recombinant staphylococcal enterotoxin type C mutant vaccine against experimental bovine infection by a strain of Staphylococcus aureus isolated from subclinical mastitis in dairy cattle.

    PubMed

    Chang, Byoung Sun; Moon, Jin San; Kang, Hyun-Mi; Kim, Young-In; Lee, Hong-Kyun; Kim, Jong-Duk; Lee, Byung-Saeng; Koo, Hye Cheong; Park, Yong Ho

    2008-04-16

    Staphylococcus aureus is one of the main etiological agents of bovine mastitis; however, antibiotics that are effective against bovine strains of S. aureus are not currently available. Staphylococcal enterotoxin type C (SEC), a superantigen, is the enterotoxin most frequently expressed by bovine strains of S. aureus and one of immunogenic determinants. The purpose of this study was to evaluate the protective effectiveness of recombinant SEC mutant vaccine (MastaVactrade mark) against experimentally induced bovine infection. Three representative SEC secreting strains were selected from 9 candidate isolates that showed various intensities of pathogenicity on mice and inoculated into 5 lactating dairy cattle at a concentration of 50-5.0x10(8) CFU per quarter. The optimal experimental bovine subclinical mastitis model was produced by inoculation with 50 CFU of S. aureus 409 per quarter, a level which was not lethal to mice. After the experimental model was determined, other 3 cattle were intramuscularly administered three doses of vaccine at day 0, at 2 wks and at 6 wks. Nine quarters of 3 vaccinated cattle and 8 quarters of 3 control cattle were then challenged with S. aureus 409. An SEC-specific ELISA test conducted at 4 wks post-immunization confirmed the presence of a high antibody titer against SEC in all vaccinated cattle. The somatic cell counts from the vaccinated group remained relatively low, whereas those of control group increased significantly after challenge with S. aureus. After challenge, S. aureus was not isolated from any cattle in the vaccinated group, whereas it was isolated from 75% of the cattle in the control group. These results indicate that recombinant SEC mutant vaccine had a protective effect against S. aureus intramammary infection in lactating cattle.

  15. Live attenuated mutants of Francisella tularensis protect rabbits against aerosol challenge with a virulent type A strain.

    PubMed

    Reed, Douglas S; Smith, Le'kneitah P; Cole, Kelly Stefano; Santiago, Araceli E; Mann, Barbara J; Barry, Eileen M

    2014-05-01

    Francisella tularensis, a Gram-negative bacterium, is the causative agent of tularemia. No licensed vaccine is currently available for protection against tularemia, although an attenuated strain, dubbed the live vaccine strain (LVS), is given to at-risk laboratory personnel as an investigational new drug (IND). In an effort to develop a vaccine that offers better protection, recombinant attenuated derivatives of a virulent type A strain, SCHU S4, were evaluated in New Zealand White (NZW) rabbits. Rabbits vaccinated via scarification with the three attenuated derivatives (SCHU S4 ΔguaBA, ΔaroD, and ΔfipB strains) or with LVS developed a mild fever, but no weight loss was detected. Twenty-one days after vaccination, all vaccinated rabbits were seropositive for IgG to F. tularensis lipopolysaccharide (LPS). Thirty days after vaccination, all rabbits were challenged with aerosolized SCHU S4 at doses ranging from 50 to 500 50% lethal doses (LD50). All rabbits developed fevers and weight loss after challenge, but the severity was greater for mock-vaccinated rabbits. The ΔguaBA and ΔaroD SCHU S4 derivatives provided partial protection against death (27 to 36%) and a prolonged time to death compared to results for the mock-vaccinated group. In contrast, LVS and the ΔfipB strain both prolonged the time to death, but there were no survivors from the challenge. This is the first demonstration of vaccine efficacy against aerosol challenge with virulent type A F. tularensis in a species other than a rodent since the original work with LVS in the 1960s. The ΔguaBA and ΔaroD SCHU S4 derivatives warrant further evaluation and consideration as potential vaccines for tularemia and for identification of immunological correlates of protection.

  16. Unusual ∆7,12,19 C35:3 Alkenone Produced by the Mutant Emiliania huxleyi strain CCMP2758 in Culture

    NASA Astrophysics Data System (ADS)

    Zheng, Y.; Huang, Y.; Zhang, Y.; Dillon, J. T.

    2015-12-01

    Alkenones with chain length ranging from C37 to C40 are highly specific biomarkers for certain haptophyte algae in ocean and lake sediments and have been widely used for paleoclimate studies. Short chain alkenones (e.g., C35 and C36) have been found in environmental and culture samples but the origin and structures of these compounds are not fully understood. The benchmark marine alkenone producer, Emiliania huxleyi CCMP2758 strain (the mutant of strain CCMP1742, NEPCC55a) was reported to make 35:2 alkenone when cultured at 15 °C (Prahl et al., 2006). Here we show, when this strain is cultured at lower temperatures (e.g., 4°C), CCMP2758 produces large amount of 35:3 alkenone with unusual double bond positions of ∆7,12,19. We determined the double bond positions of the C35:3 methyl ketonee based on GC-MS analysis of cyclobutylimine derivatives and dimethyl disulfide derivatives respectively, and provide the first temperature calibrations based on the unsaturation ratios of C35 alkenones. Previous studies have found 35:2 alkenone with three methylene interruption in the Black Sea sediment, but it is the first time that an alkenone with a mixed three and five methylene interruption is found. The discovery of short chain alkenones with unusual double bond positions may shed new light to alkenone biosynthesis.

  17. AgrAbility: Frequently Asked Questions

    MedlinePlus

    ... About AgrAbility State Projects Directory The Toolbox AT Database Resources Veterans & Beginning Farmers Communities of Interest News ... 800) 825-4264 Home About The Toolbox AT Database Resources Online Training Contact Us You are here: ...

  18. The agr function and polymorphism: impact on Staphylococcus aureus susceptibility to photoinactivation

    PubMed Central

    Grinholc, Mariusz; Nakonieczna, Joanna; Negri, Alessandro; Rapacka-Zdonczyk, Aleksandra; Motyka, Agata; Fila, Grzegorz; Kurlenda, Julianna; Leibner-Ciszak, Justyna; Otto, Michael; Bielawski, Krzysztof P.

    2016-01-01

    Staphylococcus aureus is an important human pathogen that causes healthcare-associated and community-acquired infections. Moreover, the growing prevalence of multiresistant strains requires the development of alternative methods to antibiotic therapy. One effective therapeutic option may be antimicrobial photodynamic inactivation (aPDI). Recently, S. aureus strain-dependent response to PDI was demonstrated, although the mechanism underlying this phenomenon remains unexplained. The aim of the current study was to investigate statistically relevant correlations between the functionality and polymorphisms of agr gene determined for 750 methicillin-susceptible and methicillin-resistant S. aureus strains and their responses to photodynamic inactivation using protoporphyrin IX. An AluI and RsaI digestion of the agr gene PCR product revealed existing correlations between the determined digestion profiles (designations used for the first time) and the PDI response. Moreover, the functionality of the agr system affected S. aureus susceptibility to PDI. Based on our results, we conclude that the agr gene may be a genetic factor affecting the strain dependent response to PDI. PMID:24211295

  19. The mode of cauliflower mosaic virus propagation in the plant allows rapid amplification of viable mutant strains.

    PubMed

    Riederer, M A; Grimsley, N H; Hohn, B; Jiricny, J

    1992-06-01

    We inoculated the leaves of turnip plants (Brassica campestris spp. rapa cv. Just Right) with two cauliflower mosaic viruses (CaMVs) with different small mutations in a dispensable region of the viral genome, and followed the spread of the virus infection through the plant. Surprisingly, analysis of viral DNA in single primary chlorotic lesions revealed the presence of both mutants. In contrast, the secondary chlorotic lesions and systemically infected leaves contained virus molecules of either one or the other type only. Infection of plants with different ratios of the two reporter viruses showed that this ratio is not conserved during systemic virus spread. Infection with CaMV DNA in the form of heteroduplexes containing a single mismatched base pair, in which each strand carried a distinct diagnostic marker, provided us with evidence that the mismatch was subjected to a repair process in the host plant.

  20. EXAFS of Klebsiella pneumoniae nitrogenase MoFe protein from wild-type and nif V mutant strains

    SciTech Connect

    Eidsness, M.K.; Flank, A.M.; Smith, B.E.; Flood, A.C.; Garner, C.D.; Cramer. S.P.

    1986-05-14

    The enzyme nitrogenase catalyzes the biological reduction of N/sub 2/ to NH/sub 3/. In Klebsiella pneumoniae a cluster of 17 genes in seven transcriptional units has been associated with nitrogen fixation. The nitrogenase enzyme from the nif V mutants is relatively ineffective at dinitrogen reduction, is more efficient than the wild-type enzyme at HCN reduction, and has its hydrogen evolution activity inhibited up to 80% by CO. This altered substrate specificity has been shown to be associated with the iron-molybdenum cofactor, FeMo-co, of the enzyme. X-ray absorption spectroscopy has been a valuable tool for probing the molybdenum environment of wild-type nitrogenase, and the authors report here similar studies on the Nif V/sup -/ enzyme.

  1. Novel mutant human immunodeficiency virus type 1 strains with high degree of resistance to cynomolgus macaque TRIMCyp generated by random mutagenesis.

    PubMed

    Sultana, Tahmina; Nakayama, Emi E; Tobita, Satoshi; Yokoyama, Masaru; Seki, Yohei; Saito, Akatsuki; Nomaguchi, Masako; Adachi, Akio; Akari, Hirofumi; Sato, Hironori; Shioda, Tatsuo

    2016-04-01

    Old World monkey TRIM5α strongly suppresses human immunodeficiency virus type 1 (HIV-1) replication. A fusion protein comprising cynomolgus macaque (CM) TRIM5 and cyclophilin A (CM TRIMCyp) also potently suppresses HIV-1 replication. However, CM TRIMCyp fails to suppress a mutant HIV-1 that encodes a mutant capsid protein containing a SIVmac239-derived loop between α-helices 4 and 5 (L4/5). There are seven amino acid differences between L4/5 of HIV-1 and SIVmac239. Here, we investigated the minimum numbers of amino acid substitutions that would allow HIV-1 to evade CM TRIMCyp-mediated suppression. We performed random PCR mutagenesis to construct a library of HIV-1 variants containing mutations in L4/5, and then we recovered replication-competent viruses from CD4+ MT4 cells that expressed high levels of CM TRIMCyp. CM TRIMCyp-resistant viruses were obtained after three rounds of selection in MT4 cells expressing CM TRIMCyp and these were found to contain four amino acid substitutions (H87R, A88G, P90D and P93A) in L4/5. We then confirmed that these substitutions were sufficient to confer CM TRIMCyp resistance to HIV-1. In a separate experiment using a similar method, we obtained novel CM TRIM5α-resistant HIV-1 strains after six rounds of selection and rescue. Analysis of these mutants revealed that V86A and G116E mutations in the capsid region conferred partial resistance to CM TRIM5α without substantial fitness cost when propagated in MT4 cells expressing CM TRIM5α. These results confirmed and further extended the previous notion that CM TRIMCyp and CM TRIM5α recognize the HIV-1 capsid in different manners. PMID:26795727

  2. Tubulointerstitial Nephritis Due to a Mutant Polyomavirus BK Virus Strain, BKV(Cin), Causing End-Stage Renal Disease

    PubMed Central

    Smith, R. D.; Galla, J. H.; Skahan, K.; Anderson, P.; Linnemann, C. C.; Ault, G. S.; Ryschkewitsch, C. F.; Stoner, G. L.

    1998-01-01

    A renal biopsy from a 36-year-old man with AIDS showed a severe tubulointerstitial nephritis with intranuclear inclusions in epithelial cells. Electron microscopy revealed the characteristic findings of a polyomavirus (PyV) infection, and immunofluorescence indicated the presence of BK virus (BKV) antigen. Inoculation of rhesus monkey kidney cell cultures both with urine and with buffy coat blood cells resulted in a cytopathic response which was subsequently confirmed to be due to BKV. Further characterization of the viral DNA from the kidney by PCR amplification and Southern blot analysis with PyV and strain-specific primers and probes indicated that the virus was closely related to the BK(Dun) strain but different in its apparent sequence arrangement. Subsequent cycle sequencing showed a dinucleotide mutation of TG→AA which substitutes hydrophilic Gln for hydrophobic Leu in a sequence homologous to an origin DNA-binding domain of simian virus 40 T antigen. It is suggested that the mutation and a coding region rearrangement of this strain of BKV designated BKV(Cin) has the potential to alter viral DNA replication and enhance pathogenicity. PMID:9620396

  3. AGR-1 Irradiation Experiment Test Plan

    SciTech Connect

    John T. Maki

    2009-10-01

    This document presents the current state of planning for the AGR-1 irradiation experiment, the first of eight planned irradiations for the Advanced Gas Reactor (AGR) Fuel Development and Qualification Program. The objectives of the AGR-1 experiment are: 1. To gain experience with multi-capsule test train design, fabrication, and operation with the intent to reduce the probability of capsule or test train failure in subsequent irradiation tests. 2. To irradiate fuel produced in conjunction with the AGR fuel process development effort. 3. To provide data that will support the development of an understanding of the relationship between fuel fabrication processes, fuel product properties, and irradiation performance. In order to achieve the test objectives, the AGR-1 experiment will be irradiated in the B-10 position of the Advanced Test Reactor (ATR) at the Idaho National Laboratory (INL). The test will contain six independently controlled and monitored capsules. Each capsule will contain a single type, or variant, of the AGR coated fuel. The irradiation is planned for about 700 effective full power days (approximately 2.4 calendar years) with a time-averaged, volume-average temperature of approximately 1050 °C. Average fuel burnup, for the entire test, will be greater than 17.7 % FIMA, and the fuel will experience fast neutron fluences between 2.4 and 4.5 x 1025 n/m2 (E>0.18 MeV).

  4. [Characteristics of the structural organization of the DIP1 gene in Drosophila melanogaster strains mutant for the flamenco gene].

    PubMed

    Nefedova, L N; Romanova, N I; Kim, A I

    2007-01-01

    Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flamSS (SS) and flamMS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flampy + (P) carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco+. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flamSS, flamMS, flampy +(P), and flamenco+ considerably differ from one another in the structure of DIP1. Strains flamss and flamMS have no Dral restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The Dral gene of strainsflamSS andflamMS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tcl/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIPI and, probably, neighboring genes. In strains flamenco+ and flampy + (P), the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either

  5. Multiple Mutations Lead to MexXY-OprM-Dependent Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa

    PubMed Central

    Guénard, Sophie; Muller, Cédric; Monlezun, Laura; Benas, Philippe; Broutin, Isabelle; Jeannot, Katy

    2014-01-01

    Constitutive overproduction of the pump MexXY-OprM is recognized as a major cause of resistance to aminoglycosides, fluoroquinolones, and zwitterionic cephalosporins in Pseudomonas aeruginosa. In this study, 57 clonally unrelated strains recovered from non-cystic fibrosis patients were analyzed to characterize the mutations resulting in upregulation of the mexXY operon. Forty-four (77.2%) of the strains, classified as agrZ mutants were found to harbor mutations inactivating the local repressor gene (mexZ) of the mexXY operon (n = 33; 57.9%) or introducing amino acid substitutions in its product, MexZ (n = 11; 19.3%). These sequence variations, which mapped in the dimerization domain, the DNA binding domain, or the rest of the MexZ structure, mostly affected amino acid positions conserved in TetR-like regulators. The 13 remaining MexXY-OprM strains (22.8%) contained intact mexZ genes encoding wild-type MexZ proteins. Eight (14.0%) of these isolates, classified as agrW1 mutants, overexpressed the gene PA5471, which codes for the MexZ antirepressor AmrZ, with 5 strains exhibiting growth defects at 37°C and 44°C, consistent with mutations impairing ribosome activity. Interestingly, one agrW1 mutant appeared to harbor a 7-bp deletion in the coding sequence of the leader peptide, PA5471.1, involved in ribosome-dependent, translational attenuation of PA5471 expression. Finally, DNA sequencing and complementation experiments revealed that 5 (8.8%) strains, classified as agrW2 mutants, harbored single amino acid variations in the sensor histidine kinase of ParRS, a two-component system known to positively control mexXY expression. Collectively, these results demonstrate that clinical strains of P. aeruginosa exploit different regulatory circuitries to mutationally overproduce the MexXY-OprM pump and become multidrug resistant, which accounts for the high prevalence of MexXY-OprM mutants in the clinical setting. PMID:24145539

  6. Staphylococcus aureus agr Genotypes with Enterotoxin Production Capabilities Can Resist Neutrophil Bactericidal Activity

    PubMed Central

    Mullarky, I. K.; Su, C.; Frieze, N.; Park, Y. H.; Sordillo, L. M.

    2001-01-01

    Staphylococcus aureus pathogenicity is mainly due to the production of a number of secreted and cell surface-associated proteins under the regulation of the agr gene. A region of the agr gene was used to subgroup S. aureus strains according to restriction fragment length polymorphisms. Additionally, strains were subtyped according to the coagulase gene in order to strengthen discriminatory power. Virulence capabilities of agr genotype subgroups were evaluated using an in vitro neutrophil bactericidal assay, which showed that prevalent genotypes were significantly better at evading this primary host defense. Multiplex PCR was then used to detect enterotoxin genes among the genotype subgroups in order to determine possible virulence candidates that enable strains to combat neutrophil killing. The prevalent genotype strains were found to possess higher production capabilities for enterotoxin A than did low-prevalence strains. The significance of enterotoxin A production capabilities in affecting pathogenicity of S. aureus strains was evaluated and found to have a profound effect on neutrophil killing abilities. The use of a large epidemiological database as a tool for subgrouping strains with varying degrees of pathogenicity has allowed the identification of relevant and previously undefined virulence factors that affect a pathogen's capability to overcome host immune defenses. PMID:11119487

  7. Molecular modeling of HIV-1 reverse transcriptase drug-resistant mutant strains: implications for the mechanism of polymerase action.

    PubMed

    Kroeger Smith, M B; Michejda, C J; Hughes, S H; Boyer, P L; Janssen, P A; Andries, K; Buckheit, R W; Smith, R H

    1997-12-01

    A computer model of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) either alone, or complexed with a non-nucleoside inhibitor (NNI), was constructed using crystal coordinate data from a subset of the protein surrounding the binding pocket region. Molecular mechanics calculations were carried out on solvated wild-type RT and RT that contained modifications corresponding to resistance-engendering mutations. Results from the calculations revealed that the r.m.s. difference between 12 modified proteins and that of wild-type RT could be qualitatively correlated with the measured polymerase activity of the enzyme in the presence of these mutations. In addition, the level of activity was related to the measured distance between the primer grip and dNTP binding regions of the protein. These data suggest a direct correlation between RT structure and function. Complexes of RT-8-C1 TIBO and RT-alpha-APA were also minimized in models containing modifications corresponding to key drug-resistant mutants. The variant complexes all showed weaker binding than wild-type RT, while giving rise to similar, but critical changes in the protein. Therefore, the design of new inhibitors should center on obtaining stronger binding drugs to key drug-resistant RT variants.

  8. Transcription Profiling-Based Identification of Staphylococcus aureus Genes Regulated by the agr and/or sarA Loci

    PubMed Central

    Dunman, P. M.; Murphy, E.; Haney, S.; Palacios, D.; Tucker-Kellogg, G.; Wu, S.; Brown, E. L.; Zagursky, R. J.; Shlaes, D.; Projan, S. J.

    2001-01-01

    The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the Staphylococcus aureus genome, was used to identify open reading frames that are regulated by agr and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and agr, sarA, and agr sarA mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up- or downregulated in an agr- and/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including hla (alpha-toxin) and spa (protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and protein A expression. Several putative virulence factors were also identified as regulated by agr and/or SarA. In addition, genes that are involved in several biological processes but which are difficult to reconcile as playing a direct role in the organism's pathogenesis also appeared to be regulated by each effector, suggesting that products of both the agr and the sarA locus are more-global transcription regulators than previously realized. PMID:11717293

  9. Differential activities of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives against different human immunodeficiency virus type 1 mutant strains.

    PubMed Central

    Balzarini, J; Baba, M; De Clercq, E

    1995-01-01

    A series of 23 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives that were highly potent inhibitors of wild-type human immunodeficiency virus type 1 strain IIIB (HIV-1/IIIB) replication in CEM cells were evaluated against a panel of HIV-1 mutant strains containing the replacement of leucine by isoleucine at position 100 (100-Leu-->Ile), 103-Lys-->Asn, 106-Val-->Ala, 138-Glu-->Lys, 181-Tyr-->Cys, 181-Tyr-->Ile, or 188-Tyr-->His in their reverse transcriptase (RT). A different structure-antiviral activity relationship was found, depending on the nature of the mutated amino acid in the HIV-1 RT. The results show that 5-ethyl-1-ethoxymethyl-6-(3,5-dimethylbenzyl)uracil, 5-ethyl-1-ethoxymethyl-6-(3,5-dimethylphenylthio)uracil, and 5-ethyl-1-ethoxymethyl-6-(3,5-dimethylphenylthio)-2-thiouracil remain active against the majority of viruses containing single mutations which confer resistance to nonnucleoside RT inhibitors. PMID:7540384

  10. Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

    PubMed

    Galindo-Leva, Luz Ángela; Hughes, Stephen R; López-Núñez, Juan Carlos; Jarodsky, Joshua M; Erickson, Adam; Lindquist, Mitchell R; Cox, Elby J; Bischoff, Kenneth M; Hoecker, Eric C; Liu, Siqing; Qureshi, Nasib; Jones, Marjorie A

    2016-07-01

    Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL. PMID:27130462

  11. Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

    PubMed

    Galindo-Leva, Luz Ángela; Hughes, Stephen R; López-Núñez, Juan Carlos; Jarodsky, Joshua M; Erickson, Adam; Lindquist, Mitchell R; Cox, Elby J; Bischoff, Kenneth M; Hoecker, Eric C; Liu, Siqing; Qureshi, Nasib; Jones, Marjorie A

    2016-07-01

    Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.

  12. Mapping of internal monophosphate 5′ ends of Bacillus subtilis messenger RNAs and ribosomal RNAs in wild-type and ribonuclease-mutant strains

    PubMed Central

    DiChiara, Jeanne M.; Liu, Bo; Figaro, Sabine; Condon, Ciarán; Bechhofer, David H.

    2016-01-01

    The recent findings that the narrow-specificity endoribonuclease RNase III and the 5′ exonuclease RNase J1 are not essential in the Gram-positive model organism, Bacillus subtilis, facilitated a global analysis of internal 5′ ends that are generated or acted upon by these enzymes. An RNA-Seq protocol known as PARE (Parallel Analysis of RNA Ends) was used to capture 5′ monophosphorylated RNA ends in ribonuclease wild-type and mutant strains. Comparison of PARE peaks in strains with RNase III present or absent showed that, in addition to its well-known role in ribosomal (rRNA) processing, many coding sequences and intergenic regions appeared to be direct targets of RNase III. These target sites were, in most cases, not associated with a known antisense RNA. The PARE analysis also revealed an accumulation of 3′-proximal peaks that correlated with the absence of RNase J1, confirming the importance of RNase J1 in degrading RNA fragments that contain the transcription terminator structure. A significant result from the PARE analysis was the discovery of an endonuclease cleavage just 2 nts downstream of the 16S rRNA 3′ end. This latter observation begins to answer, at least for B. subtilis, a long-standing question on the exonucleolytic versus endonucleolytic nature of 16S rRNA maturation. PMID:26883633

  13. Traumatic brain injury causes delayed motor and cognitive impairment in a mutant mouse strain known to exhibit delayed Wallerian degeneration.

    PubMed

    Fox, G B; Faden, A I

    1998-09-15

    Delayed Wallerian degeneration after neuronal injury is a feature of the C57BL/Wld(s) mouse mutant. In the present study, we examined the effect of unilateral controlled cortical impact (CCI) on motor and cognitive performance in C57BL/6 and C57BL/Wld(s) mice. Performance on a beam-walking task was impaired in both injured groups over the first 3 weeks; however, between 28 and 35 days post injury, C57BL/6 mice continued to improve whereas C57BL/Wld(s) mice showed increased footfaults. In a spatial learning task, C57BL/Wld(s) animals performed consistently better than C57BL/6 mice when tested 7-10 days and 14-17 days following CCI. C57BL/Wld(s) mice also demonstrated improved working memory performance as compared with C57BL/6 mice when trained on days 21-22 after injury; this effect was lost on days 23 and 24, and was not evident in other animals tested in the same task at 28-31 days following injury. These results indicate a marked delay in motor and cognitive impairment following CCI in C57BL/Wld(s) mice compared with injured C57BL/6 controls. This is consistent with previous work showing delayed temporal evolution of neuronal degeneration in C57BL/Wld(s) mice and suggests CCI may be a suitable model for examining the functional consequences of traumatic brain injury (TBI) in genetically altered mice.

  14. Studies on the Mechanism of Telavancin Decreased Susceptibility in a Laboratory-Derived Mutant

    PubMed Central

    Song, Yang; Lunde, Christopher S.; Benton, Bret M.

    2013-01-01

    Telavancin is a novel semisynthetic lipoglycopeptide derivative of vancomycin with a dual mode of action. This study sought to understand the mechanisms of decreased telavancin susceptibility in a laboratory-derived Staphlococcus aureus mutant TlvDSMED1952. There were extensive changes in the transcriptome of TlvDSMED1952 compared to the susceptible parent strain MED1951. Genes upregulated included cofactor biosynthesis genes, cell wall-related genes, fatty acid biosynthesis genes, and stress genes. Downregulated genes included lysine operon biosynthesis genes and lrgB, which are induced by telavancin in susceptible strains, agr and kdpDE genes, various cell surface protein genes, phenol-soluble modulin genes, several protease genes, and genes involved in anaerobic metabolism. The decreased susceptibility mutant had somewhat thicker cell walls and a decreased autolytic activity that may be related to decreased proteolytic peptidoglycan hydrolase processing. Membrane fatty acid changes correlated with increased membrane fluidity were observed. It seems likely that there are multiple genetic changes associated with the development of decreased telavancin susceptibility. The TlvDS mutant showed some similar features to vancomycin-intermediate S. aureus and decreased daptomycin susceptibility strains, but also exhibited its own unique features. PMID:23551248

  15. Studies on the mechanism of telavancin decreased susceptibility in a laboratory-derived mutant.

    PubMed

    Song, Yang; Lunde, Christopher S; Benton, Bret M; Wilkinson, Brian J

    2013-08-01

    Telavancin is a novel semisynthetic lipoglycopeptide derivative of vancomycin with a dual mode of action. This study sought to understand the mechanisms of decreased telavancin susceptibility in a laboratory-derived Staphlococcus aureus mutant Tlv(DS)MED1952. There were extensive changes in the transcriptome of Tlv(DS)MED1952 compared to the susceptible parent strain MED1951. Genes upregulated included cofactor biosynthesis genes, cell wall-related genes, fatty acid biosynthesis genes, and stress genes. Downregulated genes included lysine operon biosynthesis genes and lrgB, which are induced by telavancin in susceptible strains, agr and kdpDE genes, various cell surface protein genes, phenol-soluble modulin genes, several protease genes, and genes involved in anaerobic metabolism. The decreased susceptibility mutant had somewhat thicker cell walls and a decreased autolytic activity that may be related to decreased proteolytic peptidoglycan hydrolase processing. Membrane fatty acid changes correlated with increased membrane fluidity were observed. It seems likely that there are multiple genetic changes associated with the development of decreased telavancin susceptibility. The Tlv(DS) mutant showed some similar features to vancomycin-intermediate S. aureus and decreased daptomycin susceptibility strains, but also exhibited its own unique features.

  16. Human oral isolate Lactobacillus fermentum AGR1487 induces a pro-inflammatory response in germ-free rat colons

    PubMed Central

    Anderson, Rachel C.; Ulluwishewa, Dulantha; Young, Wayne; Ryan, Leigh J.; Henderson, Gemma; Meijerink, Marjolein; Maier, Eva; Wells, Jerry M.; Roy, Nicole C.

    2016-01-01

    Lactobacilli are thought to be beneficial for human health, with lactobacilli-associated infections being confined to immune-compromised individuals. However, Lactobacillus fermentum AGR1487 negatively affects barrier integrity in vitro so we hypothesized that it caused a pro-inflammatory response in the host. We compared germ-free rats inoculated with AGR1487 to those inoculated with another L. fermentum strain, AGR1485, which does not affect in vitro barrier integrity. We showed that rats inoculated with AGR1487 had more inflammatory cells in their colon, higher levels of inflammatory biomarkers, and increased colonic gene expression of pro-inflammatory pathways. In addition, our in vitro studies showed that AGR1487 had a greater capacity to activate TLR signaling and induce pro-inflammatory cytokines in immune cells. This study indicates the potential of strains of the same species to differentially elicit inflammatory responses in the host and highlights the importance of strain characterization in probiotic approaches to treat inflammatory disorders. PMID:26843130

  17. Hydrogen production by hup(-) mutant and wild-type strains of Rhodobacter capsulatus from dark fermentation effluent of sugar beet thick juice in batch and continuous photobioreactors.

    PubMed

    Uyar, Basar; Gürgan, Muazzez; Özgür, Ebru; Gündüz, Ufuk; Yücel, Meral; Eroglu, Inci

    2015-10-01

    Photofermentative production of hydrogen is a promising and sustainable process; however, it should be coupled to dark fermentation to become cost effective. In order to integrate dark fermentation and photofermentation, the suitability of dark fermenter effluents for the photofermentative hydrogen production must be demonstrated. In this study, thermophilic dark fermenter effluent (DFE) of sugar beet thick juice was used as a substrate in photofermentation process to compare wild-type and uptake hydrogenase-deficient (hup (-)) mutant strains of Rhodobacter capsulatus by means of hydrogen production and biomass growth. The tests were conducted in small-scale (50 mL) batch and large-scale (4 L) continuous photobioreactors in indoor conditions under continuous illumination. In small scale batch conditions, maximum cell concentrations were 0.92 gdcw/L c and 1.50 gdcw/L c, hydrogen yields were 34 % and 31 %, hydrogen productivities were 0.49 mmol/(L c·h) and 0.26 mmol/(Lc·h), for hup (-) and wild-type cells, respectively. In large-scale continuous conditions, maximum cell concentrations were 1.44 gdcw/L c and 1.87 gdcw/L c, hydrogen yields were 48 and 46 %, and hydrogen productivities were 1.01 mmol/(L c·h) and 1.05 mmol/(L c·h), for hup (-) and wild-type cells, respectively. Our results showed that Rhodobacter capsulatus hup (-) cells reached to a lower maximum cell concentration but their hydrogen yield and productivity were in the same range or superior compared to the wild-type cells in both batch and continuous operating modes. The maximum biomass concentration, yield and productivity of hydrogen were higher in continuous mode compared to the batch mode with both bacterial strains.

  18. Hydrogen production by hup(-) mutant and wild-type strains of Rhodobacter capsulatus from dark fermentation effluent of sugar beet thick juice in batch and continuous photobioreactors.

    PubMed

    Uyar, Basar; Gürgan, Muazzez; Özgür, Ebru; Gündüz, Ufuk; Yücel, Meral; Eroglu, Inci

    2015-10-01

    Photofermentative production of hydrogen is a promising and sustainable process; however, it should be coupled to dark fermentation to become cost effective. In order to integrate dark fermentation and photofermentation, the suitability of dark fermenter effluents for the photofermentative hydrogen production must be demonstrated. In this study, thermophilic dark fermenter effluent (DFE) of sugar beet thick juice was used as a substrate in photofermentation process to compare wild-type and uptake hydrogenase-deficient (hup (-)) mutant strains of Rhodobacter capsulatus by means of hydrogen production and biomass growth. The tests were conducted in small-scale (50 mL) batch and large-scale (4 L) continuous photobioreactors in indoor conditions under continuous illumination. In small scale batch conditions, maximum cell concentrations were 0.92 gdcw/L c and 1.50 gdcw/L c, hydrogen yields were 34 % and 31 %, hydrogen productivities were 0.49 mmol/(L c·h) and 0.26 mmol/(Lc·h), for hup (-) and wild-type cells, respectively. In large-scale continuous conditions, maximum cell concentrations were 1.44 gdcw/L c and 1.87 gdcw/L c, hydrogen yields were 48 and 46 %, and hydrogen productivities were 1.01 mmol/(L c·h) and 1.05 mmol/(L c·h), for hup (-) and wild-type cells, respectively. Our results showed that Rhodobacter capsulatus hup (-) cells reached to a lower maximum cell concentration but their hydrogen yield and productivity were in the same range or superior compared to the wild-type cells in both batch and continuous operating modes. The maximum biomass concentration, yield and productivity of hydrogen were higher in continuous mode compared to the batch mode with both bacterial strains. PMID:26164274

  19. Effect of multiple short highly energetic X-ray pulses on the synthesis of endoglucanase by a mutant strain of Trichoderma reesei-M7

    PubMed Central

    Gemishev, Orlin; Zapryanov, Stanislav; Blagoev, Alexander; Markova, Maya; Savov, Valentin

    2014-01-01

    Bioconversion of cellulose-containing substrate to glucose represents an important area of modern biotechnology. Enzymes for the degradation of the polysaccharide part of biomass have been produced, mostly by fungi belonging to genus Trichoderma. Studies were carried out with the mutant strain Trichoderma reesei-M7, a cellulase producer. Spores of the enzyme producer were irradiated with different doses of characteristic X-ray radiation from metallic tungsten (mainly the W Kα1 and Kα2 lines) with a high dose rate. The latter is a specific property of the dense plasma focus (DPF) device, which has pulsed operation and thus gives short and highly energetic pulses of multiple types of rays and particles. In this case, we focused our study on the influence of hard X-rays. The doses of X-rays absorbed by the spores varied in the range of approximately 5–11,000 mSv measured with thermoluminescent dosimeters (TLD). The influence of the applied doses in combination with exceptionally high dose rates (in the order of tens of millisieverts per microsecond) on the activity of the produced endoglucanase, amount of biomass and extra-cellular protein, was studied in batch cultivation conditions. In the dose range of 200–1200 mSv, some enhancement of endoglucanase activity was obtained: around 18%–32%, despite the drop of the biomass amount, compared with the untreated material. PMID:26019569

  20. Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

    PubMed Central

    Bisht, Deepali; Yadav, Santosh Kumar; Darmwal, Nandan Singh

    2013-01-01

    Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L−1; starch, 15.0 g.L−1; triton-X-100, 0.93 mL.L−1; incubation temperature, 34.12 °C and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL−1). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R2) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization. PMID:24159311

  1. Attenuated mutant strain of Salmonella Typhimurium lacking the ZnuABC transporter contrasts tumor growth promoting anti-cancer immune response

    PubMed Central

    Chirullo, Barbara; Ammendola, Serena; Leonardi, Leonardo; Falcini, Roberto; Petrucci, Paola; Pistoia, Claudia; Vendetti, Silvia; Battistoni, Andrea; Pasquali, Paolo

    2015-01-01

    Salmonella Typhimurium has been shown to be highly effective as antitumor agent. The aim of this study was to investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STM) devoid of the whole operon coding for the high-affinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens. We showed that STM is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STM in mammary adenocarcinoma mouse model led to both reduction of tumor growth and increase in life expectancy of STM treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STM stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors. PMID:26158862

  2. Estudio de Salud Agrícola

    Cancer.gov

    En 1993, científicos del Instituto Nacional del Cáncer, Instituto Nacional de Ciencias Ambientales y la Agencia de Protección Ambiental de Estados Unidos iniciaron un estudio conocido como Estudio de Salud Agrícola (AHS).

  3. Comparative Studies of Mutations in Animal Isolates and Experimental In Vitro- and In Vivo-Selected Mutants of Salmonella spp. Suggest a Counterselection of Highly Fluoroquinolone-Resistant Strains in the Field

    PubMed Central

    Giraud, Etienne; Brisabois, Anne; Martel, Jean-Louis; Chaslus-Dancla, Elisabeth

    1999-01-01

    The occurrence of mutations in the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) of Salmonella typhimurium experimental mutants selected in vitro and in vivo and of 138 nalidixic acid-resistant Salmonella field isolates was investigated. The sequencing of the quinolone resistance-determining region of these genes in highly fluoroquinolone-resistant mutants (MICs of 4 to 16 μg/ml) revealed the presence of gyrA mutations at codons corresponding to Gly-81 or Ser-83, some of which were associated with a mutation at Asp-87. No mutations were found in the gyrB, parC, and parE genes. An assay combining allele-specific PCR and restriction fragment length polymorphism was developed to rapidly screen mutations at codons 81, 83, and 87 of gyrA. The MICs of ciprofloxacin for the field isolates reached only 2 μg/ml, versus 16 μg/ml for some in vitro-selected mutants. The field isolates, like the mutants selected in vivo, had only a single gyrA mutation at codon 83 or 87. Single gyrA mutations were also found in highly resistant in vitro-selected mutants (MIC of ciprofloxacin, 8 μg/ml), which indicates that mechanisms other than the unique modification of the intracellular targets could participate in fluoroquinolone resistance in Salmonella spp. A comparison of experimental mutants selected in vitro, field strains, and mutants selected in vivo suggests that highly fluoroquinolone-resistant strains are counterselected in field conditions in the absence of selective pressure. PMID:10471553

  4. AGR-2 Data Qualification Interim Report

    SciTech Connect

    Michael L. Abbott

    2010-09-01

    Projects for the very high temperature reactor (VHTR) Technology Development Office program provide data in support of Nuclear Regulatory Commission licensing of the VHTR. Fuel and materials to be used in the reactor are tested and characterized to quantify performance in high temperature and high fluence environments. The VHTR program established the NGNP Data Management and Analysis System (NDMAS) to manage and document VHTR data qualification, for storage of the data in a readily accessible electronic form, and to assist in the analysis and presentation of the data. This document gives the status of NDMAS processing and qualification of data associated with the initial reactor cycle (147A) of the second Advanced Gas Reactor (AGR-2) experiment which began on June 21, 2010. Because it is early in the AGR-2 experiment, data from only two AGR-2 data streams are reported on: Fuel Fabrication and Fuel Irradiation data. As of August 1, 2010, approximately 311,000 irradiation data records have been stored in NDMAS, and qualification tests are in progress. Preliminary information indicates that TC 2 in Capsule 2 failed prior to start of the experiment, and NDMAS testing has thus far identified only two invalid data values from the METSO data collection system Data from the Fission Product Monitoring System (FPMS) are not currently processed until after reactor cycle shutdown and have not yet been received. A description of the ATR operating conditions data associated with the AGR-2 experiment (e.g., power levels) are summarized in the AGR-1 data qualification report (INL/EXT-09-16460). Since ATR data are collected under ATR program data quality requirements (i.e., outside the VHTR program), the NGNP program and NDMAS do not take additional actions to qualify these data other than NDMAS capture testing. Data qualification of graphite characterization data collected under the Graphite Technology Development Project is reported in a separate status report (Hull 2010).

  5. Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803.

    PubMed Central

    Valverde, F; Losada, M; Serrano, A

    1997-01-01

    The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD- to NADP-dependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis

  6. Visualization analysis of the vacuole-targeting fungicidal activity of amphotericin B against the parent strain and an ergosterol-less mutant of Saccharomyces cerevisiae.

    PubMed

    Kang, Chang-Kyung; Yamada, Keiichi; Usuki, Yoshinosuke; Ogita, Akira; Fujita, Ken-ichi; Tanaka, Toshio

    2013-05-01

    Here, we sought to investigate the vacuole-targeting fungicidal activity of amphotericin B (AmB) in the parent strain and AmB-resistant mutant of Saccharomyces cerevisiae and elucidate the mechanisms involved in this process. Our data demonstrated that the vacuole-targeting fungicidal activity of AmB was markedly enhanced by N-methyl-N″-dodecylguanidine (MC12), a synthetic analogue of the alkyl side chain in niphimycin, as represented by the synergy in their antifungal activities against parent cells of S. cerevisiae. Indifference was observed only with Δerg3 cells, indicating that the replacement of ergosterol with episterol facilitated their resistance to the combined lethal actions of AmB and MC12. Dansyl-labelled amphotericin B (AmB-Ds) was concentrated into normal rounded vacuoles when parent cells were treated with AmB-Ds alone, even at a non-lethal concentration. The additional supplementation of MC12 resulted in a marked loss of cell viability and vacuole disruption, as judged by the fluorescence from AmB-Ds scattered throughout the cytoplasm. In Δerg3 cells, AmB-Ds was scarcely detected in the cytoplasm, even with the addition of MC12, reflecting its failure to normally incorporate across the plasma membrane into the vacuole. Thus, this study supported the hypothesis that ergosterol is involved in the mobilization of AmB into the vacuolar membrane so that AmB-dependent vacuole disruption can be fully enhanced by cotreatment with MC12.

  7. Identification of novel thiocarboxanilide derivatives that suppress a variety of drug-resistant mutant human immunodeficiency virus type 1 strains at a potency similar to that for wild-type virus.

    PubMed Central

    Balzarini, J; Brouwer, W G; Dao, D C; Osika, E M; De Clercq, E

    1996-01-01

    A large variety of carboxanilide and thiocarboxanilide derivatives in which the original oxathiin or aliphatic moieties present in the prototype compounds UC84 and UC38 were replaced by an (un) substituted furanyl, thienyl, phenyl, or pyrrole entity have been evaluated for activity against wild-type human immunodeficiency virus type 1 strain IIIB [HIV-1 (IIIB)] and a series of mutant virus strains derived thereof. The mutant viruses contained either the Leu-100-->Ile, Lys-103-->Asn, Val-106-->Ala, Glu-138-->Lys, Tyr-181-->Cys, or Tyr-188-->Leu mutation in their reverse transcriptase. Several 3-(2-methylfuranyl)- and 3-(2-methylthienyl)-thiocarboxanilide ester, (thio)ether, and oxime ether derivatives showed exquisitely potent antiviral activity against wild-type HIV-1 (50% effective concentration, 0.009 to 0.021 microM). The pentenylethers of the 2-methylfuranyl and 2-methylthienyl derivatives (i.e., 313, N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl]- 2-methyl-3-furancarbothioamide or UC-781, and 314, N-[4-chloro-3-(3-methyl-2-butenyloxy)phenyl] -2-methyl-3-thiophenecarbothioamide or UC-82) proved virtually equally inhibitory for wild-type and the Ile-100, Ala-106, and Lys-138 mutant virus strains (50% effective concentration, 0.015 to 0.021 microM). Their inhibitory effect against the Asn-103 and Cys-181 reverse transcriptase mutant virus strains was decreased only four- to sevenfold compared with wildtype virus. UC-781 and UC-82 should be considered potential candidate drugs for the treatment of HIV-1-infected individuals. PMID:8726019

  8. Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics

    PubMed Central

    Curiao, Tânia; Marchi, Emmanuela; Viti, Carlo; Oggioni, Marco R.; Baquero, Fernando; Martinez, José Luis

    2015-01-01

    Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRIr) and triclosan-hypersusceptible (TRIhs) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRIr mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRIr mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRIr mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRIr mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRIr mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive traits. PMID

  9. AGR-2 and AGR-3/4 Release-to-Birth Ratio Data Analysis

    SciTech Connect

    Pham, Binh T.; Einerson, Jeffrey J.; Scates, Dawn M.; Maki, John T.; Petti, David A.

    2014-09-01

    A series of Advanced Gas Reactor (AGR) irradiation tests is being conducted in the Advanced Test Reactor (ATR) at Idaho National Laboratory (INL) in support of development and qualification of tristructural isotropic (TRISO) low enriched fuel used in the High Temperature Gas-cooled Reactor (HTGR). Each AGR test consists of multiple independently controlled and monitored capsules containing fuel compacts placed in a graphite cylinder shrouded by a steel shell. These capsules are instrumented with thermocouples embedded in the graphite enabling temperature control. AGR configuration and irradiation conditions are based on prismatic HTGR technology that is distinguished primarily through use of helium coolant, a low-power-density ceramic core capable of withstanding very high temperatures, and TRISO coated particle fuel. Thus, these tests provide valuable irradiation performance data to support fuel process development, qualify fuel for normal operating conditions, and support development and validation of fuel performance and fission product transport models and codes.

  10. Sustained photoevolution of molecular hydrogen in a mutant of Synechocystis sp. strain PCC 6803 deficient in the type I NADPH-dehydrogenase complex.

    PubMed

    Cournac, Laurent; Guedeney, Geneviève; Peltier, Gilles; Vignais, Paulette M

    2004-03-01

    The interaction between hydrogen metabolism, respiration, and photosynthesis was studied in vivo in whole cells of Synechocystis sp. strain PCC 6803 by continuously monitoring the changes in gas concentrations (H2, CO2, and O2) with an online mass spectrometer. The in vivo activity of the bidirectional [NiFe]hydrogenase [H2:NAD(P) oxidoreductase], encoded by the hoxEFUYH genes, was also measured independently by the proton-deuterium (H-D) exchange reaction in the presence of D2. This technique allowed us to demonstrate that the hydrogenase was insensitive to light, was reversibly inactivated by O2, and could be quickly reactivated by NADH or NADPH (+H2). H2 was evolved by cells incubated anaerobically in the dark, after an adaptation period. This dark H2 evolution was enhanced by exogenously added glucose and resulted from the oxidation of NAD(P)H produced by fermentation reactions. Upon illumination, a short (less than 30-s) burst of H2 output was observed, followed by rapid H2 uptake and a concomitant decrease in CO2 concentration in the cyanobacterial cell suspension. Uptake of both H2 and CO2 was linked to photosynthetic electron transport in the thylakoids. In the ndhB mutant M55, which is defective in the type I NADPH-dehydrogenase complex (NDH-1) and produces only low amounts of O2 in the light, H2 uptake was negligible during dark-to-light transitions, allowing several minutes of continuous H2 production. A sustained rate of photoevolution of H2 corresponding to 6 micro mol of H2 mg of chlorophyll(-1) h(-1) or 2 ml of H2 liter(-1) h(-1) was observed over a longer time period in the presence of glucose and was slightly enhanced by the addition of the O2 scavenger glucose oxidase. By the use of the inhibitors DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] and DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), it was shown that two pathways of electron supply for H2 production operate in M55, namely photolysis of water at the level of photosystem II and

  11. Mutant USA strain of porcine circovirus type 2 (mPCV2) exhibits similar virulence to the classical PCV2a and PCV2b strains in caesarean-derived, colostrum-deprived pigs.

    PubMed

    Opriessnig, Tanja; Xiao, Chao-Ting; Gerber, Priscilla F; Halbur, Patrick G; Matzinger, Shannon R; Meng, Xiang-Jin

    2014-11-01

    In 2012, a mutant porcine circovirus type 2 (mPCV2) strain was identified in cases of PCV-associated disease (PCVAD) in the USA. The mPCV2 had an additional amino acid, lysine (K), in the capsid at position 234. The objectives of this study were to compare the pathogenicity of mPCV2, PCV2a and PCV2b in pigs using biologically pure infectious virus stocks derived from respective infectious DNA clones, and to investigate the importance of genotype-specific ORF2 and the presence of lysine at position 234 of the capsid. A total of 47, 2-week-old, caesarean-derived, colostrum-deprived (CDCD) pigs were assigned to one of seven groups. At 3 weeks of age, the pigs were experimentally inoculated with saline, PCV2a, PCV2b, mPCV2, PCV2b-234-K (lysine addition in ORF2), chimeric PCV2b-ORF1/mPCV2-ORF2 or reciprocal chimeric mPCV2-ORF1/PCV2b-ORF2. All pigs were necropsied 21 days post-infection (p.i.). Gross lesions were limited to visible icterus and loss of body condition in a portion of the mPCV2 pigs. The amount of PCV2 DNA was significantly higher in pigs inoculated with mPCV2 compared with PCV2b in sera at 7 days p.i. and faecal swabs at 14 days p.i. Based on lymphoid lesions, a higher prevalence of PCVAD was seen in pigs infected with PCV2s containing the additional 234-K (64.3 %) compared with those infected with a PCV2 with the regular 233 bp ORF2 (40 %). Results indicated that all PCV2 isolates were capable of inducing severe lesions and disease in the CDCD pig model, and there was no significant difference in virulence.

  12. A West Nile virus NS4B-P38G mutant strain induces cell intrinsic innate cytokine responses in human monocytic and macrophage cells

    PubMed Central

    Xie, Guorui; Luo, Huanle; Tian, Bing; Mann, Brian; Bao, Xiaoyong; McBride, Jere; Tesh, Robert; Barrett, Alan D; Wang, Tian

    2015-01-01

    Previous studies have shown that an attenuated West Nile virus (WNV) nonstructural (NS) 4B-P38G mutant induces stronger innate and adaptive immune responses than wild-type WNV in mice, which has important applications to vaccine development. To investigate the mechanism of immunogenicity, we characterized WNV NS4B-P38G mutant infection in two human cell lines--THP-1 cells and THP-1 macrophages. Although the NS4B-P38G mutant produced more viral RNA than the parental WNV NY99 in both cell types, there was no detectable infectious virus in the supernatant of either cell type. Nonetheless, the attenuated mutant boosted higher innate cytokine responses than virulent parental WNV NY99 in these cells. The NS4B-P38G mutant infection of THP-1 cells led to more diverse and robust innate cytokine responses than that seen in THP-1 macrophages, which were mediated by toll-like receptor (TLR)7 and retinoic acid-inducible gene 1(RIG-I) signaling pathways. Overall, these results suggest that a defective viral life cycle during NS4B-P38G mutant infection in human monocytic and macrophage cells leads to more potent cell intrinsic innate cytokine responses. PMID:25562791

  13. Polymorphic variation in susceptibility and metabolism of triclosan-resistant mutants of Escherichia coli and Klebsiella pneumoniae clinical strains obtained after exposure to biocides and antibiotics.

    PubMed

    Curiao, Tânia; Marchi, Emmanuela; Viti, Carlo; Oggioni, Marco R; Baquero, Fernando; Martinez, José Luis; Coque, Teresa M

    2015-01-01

    Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRI(r)) and triclosan-hypersusceptible (TRI(hs)) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRI(r) mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRI(r) mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRI(r) mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRI(r) mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRI(r) mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive

  14. AGR-1 Data Qualification Interim Report

    SciTech Connect

    Machael Abbott

    2009-08-01

    Projects for the very-high-temperature reactor (VHTR) program provide data in support of Nuclear Regulatory Commission licensing of the VHTR. Fuel and materials to be used in the reactor are tested and characterized to quantify performance in high temperature and high fluence environments. The VHTR Program has established the NGNP Data Management and Analysis System (NDMAS) to ensure that VHTR data are (1) qualified for use, (2) stored in a readily accessible electronic form, and (3) analyzed to extract useful results. This document focuses on the first NDMAS objective. It describes the data streams associated with the first Advanced Gas Reactor (AGR-1) experiment, the processing of these data within NDMAS, and reports the interim FY09 qualification status of the AGR-1 data to date. Data qualification activities within NDMAS for specific types of data are determined by the data qualification category, which is assigned by the data generator, and include: (1) capture testing, to confirm that the data stored within NDMAS are identical to the raw data supplied, (2) accuracy testing, to confirm that the data are an accurate representation of the system or object being measured, and (3) documentation that the data were collected under an NQA-1 or equivalent QA program. The interim qualification status of the following four data streams is reported in this document: (1) fuel fabrication data, (2) fuel irradiation data, (3) fission product monitoring system (FPMS) data, and (4) Advanced Test Reactor (ATR) operating conditions data. A final report giving the NDMAS qualification status of all AGR-1 data (including cycle 145A) is planned for February 2010.

  15. AGR-1 Post Irradiation Examination Final Report

    SciTech Connect

    Demkowicz, Paul Andrew

    2015-08-01

    The post-irradiation examination (PIE) of the Advanced Gas Reactor (AGR)-1 experiment was a multi-year, collaborative effort between Idaho National Laboratory (INL) and Oak Ridge National Laboratory (ORNL) to study the performance of UCO (uranium carbide, uranium oxide) tristructural isotropic (TRISO) coated particle fuel fabricated in the U.S. and irradiated at the Advanced Test Reactor at INL to a peak burnup of 19.6% fissions per initial metal atom. This work involved a broad array of experiments and analyses to evaluate the level of fission product retention by the fuel particles and compacts (both during irradiation and during post-irradiation heating tests to simulate reactor accident conditions), investigate the kernel and coating layer morphology evolution and the causes of coating failure, and explore the migration of fission products through the coating layers. The results have generally confirmed the excellent performance of the AGR-1 fuel, first indicated during the irradiation by the observation of zero TRISO coated particle failures out of 298,000 particles in the experiment. Overall release of fission products was determined by PIE to have been relatively low during the irradiation. A significant finding was the extremely low levels of cesium released through intact coatings. This was true both during the irradiation and during post-irradiation heating tests to temperatures as high as 1800°C. Post-irradiation safety test fuel performance was generally excellent. Silver release from the particles and compacts during irradiation was often very high. Extensive microanalysis of fuel particles was performed after irradiation and after high-temperature safety testing. The results of particle microanalysis indicate that the UCO fuel is effective at controlling the oxygen partial pressure within the particle and limiting kernel migration. Post-irradiation examination has provided the final body of data that speaks to the quality of the AGR-1 fuel, building

  16. A Salmonella Enteritidis hilAssrAfliG deletion mutant is a safe live vaccine strain that confers protection against colonization by Salmonella Enteritidis in broilers.

    PubMed

    De Cort, W; Geeraerts, S; Balan, V; Elroy, M; Haesebrouck, F; Ducatelle, R; Van Immerseel, F

    2013-10-17

    Consumption of contaminated poultry meat is an important cause of Salmonella infections in humans. Therefore, there is a need for control methods that protect broilers from day-of-hatch until slaughter age against infection with Salmonella. Colonization-inhibition, a concept in which a live Salmonella strain is orally administered to day-old chickens and protects against subsequent challenge, can potentially be used as control method. In this study, the safety and efficacy of a Salmonella Enteritidis ΔhilAssrAfliG strain as a colonization-inhibition strain for protection of broilers against Salmonella Enteritidis was evaluated. After administration of the Salmonella Enteritidis ΔhilAssrAfliG strain to day-old chickens, this strain could not be isolated from the gut, internal organs or faeces after 21 days of age. In addition, administration of this strain to one-day-old broiler chickens decreased faecal shedding and caecal and internal organ colonization of a Salmonella Enteritidis challenge strain administered one day later using a seeder bird model. To our knowledge, this is the first report of an attenuated Salmonella strain for which both the safety and efficacy has been shown in long-term experiments (until slaughter age) in broiler strain can potentially be used as a live colonization-inhibition strain for controlling Salmonella Enteritidis infections in broilers. PMID:24012569

  17. Identification of the putative staphylococcal AgrB catalytic residues involving the proteolytic cleavage of AgrD to generate autoinducing peptide.

    PubMed

    Qiu, Rongde; Pei, Wuhong; Zhang, Linsheng; Lin, Jianqun; Ji, Guangyong

    2005-04-29

    The P2 operon of the staphylococcal accessory gene regulator (agr) encodes four genes (agrA, -B, -C, and -D) whose products compose a quorum sensing system: AgrA and AgrC resemble a two-component signal transduction system of which AgrC is a sensor kinase and AgrA is a response regulator; AgrD, a polypeptide that is integrated into the cytoplasmic membrane via an amphipathic alpha-helical motif in its N-terminal region, is the propeptide for an autoinducing peptide that is the ligand for AgrC; and AgrB is a novel membrane protein that involves in the processing of AgrD propeptide and possibly the secretion of the mature autoinducing peptide. In this study, we demonstrated that AgrB had endopeptidase activity, and identified 2 amino acid residues in AgrB (cysteine 84 and histidine 77) that might form a putative cysteine endopeptidase catalytic center in the proteolytic cleavage of AgrD at its C-terminal processing site. Computer analysis revealed that the cysteine and histidine residues were conserved among the potential AgrB homologous proteins, suggesting that the Agr quorum sensing system homologues might also exist in other Gram-positive bacteria.

  18. AGR-1 Safety Test Predictions using the PARFUME code

    SciTech Connect

    Blaise Collin

    2012-05-01

    The PARFUME modeling code was used to predict failure probability of TRISO-coated fuel particles and diffusion of fission products through these particles during safety tests following the first irradiation test of the Advanced Gas Reactor program (AGR-1). These calculations support the AGR-1 Safety Testing Experiment, which is part of the PIE effort on AGR-1. Modeling of the AGR-1 Safety Test Predictions includes a 620-day irradiation followed by a 300-hour heat-up phase of selected AGR-1 compacts. Results include fuel failure probability, palladium penetration, and fractional release of fission products. Results show that no particle failure is predicted during irradiation or heat-up, and that fractional release of fission products is limited during irradiation but that it significantly increases during heat-up.

  19. Isolation of an Escherichia coil strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase.

    PubMed

    Di Martino, P; Merieau, A; Phillips, R; Orange, N; Hulen, C

    2002-02-01

    Escherichia coli adherence to biotic and abiotic surfaces constitutes the first step of infection by promoting colonization and biofilm formation. The aim of this study was to gain a better understanding of the relationship between E. coli adherence to different biotic surfaces and biofilm formation on abiotic surfaces. We isolated mutants defective in A549 pneumocyte cells adherence, fibronectin adherence, and biofilm formation by random transposition mutagenesis and sequential passages over A549 cell monolayers. Among the 97 mutants tested, 80 were decreased in biofilm formation, 8 were decreased in A549 cells adherence, 7 were decreased in their adherence to fibronectin, and 17 had no perturbations in either of the three phenotypes. We observed a correlation between adherence to fibronectin or A549 cells and biofilm formation, indicating that biotic adhesive factors are involved in biofilm formation by E. coli. Molecular analysis of the mutants revealed that a transposon insertion in the tnaA gene encoding for tryptophanase was associated with a decrease in both A549 cells adherence and biofilm formation by E. coli. The complementation of the tnaA mutant with plasmid-located wild-type tnaA restored the tryptophanase activity, epithelial cells adherence, and biofilm formation on polystyrene. The possible mechanism of tryptophanase involvement in E. coli adherence and biofilm formation is discussed.

  20. A West Nile virus NS4B-P38G mutant strain induces adaptive immunity via TLR7-MyD88-dependent and independent signaling pathways.

    PubMed

    Xie, Guorui; Welte, Thomas; Wang, Jia; Whiteman, Melissa C; Wicker, Jason A; Saxena, Vandana; Cong, Yingzi; Barrett, Alan D T; Wang, Tian

    2013-08-28

    Prior work shows that an attenuated West Nile virus (WNV), the nonstructural (NS)4B-P38G mutant infection in mice induced strong immune responses and protected host from subsequent lethal wild-type WNV infection. Here, we investigated NS4B-P38G mutant infection in myeloid differentiation factor 88-deficient (MyD88(-/-)) and Toll-like receptor 7-deficient (TLR7(-/-)) mice and found they had enhanced susceptibility compared to wild-type mice. Both groups had lower WNV-specific IgM response and reduced effector T cell functions. Dendritic cells (DCs) also exhibited a reduced maturation and impaired antigen-presenting functions compared to wild-type DCs. Moreover, infection with NS4B-P38G mutant in TLR7(-/-) and MyD88(-/-) mice provided full and partial protection respectively from subsequent challenge with lethal wild-type WNV. There were reduced T cell responses in MyD88(-/-) and interleukin-1 receptor deficient (IL-1R(-/-)) mice during secondary challenge with wild-type WNV. In contrast, TLR7(-/-) mice displayed normal T cell functions. Collectively, these results suggest that TLR7-dependent MyD88 signaling is required for T cell priming during NS4B-P38G mutant infection, whereas the TLR7-independent MyD88 signaling pathways are involved in memory T cell development, which may contribute to host protection during secondary challenge with wild-type WNV. PMID:23845800

  1. The effects of modeled microgravity on growth kinetics, antibiotic susceptibility, cold growth, and the virulence potential of a Yersinia pestis ymoA-deficient mutant and its isogenic parental strain.

    PubMed

    Lawal, Abidat; Kirtley, Michelle L; van Lier, Christina J; Erova, Tatiana E; Kozlova, Elena V; Sha, Jian; Chopra, Ashok K; Rosenzweig, Jason A

    2013-09-01

    Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered. To better understand why CR did not enhance YP's virulence potential as it did for other bacterial pathogens, a YP ΔymoA isogenic mutant in the KIM/D27 background strain that is unable to produce the histone-like YmoA protein and influences DNA topography was used in both cell culture and murine models of infection. YmoA represses type three secretion system (T3SS) virulence gene expression in the yersiniae. Similar to our CR-grown parental YP strain data, the CR-grown ΔymoA mutant induced reduced HeLa cell cytotoxicity with concomitantly decreased Yersinia outer protein E (YopE) and low calcium response V (LcrV) antigen production and secretion. Important, however, were our findings that, although no significant differences were observed in survival of mice infected intraperitoneally with either normal gravity (NG)- or CR-grown parental YP, the ΔymoA mutant induced significantly more mortality in infected mice than did the parental strain following CR growth. Taken together, our data demonstrate that CR did enhance the virulence potential of the YP ΔymoA mutant in a murine infection model (relative to the CR-grown parental strain), despite inducing less HeLa cell rounding in our cell culture infection assay due to reduced T3SS activity. Therefore, CR, which induces a unique type of bacterial stress, might be enhancing YP's virulence potential in vivo through a T3SS-independent mechanism when the histone-like YmoA protein is absent.

  2. The Effects of Modeled Microgravity on Growth Kinetics, Antibiotic Susceptibility, Cold Growth, and the Virulence Potential of a Yersinia pestis ymoA-Deficient Mutant and Its Isogenic Parental Strain

    PubMed Central

    Lawal, Abidat; Kirtley, Michelle L.; van Lier, Christina J.; Erova, Tatiana E.; Kozlova, Elena V.; Sha, Jian; Chopra, Ashok K.

    2013-01-01

    Abstract Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered. To better understand why CR did not enhance YP's virulence potential as it did for other bacterial pathogens, a YP ΔymoA isogenic mutant in the KIM/D27 background strain that is unable to produce the histone-like YmoA protein and influences DNA topography was used in both cell culture and murine models of infection. YmoA represses type three secretion system (T3SS) virulence gene expression in the yersiniae. Similar to our CR-grown parental YP strain data, the CR-grown ΔymoA mutant induced reduced HeLa cell cytotoxicity with concomitantly decreased Yersinia outer protein E (YopE) and low calcium response V (LcrV) antigen production and secretion. Important, however, were our findings that, although no significant differences were observed in survival of mice infected intraperitoneally with either normal gravity (NG)- or CR-grown parental YP, the ΔymoA mutant induced significantly more mortality in infected mice than did the parental strain following CR growth. Taken together, our data demonstrate that CR did enhance the virulence potential of the YP ΔymoA mutant in a murine infection model (relative to the CR-grown parental strain), despite inducing less HeLa cell rounding in our cell culture infection assay due to reduced T3SS activity. Therefore, CR, which induces a unique type of bacterial stress, might be enhancing YP's virulence potential in vivo through a T3SS-independent mechanism when the histone-like YmoA protein is absent. Key Words: Type three secretion system (T3SS

  3. Complete Genome Sequence of Klebsiella pneumoniae Strain ATCC 43816 KPPR1, a Rifampin-Resistant Mutant Commonly Used in Animal, Genetic, and Molecular Biology Studies.

    PubMed

    Broberg, Christopher A; Wu, Weisheng; Cavalcoli, James D; Miller, Virginia L; Bachman, Michael A

    2014-09-25

    Klebsiella pneumoniae is an urgent public health threat due to the spread of carbapenem-resistant strains causing serious, and frequently fatal, infections. To facilitate genetic, molecular, and immunological studies of this pathogen, we report the complete chromosomal sequence of a genetically tractable, prototypical strain used in animal models.

  4. Teichoic acids and lipids associated with the membrane of a Bacillus licheniformis mutant and the membrane lipids of the parental strain.

    PubMed Central

    Button, D; Hemmings, N L

    1976-01-01

    Bacillus licheniformis 6346 MH-1 and a phosphoglucomutase-deficient poorly lytic mutant, B. licheniformis 6346 MH-5, both contain cardiolipin, phosphatidyl ethanolamine, and phosphatidyl glycerol but are devoid of phosphoglycolipids. Gentiobiosyl diglyceride is present in the parent organism but glycolipids are absent from the mutant. Lipoteichoic acid was extracted from the whole cells of MH-5 with hot aqueous phenol and contained fatty acids, glucosamine, and 1,3-polyglycerol phosphate. The fatty acids were predominantly of the branched-chain type and were esterified to hydroxyl groups of a terminal glycerol residue. The polyglycerol phosphate chains contained, on average, 32 to 40 glycerol residues, some of which were substituted at the secondary hydroxyl group with alpha-N-acylglucosaminyl residues. Phenol extraction of the supernatant fluid that remained when walls were removed from preparations of disrupted cells of MH-5 yielded membrane teichoic acid, which consisted of substituted polyglycerol phosphate but was devoid of fatty acids. PMID:977537

  5. Anthracycline metabolites from Streptomyces violaceus A262. II. New anthracycline epelmycins produced by a blocked mutant strain SU2-730.

    PubMed

    Johdo, O; Watanabe, Y; Ishikura, T; Yoshimoto, A; Naganawa, H; Sawa, T; Takeuchi, T

    1991-10-01

    New anthracycline antibiotics, identified as epsilon-rhodomycinone glycosides, were isolated from the culture broth of a blocked mutant of beta-rhodomycin-producing Streptomyces violaceus A262. They were designated as epelmycins A, B, C, D and E, and assayed for their in vitro cytotoxicities against murine leukemic L1210 cell culture and the antimicrobial activities in comparison with known anthracycline antibiotics. PMID:1955395

  6. Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra- Mutant Strain K1026

    PubMed Central

    Vicedo, Begonya; Peñalver, Ramón; Asins, María José; López, María M.

    1993-01-01

    The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach × almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall. Images PMID:16348854

  7. Locating a modifier gene of Ovum mutant through crosses between DDK and C57BL/6J inbred strains in mice.

    PubMed

    Tan, Jing; Song, Gen Di; Song, Jia Sheng; Ren, Shi Hao; Li, Chun Li; Zheng, Zhen Yu; Zhao, Wei Dong

    2016-06-01

    A striking infertile phenotype has been discovered in the DDK strain of mouse. The DDK females are usually infertile when crossed with males of other inbred strains, whereas DDK males exhibit normal fertility in reciprocal crosses. This phenomenon is caused by mutation in the ovum (Om) locus on chromosome 11 and known as the DDK syndrome. Previously, some research groups reported that the embryonic mortality deviated from the semilethal rate in backcrosses between heterozygous (Om/+) females and males of other strains. This embryonic mortality exhibited an aggravated trend with increasing background genes of other strains. These results indicated that some modifier genes of Om were present in other strains. In the present study, a population of N₂2 (Om/+) females from the backcrosses between C57BL/6J (B6) and F₁ (B6♀ × DDK♂) was used to map potential modifier genes of Om. Quantitative trait locus showed that a major locus, namely Amom1 (aggravate modifier gene of Om 1), was located at the middle part of chromosome 9 in mice. The Amom1 could increase the expressivity of Om gene, thereby aggravating embryonic lethality when heterozygous (Om/+) females mated with males of B6 strain. Further, the 1.5 LOD-drop analysis indicated that the confidence interval was between 37.54 and 44.46 cM, ~6.92 cM. Amom1 is the first modifier gene of Om in the B6 background.

  8. Locating a modifier gene of Ovum mutant through crosses between DDK and C57BL/6J inbred strains in mice.

    PubMed

    Tan, Jing; Song, Gen Di; Song, Jia Sheng; Ren, Shi Hao; Li, Chun Li; Zheng, Zhen Yu; Zhao, Wei Dong

    2016-06-01

    A striking infertile phenotype has been discovered in the DDK strain of mouse. The DDK females are usually infertile when crossed with males of other inbred strains, whereas DDK males exhibit normal fertility in reciprocal crosses. This phenomenon is caused by mutation in the ovum (Om) locus on chromosome 11 and known as the DDK syndrome. Previously, some research groups reported that the embryonic mortality deviated from the semilethal rate in backcrosses between heterozygous (Om/+) females and males of other strains. This embryonic mortality exhibited an aggravated trend with increasing background genes of other strains. These results indicated that some modifier genes of Om were present in other strains. In the present study, a population of N₂2 (Om/+) females from the backcrosses between C57BL/6J (B6) and F₁ (B6♀ × DDK♂) was used to map potential modifier genes of Om. Quantitative trait locus showed that a major locus, namely Amom1 (aggravate modifier gene of Om 1), was located at the middle part of chromosome 9 in mice. The Amom1 could increase the expressivity of Om gene, thereby aggravating embryonic lethality when heterozygous (Om/+) females mated with males of B6 strain. Further, the 1.5 LOD-drop analysis indicated that the confidence interval was between 37.54 and 44.46 cM, ~6.92 cM. Amom1 is the first modifier gene of Om in the B6 background. PMID:27350672

  9. Uncertainty Quantification of Calculated Temperatures for the AGR-1 Experiment

    SciTech Connect

    Binh T. Pham; Jeffrey J. Einerson; Grant L. Hawkes

    2012-04-01

    This report documents an effort to quantify the uncertainty of the calculated temperature data for the first Advanced Gas Reactor (AGR-1) fuel irradiation experiment conducted in the INL's Advanced Test Reactor (ATR) in support of the Next Generation Nuclear Plant (NGNP) R&D program. Recognizing uncertainties inherent in physics and thermal simulations of the AGR-1 test, the results of the numerical simulations can be used in combination with the statistical analysis methods to improve qualification of measured data. Additionally, the temperature simulation data for AGR tests can be used for validation of the fuel transport and fuel performance simulation models. The crucial roles of the calculated fuel temperatures in ensuring achievement of the AGR experimental program objectives require accurate determination of the model temperature uncertainties. The report is organized into three chapters. Chapter 1 introduces the AGR Fuel Development and Qualification program and provides overviews of AGR-1 measured data, AGR-1 test configuration and test procedure, and thermal simulation. Chapters 2 describes the uncertainty quantification procedure for temperature simulation data of the AGR-1 experiment, namely, (i) identify and quantify uncertainty sources; (ii) perform sensitivity analysis for several thermal test conditions; (iii) use uncertainty propagation to quantify overall response temperature uncertainty. A set of issues associated with modeling uncertainties resulting from the expert assessments are identified. This also includes the experimental design to estimate the main effects and interactions of the important thermal model parameters. Chapter 3 presents the overall uncertainty results for the six AGR-1 capsules. This includes uncertainties for the daily volume-average and peak fuel temperatures, daily average temperatures at TC locations, and time-average volume-average and time-average peak fuel temperatures.

  10. Uncertainty Quantification of Calculated Temperatures for the AGR-1 Experiment

    SciTech Connect

    Binh T. Pham; Jeffrey J. Einerson; Grant L. Hawkes

    2013-03-01

    This report documents an effort to quantify the uncertainty of the calculated temperature data for the first Advanced Gas Reactor (AGR-1) fuel irradiation experiment conducted in the INL’s Advanced Test Reactor (ATR) in support of the Next Generation Nuclear Plant (NGNP) R&D program. Recognizing uncertainties inherent in physics and thermal simulations of the AGR-1 test, the results of the numerical simulations can be used in combination with the statistical analysis methods to improve qualification of measured data. Additionally, the temperature simulation data for AGR tests can be used for validation of the fuel transport and fuel performance simulation models. The crucial roles of the calculated fuel temperatures in ensuring achievement of the AGR experimental program objectives require accurate determination of the model temperature uncertainties. The report is organized into three chapters. Chapter 1 introduces the AGR Fuel Development and Qualification program and provides overviews of AGR-1 measured data, AGR-1 test configuration and test procedure, and thermal simulation. Chapters 2 describes the uncertainty quantification procedure for temperature simulation data of the AGR-1 experiment, namely, (i) identify and quantify uncertainty sources; (ii) perform sensitivity analysis for several thermal test conditions; (iii) use uncertainty propagation to quantify overall response temperature uncertainty. A set of issues associated with modeling uncertainties resulting from the expert assessments are identified. This also includes the experimental design to estimate the main effects and interactions of the important thermal model parameters. Chapter 3 presents the overall uncertainty results for the six AGR-1 capsules. This includes uncertainties for the daily volume-average and peak fuel temperatures, daily average temperatures at TC locations, and time-average volume-average and time-average peak fuel temperatures.

  11. Levels of alpha-toxin correlate with distinct phenotypic response profiles of blood mononuclear cells and with agr background of community-associated Staphylococcus aureus isolates.

    PubMed

    Mairpady Shambat, Srikanth; Haggar, Axana; Vandenesch, Francois; Lina, Gerard; van Wamel, Willem J B; Arakere, Gayathri; Svensson, Mattias; Norrby-Teglund, Anna

    2014-01-01

    Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of α-toxin than did the proliferative supernatants. Addition of α-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, δ-toxin or phenol soluble modulin α-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.

  12. Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

    PubMed Central

    Mairpady Shambat, Srikanth; Haggar, Axana; Vandenesch, Francois; Lina, Gerard; van Wamel, Willem J. B.; Arakere, Gayathri; Svensson, Mattias; Norrby-Teglund, Anna

    2014-01-01

    Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of α-toxin than did the proliferative supernatants. Addition of α-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, δ-toxin or phenol soluble modulin α-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes. PMID:25166615

  13. Cloning of an agr homologue of Staphylococcus saprophyticus.

    PubMed

    Sakinc, Türkan; Kulczak, Pawel; Henne, Karsten; Gatermann, Sören G

    2004-08-01

    An agr homologue of Staphylococcus saprophyticus was identified, cloned and sequenced. The gene locus shows homologies to other staphylococcal agr systems, especially to those of S. epidermidis and S. lugdunensis. A putative RNAIII was identified and found to be differentially expressed during the growth phases. In contrast to the RNAIII molecules of S. epidermidis and S. aureus it does not contain an open reading frame that codes for a protein with homologies to the delta-toxin. Using PCR, the agr was found to be present in clinical isolates of S. saprophyticus.

  14. Cultivar-Dependent Transcript Accumulation in Wheat Roots Colonized by Pseudomonas fluorescens Q8r1-96 Wild Type and Mutant Strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In Triticum aestivum L. (wheat), the root-colonizing bacterium Pseudomonas fluorescens strain Q8r1-96 produces the antifungal metabolite 2,4-diacetylphloroglucinol (DAPG), suppresses damage caused by soilborne root pathogens, and modulates multiple stress or defense pathways in wheat roots. To test...

  15. A specialized host-vector system for the in vivo cloning of the trp operon of wild-type and mutant strains of Salmonella typhimurium by generalized transduction.

    PubMed

    Patterson, T; Bauerle, R

    1984-11-01

    Using in vitro methods, a 14.2-kb EcoRI fragment of the Salmonella typhimurium chromosome containing the trp operon plus associated flanking sequences from deletion mutant delta trpDCB763 was cloned into the EcoRI site of plasmid pBR322 in a S. typhimurium host. An in vivo cloning vector was constructed from the recombinant plasmid by the in vitro excision of a SalI fragment that contains the entire trp operon. The derived plasmid (pSTP21) carries a hybrid insert made up of the 5.4-kb EcoRI-SalI upstream flanking sequence and the 3.2-kb SalI-EcoRI downstream flanking sequence. Plasmid pSTP21 has been used as a receptor plasmid to clone a variety of mutant and wild-type trp operons by RecA-dependent in vivo recombination between the insert DNA of the plasmid and the homologous trp flanking sequences of transducing DNA fragments transferred into the cell by bacteriophage P22. The host-vector system developed for the in vivo cloning permits the differentiation of plasmid transductants from chromosomal transductants on the primary selective medium. Expression of the cloned trp operons is regulated normally by tryptophan. A substantial amplification of trp enzymes is attainable upon derepression. The recombinant plasmids are stably inherited in RecA+ and RecA- S. typhimurium hosts. However, conditions of high expression of the trp operon lead to a rapid loss of cellular viability and of plasmid stability.

  16. Breeding L-arginine-producing strains by a novel mutagenesis method: Atmospheric and room temperature plasma (ARTP).

    PubMed

    Cheng, Gong; Xu, Jianzhong; Xia, Xiuhua; Guo, Yanfeng; Xu, Kai; Su, Cunsheng; Zhang, Weiguo

    2016-07-01

    A plasma jet, driven by an active helium atom supplied with an atmospheric and room temperature plasma (ARTP) biological breeding system, was used as a novel method to breed L-arginine high-yielding strains. A mutant with resistance to L-homoarginine and 8-azaguaine, ARG 3-15 (L-HA(r), 8-AG(r), L-His(-)), was screened after several rounds of screening. The L-arginine production of these mutants was more than that of the original strain, increased by 43.79% for ARG 3-15. Moreover, N-acetyl-L-glutamate synthase activity of these mutants was also increased. After a series of passages, the hereditary properties of these mutants were found to be stable. Interestingly, beet molasses was utilized in a co-feeding fermentation and benefited to increase the productivity by 5.88%. Moreover, the fermentation with 1.0 g/L betaine could produce 9.33% more L-arginine than without betaine. In fed-batch fermentation, C. glutamicum ARG 3-15 began to produce L-arginine at the initial of logarithmic phase, and continuously increased over 24 hr to a final titer of 45.36 ± 0.42 g/L. The L-arginine productivity was 0.571 g/L/hr and the conversion of glucose (α) was 32.4% after 96 hr. These results indicated that C. glutamicum ARG 3-15 is a promising industrial producer.

  17. Factors affecting growth and antibiotic susceptibility of Helicobacter pylori: effect of pH and urea on the survival of a wild-type strain and a urease-deficient mutant.

    PubMed

    Sjöström, J E; Larsson, H

    1996-06-01

    This study investigated how pH and the presence of urea affect the survival and growth of Helicobacter pylori and whether these factors affect susceptibility to antibiotics in vitro. The viability of a wild-type strain and a urease-deficient mutant of H. pylori was studied after incubation for 1 h in buffers at different pH values at 37 degrees C under microaerophilic conditions. Viable counts were not affected at pH 5 and pH 7. In buffer at pH 3, there were no viable organisms, but urea (6.25 mM) protected the wild-type strain, which survived well. At pH 9, urea further reduced the viability of H. pylori and flurofamide almost abolished the effect of urea on the wild-type strain. Neither urea nor flurofamide affected the viability of the urease-deficient mutant under the same conditions. Growth was also pH dependent and was enhanced in shake-cultures. At pH 5, urea supported growth of the wild-type strain, but at pH 7 a toxic effect on the bacteria was observed. Growth of H. pylori at pH 5.9 was poor, and susceptibility to amoxycillin, erythromycin and clarithromycin was markedly less than at pH 7.2 and 7.9. The bactericidal activities of metronidazole and tetracycline were similar at the different pH values studied. At neutral pH the killing rates of amoxycillin and clarithromycin were growth rate dependent. Susceptibility to metronidazole was enhanced in stationary cultures. The interaction obtained between the proton pump inhibitor, omeprazole, and amoxycillin at pH 7 was of additive type. These results suggest that pH and growth conditions may be important in the antibacterial efficacy of different antibiotics in vivo and also provide a possible explanation for the potentiating effect of omeprazole with antibiotics in the treatment of H. pylori infections.

  18. Growth of wildtype and mutant E. coli strains in minimal media for optimal production of nucleic acids for preparing labeled nucleotides

    PubMed Central

    Thakur, Chandar S.; Brown, Margaret E.; Sama, Jacob N.; Jackson, Melantha E.

    2010-01-01

    Since RNAs lie at the center of most cellular processes, there is a need for synthesizing large amounts of RNAs made from stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (NMR) spectroscopy. A particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria grown in defined minimal media supplemented with 15NH4Cl and various carbon sources. Given the high cost of carbon precursors required for labeling nucleic acids for NMR studies, it becomes important to evaluate the optimal growth for commonly used strains under standard minimal media conditions. Such information is lacking. In this study, we characterize the growth for Escherichia coli strains K12, K10zwf, and DL323 in three minimal media with isotopic-labeled carbon sources of acetate, glycerol, and glycerol combined with formate. Of the three media, the LeMaster-Richards and the Studier media outperform the commonly used M9 media and both support optimal growth of E. coli for the production of nucleotides. However, the growth of all three E. coli strains in acetate is reduced almost twofold compared to growth in glycerol. Analysis of the metabolic pathway and previous gene array studies help to explain this differential growth in glycerol and acetate. These studies should benefit efforts to make selective 13C-15N isotopic-labeled nucleotides for synthesizing biologically important RNAs. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2813-y) contains supplementary material, which is available to authorized users. PMID:20730533

  19. The agr Inhibitors Solonamide B and Analogues Alter Immune Responses to Staphylococccus aureus but Do Not Exhibit Adverse Effects on Immune Cell Functions.

    PubMed

    Baldry, Mara; Kitir, Betül; Frøkiær, Hanne; Christensen, Simon B; Taverne, Nico; Meijerink, Marjolein; Franzyk, Henrik; Olsen, Christian A; Wells, Jerry M; Ingmer, Hanne

    2016-01-01

    Staphylococcus aureus infections are becoming increasingly difficult to treat due to antibiotic resistance with the community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 being of particular concern. The inhibition of bacterial virulence has been proposed as an alternative approach to treat multi-drug resistant pathogens. One interesting anti-virulence target is the agr quorum-sensing system, which regulates virulence of CA-MRSA in response to agr-encoded autoinducing peptides. Agr regulation confines exotoxin production to the stationary growth phase with concomitant repression of surface-expressed adhesins. Solonamide B, a non-ribosomal depsipeptide of marine bacterial origin, was recently identified as a putative anti-virulence compound that markedly reduced expression of α-hemolysin and phenol-soluble modulins. To further strengthen solonamide anti-virulence candidacy, we report the chemical synthesis of solonamide analogues, investigation of structure-function relationships, and assessment of their potential to modulate immune cell functions. We found that structural differences between solonamide analogues confer significant differences in interference with agr, while immune cell activity and integrity is generally not affected. Furthermore, treatment of S. aureus with selected solonamides was found to only marginally influence the interaction with fibronectin and biofilm formation, thus addressing the concern that application of compounds inducing an agr-negative state may have adverse interactions with host factors in favor of host colonization. PMID:26731096

  20. The agr Inhibitors Solonamide B and Analogues Alter Immune Responses to Staphylococccus aureus but Do Not Exhibit Adverse Effects on Immune Cell Functions

    PubMed Central

    Baldry, Mara; Kitir, Betül; Frøkiær, Hanne; Christensen, Simon B.; Taverne, Nico; Meijerink, Marjolein; Franzyk, Henrik; Olsen, Christian A.; Wells, Jerry M.; Ingmer, Hanne

    2016-01-01

    Staphylococcus aureus infections are becoming increasingly difficult to treat due to antibiotic resistance with the community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 being of particular concern. The inhibition of bacterial virulence has been proposed as an alternative approach to treat multi-drug resistant pathogens. One interesting anti-virulence target is the agr quorum-sensing system, which regulates virulence of CA-MRSA in response to agr-encoded autoinducing peptides. Agr regulation confines exotoxin production to the stationary growth phase with concomitant repression of surface-expressed adhesins. Solonamide B, a non-ribosomal depsipeptide of marine bacterial origin, was recently identified as a putative anti-virulence compound that markedly reduced expression of α-hemolysin and phenol-soluble modulins. To further strengthen solonamide anti-virulence candidacy, we report the chemical synthesis of solonamide analogues, investigation of structure–function relationships, and assessment of their potential to modulate immune cell functions. We found that structural differences between solonamide analogues confer significant differences in interference with agr, while immune cell activity and integrity is generally not affected. Furthermore, treatment of S. aureus with selected solonamides was found to only marginally influence the interaction with fibronectin and biofilm formation, thus addressing the concern that application of compounds inducing an agr-negative state may have adverse interactions with host factors in favor of host colonization. PMID:26731096

  1. Safety, infectivity, immunogenicity, and in vivo stability of two attenuated auxotrophic mutant strains of Salmonella typhi, 541Ty and 543Ty, as live oral vaccines in humans.

    PubMed Central

    Levine, M M; Herrington, D; Murphy, J R; Morris, J G; Losonsky, G; Tall, B; Lindberg, A A; Svenson, S; Baqar, S; Edwards, M F

    1987-01-01

    Two Salmonella typhi mutants, 541Ty (Vi+) and 543Ty (Vi-), auxotrophic for p-aminobenzoate and adenine, were evaluated as live oral vaccines. 33 volunteers ingested single doses of 10(8), 10(9), or 10(10) vaccine organisms, while four others received two 2 X 10(9) organism doses 4 d apart. No adverse reactions were observed. Vaccine was recovered from coprocultures of 29 of 37 vaccinees (78%) and from duodenal string cultures of two; repeated blood cultures were negative. The humoral antibody response to S. typhi O, H, Vi, and lysate antigens in serum and intestinal fluid was meager. In contrast, all vaccinees manifested cell-mediated immune responses. After vaccination, 69% of vaccinees overall and 89% of recipients of doses greater than or equal to 10(9) responded to S. typhi particulate or purified O polysaccharide antigens in lymphocyte replication studies but not to antigens of other Salmonella or Escherichia coli. All individuals, postvaccination, demonstrated a significant plasma-dependent mononuclear cell inhibition of wild S. typhi. PMID:3818953

  2. Comparison of the Exposure Time Dependence of the Activities of Synthetic Ozonide Antimalarials and Dihydroartemisinin against K13 Wild-Type and Mutant Plasmodium falciparum Strains.

    PubMed

    Yang, Tuo; Xie, Stanley C; Cao, Pengxing; Giannangelo, Carlo; McCaw, James; Creek, Darren J; Charman, Susan A; Klonis, Nectarios; Tilley, Leann

    2016-08-01

    Fully synthetic endoperoxide antimalarials, namely, OZ277 (RBx11160; also known as arterolane) and OZ439 (artefenomel), have been approved for marketing or are currently in clinical development. We undertook an analysis of the kinetics of the in vitro responses of Plasmodium falciparum to the new ozonide antimalarials. For these studies we used a K13 mutant (artemisinin resistant) isolate from a region in Cambodia and a genetically matched (artemisinin sensitive) K13 revertant. We used a pulsed-exposure assay format to interrogate the time dependence of the response. Because the ozonides have physicochemical properties different from those of the artemisinins, assay optimization was required to ensure that the drugs were completely removed following the pulsed exposure. Like that of artemisinins, ozonide activity requires active hemoglobin degradation. Short pulses of the ozonides were less effective than short pulses of dihydroartemisinin; however, when early-ring-stage parasites were exposed to drugs for periods relevant to their in vivo exposure, the ozonide antimalarials were markedly more effective. PMID:27161632

  3. Comparison of the Exposure Time Dependence of the Activities of Synthetic Ozonide Antimalarials and Dihydroartemisinin against K13 Wild-Type and Mutant Plasmodium falciparum Strains

    PubMed Central

    Yang, Tuo; Xie, Stanley C.; Cao, Pengxing; Giannangelo, Carlo; McCaw, James; Creek, Darren J.; Charman, Susan A.; Klonis, Nectarios

    2016-01-01

    Fully synthetic endoperoxide antimalarials, namely, OZ277 (RBx11160; also known as arterolane) and OZ439 (artefenomel), have been approved for marketing or are currently in clinical development. We undertook an analysis of the kinetics of the in vitro responses of Plasmodium falciparum to the new ozonide antimalarials. For these studies we used a K13 mutant (artemisinin resistant) isolate from a region in Cambodia and a genetically matched (artemisinin sensitive) K13 revertant. We used a pulsed-exposure assay format to interrogate the time dependence of the response. Because the ozonides have physicochemical properties different from those of the artemisinins, assay optimization was required to ensure that the drugs were completely removed following the pulsed exposure. Like that of artemisinins, ozonide activity requires active hemoglobin degradation. Short pulses of the ozonides were less effective than short pulses of dihydroartemisinin; however, when early-ring-stage parasites were exposed to drugs for periods relevant to their in vivo exposure, the ozonide antimalarials were markedly more effective. PMID:27161632

  4. Starch mutants of Chlamydomonas

    SciTech Connect

    Berry-Lowe, S.L.; Schmidt, G.W. )

    1990-05-01

    Wild type Chlamydomonas accumulates starch and triglycerides when grown under nitrogen limiting conditions. Toward elucidation of the mechanisms for control of starch biosynthesis, we isolated mutants impaired int he accumulation of storage carbohydrates. Chlamydomonas reinhardtii (strain ya-12) was mutagenized by UV irradiation and colonies were screened by iodine staining after growth in darkness. Mutants, denoted ais for altered in iodine staining, have been characterized by electron microscopy and assays for starch synthease, ADPG-pyrophosphorylase, phosphoglucose isomerase (PGI), phosphoglucomutase and fructose 1,6-bisphosphatase, and amylase activities. Transcript analysis of wild type and mutant RNAs with PGI, ADPG-pyrophosphorylase, and waxy probes have also been carried out. No deficiencies of any of these components have been detected. Furthermore, long-term cultures of ya-12 and ais-1d in nitrogen-limited chemostats have been studied; starch also does not accumulate in ais-1d under these conditions. Thus, the lesion affects an essential factor of unknown identity that is required for starch synthesis.

  5. The Live Attenuated Actinobacillus pleuropneumoniae Triple-Deletion Mutant ΔapxIC ΔapxIIC ΔapxIV-ORF1 Strain, SLW05, Immunizes Pigs against Lethal Challenge with Haemophilus parasuis

    PubMed Central

    Fu, Shulin; Ou, Jiwen; Zhang, Minmin; Xu, Juan; Liu, Huazhen; Liu, Jinlin; Yuan, Fangyan; Chen, Huanchun

    2013-01-01

    Haemophilus parasuis and Actinobacillus pleuropneumoniae both belong to the family Pasteurellaceae and are major respiratory pathogens that cause large economic losses in the pig industry worldwide. We previously constructed an attenuated A. pleuropneumoniae serovar 1 live vaccine prototype, SLW05 (ΔapxIC ΔapxIIC ΔapxIV-ORF1), which is able to produce nontoxic but immunogenic ApxIA, ApxIIA, and ApxIVA. This triple-deletion mutant strain was shown to elicit protective immunity against virulent A. pleuropneumoniae. In the present study, we investigated whether immunization with SLW05 could also protect against lethal challenge with virulent H. parasuis SH0165 (serovar 5) or MD0322 (serovar 4). The SLW05 strain was found to elicit a strong humoral antibody response in pigs and to confer significant protection against challenge with a lethal dose of H. parasuis SH0165 or MD0322. IgG subtype analysis revealed that SLW05 induces a bias toward a Th1-type immune response and stimulates interleukin 2 (IL-2) and gamma interferon (IFN-γ) production. Moreover, antisera from SLW05-vaccinated pigs efficiently inhibited both A. pleuropneumoniae and H. parasuis growth in a whole-blood assay. This is the first report that a live attenuated A. pleuropneumoniae vaccine with SLW05 can protect against lethal H. parasuis infection, which provides a novel approach for developing an attenuated H. parasuis vaccine. PMID:23220998

  6. A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain.

    PubMed

    Gutgsell, N S; Del Campo, M; Raychaudhuri, S; Ofengand, J

    2001-07-01

    This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines. Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.

  7. Altered antibiotic transport in OmpC mutants isolated from a series of clinical strains of multi-drug resistant E. coli.

    PubMed

    Lou, Hubing; Chen, Min; Black, Susan S; Bushell, Simon R; Ceccarelli, Matteo; Mach, Tivadar; Beis, Konstantinos; Low, Alison S; Bamford, Victoria A; Booth, Ian R; Bayley, Hagan; Naismith, James H

    2011-01-01

    Antibiotic-resistant bacteria, particularly gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane.

  8. AGR-2 Data Qualification Report for ATR Cycle 154B

    SciTech Connect

    Binh Pham; Jeff Einerson

    2014-01-01

    This report provides the data qualification status of Advanced Gas Reactor-2 (AGR-2) fuel irradiation experimental data from Advanced Test Reactor (ATR) Cycle 154B as recorded in the Nuclear Data Management and Analysis System (NDMAS). This is the last cycle of AGR-2 irradiation, as the test train was pulled from the ATR core during the outage portion of ATR Cycle 155A. The AGR-2 data streams addressed in this report include thermocouple (TC) temperatures, sweep gas data (flow rates including new Fission Product Monitoring (FPM) downstream flows from Fission Product Monitoring System (FPMS) detectors, pressure, and moisture content), and FPMS data (release rates and release-to-birth rate ratios [R/Bs]) for each of the six capsules in the AGR-2 experiment. The final data qualification status for these data streams is determined by a Data Review Committee (DRC) comprised of AGR technical leads, Sitewide Quality Assurance (QA), and NDMAS analysts. The Data Review Committee reviewed the data acquisition process, considered whether the data met the requirements for data collection as specified in QA-approved Very High Temperature Reactor (VHTR) data collection plans, examined the results of NDMAS data testing and statistical analyses, and confirmed the qualification status of the data as given in this report.

  9. Secretion of protein disulphide isomerase AGR2 confers tumorigenic properties

    PubMed Central

    Fessart, Delphine; Domblides, Charlotte; Avril, Tony; Eriksson, Leif A; Begueret, Hugues; Pineau, Raphael; Malrieux, Camille; Dugot-Senant, Nathalie; Lucchesi, Carlo; Chevet, Eric; Delom, Frederic

    2016-01-01

    The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis. DOI: http://dx.doi.org/10.7554/eLife.13887.001 PMID:27240165

  10. Alterations of nuclear DNA synthesis after irradiation of the cellular slime mold Dictyostelium discoideum: studies performed in a mutant strain displaying enhanced thymidine uptake

    SciTech Connect

    Hurley, D.L.

    1986-01-01

    The auxotrophic Dictyostelium discoideum strain HPS 401 was studied. Thymidine at 8 ..mu..g/ml or thymidylate at 50 ..mu..g/ml supported growth to maximal cell densities. Thin layer chromatography of cell extracts showed rapid intracellular accumulation of thymidine in HPS 401 vs slightly detectable accumulation in wild-type cells. Measurements showed that methionine and thymidylate were taken into all strains at a low rate, but HPS 401 had enhanced uptake of thymidine and uridine compared to wild-type. The HPS 401 phenotype is due to the efficient utilization of thymidine as a result of increased nucleoside uptake. Rapid nuclear purification removed mitochondrial DNA without decreasing the single-strand molecular weight of the nuclear DNA. The nuclear DNA peaks on alkaline sucrose gradients were identified using filter hybridization to cloned probes. As measured by pulse-chase labelling, production of full-sized main band DNA required 45-50 minutes. Pulse labelling of the cells immediately after ultraviolet irradiation caused the single-strand molecular weight of the DNA synthesized to decrease from 8 x 10/sup 6/ daltons at O J/m/sup 2/ to 3.9 x 10/sup 6/ daltons at 50 J/m/sup 2/ to 2.6 x 10/sup 6/ daltons at 200 J/m/sup 2/. The time required for maturation into full-sized DNA increased from 1 hour at O J/m/sup 2/ to 4 hours at 20 J/m/sup 2/ and to 21 hours at 200 J/m/sup 2/. Measured amounts of DNA synthesis at times after ultraviolet irradiation showed a period of reduced incorporation, followed by the resumption of control levels. The lag period ended at the same time as the production of full-sized DNA resumed.

  11. Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2.

    PubMed

    Ho, Melissa E; Quek, Sue-Ing; True, Lawrence D; Seiler, Roland; Fleischmann, Achim; Bagryanova, Lora; Kim, Sara R; Chia, David; Goodglick, Lee; Shimizu, Yoshiko; Rosser, Charles J; Gao, Yuqian; Liu, Alvin Y

    2016-03-29

    Anterior gradient 2 (AGR2) is a cancer-associated secreted protein found predominantly in adenocarcinomas. Given its ubiquity in solid tumors, cancer-secreted AGR2 could be a useful biomarker in urine or blood for early detection. However, normal organs express and might also secrete AGR2, which would impact its utility as a cancer biomarker. Uniform AGR2 expression is found in the normal bladder urothelium. Little AGR2 is secreted by the urothelial cells as no measurable amounts could be detected in urine. The urinary proteomes of healthy people contain no listing for AGR2. Likewise, the blood proteomes of healthy people also contain no significant peptide counts for AGR2 suggesting little urothelial secretion into capillaries of the lamina propria. Expression of AGR2 is lost in urothelial carcinoma, with only 25% of primary tumors observed to retain AGR2 expression in a cohort of lymph node-positive cases. AGR2 is secreted by the urothelial carcinoma cells as urinary AGR2 was measured in the voided urine of 25% of the cases analyzed in a cohort of cancer vs. non-cancer patients. The fraction of AGR2-positive urine samples was consistent with the fraction of urothelial carcinoma that stained positive for AGR2. Since cancer cells secrete AGR2 while normal cells do not, its measurement in body fluids could be used to indicate tumor presence. Furthermore, AGR2 has also been found on the cell surface of cancer cells. Taken together, secretion and cell surface localization of AGR2 are characteristic of cancer, while expression of AGR2 by itself is not. PMID:26894971

  12. Bladder cancer cells secrete while normal bladder cells express but do not secrete AGR2

    PubMed Central

    Ho, Melissa E.; Quek, Sue-Ing; True, Lawrence D.; Seiler, Roland; Fleischmann, Achim; Bagryanova, Lora; Kim, Sara R.; Chia, David; Goodglick, Lee; Shimizu, Yoshiko; Rosser, Charles J.; Gao, Yuqian; Liu, Alvin Y.

    2016-01-01

    Anterior gradient 2 (AGR2) is a cancer-associated secreted protein found predominantly in adenocarcinomas. Given its ubiquity in solid tumors, cancer-secreted AGR2 could be a useful biomarker in urine or blood for early detection. However, normal organs express and might also secrete AGR2, which would impact its utility as a cancer biomarker. Uniform AGR2 expression is found in the normal bladder urothelium. Little AGR2 is secreted by the urothelial cells as no measurable amounts could be detected in urine. The urinary proteomes of healthy people contain no listing for AGR2. Likewise, the blood proteomes of healthy people also contain no significant peptide counts for AGR2 suggesting little urothelial secretion into capillaries of the lamina propria. Expression of AGR2 is lost in urothelial carcinoma, with only 25% of primary tumors observed to retain AGR2 expression in a cohort of lymph node-positive cases. AGR2 is secreted by the urothelial carcinoma cells as urinary AGR2 was measured in the voided urine of 25% of the cases analyzed in a cohort of cancer vs. non-cancer patients. The fraction of AGR2-positive urine samples was consistent with the fraction of urothelial carcinoma that stained positive for AGR2. Since cancer cells secrete AGR2 while normal cells do not, its measurement in body fluids could be used to indicate tumor presence. Furthermore, AGR2 has also been found on the cell surface of cancer cells. Taken together, secretion and cell surface localization of AGR2 are characteristic of cancer, while expression of AGR2 by itself is not. PMID:26894971

  13. Formation of a host range mutant of the lymphotropic strain of minute virus of mice during persistent infection in mouse L cells.

    PubMed Central

    Ron, D; Tattersall, P; Tal, J

    1984-01-01

    Minute virus of mice (i), the lymphotropic strain of minute virus of mice, established a persistent infection in normally restrictive L cells. The carrier state, which lasted 150 days, exhibited three clearly distinguishable stages. During the early stage (days 1 to 10 postinfection), small amounts of virus were formed. A "crisis" then developed that lasted 50 to 60 days and was characterized by massive cell lysis and high titers of virus. This was followed by a 70- to 80-day period in which small but stable quantities of virus were produced. Virus shed by the carrier culture during the latter phase had acquired an altered host range, namely, it had lost its ability to replicate in T-lymphocyte cell lines and had adapted to growth in L cells. Virus isolated at this time from a single plaque in L cells, designated hr301, was shown to possess similar host range properties. No differences, however, could be found between the DNAs of minute virus of mice (i) and of hr301 by restriction enzyme analysis, suggesting that the mutation that affected the viral host range did not involve an extensive region of the viral genome. Images PMID:6090711

  14. Proteomic and transcriptomic profiling of Staphylococcus aureus surface LPXTG-proteins: correlation with agr genotypes and adherence phenotypes.

    PubMed

    Ythier, Mathilde; Resch, Grégory; Waridel, Patrice; Panchaud, Alexandre; Gfeller, Aurélie; Majcherczyk, Paul; Quadroni, Manfredo; Moreillon, Philippe

    2012-11-01

    Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence

  15. Proteomic and Transcriptomic Profiling of Staphylococcus aureus Surface LPXTG-proteins: Correlation with agr Genotypes and Adherence Phenotypes*

    PubMed Central

    Ythier, Mathilde; Resch, Grégory; Waridel, Patrice; Panchaud, Alexandre; Gfeller, Aurélie; Majcherczyk, Paul; Quadroni, Manfredo; Moreillon, Philippe

    2012-01-01

    Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence

  16. New insight into transmembrane topology of Staphylococcus aureus histidine kinase AgrC.

    PubMed

    Wang, Lina; Quan, Chunshan; Xiong, Wen; Qu, Xiaojing; Fan, Shengdi; Hu, Wenzhong

    2014-03-01

    Staphylococcus aureus accessory gene regulator (agr) locus controls the expression of virulence factors through a classical two-component signal transduction system that consists of a receptor histidine protein kinase AgrC and a cytoplasmic response regulator AgrA. An autoinducing peptide (AIP) encoded by agr locus activates AgrC, which transduces extracellular signals into the cytoplasm. Despite extensive investigations to identify AgrC-AIP interaction sites, precise signal recognition mechanisms remain unknown. This study aims to clarify the membrane topology of AgrC by applying the green fluorescent protein (GFP) fusion technique and the substituted cysteine accessibility method (SCAM). However, our findings were inconsistent with profile obtained previously by alkaline phosphatase. We report the topology of AgrC shows seven transmembrane segments, a periplasmic N-terminus, and a cytoplasmic C-terminus.

  17. Comparative Analysis of kdp and ktr Mutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

    PubMed Central

    Nanatani, Kei; Shijuku, Toshiaki; Takano, Yousuke; Zulkifli, Lalu; Yamazaki, Tomoko; Tominaga, Akira; Souma, Satoshi; Onai, Kiyoshi; Morishita, Megumi; Ishiura, Masahiro; Hagemann, Martin; Suzuki, Iwane; Maruyama, Hisataka; Arai, Fumihito

    2014-01-01

    Photoautotrophic bacteria have developed mechanisms to maintain K+ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K+ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K+ (Kdp). The contributions of each of these K+ transport systems to cellular K+ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K+ uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K+ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K+ depletion. Correspondingly, Kdp-mediated K+ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K+ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K+ concentration under conditions of limited K+ in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K+ availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K+ levels under fluctuating environmental conditions. PMID:25313394

  18. Identification of ligand specificity determinants in AgrC, the Staphylococcus aureus quorum-sensing receptor.

    PubMed

    Geisinger, Edward; George, Elizabeth A; Chen, John; Muir, Tom W; Novick, Richard P

    2008-04-01

    Activation of the agr system, a major regulator of staphylococcal virulence, is initiated by the binding of a specific autoinducing peptide (AIP) to the extracellular domain of AgrC, a classical receptor histidine protein kinase. There are four known agr specificity groups in Staphylococcus aureus, and we have previously localized the determinant of AIP receptor specificity to the C-terminal half of the AgrC sensor domain. We have now identified the specific amino acid residues that determine ligand activation specificity for agr groups I and IV, the two most closely related. Comparison of the AgrC-I and AgrC-IV sequences revealed a set of five divergent residues in the region of the second extracellular loop of the receptor that could be responsible. Accordingly, we exchanged these residues between AgrC-I and AgrC-IV and tested the resulting constructs for activation by the respective AIPs, measuring activation kinetics with a transcriptional fusion of blaZ to the principal agr promoter, P3. Exchange of all five residues caused a complete switch in receptor specificity. Replacement of two of the AgrC-IV residues by the corresponding residues in AgrC-I caused the receptor to be activated by AIP-I nearly as well as the wild type AgrC-I receptor. Replacement of two different AgrC-I residues by the corresponding AgrC-IV residues broadened receptor recognition specificity to include both AIPs. Various types of intermediate activity were observed with other replacement mutations. Preliminary characterization of the AgrC-I-AIP-I interaction suggests that ligand specificity may be sterically determined.

  19. 15 CFR 740.18 - Agricultural commodities (AGR).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... notification or verify the status in BIS's Simplified Network Applications Processing Redesign (SNAP-R) System... confirm with either STELA or SNAP-R that no objection has been raised. If an objection is raised, STELA and SNAP-R will indicate that a license is required. The AGR notification will then be processed as...

  20. 15 CFR 740.18 - Agricultural commodities (AGR).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... notification or verify the status in BIS's Simplified Network Applications Processing Redesign (SNAP-R) System... confirm with either STELA or SNAP-R that no objection has been raised. If an objection is raised, STELA and SNAP-R will indicate that a license is required. The AGR notification will then be processed as...

  1. 15 CFR 740.18 - Agricultural commodities (AGR).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... notification or verify the status in BIS's Simplified Network Applications Processing Redesign (SNAP-R) System... confirm with either STELA or SNAP-R that no objection has been raised. If an objection is raised, STELA and SNAP-R will indicate that a license is required. The AGR notification will then be processed as...

  2. Efflux Pump Overexpression in Multiple-Antibiotic-Resistant Mutants of Bacteroides fragilis

    PubMed Central

    Pumbwe, Lilian; Glass, Daniel; Wexler, Hannah M.

    2006-01-01

    Multidrug-resistant mutants of a wild-type Bacteroides fragilis strain (strain ADB77) and a quadruple resistance nodulation division family efflux pump deletion mutant (ADB77 ΔbmeB1 ΔbmeB3 ΔbmeB12 ΔbmeB15) were selected with antimicrobials. Ampicillin, doripenem, imipenem, levofloxacin, and metronidazole selected for mutants from both strains; cefoxitin selected for mutants from strain ADB77 only; and sodium dodecyl sulfate selected mutants from ADB77ΔbmeB1 ΔbmeB3 ΔbmeB12 ΔbmeB15 only. The mutants overexpressed one or more efflux pumps. PMID:16940115

  3. AGR-2 Irradiation Test Final As-Run Report

    SciTech Connect

    Collin, Blaise P.

    2014-08-01

    This document presents the as-run analysis of the AGR-2 irradiation experiment. AGR-2 is the second of the planned irradiations for the Advanced Gas Reactor (AGR) Fuel Development and Qualification Program. Funding for this program is provided by the U.S. Department of Energy as part of the Very High Temperature Reactor (VHTR) Technology Development Office (TDO) program. The objectives of the AGR-2 experiment are to: 1. Irradiate UCO (uranium oxycarbide) and UO2 (uranium dioxide) fuel produced in a large coater. Fuel attributes are based on results obtained from the AGR-1 test and other project activities. 2. Provide irradiated fuel samples for post-irradiation experiment (PIE) and safety testing. 3. Support the development of an understanding of the relationship between fuel fabrication processes, fuel product properties, and irradiation performance. The primary objective of the test was to irradiate both UCO and UO2 TRISO (tristructural isotropic) fuel produced from prototypic scale equipment to obtain normal operation and accident condition fuel performance data. The UCO compacts were subjected to a range of burnups and temperatures typical of anticipated prismatic reactor service conditions in three capsules. The test train also includes compacts containing UO2 particles produced independently by the United States, South Africa, and France in three separate capsules. The range of burnups and temperatures in these capsules were typical of anticipated pebble bed reactor service conditions. The results discussed in this report pertain only to U.S.-produced fuel.

  4. AGR-2 Irradiation Test Final As-Run Report

    SciTech Connect

    Collin, Blaise P.

    2014-08-01

    This document presents the as-run analysis of the AGR-2 irradiation experiment. AGR-2 is the second of the planned irradiations for the Advanced Gas Reactor (AGR) Fuel Development and Qualification Program. Funding for this program is provided by the U.S. Department of Energy as part of the Very High Temperature Reactor (VHTR) Technical Development Office (TDO) program. The objectives of the AGR-2 experiment are to: (a) Irradiate UCO (uranium oxycarbide) and UO2 (uranium dioxide) fuel produced in a large coater. Fuel attributes are based on results obtained from the AGR-1 test and other project activities. (b) Provide irradiated fuel samples for post-irradiation experiment (PIE) and safety testing. (c) Support the development of an understanding of the relationship between fuel fabrication processes, fuel product properties, and irradiation performance. The primary objective of the test was to irradiate both UCO and UO2 TRISO (tri-structural isotropic) fuel produced from prototypic scale equipment to obtain normal operation and accident condition fuel performance data. The UCO compacts were subjected to a range of burnups and temperatures typical of anticipated prismatic reactor service conditions in three capsules. The test train also includes compacts containing UO2 particles produced independently by the United States, South Africa, and France in three separate capsules. The range of burnups and temperatures in these capsules were typical of anticipated pebble bed reactor service conditions. The results discussed in this report pertain only to U.S. produced fuel.

  5. Recombination-deficient mutant of Streptococcus faecalis

    SciTech Connect

    Yagi, Y.; Clewell, D.B.

    1980-08-01

    An ultraviolet radiation-sensitive derivative of Streptococcus faecalis strain JH2-2 was isolated and found to be deficient in recombination, using a plasmid-plasmid recombination system. The strain was sensitive to chemical agents which interact with deoxyribonucleic acid and also underwent deoxyribonucleic acid degradation after ultraviolet irradiation. Thus, the mutant has properties similar to those of recA strains of Escherichia coli.

  6. AGR-2 safety test predictions using the PARFUME code

    SciTech Connect

    Collin, Blaise P.

    2014-09-01

    This report documents calculations performed to predict failure probability of TRISO-coated fuel particles and diffusion of fission products through these particles during safety tests following the second irradiation test of the Advanced Gas Reactor program (AGR-2). The calculations include the modeling of the AGR-2 irradiation that occurred from June 2010 to October 2013 in the Advanced Test Reactor (ATR) and the modeling of a safety testing phase to support safety tests planned at Oak Ridge National Laboratory and at Idaho National Laboratory (INL) for a selection of AGR-2 compacts. The heat-up of AGR-2 compacts is a critical component of the AGR-2 fuel performance evaluation, and its objectives are to identify the effect of accident test temperature, burnup, and irradiation temperature on the performance of the fuel at elevated temperature. Safety testing of compacts will be followed by detailed examinations of the fuel particles to further evaluate fission product retention and behavior of the kernel and coatings. The modeling was performed using the particle fuel model computer code PARFUME developed at INL. PARFUME is an advanced gas-cooled reactor fuel performance modeling and analysis code (Miller 2009). It has been developed as an integrated mechanistic code that evaluates the thermal, mechanical, and physico-chemical behavior of fuel particles during irradiation to determine the failure probability of a population of fuel particles given the particle-to-particle statistical variations in physical dimensions and material properties that arise from the fuel fabrication process, accounting for all viable mechanisms that can lead to particle failure. The code also determines the diffusion of fission products from the fuel through the particle coating layers, and through the fuel matrix to the coolant boundary. The subsequent release of fission products is calculated at the compact level (release of fission products from the compact). PARFUME calculates the

  7. Methods of producing protoporphyrin IX and bacterial mutants therefor

    DOEpatents

    Zhou, Jizhong; Qiu, Dongru; He, Zhili; Xie, Ming

    2016-03-01

    The presently disclosed inventive concepts are directed in certain embodiments to a method of producing protoporphyrin IX by (1) cultivating a strain of Shewanella bacteria in a culture medium under conditions suitable for growth thereof, and (2) recovering the protoporphyrin IX from the culture medium. The strain of Shewanella bacteria comprises at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX. In certain embodiments of the method, the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or of shew_1140. In other embodiments, the presently disclosed inventive concepts are directed to mutant strains of Shewanella bacteria having at least one mutant hemH gene which is incapable of normal expression, thereby causing an accumulation of protoporphyrin IX during cultivation of the bacteria. In certain embodiments the strain of Shewanella bacteria is a strain of S. loihica, and more specifically may be S. loihica PV-4. In certain embodiments, the mutant hemH gene of the strain of Shewanella bacteria may be a mutant of shew_2229 and/or shew_1140.

  8. Safety testing of AGR-2 UO2 compacts 3-3-2 and 3-4-2

    SciTech Connect

    Hunn, John D.; Morris, Robert Noel; Baldwin, Charles A.; Montgomery, Fred C.

    2015-09-01

    Post-irradiation examination (PIE) is in progress on tristructural-isotropic (TRISO) coated-particle fuel compacts from the Advanced Gas Reactor (AGR) Fuel Development and Qualification Program second irradiation experiment (AGR-2) [Collin 2014]. The AGR-2 PIE will build upon new information and understanding acquired throughout the recently-concluded six-year AGR-1 PIE campaign [Demkowicz et al. 2015] and establish a database for the different AGR-2 fuel designs.

  9. Mutants of yeast sensitive to ultraviolet light.

    PubMed

    Snow, R

    1967-09-01

    Six uvr mutants of Saccharomyces cerevisiae with hypersensitivity to ultraviolet (UV) light were isolated after mutagen treatment with ethylmethanesulfonate. UV sensitivity ranges from moderate to extreme, and four of the mutants are also sensitive to nitrous acid. Ranking in terms of UV sensitivity does not parallel ranking in terms of nitrous acid sensitivity. Homozygous diploid mutant strains are somewhat less sensitive than the corresponding haploids. All mutations are recessive. None of the mutants is sensitive to gamma rays, and each shows photoreactivation after UV radiation. Complementation tests and tetrad analysis indicate that each strain represents mutation in a different gene. Two of the uvr genes are linked, and two others are centromere-linked.

  10. (1,3)-beta-D-glucan synthase activity in mycelial and cell wall-less phenotypes of the fz, sg, os-1 ("slime") mutant strain of Neurospora crassa.

    PubMed

    Polizeli, M del L; Noventa-Jordão, M A; da Silva, M M; Jorge, J A; Terenzi, H F

    1995-03-01

    The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-beta-D-glucan synthase activity. fz, sg, os-1 segregants from slime x wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-beta-glucan, chitin, and other polysaccharides and possess (1,3)-beta-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, Km app, Vmax, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate x wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual

  11. AGR-1 Irradiation Test Final As-Run Report

    SciTech Connect

    Blaise P. Collin

    2012-06-01

    This document presents the as-run analysis of the AGR-1 irradiation experiment. AGR-1 is the first of eight planned irradiations for the Advanced Gas Reactor (AGR) Fuel Development and Qualification Program. Funding for this program is provided by the US Department of Energy (DOE) as part of the Next-Generation Nuclear Plant (NGNP) project. The objectives of the AGR-1 experiment are: 1. To gain experience with multi-capsule test train design, fabrication, and operation with the intent to reduce the probability of capsule or test train failure in subsequent irradiation tests. 2. To irradiate fuel produced in conjunction with the AGR fuel process development effort. 3. To provide data that will support the development of an understanding of the relationship between fuel fabrication processes, fuel product properties, and irradiation performance. In order to achieve the test objectives, the AGR-1 experiment was irradiated in the B-10 position of the Advanced Test Reactor (ATR) at the Idaho National Laboratory (INL) for a total duration of 620 effective full power days of irradiation. Irradiation began on December 24, 2006 and ended on November 6, 2009 spanning 13 ATR cycles and approximately three calendar years. The test contained six independently controlled and monitored capsules. Each capsule contained 12 compacts of a single type, or variant, of the AGR coated fuel. No fuel particles failed during the AGR-1 irradiation. Final burnup values on a per compact basis ranged from 11.5 to 19.6 %FIMA, while fast fluence values ranged from 2.21 to 4.39 ?1025 n/m2 (E >0.18 MeV). We’ll say something here about temperatures once thermal recalc is done. Thermocouples performed well, failing at a lower rate than expected. At the end of the irradiation, nine of the originally-planned 19 TCs were considered functional. Fission product release-to-birth (R/B) ratios were quite low. In most capsules, R/B values at the end of the irradiation were at or below 10-7 with only one

  12. Mutants of thermotaxis in Dictyostelium discoideum

    SciTech Connect

    Schneider, M.J.; Fontana, D.R.; Poff, K.L.

    1982-08-01

    Amoebae of Dictyostelium discoideum, strain HL50 were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, cloned, allowed to form pseudoplasmodia and screened for aberrant positive and negative thermotaxis. Three types of mutants were found. Mutant HO428 exhibits only positive thermotaxis over the entire temperature range (no negative thermotaxis). HO596 and HO813 exhibit weakened positive thermotaxis and normal negative thermotaxis. The weakened positive thermotactic response results in a shift toward warmer temperatures in the transition temperature from negative to positive thermotaxis. Mutant HO209 exhibits weakened positive and negative thermotactic responses and has a transition temperature similar to the 'wild type' (HL50).The two types of mutants represented by HO428, HO596 and HO813 support the model that positive and negative thermotaxis have separate pathways for temperature sensing. The type of mutants which contains HO209 suggests that those two pathways converge at some point before the response.

  13. Targeting agr- and agr-Like Quorum Sensing Systems for Development of Common Therapeutics to Treat Multiple Gram-Positive Bacterial Infections

    PubMed Central

    Gray, Brian; Hall, Pamela; Gresham, Hattie

    2013-01-01

    Invasive infection by the Gram-positive pathogen Staphylococcus aureus is controlled by a four gene operon, agr that encodes a quorum sensing system for the regulation of virulence. While agr has been well studied in S. aureus, the contribution of agr homologues and analogues in other Gram-positive pathogens is just beginning to be understood. Intriguingly, other significant human pathogens, including Clostridium perfringens, Listeria monocytogenes, and Enterococcus faecalis contain agr or analogues linked to virulence. Moreover, other significant human Gram-positive pathogens use peptide based quorum sensing systems to establish or maintain infection. The potential for commonality in aspects of these signaling systems across different species raises the prospect of identifying therapeutics that could target multiple pathogens. Here, we review the status of research into these agr homologues, analogues, and other peptide based quorum sensing systems in Gram-positive pathogens as well as the potential for identifying common pathways and signaling mechanisms for therapeutic discovery. PMID:23598501

  14. Separation of prodigiosenes and identification as prodigiosin syntrophic pigment from mutant pairs of Serratia marcescens.

    PubMed

    Hearn, W R; Williams, R H; Burgus, R C; Williams, R P

    1972-10-01

    Countercurrent distribution is capable of resolving mixtures of closely related prodigiosene pigments. Syntrophic pigment produced by several pairs of Serratia marcescens color mutants was identified as prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) by countercurrent distribution, soda lime pyrolysis, and other techniques. The metabolic block of mutant strain H-462, derived from parent strain HY, was located between the blocks of mutant strains OF and WF, both derived from parent strain Nima.

  15. Behavioral characterization of system xc- mutant mice.

    PubMed

    McCullagh, Elizabeth A; Featherstone, David E

    2014-05-15

    The slc7a11 gene encodes xCT, an essential component of 'system xc-', a plasma membrane exchanger that imports cystine and exports glutamate. Slc7a11 is expressed primarily in the brain, but its role there is not clear. We performed behavioral tests on two different strains of homozygous slc7a11 mutant mice ('sut' and 'xCT'), as well as heteroallelic offspring of these two strains ('xCT/sut') and their associated genetic backgrounds. Homozygous sut mutant males showed reduced spontaneous alternation in spontaneous alternation tasks as well as reduced movement in an open field maze, but xCT and xCT/sut strains did not show significant changes in these tasks compared to appropriate controls. Neither xCT nor sut mutants showed differences from controls in rotarod tests. Female behavioral phenotypes were independent of estrus cycle stage. To ensure that homozygous xCT, sut, and xCT/sut strains all represent protein null alleles, we measured whole brain xCT protein levels using immunoblots. xCT, sut and xCT/sut strains showed no detectable xCT protein expression, confirming them as null alleles. Previously published microdialysis experiments showed reduced striatal glutamate in xCT mutants. Using the same methods, we measured reduced interstitial glutamate levels in the striatum but not cerebellum of sut mutants. However, we detected no glutamate change in the striatum or cerebellum of sut/xCT mice. We detected no changes in whole brain EAAT-1, -2, or -3 expression. We conclude that the behavioral and chemical differences exist between slc7a11 mutant strains, but we were unable to definitively attribute any of these differences to loss of system xc-.

  16. AGR 3/4 Irradiation Test Final As Run Report

    SciTech Connect

    Collin, Blaise P.

    2015-06-01

    Several fuel and material irradiation experiments have been planned for the Idaho National Laboratory Advanced Reactor Technologies Technology Development Office Advanced Gas Reactor Fuel Development and Qualification Program (referred to as the INL ART TDO/AGR fuel program hereafter), which supports the development and qualification of tristructural-isotropic (TRISO) coated particle fuel for use in HTGRs. The goals of these experiments are to provide irradiation performance data to support fuel process development, qualify fuel for normal operating conditions, support development and validation of fuel performance and fission product transport models and codes, and provide irradiated fuel and materials for post irradiation examination and safety testing (INL 05/2015). AGR-3/4 combined the third and fourth in this series of planned experiments to test TRISO coated low enriched uranium (LEU) oxycarbide fuel. This combined experiment was intended to support the refinement of fission product transport models and to assess the effects of sweep gas impurities on fuel performance and fission product transport by irradiating designed-to-fail fuel particles and by measuring subsequent fission metal transport in fuel-compact matrix material and fuel-element graphite. The AGR 3/4 fuel test was successful in irradiating the fuel compacts to the burnup and fast fluence target ranges, considering the experiment was terminated short of its initial 400 EFPD target (Collin 2015). Out of the 48 AGR-3/4 compacts, 42 achieved the specified burnup of at least 6% fissions per initial heavy-metal atom (FIMA). Three capsules had a maximum fuel compact average burnup < 10% FIMA, one more than originally specified, and the maximum fuel compact average burnup was <19% FIMA for the remaining capsules, as specified. Fast neutron fluence fell in the expected range of 1.0 to 5.5×1025 n/m2 (E >0.18 MeV) for all compacts. In addition, the AGR-3/4 experiment was globally successful in keeping the

  17. Neurospora crassa mutants deficient in asparagine synthetase.

    PubMed Central

    MacPhee, K G; Nelson, R E; Schuster, S M

    1983-01-01

    Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase. PMID:6137480

  18. The 2009 Lindau Nobel Laureate Meeting: Peter Agre, Chemistry 2003

    PubMed Central

    Agre, Peter

    2009-01-01

    Peter Agre, born in 1949 in Northfield Minnesota, shared the 2003 Nobel Prize in Chemistry with Roderick MacKinnon for his discovery of aquaporins, the channel proteins that allow water to cross the cell membrane. Agre's interest medicine was inspired by the humanitarian efforts of the Medical Missionary program run by the Norwegians of his home community in Minnesota. Hoping to provide new treatments for diseases affecting the poor, he joined a cholera laboratory during medical school at Johns Hopkins. He found that he enjoyed biomedical research, and continued his laboratory studies for an additional year after medical school. Agre completed his clinical training at Case Western Hospitals of Cleveland and the University of North Carolina, and returned to Johns Hopkins in 1981. There, his serendipitous discovery of aquaporins was made while pursuing the identity of the Rhesus (Rh) antigen. For a century, physiologists and biophysicists had been trying to understand the mechanism by which fluid passed across the cell's plasma membrane. Biophysical evidence indicated a limit to passive diffusion of water, suggesting the existence of another mechanism for water transport across the membrane. The putative "water channel," however, could not be identified. In 1988, while attempting to purify the 30kDa Rh protein, Agre and colleagues began investigating a 28 kDa contaminant that they believed to be a proteolytic fragment of the Rh protein. Subsequent studies over the next 3-4 years revealed that the contaminant was a membrane-spanning oligomeric protein, unrelated to the Rh antigen, and that it was highly abundant in renal tubules and red blood cells. Still, they could not assign a function to it. The breakthrough came following a visit with his friend and former mentor John Parker. After Agre described the properties of the mysterious 28 kDa protein, Parker suggested that it might be the long-sought-after water channel. Agre and colleagues tested this idea by

  19. Selection and characterization of L-ethionine resistant mutants of Trichosporon cutaneum.

    PubMed

    Georgieva, Nelly; Alexieva, Zlatka

    2005-01-01

    Trichosporon cutaneum R57 and its L-ethionine resistant mutant NZ94 strain were investigated. The amino acid analyses of cell content of both strains were carried out. The pool of free methionine in the mutant strain is enhanced 16.5 times. The total amount of sulphur-containing amino acids in the mutant cells was significantly increased from 36.8 in the wild strain to 113.4 mg/g protein in the mutant strain. In the process of mutant strain cultivation there was found a high excretion of free methionine (259 microg/ml) in the medium. It was shown that the amino acid content of both wild and mutant strains would be helpful for formulating of new improved animal nutritional diets.

  20. Design and Expected Performance of the AGR-1 Fission Product Monitoring System (FPMS)

    SciTech Connect

    John K. Hartwell; Dawn M. Scates

    2005-09-01

    The effluent from each test capsule of the AGR-1 experiment will be monitored by a detector system consisting of a gamma-ray spectrometer and a gross radiation detector. This collection of radiation measurement systems will be known as the AGR-1 Fission Product Monitoring System (FPMS). Proper design and functioning of the FPMS is critical to the success of the AGR-1 fuel test experiment.This document describes the AGR-1 FPMS and presents calculations indicating that this design will meet the pertinent test requirements.

  1. Microscopic analysis of irradiated AGR-1 coated particle fuel compacts

    SciTech Connect

    Scott A. Ploger; Paul A. Demkowicz; John D. Hunn; Jay S. Kehn

    2014-05-01

    The AGR-1 experiment involved irradiation of 72 TRISO-coated particle fuel compacts to a peak compact-average burnup of 19.5% FIMA with no in-pile failures observed out of 3 x 105 total particles. Irradiated AGR-1 fuel compacts have been cross-sectioned and analyzed with optical microscopy to characterize kernel, buffer, and coating behavior. Six compacts have been examined, spanning a range of irradiation conditions (burnup, fast fluence, and irradiation temperature) and including all four TRISO coating variations irradiated in the AGR-1 experiment. The cylindrical specimens were sectioned both transversely and longitudinally, then polished to expose from 36 to 79 individual particles near midplane on each mount. The analysis focused primarily on kernel swelling and porosity, buffer densification and fracturing, buffer–IPyC debonding, and fractures in the IPyC and SiC layers. Characteristic morphologies have been identified, 981 particles have been classified, and spatial distributions of particle types have been mapped. No significant spatial patterns were discovered in these cross sections. However, some trends were found between morphological types and certain behavioral aspects. Buffer fractures were found in 23% of the particles, and these fractures often resulted in unconstrained kernel protrusion into the open cavities. Fractured buffers and buffers that stayed bonded to IPyC layers appear related to larger pore size in kernels. Buffer–IPyC interface integrity evidently factored into initiation of rare IPyC fractures. Fractures through part of the SiC layer were found in only four classified particles, all in conjunction with IPyC–SiC debonding. Compiled results suggest that the deliberate coating fabrication variations influenced the frequencies of IPyC fractures and IPyC–SiC debonds.

  2. Ceramographic Examinations of Irradiated AGR-1 Fuel Compacts

    SciTech Connect

    Paul Demkowicz; Scott Ploger; John Hunn

    2012-05-01

    The AGR 1 experiment involved irradiating 72 cylindrical fuel compacts containing tri-structural isotropic (TRISO)-coated particles to a peak burnup of 19.5% fissions per initial metal atom with no in-pile failures observed out of almost 300,000 particles. Five irradiated AGR 1 fuel compacts were selected for microscopy that span a range of irradiation conditions (temperature, burnup, and fast fluence). These five compacts also included all four TRISO coating variations irradiated in the AGR experiment. The five compacts were cross-sectioned both transversely and longitudinally, mounted, ground, and polished after development of careful techniques for preserving particle structures against preparation damage. Approximately 40 to 80 particles within each cross section were exposed near enough to mid-plane for optical microscopy of kernel, buffer, and coating behavior. The microstructural analysis focused on kernel swelling and porosity, buffer densification and fracture, debonding between the buffer and inner pyrolytic carbon (IPyC) layers, and fractures in the IPyC and SiC layers. Three basic particle morphologies were established according to the extent of bonding between the buffer and IPyC layers: complete debonding along the interface (Type A), no debonding along the interface (Type B), and partial debonding (Type AB). These basic morphologies were subdivided according to whether the buffer stayed intact or fractured. The resulting six characteristic morphologies were used to classify particles within each cross section, but no spatial patterns were clearly observed in any of the cross-sectional morphology maps. Although positions of particle types appeared random within compacts, examining a total of 830 classified particles allowed other relationships among morphological types to be established.

  3. Ceramographic Examinations of Irradiated AGR-1 Fuel Compacts

    SciTech Connect

    Paul Demkowicz; Scott Ploger; John Hunn; Jay S. Kehn

    2012-09-01

    The AGR 1 experiment involved irradiating 72 cylindrical fuel compacts containing tri-structural isotropic (TRISO)-coated particles to a peak burnup of 19.5% fissions per initial metal atom with no in-pile failures observed out of almost 300,000 particles. Six irradiated AGR 1 fuel compacts were selected for microscopy that span a range of irradiation conditions (temperature, burnup, and fast fluence). These six compacts also included all four TRISO coating variations irradiated in the AGR experiment. The six compacts were cross-sectioned both transversely and longitudinally, mounted, ground, and polished after development of careful techniques for preserving particle structures against preparation damage. From 36 to 79 particles within each cross section were exposed near enough to midplane for optical microscopy of kernel, buffer, and coating behavior. The microstructural analysis focused on kernel swelling and porosity, buffer densification and fracture, debonding between the buffer and inner pyrolytic carbon (IPyC) layers, and fractures in the IPyC and SiC layers. Three basic particle morphologies were established according to the extent of bonding between the buffer and IPyC layers: complete debonding along the interface (Type A), no debonding along the interface (Type B), and partial debonding (Type AB). These basic morphologies were subdivided according to whether the buffer stayed intact or fractured. The resulting six characteristic morphologies were used to classify particles within each cross section, but no spatial patterns were clearly observed in any of the cross-sectional morphology maps. Although positions of particle types appeared random within compacts, examining a total of 931 classified particles allowed other relationships among morphological types to be established.

  4. Microscopic analysis of irradiated AGR-1 coated particle fuel compacts

    SciTech Connect

    Scott Ploger; Paul Demkowicz; John Hunn; Robert Morris

    2012-10-01

    The AGR-1 experiment involved irradiation of 72 TRISO-coated particle fuel compacts to a peak burnup of 19.5% FIMA with no in-pile failures observed out of 3×105 total particles. Irradiated AGR-1 fuel compacts have been cross-sectioned and analyzed with optical microscopy to characterize kernel, buffer, and coating behavior. Five compacts have been examined so far, spanning a range of irradiation conditions (burnup, fast fluence, and irradiation temperature) and including all four TRISO coating variations irradiated in the AGR-1 experiment. The cylindrical specimens were sectioned both transversely and longitudinally, then polished to expose between approximately 40-80 individual particles on each mount. The analysis focused primarily on kernel swelling and porosity, buffer densification and fracturing, buffer-IPyC debonding, and fractures in the IPyC and SiC layers. Characteristic morphologies have been identified, over 800 particles have been classified, and spatial distributions of particle types have been mapped. No significant spatial patterns were discovered in these cross sections. However, some trends were found between morphological types and certain behavioral aspects. Buffer fractures were found in approximately 23% of the particles, and these fractures often resulted in unconstrained kernel swelling into the open cavities. Fractured buffers and buffers that stayed bonded to IPyC layers appear related to larger pore size in kernels. Buffer-IPyC interface integrity evidently factored into initiation of rare IPyC fractures. Fractures through part of the SiC layer were found in only three particles, all in conjunction with IPyC-SiC debonding. Compiled results suggest that the deliberate coating fabrication variations influenced the frequencies of IPyC fractures, IPyC-SiC debonds, and SiC fractures.

  5. Isolation of Pasteurella haemolytica leukotoxin mutants.

    PubMed Central

    Chidambaram, M; Sharma, B; Petras, S F; Reese, C P; Froshauer, S; Weinstock, G M

    1995-01-01

    Two mutants of Pasteurella haemolytica A1 that do not produce leukotoxin were isolated. Following mutagenesis, colonies were screened with antiserum by a filter assay for absence of the secreted leukotoxin. The two mutants both appeared to produce normal amounts of other antigens, as judged by reactivity with polyclonal serum from an animal with pasteurellosis, and were not altered in beta-hemolytic activity as seen on blood agar plates. There was no evidence of either cell-associated or secreted leukotoxin protein when Western blots (immunoblots) were carried out with the polyclonal serum or with a monoclonal antibody directed against the leukotoxin. Southern blots revealed that both mutants show the wild-type restriction pattern at the leukotoxin locus, although the strain with the lktA2 mutation showed differences in other regions of the chromosome on analysis by pulsed-field gel electrophoresis. The strain with the lktA2 mutation grew more slowly than did the wild-type strain, while the strain with the lktA1 mutation was indistinguishable from the wild-type strain in its growth properties. The strain with the lktA1 mutation should be valuable in determining the role of the leukotoxin in virulence as well as in identifying other virulence factors of P. haemolytica. PMID:7868223

  6. Isolation and characterization of Klebsiella pneumoniae unencapsulated mutants

    SciTech Connect

    Benedi, V.J.; Ciurana, B.; Tomas, J.M.

    1989-01-01

    Klebsiella pneumoniae mutants were obtained after UV irradiation and negative selection with anticapsular serum. Unencapsulation, rather than expression of a structurally altered capsule, was found in the mutants. The mutant strains showed no alterations in their outer membrane proteins and lipopolysaccharide, and a great similarity with the wild type in the properties tested (serum resistance, antimicrobial sensitivity, and lipopolysaccharide-specific bacteriophage sensitivity), with the exception of a higher cell surface hydrophobicity and resistance to bacteriophage FC3-9.

  7. Structurally altered capsular polysaccharides produced by mutant bacteria

    NASA Technical Reports Server (NTRS)

    Kern, Roger G. (Inventor); Petersen, Gene R. (Inventor); Richards, Gil F. (Inventor)

    1995-01-01

    Structurally altered capsular polysaccharides are produced by mutant bacteria. These polysaccharides are isolated by selecting a wild type bacterial strain and a phage producing degradative enzymes that have substrate specificity for the capsular polysaccharides produced by the wild type bacteria. Phage-resistant mutants producing capsular polysaccharides are selected and the structurally altered capsular polysaccharide is isolated therefrom.

  8. A Porcine Circovirus Type 2 (PCV2) Mutant with 234 Amino Acids in Capsid Protein Showed More Virulence In Vivo, Compared with Classical PCV2a/b Strain

    PubMed Central

    Guo, Longjun; Fu, Yujie; Wang, Yiping; Lu, Yuehua; Wei, Yanwu; Tang, Qinghai; Fan, Peihu; Liu, Jianbo; Zhang, Long; Zhang, Feiyan; Huang, Liping; Liu, Dan; Li, Shengbin; Wu, Hongli; Liu, Changming

    2012-01-01

    Background Porcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness. Methodology/Principal Findings Twenty healthy, 30-day-old, commercial piglets served as controls or were challenged with PCV2a, PCV2b and the newly emerging mutant virus. A series of indexes representing different parameters were adopted to evaluate virulence, including clinical signs, serological detection, viral load and distribution, changes in immune cell subsets in the peripheral blood, and evaluation of pathological lesions. The newly emerging PCV2 mutant demonstrated more severe signs compatible with PMWS, characterized by wasting, coughing, dyspnea, diarrhea, rough hair-coat and depression. Moreover, the pathological lesions and viremia, as well as the viral loads in lymph nodes, tonsils and spleen, were significantly more severe (P<0.05) for piglets challenged with the newly emerging mutant compared with those in the groups challenged with PCV2a and PCV2b. In addition, a significantly lower average daily weight gain (P<0.05) was recorded in the group challenged with the newly emerging PCV2 mutant than in the groups challenged with the prevailing PCV2a and PCV2b. Conclusions This is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo. PMID:22829951

  9. Repair effects of laser on mutants of filamentous fungi

    NASA Astrophysics Data System (ADS)

    Zhao, Yansheng; Xiao, Canpeng; Qian, Hailun; Su, Baoliang; Hu, Yujun; Deng, Jianhui

    1999-09-01

    The paper reports that penicillin-producing strains and lovastatin-producing strains were irradiated by UV and subsequently by laser (632.8 nm), and the reparation rate reached 297% and 264%. High-yield mutant was selected with improved potency of 24.5% and 30%, respectively; Gibberellin producing strains were treated with chemical agent LiCl, and then irradiated with 632.8 nm laser. One mutant with 189.6% increased potency was obtained. The experimental results indicated that using laser irradiation after UV or chemical agent mutation was a new useful method in breeding high-yield strains.

  10. AGR2 is associated with gastric cancer progression and poor survival

    PubMed Central

    ZHANG, JUN; JIN, YONGMING; XU, SHAONAN; ZHENG, JIAYIN; ZHANG, QI; WANG, YUANYU; CHEN, JINPING; HUANG, YAZENG; HE, XUJUN; ZHAO, ZHONGSHENG

    2016-01-01

    Anterior gradient protein 2 (AGR2) has been reported as a novel biomarker with a potential oncogenic role. However, its association with the prognosis and survival rate of gastric cancer (GC) has not yet been determined. Therefore, the present study aimed to examine the expression and prognostic significance of AGR2 in patients with GC. Immunohistochemistry was used to analyze AGR2 and cathepsin D (CTSD) protein expression in 436 clinicopathologically characterized GC cases and 92 noncancerous tissue samples. AGR2 and CTSD expression were both elevated in GC lesions compared with noncancerous tissues. In 204/436 (46.8%) GC patients, high expression of AGR2 was positively correlated with the expression of CTSD (r=0.577, P<0.01). Furthermore, several clinicopathological parameters were significantly associated with AGR2 expression level, including tumor size, depth of invasion and TNM stage (P<0.05). Using Kaplan-Meier survival analysis, it was determined that the mean survival time of patients with low levels of AGR2 expression was significantly longer than those with high ARG2 expression (in stages I, II and III; P<0.05). For stage IV disease, no significant difference in survival time was identified. Multivariate survival analysis demonstrated that AGR2 was an independent prognostic factor and was associated in the progression of GC. The findings of the present study indicate that AGR2 expression is significantly associated with location and size of GC, depth of invasion, TNM stage, lymphatic metastasis, vessel invasion, distant metastasis, Lauren's classification, high CTSD expression and poor prognosis. Thus, AGR2 may be a novel GC marker and may present a potential therapeutic target for GC. PMID:26998125

  11. Identification of agr-positive methicillin-resistant Staphylococcus aureus harbouring the class A mec complex by MALDI-TOF mass spectrometry.

    PubMed

    Josten, Michaele; Dischinger, Jasmin; Szekat, Christiane; Reif, Marion; Al-Sabti, Nahed; Sahl, Hans-Georg; Parcina, Marijo; Bekeredjian-Ding, Isabelle; Bierbaum, Gabriele

    2014-11-01

    A small peptide called PSM-mec is encoded on the type II, III and VIII SCCmec cassettes present in the genomes of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains. This peptide is excreted by agr-positive strains, which represent about 89% of the strains of our collection and can be identified by the presence of delta toxin in mass spectrometry. The presence of the peptide in the MALDI-TOF MS spectra of whole cells was proved by a knock-down experiment employing a clone that expressed antisense RNA to psm-mec. Furthermore, evaluation of a collection of clinical agr-positive MRSA and MSSA isolates and type strains showed that, using a detection window of m/z 2411-2419, the PSM-mec is detected by mass spectrometry of whole cells with a sensitivity of 0.95 and a specificity of 1, thereby enabling rapid identification of a subgroup of MRSA with a method that is used during routine identification procedures.

  12. Antigenic and virulence properties of Pasteurella haemolytica leukotoxin mutants.

    PubMed Central

    Petras, S F; Chidambaram, M; Illyes, E F; Froshauer, S; Weinstock, G M; Reese, C P

    1995-01-01

    Antigenic properties of two mutants of Pasteurella haemolytica, strains 59B0071 and 59B0072, that do not produce detectable leukotoxin were investigated. Western blot (immunoblot) analysis with a number of polyclonal sera from animals recovering from pasteurellosis revealed that both mutants secreted a variety of antigens that were also present in cultures of several wild-type strains. These antigens ranged from about 100 to 15 kDa. Mutant strain 59B0071 was found to be totally deficient in leukotoxin, as judged not only by Western blotting but also by cytotoxicity assays with bovine lymphoma (BL-3) cells or bovine polymorphonuclear cells as targets. The mutant strain 59B0071 had normal levels of a secreted sialylglycoprotease, however. When strains were tested for virulence in goat and cattle challenge experiments, a reduction in mortality and lung lesions was observed with the mutant 59B0071 in comparison with results obtained with wild-type strains. These results are consistent with an important role for leukotoxin in P. haemolytica virulence and suggest that leukotoxin-negative mutants may be useful tools in the investigation of other virulence properties involved in P. haemolytica infections. PMID:7868224

  13. Quantity of 135I released from the AGR-1, AGR-2, and AGR-3/4 experiments and discovery of 131I at the FPMS traps during the AGR-3/4 experiment

    SciTech Connect

    Scates, Dawn M.

    2014-09-01

    A series of three Advanced Gas Reactor (AGR) experiments have been conducted in the Advanced Test Reactor (ATR) at Idaho National Laboratory (INL). From 2006 through 2014, these experiments supported the development and qualification of the new U.S. tristructural isotropic (TRISO) particle fuel for Very High Temperature Reactors (VHTR). Each AGR experiment consisted of multiple fueled capsules, each plumbed for independent temperature control using a mix of helium and neon gases. The gas leaving a capsule was routed to individual Fission Product Monitor (FPM) detectors. For intact fuel particles, the TRISO particle coatings provide a substantial barrier to fission product release. However, particles with failed coatings, whether because of a minute percentage of initially defective particles, those which fail during irradiation, or those designed to fail (DTF) particles, can release fission products to the flowing gas stream. Because reactive fission product elements like iodine and cesium quickly deposit on cooler capsule components and piping structures as the effluent gas leaves the reactor core, only the noble fission gas isotopes of Kr and Xe tend to reach FPM detectors. The FPM system utilizes High Purity Germanium (HPGe) detectors coupled with a thallium activated sodium iodide NaI(Tl) scintillator. The HPGe detector provides individual isotopic information, while the NaI(Tl) scintillator is used as a gross count rate meter. During irradiation, the 135mXe concentration reaching the FPM detectors is from both direct fission and by decay of the accumulated 135I. About 2.5 hours after irradiation (ten 15.3 minute 135mXe half lives) the directly produced 135mXe has decayed and only the longer lived 135I remains as a source. Decay systematics dictate that 135mXe will be in secular equilibrium with its 135I parent, such that its production rate very nearly equals the decay rate of the

  14. Production and characterization of streptomycin dependent mutants of Pasteurella multocida from bovine haemorrhagic septicaemia.

    PubMed Central

    de Alwis, M C; Carter, G R; Chengappa, M M

    1980-01-01

    A large number of streptomycin dependent mutants were produced from bovine haemorrhagic septicaemia strains of Pasteurella multocida. The mutants required a minimum concentration of 25-50 microgram/mL streptomycin for growth and tolerated a concentration of 200 mg/mL. These mutants were avirulent to mice, when inoculated alone, but some mutants killed mice when inoculated with streptomycin. Biochemically all mutants were uniform and similar to the wild type. Most mutants were stable, but a few produced streptomycin independent revertants. The rate of reversion varied with each mutant. Most revertants were highly virulent for mice, some totally avirulant and a few relatively avirulent. PMID:6778598

  15. AGR-5/6/7 LEUCO Kernel Fabrication Readiness Review

    SciTech Connect

    Marshall, Douglas W.; Bailey, Kirk W.

    2015-02-01

    In preparation for forming low-enriched uranium carbide/oxide (LEUCO) fuel kernels for the Advanced Gas Reactor (AGR) fuel development and qualification program, Idaho National Laboratory conducted an operational readiness review of the Babcock & Wilcox Nuclear Operations Group – Lynchburg (B&W NOG-L) procedures, processes, and equipment from January 14 – January 16, 2015. The readiness review focused on requirements taken from the American Society Mechanical Engineers (ASME) Nuclear Quality Assurance Standard (NQA-1-2008, 1a-2009), a recent occurrence at the B&W NOG-L facility related to preparation of acid-deficient uranyl nitrate solution (ADUN), and a relook at concerns noted in a previous review. Topic areas open for the review were communicated to B&W NOG-L in advance of the on-site visit to facilitate the collection of objective evidences attesting to the state of readiness.

  16. Nondisjunction Mutants of the Nematode CAENORHABDITIS ELEGANS

    PubMed Central

    Hodgkin, Jonathan; Horvitz, H. Robert; Brenner, Sydney

    1979-01-01

    The frequency of males (5AA; XO) among the self progeny of wild-type Caenorhabditis elegans hermaphrodites (5AA; XX) is about one in 500. Fifteen him (for "high incidence of males") mutations have been identified that increase this frequency by a factor of ten to 150, as a result of increased X-chromosome nondisjunction. The mutations define ten complementation groups, which have been mapped: nine are autosomal, and one sex linked. Most of the mutants are superficially wild type in anatomy and behavior; however, him-4 mutants display gonadal abnormalities, and unc-86 mutants, which have a Him phenotype, exhibit a variety of anatomical and behavioral abnormalities. All the mutants segregate fertile 3X hermaphrodite progeny as well as XO male progeny. Some produce large numbers of inviable zygotes. Mutants in all ten genes produce diplo-X and nullo-X exceptional ova, and in the four strains tested, diplo-X and nullo-X exceptional sperm are produced by 2X "transformed" males. It appears likely that most of the mutants have defects in both gamete lines of the hermaphrodite. XO males of him strains other than him-4 and unc-86 are similar to wild-type males in anatomy and behavior, and all produce equal or almost equal numbers of haplo-X and nullo-X sperm, and no diplo-X sperm. Male fertility is reduced to varying extents in all him mutants. In four of the strains, nondisjunction during oogenesis has been shown to occur at a reductional division, and in three of these strains, abnormalities in recombination have been demonstrated. One mutant also exhibits autosomal nondisjunction, but many of the others probably do not. Therefore, the X chromosome of C. elegans may differ from the autosomes in the mechanisms controlling its meiotic behavior.——3X hermaphrodites are shorter and less fertile than 2X hermaphrodites, and they produce many inviable zygotes among their self progeny: these are probably 4X zygotes. Haplo-X and diplo-X ova are produced in 2:1 ratio by 3X

  17. Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.

    PubMed Central

    Hauben, K J; Bartlett, D H; Soontjens, C C; Cornelis, K; Wuytack, E Y; Michiels, C W

    1997-01-01

    Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing. PMID:9055412

  18. Selection and properties of Streptococcus thermophilus mutants deficient in urease.

    PubMed

    Monnet, C; Pernoud, S; Sepulchre, A; Fremaux, C; Corrieu, G

    2004-06-01

    Natural variations of the urea content of milk have a detrimental effect on the regularity of acidification by Streptococcus thermophilus strains used in dairy processes. The aim of the present study was to select urease-deficient mutants of S. thermophilus and to investigate their properties. Using an improved screening medium on agar plates, mutants were selected from 4 different parent strains after mutagen treatment and by spontaneous mutation. Most mutants were stable and had a phage sensitivity profile similar to that of their parent strain. Some of them contained detrimental secondary mutations, as their acidifying activity was lower than that of the parent strain cultivated in the presence of the urease inhibitor flurofamide. The proportion of this type of mutant was much lower among spontaneous mutants than among mutants selected after mutagen treatment. Utilization of urease-deficient mutants in dairy processes may have several advantages, such as an increase in acidification, an improved regularity of acidification, and a lower production of ammonia in whey.

  19. Disruption of Paneth and goblet cell homeostasis and increased endoplasmic reticulum stress in Agr2−/− mice

    PubMed Central

    Zhao, Fang; Edwards, Robert; Dizon, Diana; Mastroianni, Jennifer R.; Geyfman, Mikhail; Ouellette, André J.; Andersen, Bogi; Lipkin, Steven M

    2010-01-01

    Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that plays important roles in diverse processes in multiple cell lineages as a developmental regulator, survival factor and susceptibility gene for inflammatory bowel disease. Here, we show using germline and inducible Agr2−/− mice that Agr2 plays important roles in intestinal homeostasis. Agr2−/− intestine has decreased goblet cell Mucin 2, dramatic expansion of the Paneth cell compartment, abnormal Paneth cell localization, elevated endoplasmic reticulum (ER) stress, severe terminal ileitis and colitis. Cell culture experiments show that Agr2 expression is induced by ER stress, and that siRNA knockdown of Agr2 increases ER stress response. These studies implicate Agr2 in intestinal homeostasis and ER stress and suggest a role in the etiology of inflammatory bowel disease. PMID:20025862

  20. Mutants of Cercospora kikuchii Altered in Cercosporin Synthesis and Pathogenicity.

    PubMed

    Upchurch, R G; Walker, D C; Rollins, J A; Ehrenshaft, M; Daub, M E

    1991-10-01

    We have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus.

  1. Mutants of Cercospora kikuchii Altered in Cercosporin Synthesis and Pathogenicity

    PubMed Central

    Upchurch, R. G.; Walker, D. C.; Rollins, J. A.; Ehrenshaft, M.; Daub, M. E.

    1991-01-01

    We have obtained spontaneous and UV-induced stable mutants, altered in the synthesis of cercosporin, of the fungal soybean pathogen Cercospora kikuchii. The mutants were isolated on the basis of colony color on minimal medium. The UV-induced mutants accumulated, at most, 2% of wild-type cercosporin levels on all media tested. In contrast, cercosporin accumulation by the spontaneous mutants was strongly medium regulated, occurring only on potato dextrose medium but at concentrations comparable to those produced by the wild-type strain. UV-induced mutants unable to synthesize cercosporin on any medium were unable to incite lesions when inoculated onto the soybean host. Cercosporin was reproducibly isolated from all inoculated leaves showing lesions. Although cercosporin involvement in disease has been indirectly suggested by many previous studies, this is the first report in which mutants blocked in cercosporin synthesis have been used to demonstrate that cercosporin is a crucial pathogenicity factor for this fungal genus. Images PMID:16348567

  2. Superior triacylglycerol (TAG) accumulation in starchless mutants of Scenedesmus obliquus: (I) mutant generation and characterization

    PubMed Central

    2014-01-01

    Background Microalgae are a promising platform for producing neutral lipids, to be used in the application for biofuels or commodities in the feed and food industry. A very promising candidate is the oleaginous green microalga Scenedesmus obliquus, because it accumulates up to 45% w/w triacylglycerol (TAG) under nitrogen starvation. Under these conditions, starch is accumulated as well. Starch can amount up to 38% w/w under nitrogen starvation, which is a substantial part of the total carbon captured. When aiming for optimized TAG production, blocking the formation of starch could potentially increase carbon allocation towards TAG. In an attempt to increase TAG content, productivity and yield, starchless mutants of this high potential strain were generated using UV mutagenesis. Previous studies in Chlamydomonas reinhardtii have shown that blocking the starch synthesis yields higher TAG contents, although these TAG contents do not surpass those of oleaginous microalgae yet. So far no starchless mutants in oleaginous green microalgae have been isolated that result in higher TAG productivities. Results Five starchless mutants have been isolated successfully from over 3,500 mutants. The effect of the mutation on biomass and total fatty acid (TFA) and TAG productivity under nitrogen-replete and nitrogen-depleted conditions was studied. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion, reached a TAG content of 49.4% (% of dry weight) and had no substantial change in biomass productivity compared to the wild type. Conclusions The improved S. obliquus TAG production strains are the first starchless mutants in an oleaginous green microalga that show enhanced TAG content under

  3. Localization of transposon insertions in pathogenicity mutants of Erwinia amylovora and their biochemical characterization.

    PubMed

    Bellemann, P; Geider, K

    1992-05-01

    Transposon Tn5, on a mobilizable ColE1 plasmid, on a Ti plasmid derepressed for bacterial transfer, and on the bacteriophage fd genome, was used to construct pathogenicity mutants of the fire blight pathogen Erwinia amylovora. Eleven nonpathogenic mutants were isolated from 1600 independent mutants screened. These mutants were divided into three types: auxotrophs, exopolysaccharide (EPS)-deficient mutants and a mutant of the dsp phenotype. According to their insertion sites the Tn5 mutants were mapped into several classes. Some of the mutants could be complemented with cosmid clones from a genomic library of the parent strain for EPS production on minimal agar. EPS-deficient mutants and the dsp mutant could complement each other to produce virulence symptoms on pear slices.

  4. Crystal Structure of AGR_C_4470p from Agrobacterium tumefaciens

    SciTech Connect

    Vorobiev,S.; Neely, H.; Seetharaman, J.; Ma, L.; Xiao, R.; Acton, T.; Montelione, G.; Tong, L.

    2007-01-01

    We report here the crystal structure at 2.0 {angstrom} resolution of the AGR{_}C{_}4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR{_}C{_}4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR{_}C{_}4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR{_}C{_}4470p in E. coli, in addition to the ChuS protein.

  5. AGR-2 Irradiated Test Train Preliminary Inspection and Disassembly First Look

    SciTech Connect

    Ploger, Scott; Demkowciz, Paul; Harp, Jason

    2015-05-01

    The AGR 2 irradiation experiment began in June 2010 and was completed in October 2013. The test train was shipped to the Materials and Fuels Complex in July 2014 for post-irradiation examination (PIE). The first PIE activities included nondestructive examination of the test train, followed by disassembly of the test train and individual capsules and detailed inspection of the capsule contents, including the fuel compacts and their graphite fuel holders. Dimensional metrology was then performed on the compacts, graphite holders, and steel capsule shells. AGR 2 disassembly and metrology were performed with the same equipment used successfully on AGR 1 test train components. Gamma spectrometry of the intact test train gave a preliminary look at the condition of the interior components. No evidence of damage to compacts or graphite components was evident from the isotopic and gross gamma scans. Disassembly of the AGR 2 test train and its capsules was conducted rapidly and efficiently by employing techniques refined during the AGR 1 disassembly campaign. Only one major difficulty was encountered while separating the test train into capsules when thermocouples (of larger diameter than used in AGR 1) and gas lines jammed inside the through tubes of the upper capsules, which required new tooling for extraction. Disassembly of individual capsules was straightforward with only a few minor complications. On the whole, AGR 2 capsule structural components appeared less embrittled than their AGR 1 counterparts. Compacts from AGR 2 Capsules 2, 3, 5, and 6 were in very good condition upon removal. Only relatively minor damage or markings were visible using high resolution photographic inspection. Compact dimensional measurements indicated radial shrinkage between 0.8 to 1.7%, with the greatest shrinkage observed on Capsule 2 compacts that were irradiated at higher temperature. Length shrinkage ranged from 0.1 to 0.9%, with by far the lowest axial shrinkage on Capsule 3 compacts

  6. Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold

    SciTech Connect

    Kalayanamitr, A.; Bhumiratana, A.; Flegel, T.W.; Glinsukon, T.; Shinmyo, A.

    1987-08-01

    A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production.

  7. Mutants of Arabidopsis thaliana with altered phototropism

    NASA Technical Reports Server (NTRS)

    Khurana, J. P.; Poff, K. L.

    1989-01-01

    Thirty five strains of Arabidopsis thaliana (L.) Heynh. have been identified with altered phototropic responses to 450-nm light. Four of these mutants have been more thoroughly characterized. Strain JK224 shows normal gravitropism and "second positive" phototropism. However, while the amplitude for "first positive" phototropism is the same as that in the wild-type, the threshold and fluence for the maximum response in "first positive" phototropism are shifted to higher fluence by a factor of 20-30. This mutant may represent an alteration in the photoreceptor pigment for phototropism. Strain JK218 exhibits no curvature to light at any fluence from 1 micromole m-2 to 2700 micromoles m-2, but shows normal gravitropism. Strain JK345 shows no "first positive" phototropism, and reduced gravitropism and "second positive" phototropism. Strain JK229 shows no measurable "first positive" phototropism, but normal gravitropism and "second positive" phototropism. Based on these data, it is suggested that: 1. gravitropism and phototropism contain at least one common element; 2. "first positive" and "second positive" phototropism contain at least one common element; and 3. "first positive" phototropism can be substantially altered without any apparent alteration of "second positive" phototropism.

  8. Data Compilation for AGR-1 Variant 3 Compact Lot LEU01-49T-Z

    SciTech Connect

    Hunn, John D; Montgomery, Fred C; Pappano, Peter J

    2006-08-01

    This document is a compilation of characterization data for the AGR-1 vriant 3 fuel compact lot LEU01-49T-Z. The compacts were produced by ORNL for the Advanced Gas Reactor Fuel Development and Qualification (AGR) program for the first AGR irradiation test train (AGR-1). This compact lot was fabricated using particle composite LEU01-49T, which was a composite of three batches of TRISO-coated 350 {micro}m diameter 19.7% low enrichment uranium oxide/uranium carbide kernels (LEUCO). The AGR-1 TRISO-coated particles consist of a spherical kernel coated with an {approx} 50% dense carbon buffer layer (100 {micro}m nominal thickness), followed by a dense inner pyrocarbon layer (40 {micro}m nominal thickness), followed by a SiC layer (35 {micro}m nominal thickness), followed by another dense outer pyrocarbon layer (40 {micro}m nominal thickness). The kernels were obtained from BWXT and identified as composite G73D-20-69302. The BWXT kernel lot G73D-20-69302 was riffled into sublots for characterization and coating by ORNL and identified as LEU01-?? (where ?? is a series of integers beginning with 01). A data compilation for the AGR-1 variant 3 coated particle composite LEU01-49t CAN BE FOUND IN ornl/tm-2006/022.

  9. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  10. Genetic analysis of yeast strains lacking negative feedback control: a one-step method for positive selection and cloning of carbamoylphosphate synthetase-aspartate transcarbamoylase mutants unable to respond to UTP.

    PubMed

    Jaquet, L; Lollier, M; Souciet, J L; Potier, S

    1993-10-01

    We have undertaken an in vivo genetic approach to the analysis of negative feedback control by uridine triphosphate (UTP) of the yeast carbamoylphosphate synthetase-aspartate transcarbamoylase multifunctional protein (CPSase-ATCase). Using an analog of uracil, 5-fluorouracil, we have constructed a screening system leading, in one step, to selection and cloning of a functional aspartate transcarbamoylase that is defective in negative feedback control by UTP. Due to the nature of the screen, spontaneous or UV-induced mutants could be recovered. Well-characterized cloned mutants have been sequenced and reveal one or two modifications in single codons leading to single amino acid replacements. These amino acid changes occurred either in the CPSase or ATCase domains, abolishing their sensitivity to regulation but not their catalytic activities. Hence the regulatory and catalytic sites are distinct. With the same screening system, it may also be possible to enlarge the scope of the molecular study of the feedback processes to include equivalent proteins in fungi as well as higher eukaryotes. PMID:8232215

  11. Isolation of mutants of the nitrogen-fixing actinomycete Frankia.

    PubMed

    Kakoi, Kentaro; Yamaura, Masatoshi; Kamiharai, Toshihito; Tamari, Daiki; Abe, Mikiko; Uchiumi, Toshiki; Kucho, Ken-Ichi

    2014-01-01

    Frankia is a nitrogen (N)-fixing multicellular actinomycete which establishes root-nodule symbiosis with actinorhizal plants. Several aspects of Frankia N fixation and symbiosis are distinct, but genes involved in the specific features are largely unknown because of the lack of an efficient mutant screening method. In this study, we isolated mutants of Frankia sp. strain CcI3 using hyphae fragments mutagenized by chemical mutagens. Firstly, we isolated uracil auxotrophs as gain-of-function mutants resistant to 5-fluoroorotic acid (5-FOA). We obtained seven 5-FOA resistant mutants, all of which required uracil for growth. Five strains carried a frame shift mutation in orotidine-5'-phosphate decarboxylase gene and two carried an amino acid substitution in the orotate phosphoribosyltransferase gene. Secondly, we isolated mutants showing loss-of-function phenotypes. Mutagenized hyphae were fragmented by ultrasound and allowed to multiply at their tips. Hyphae were fragmented again and short fragments were enriched by filtration through 5 μm pores filters. Next-generation and Sanger sequencing revealed that colonies formed from the short hyphae fragments consisted of cells with an identical genotype. From the mutagenized colony population, we isolated three pigmentation mutants and a mutant with reduced N-fixation activity. These results indicate that our procedure is useful for the isolation of loss-of-function mutants using hyphae of Frankia. PMID:24389412

  12. Isolation of Mutants of the Nitrogen-Fixing Actinomycete Frankia

    PubMed Central

    Kakoi, Kentaro; Yamaura, Masatoshi; Kamiharai, Toshihito; Tamari, Daiki; Abe, Mikiko; Uchiumi, Toshiki; Kucho, Ken-Ichi

    2014-01-01

    Frankia is a nitrogen (N)-fixing multicellular actinomycete which establishes root-nodule symbiosis with actinorhizal plants. Several aspects of Frankia N fixation and symbiosis are distinct, but genes involved in the specific features are largely unknown because of the lack of an efficient mutant screening method. In this study, we isolated mutants of Frankia sp. strain CcI3 using hyphae fragments mutagenized by chemical mutagens. Firstly, we isolated uracil auxotrophs as gain-of-function mutants resistant to 5-fluoroorotic acid (5-FOA). We obtained seven 5-FOA resistant mutants, all of which required uracil for growth. Five strains carried a frame shift mutation in orotidine-5′-phosphate decarboxylase gene and two carried an amino acid substitution in the orotate phosphoribosyltransferase gene. Secondly, we isolated mutants showing loss-of-function phenotypes. Mutagenized hyphae were fragmented by ultrasound and allowed to multiply at their tips. Hyphae were fragmented again and short fragments were enriched by filtration through 5 μm pores filters. Next-generation and Sanger sequencing revealed that colonies formed from the short hyphae fragments consisted of cells with an identical genotype. From the mutagenized colony population, we isolated three pigmentation mutants and a mutant with reduced N-fixation activity. These results indicate that our procedure is useful for the isolation of loss-of-function mutants using hyphae of Frankia. PMID:24389412

  13. Lesion mimic mutants

    PubMed Central

    Moeder, Wolfgang

    2008-01-01

    Over the last decade a substantial number of lesion mimic mutants (LMM) have been isolated and a growing number of the genes have been cloned. It is now becoming clear that these mutants are valuable tools to dissect various aspects of programmed cell death (PCD) and pathogen resistance pathways in plants. Together with other forward genetics approaches LMMs shed light on the PCD machinery in plant cells and revealed important roles for sphingolipids, Ca2+ and chloroplast-derived porphyrin-metabolites during cell death development. PMID:19513227

  14. Data Compilation for AGR-1 Variant 1 Compact Lot LEU01-47T-Z

    SciTech Connect

    Hunn, John D; Montgomery, Fred C; Pappano, Peter J

    2006-08-01

    This document is a compilation of characterization data for the AGR-1 variant 1 compact lot LEU01-47T-Z. The compacts were produced by ORNL for the ADvanced Gas Reactor Fuel Development and Qualification (AGR) program for the first AGR irradiation test train (AGR-1). This compact lot was fabricated using particle composite LEU01-47T, which was a composite of three batches of TRISO-coated 350 {micro}m diameter 19.7% low enrichment uranium oxide/uranium carbide kernels (LEUCO). The AGR-1 TRISO-coated particles consist of a spherical kernel coated with an {approx} 50% dense carbon buffer layer (100 {micro}m nominal thickness), followed by a dense inner pyrocarbon layer (40 {micro}m nominal thickness), followed by a SiC layer (35 {micro}m nominal thickness), followed by another dense outer pyrcoarbon layer (40 {micro}m nominal thickness). The kernels were obtained from BWXT and identified as composite G73D-20-69302. The BWXT kernel lot G73D-20-69302 was riffled into sublots for characterization and coating by ORNL and identified at LEU01-?? (where ?? is a series of integers beginning with 01). A data compilation for the AGR-1 variant 1 coated particle composite LEU01-47T can be found in ORNL/TM-2006/020. The AGR-1 Fuel Product Specification and Characterization Guidance (INL EDF-4380) provides the requirements necessary for acceptance of the fuel manufactured for the AGR-1 irradiation test. Section 6.2 of EDF-4380 provides the property requirements for the heat treated compacts. The Statistical Sampling Plan for AGR Fuel Materials (INL EDF-4542) provides additional guidance regarding statistical methods for product acceptance and recommended sample sizes. The procedures for characterizing and qualifying the compacts are outlined in ORNL product inspection plan AGR-CHAR-PIP-05. The inspection report forms generated by this product inspection plan document the product acceptance for the property requirements listed in section 6.2 of EDF-4380.

  15. Data Compilation for AGR-1 Variant 2 Compact Lot LEU01-48T-Z

    SciTech Connect

    Hunn, John D; Montgomery, Fred C; Pappano, Peter J

    2006-08-01

    This document is a compilation of characterization data for the AGR-1 variant 2 compact lot LEU01-48T-Z. The compacts were produced by ORNL for the Advanced Gas Reactor Fuel Development and Qualification (AGR) program for the first AGR irradiation test train (AGR-1). This compact lot was fabricated using particle composite LEU01-48T, which was a composite of three batches of TRISO-coated 350 {micro}m diameter 19.7% low enrichment uranium oxide/uranium carbide kernels (LEUCO). The AGR-1 TRISO-coated particles consist of a spherical kernel coated with an {approx} 50% dense carbon buffer layer (100 {micro}m nominal thickness), followed by a dense inner pyrocarbon layer (40 {micro}m nominal thickness), followed by a SiC layer (35 {micro}m nominal thickness), followed by another dense outer pyrocarbon layer (40 {micro}m nominal thickness). The kernels were obtained from BWXT and identified as composite G73D-20-69302. The BWXT kernel lot G73D-20-69302 was riffled into sublots for characterization and coating by ORNL and identified as LEU01-?? (where ?? is a series of integers beginning with 01). A data compilation for the AGR-1 variant 2 coated particle composite LEU01-48T can be found in ORNL/TM-2006/021. The AGR-1 Fuel Product Specification and Characterization Guidance (INL EDF-4380) provides the requirements necessary for acceptance of the fuel manufactured for the AGR-1 irradiation test. Section 6.2 of EDF-4380 provides the property requirements for the heat treated compacts. The Statistical Sampling Plan for AGR Fuel materials (INL EDF-4542) provides additional guidance regarding statistical methods for product acceptance and recommended sample sizes. The procedures for characterizing and qualifying the compacts are outlined in ORNL product inspection plan AGR-CHAR-PIP-05. The inspection report forms generated by this product inspection plan document the product acceptance for the property requirements listed in section 6.2 of EDF-4380.

  16. Data Compilation for AGR-1 Baseline Compact Lot LEU01-46T-Z

    SciTech Connect

    Hunn, John D; Montgomery, Fred C; Pappano, Peter J

    2006-08-01

    This document is a compilation of characterization data for the AGR-1 baseline compact lot LEU01-46T-Z. The compacts were produced by ORNL for the Advanced Gas Reactor Fuel Development and Qualification (AGR) program for the first AGR irradiation test train (AGR-1). This compact lot was fabricated using particle composite LEU01-46T, which was a composite of four batches of TRISO-coated 350 {micro}m diameter 19.7% low enrichment uranium oxide/uranium carbide kernels (LEUCO). The AGR-1 TRISO-coated particles consist of a spherical kernel coated with an {approx} 50% dense carbon buffer layer (100 {micro}m nominal thickness), followed by a dense inner pyrocarbon layer (40 {micro}m nominal thickness), followed by a SiC layer (35 {micro}m nominal thickness), followed by another dense outer pyrocarbon layer (40 {micro}m nominal thickness). The kernels were obtained from BWXT and identified as composite G73D-20-69302. The BWXT kernel lot G73D-20-69302 was riffled into sublots for characterization and coating by ORNL and identified as LEU01-?? (where ?? is a series of integers beginning with 01). A data compilation for the AGR-1 baseline coated particle composite LEU01-46T can be found in ORNL/TM-2006/019. The AGR-1 Fuel product Specification and Characterization Guidance (INL EDF-4380) provides the requirements necessary for acceptance of the fuel manufactured for the AGR-1 irradiation test. Section 6.2 of EDF-4380 provides the property requirements for the heat treated compacts. The Statistical Sampling Plan for AGR Fuel materials (INL EDF-4542) provides additional guidance regarding statistical methods for product acceptance and recommended sample sizes. The procedures for characterizing and qualifying the compacts are outlined in ORNL product inspection plan AGR-CHAR-PIP-05. the inspection report forms generated by this product inspection plan document the product acceptance for the property requirements listed in section 6.2 of EDF-4380.

  17. SiC layer microstructure in AGR-1 and AGR-2 TRISO fuel particles and the influence of its variation on the effective diffusion of key fission products

    DOE PAGES

    Gerczak, Tyler J.; Hunn, John D.; Lowden, Richard A.; Allen, Todd R.

    2016-08-15

    Tristructural isotropic (TRISO) coated particle fuel is a promising fuel form for advanced reactor concepts such as high temperature gas-cooled reactors (HTGR) and is being developed domestically under the US Department of Energy’s Nuclear Reactor Technologies Initiative in support of Advanced Reactor Technologies. The fuel development and qualification plan includes a series of fuel irradiations to demonstrate fuel performance from the laboratory to commercial scale. The first irradiation campaign, AGR-1, included four separate TRISO fuel variants composed of multiple, laboratory-scale coater batches. The second irradiation campaign, AGR-2, included TRISO fuel particles fabricated by BWX Technologies with a larger coater representativemore » of an industrial-scale system. The SiC layers of as-fabricated particles from the AGR-1 and AGR-2 irradiation campaigns have been investigated by electron backscatter diffraction (EBSD) to provide key information about the microstructural features relevant to fuel performance. The results of a comprehensive study of multiple particles from all constituent batches are reported. The observations indicate that there were microstructural differences between variants and among constituent batches in a single variant. Finally, insights on the influence of microstructure on the effective diffusivity of key fission products in the SiC layer are also discussed.« less

  18. SiC layer microstructure in AGR-1 and AGR-2 TRISO fuel particles and the influence of its variation on the effective diffusion of key fission products

    NASA Astrophysics Data System (ADS)

    Gerczak, Tyler J.; Hunn, John D.; Lowden, Richard A.; Allen, Todd R.

    2016-11-01

    Tristructural isotropic (TRISO) coated particle fuel is a promising fuel form for advanced reactor concepts such as high temperature gas-cooled reactors (HTGR) and is being developed domestically under the US Department of Energy's Nuclear Reactor Technologies Initiative in support of Advanced Reactor Technologies. The fuel development and qualification plan includes a series of fuel irradiations to demonstrate fuel performance from the laboratory to commercial scale. The first irradiation campaign, AGR-1, included four separate TRISO fuel variants composed of multiple, laboratory-scale coater batches. The second irradiation campaign, AGR-2, included TRISO fuel particles fabricated by BWX Technologies with a larger coater representative of an industrial-scale system. The SiC layers of as-fabricated particles from the AGR-1 and AGR-2 irradiation campaigns have been investigated by electron backscatter diffraction (EBSD) to provide key information about the microstructural features relevant to fuel performance. The results of a comprehensive study of multiple particles from all constituent batches are reported. The observations indicate that there were microstructural differences between variants and among constituent batches in a single variant. Insights on the influence of microstructure on the effective diffusivity of key fission products in the SiC layer are also discussed.

  19. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  20. Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

    PubMed Central

    Hurst, Sheldon; Rowedder, Holli; Michaels, Brandye; Bullock, Hannah; Jackobeck, Ryan; Abebe-Akele, Feseha; Durakovic, Umjia; Gately, Jon; Janicki, Erik

    2015-01-01

    ABSTRACT The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions. IMPORTANCE The relationship between Photorhabdus and entomopathogenic nematode Heterorhabditis represents a well-known mutualistic system that has potential as a biological control agent. The elucidation of the genome of the bacterial partner and role that RNase II plays in its life cycle has provided a greater understanding of Photorhabdus as both an insect pathogen and a nematode symbiont. PMID

  1. Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

    PubMed Central

    Noel, K D; Sanchez, A; Fernandez, L; Leemans, J; Cevallos, M A

    1984-01-01

    Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained. Images PMID:6325385

  2. Selection of hypercellulolytic mutants of Trichoderma reesei based on resistance to nystatin

    SciTech Connect

    Schimenti, J.; Garrett, T.; Montenecourt, B.S.; Eveleigh, D.E.

    1983-01-01

    Nystatin-resistant mutants of Trichoderma reesei were isolated, and several derived from the wild strain showed increased production of cellulase. Thus, the concept of gaining greater enzyme release through interference with membrane function was confirmed. Certain nystatin-resistant mutants derived from the hypercellulolytic strain RUT-C30 also showed increased cellulase yields, but these strains proved unstable and reverted to giving basal enzyme yields.

  3. Selection of a recombinant Marek's disease virus in vivo through expression of the Marek's EcoRI-Q (Meq)-encoded oncoprotein: characterization of an rMd5-based mutant expressing the Meq of strain RB-1B.

    PubMed

    Kumar, Pankaj; Dong, Huimin; Lenihan, Dawn; Gaddamanugu, Syamsundar; Katneni, Upendra; Shaikh, Shireen; Tavlarides-Hontz, Phaedra; Reddy, Sanjay M; Peters, Wachen; Parcells, Mark S

    2012-06-01

    Marek's disease (MD) is a highly contagious viral disease of chickens (Gallus gallus domesticus) caused by MD virus (MDV), characterized by paralysis, neurologic signs, and the rapid onset of T-cell lymphomas. MDV-induced T-cell transformation requires a basic leucine zipper protein called Marek's EcoRI-Q-encoded protein (Meq). We have identified mutations in the coding sequence of Meq that correlated with virus pathotype (virulent, very virulent, and very virulent plus). The aim of this study was to determine whether recombinant viruses could be isolated based on Meq expression through in vivo selection. Chicken embryo fibroblasts (CEFs) were cotransfected with an rMd5 strain-based Meq deletion virus (rMd5deltaMeq) and meq loci from strains representing different pathotypes of MDV. Transfected CEFs were inoculated into chickens in two independent studies. We were able to isolate a single recombinant virus, rMDV-1137, in a contact-exposed chicken. rMDV-1137 had recombined two copies of the meq gene of RB-1B and was found to have pathogenicity similar to both RB-1B and rMd5 parental strains. We found the RB-1B- and rMd5-induced lymphomas showed differences in composition and that rMDV-1137-induced lymphomas were intermediate in their composition. We were able to establish cell lines from both RB-1B- (MDCC-UD35, -UD37) and rMDV-1137 (MDCC-UD36, -UD38)-induced, but not rMd5-induced, lymphomas. To date, no rMd5- or parent Md5-transformed T-cell lines have been reported. Our results suggest that 1) a recombinant MDV can be selected on the basis of oncogenicity; 2) changes in Meq sequence seem to affect tumor composition and the ability to establish cell lines; and 3) in addition to meq, other genomic loci affect MDV pathogenicity and oncogenicity.

  4. Isolation and characterization of Rhizobium meliloti mutants affected in exopolysaccharide production.

    PubMed

    Rodríguez-Navarro, D N; Palomares, A J; Casadesús, J

    1991-06-01

    Rhizobium meliloti mutants affected in the production of exopolysaccharide (EPS) were isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutants were classified into three phenotypic classes: (I) Exo-, rough mutants lacking exopolysaccharide; (II) Exos (for "small") which form tiny, compact colonies and synthesize reduced amounts of EPS; and (III) Exoc (for "constitutive"), hypermucoid mutants which overproduce EPS. Hypermucoid strains showed increased resistance to desiccation. All the mutants were able to nodulate, although a significant decrease in infectivity degree and/or competitiveness was found in rough and compact strains. Two mutants proved to be deficient in nitrogen fixation. Complementation analysis with cloned R. meliloti exo genes could not be applied to the study of these Fix- mutants because introduction of plasmids derived from cosmid vector pLAFR1 caused loss of nodulating ability. However, complementation of calcofluor staining and EPS production was observed. Complementation with certain exo genes also caused a marked increase in motility.

  5. Isolation and characterization of tox mutants of corynebacteriophage beta.

    PubMed Central

    Laird, W; Groman, N

    1976-01-01

    Seventeen nontoxinogenic (tox) mutants of corynebacteriophage beta have been isolated by using a tissue culture screening technique. The mutants fall into four major classes. Two of the classes, I and II, appear to contain missense and nonsense mutants, respectively. However, classes III and IV have not been previously described. Class III mutants produce two proteins (CRMs) seriologically related to diphtheria toxin, but efforts to demonstrate the presence of more than one tox gene have been successful. Class IV mutants are phenotypically CRM-, failing to produce any detectable protein serologically related to diphtheria toxin. Genetic studies indicate that the mutations in class IV strains are not in a gene distinct form the structural gene for toxin, and that the CRM- strains retain at least a portion of that gene. A natural phage isolate, gamma, behaves in a completely parallel fashion to the class IV mutants. The production of tox+ recombinants through recombination of various pairs of tox phage mutants has been demonstrated. The implications of these findings for the natural history of diphtheria are discussed. Images PMID:820873

  6. COAGULASE-NEGATIVE MUTANTS OF STAPHYLOCOCCUS AUREUS: GENETIC STUDIES.

    PubMed

    KORMAN, R Z

    1963-09-01

    Korman, Ruth Z. (Cornell University, Ithaca, N.Y.). Coagulase-negative mutants of Staphylococcus aureus: genetic studies. J. Bacteriol. 86:363-369. 1963.-The behavior in mutation and transduction of pleiotropic coagulase-negative mutants of Staphylococcus aureus PS 53 (NCTC 8511) was analyzed. Coagulase-positive colonies were recovered, as well as a novel phenotype resistant to some cell-wall inhibitors and differing in sugar fermentation pattern. The hypothesis that the coagulase-negative strains differ from the original propagating strain in the structure or organization of the cell wall is discussed.

  7. Irradiation performance of AGR-1 high temperature reactor fuel

    SciTech Connect

    Demkowicz, Paul A.; Hunn, John D.; Ploger, Scott A.; Morris, Robert N.; Baldwin, Charles A.; Harp, Jason M.; Winston, Philip L.; Gerczak, Tyler J.; van Rooyen, Isabella J.; Montgomery, Fred C.; Silva, Chinthaka M.

    2015-10-23

    The AGR-1 experiment contained 72 low-enriched uranium oxide/uranium carbide TRISO coated particle fuel compacts in six capsules irradiated to burnups of 11.2 to 19.6% FIMA, with zero TRISO coating failures detected during the irradiation. The irradiation performance of the fuel including the extent of fission product release and the evolution of kernel and coating microstructures was evaluated based on detailed examination of the irradiation capsules, the fuel compacts, and individual particles. Fractional release of 110mAg from the fuel compacts was often significant, with capsule-average values ranging from 0.01 to 0.38. Analysis of silver release from individual compacts indicated that it was primarily dependent on fuel temperature history. Europium and strontium were released in small amounts through intact coatings, but were found to be significantly retained in the outer pyrocarbon and compact matrix. The capsule-average fractional release from the compacts was 1 × 10–4 to 5 × 10–4 for 154Eu and 8 × 10–7 to 3 × 10–5 for 90Sr. The average 134Cs fractional release from compacts was <3 × 10–6 when all particles maintained intact SiC. An estimated four particles out of 2.98 × 105 in the experiment experienced partial cesium release due to SiC failure during the irradiation, driving 134Cs fractional release in two capsules to approximately 10–5. Identification and characterization of these particles has provided unprecedented insight into the nature and causes of SiC coating failure in high-quality TRISO fuel. In general, changes in coating morphology were found to be dominated by the behavior of the buffer and inner pyrolytic carbon (IPyC), and infrequently observed SiC layer damage was usually related to cracks in the IPyC. Palladium attack of the SiC layer was relatively minor, except for the particles that

  8. Irradiation performance of AGR-1 high temperature reactor fuel

    SciTech Connect

    Paul A. Demkowicz; John D. Hunn; Robert N. Morris; Charles A. Baldwin; Philip L. Winston; Jason M. Harp; Scott A. Ploger; Tyler Gerczak; Isabella J. van Rooyen; Fred C. Montgomery; Chinthaka M. Silva

    2014-10-01

    The AGR-1 experiment contained 72 low-enriched uranium oxide/uranium carbide TRISO-coated particle fuel compacts in six capsules irradiated to burnups of 11.2 to 19.5% FIMA, with zero TRISO coating failures detected during the irradiation. The irradiation performance of the fuel–including the extent of fission product release and the evolution of kernel and coating microstructures–was evaluated based on detailed examination of the irradiation capsules, the fuel compacts, and individual particles. Fractional release of 110mAg from the fuel compacts was often significant, with capsule-average values ranging from 0.01 to 0.38. Analysis of silver release from individual compacts indicated that it was primarily dependent on fuel temperature history. Europium and strontium were released in small amounts through intact coatings, but were found to be significantly retained in the outer pyrocrabon and compact matrix. The capsule-average fractional release from the compacts was 1×10 4 to 5×10 4 for 154Eu and 8×10 7 to 3×10 5 for 90Sr. The average 134Cs release from compacts was <3×10 6 when all particles maintained intact SiC. An estimated four particles out of 2.98×105 experienced partial cesium release due to SiC failure during the irradiation, driving 134Cs release in two capsules to approximately 10 5. Identification and characterization of these particles has provided unprecedented insight into the nature and causes of SiC coating failure in high-quality TRISO fuel. In general, changes in coating morphology were found to be dominated by the behavior of the buffer and inner pyrolytic carbon (IPyC), and infrequently observed SiC layer damage was usually related to cracks in the IPyC. Palladium attack of the SiC layer was relatively minor, except for the particles that released cesium during irradiation, where SiC corrosion was found adjacent to IPyC cracks. Palladium, silver, and uranium were found in the SiC layer of irradiated particles, and characterization

  9. Irradiation performance of AGR-1 high temperature reactor fuel

    DOE PAGES

    Demkowicz, Paul A.; Hunn, John D.; Ploger, Scott A.; Morris, Robert N.; Baldwin, Charles A.; Harp, Jason M.; Winston, Philip L.; Gerczak, Tyler J.; van Rooyen, Isabella J.; Montgomery, Fred C.; et al

    2015-10-23

    The AGR-1 experiment contained 72 low-enriched uranium oxide/uranium carbide TRISO coated particle fuel compacts in six capsules irradiated to burnups of 11.2 to 19.6% FIMA, with zero TRISO coating failures detected during the irradiation. The irradiation performance of the fuel including the extent of fission product release and the evolution of kernel and coating microstructures was evaluated based on detailed examination of the irradiation capsules, the fuel compacts, and individual particles. Fractional release of 110mAg from the fuel compacts was often significant, with capsule-average values ranging from 0.01 to 0.38. Analysis of silver release from individual compacts indicated that itmore » was primarily dependent on fuel temperature history. Europium and strontium were released in small amounts through intact coatings, but were found to be significantly retained in the outer pyrocarbon and compact matrix. The capsule-average fractional release from the compacts was 1 × 10–4 to 5 × 10–4 for 154Eu and 8 × 10–7 to 3 × 10–5 for 90Sr. The average 134Cs fractional release from compacts was <3 × 10–6 when all particles maintained intact SiC. An estimated four particles out of 2.98 × 105 in the experiment experienced partial cesium release due to SiC failure during the irradiation, driving 134Cs fractional release in two capsules to approximately 10–5. Identification and characterization of these particles has provided unprecedented insight into the nature and causes of SiC coating failure in high-quality TRISO fuel. In general, changes in coating morphology were found to be dominated by the behavior of the buffer and inner pyrolytic carbon (IPyC), and infrequently observed SiC layer damage was usually related to cracks in the IPyC. Palladium attack of the SiC layer was relatively minor, except for the particles that released cesium during irradiation, where SiC corrosion was found adjacent to IPyC cracks. In conclusion, palladium, silver, and

  10. Analysis of Fission Products on the AGR-1 Capsule Components

    SciTech Connect

    Paul A. Demkowicz; Jason M. Harp; Philip L. Winston; Scott A. Ploger

    2013-03-01

    The components of the AGR-1 irradiation capsules were analyzed to determine the retained inventory of fission products in order to determine the extent of in-pile fission product release from the fuel compacts. This includes analysis of (i) the metal capsule components, (ii) the graphite fuel holders, (iii) the graphite spacers, and (iv) the gas exit lines. The fission products most prevalent in the components were Ag-110m, Cs 134, Cs 137, Eu-154, and Sr 90, and the most common location was the metal capsule components and the graphite fuel holders. Gamma scanning of the graphite fuel holders was also performed to determine spatial distribution of Ag-110m and radiocesium. Silver was released from the fuel components in significant fractions. The total Ag-110m inventory found in the capsules ranged from 1.2×10 2 (Capsule 3) to 3.8×10 1 (Capsule 6). Ag-110m was not distributed evenly in the graphite fuel holders, but tended to concentrate at the axial ends of the graphite holders in Capsules 1 and 6 (located at the top and bottom of the test train) and near the axial center in Capsules 2, 3, and 5 (in the center of the test train). The Ag-110m further tended to be concentrated around fuel stacks 1 and 3, the two stacks facing the ATR reactor core and location of higher burnup, neutron fluence, and temperatures compared with Stack 2. Detailed correlation of silver release with fuel type and irradiation temperatures is problematic at the capsule level due to the large range of temperatures experienced by individual fuel compacts in each capsule. A comprehensive Ag 110m mass balance for the capsules was performed using measured inventories of individual compacts and the inventory on the capsule components. For most capsules, the mass balance was within 11% of the predicted inventory. The Ag-110m release from individual compacts often exhibited a very large range within a particular capsule.

  11. Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

    PubMed Central

    Gottschalk, Leda Maria Fortes; de Sousa Paredes, Raquel; Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant’Ana; da Silva Bon, Elba Pinto

    2013-01-01

    The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 °C and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture. PMID:24294256

  12. [Isolation of a high hydrogen-producing mutant TB34 generated by transposon insertion and analysis of hydrogen production].

    PubMed

    Liu, Hong-Yan; Wang, Guang-Ce; Shi, Liu-Yang; Zhu, Da-Ling

    2012-07-01

    To increase the hydrogen-producing capacity of Pantoea agglomerans BH18, isolated from mangrove sludge, we constructed a stable transposon mutagenesis library of this strain. A Tn7-based transposon was randomly inserted into the genomic DNA. Mutants were screened by kanamycin resistance and identified by amplification of the inserted transposon sequences. A mutant strain TB34 was isolated, whose hydrogen production capacity was significantly improved compared to the wild type strain. In seawater-containing medium supplemented with 10 g x L(-1) glucose and had an initial pH of 7.0, the hydrogen yield (H2/glucose) of the mutant strain was (2.04 +/- 0.04) mol x mol(-1), which was 43% higher than that of the wild type strain. The mutant TB34 showed steady hydrogen production capacity for five consecutive passages. Different carbon sources were tested in the hydrogen production by the mutant TB34 and the results showed that both the mutant strain TB34 and the wild type strain BH18 were able to produce hydrogen on sucrose, glucose and fructose. However, different from the wild type strain, the mutant strain TB34 was also able to produce hydrogen using xylose as substrate, with a hydrogen yield (H2/xylose) of (1.34 +/- 0.09) mol x mol(-1), indicating a broader substrate spectrum in the mutant.

  13. Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis

    SciTech Connect

    Bame, K.J.; Kiser, C.S.; Esko, J.D.

    1987-05-01

    The authors have isolated Chinese hamster ovary cell mutants defective in proteoglycan synthesis by radiographic screening for cells unable to incorporate TVSO4 into acid-precipitable material. Some mutants did not incorporate TVSO4 into acid-precipitable material, whereas others incorporated about 3-fold less radioactivity. HPLC anion exchange chromatographic analysis of radiolabelled glycosaminoglycans isolated from these mutants revealed many are defective in heparan sulfate biosynthesis. Mutants 803 and 677 do not synthesize heparan sulfate, although they produce chondroitin sulfate: strain 803 makes chondroitin sulfate normally, whereas 677 overaccumulates chondroitin sulfate by a factor of three. These mutants fall into the same complementation group, suggesting that the mutations are allelic. A second group of heparan sulfate biosynthetic mutants, consisting of cell lines 625, 668 and 679, produce undersulfated heparan sulfate and normal chondroitin sulfate. Treatment of the chains with nitrous acid should determine the position of the sulfate groups along the chain. These mutants may define a complementation group that is defective in the enzymes which modify the heparan sulfate chain. To increase the authors repertoire of heparan sulfate mutants, they are presently developing an in situ enzyme assay to screen colonies replica plated on filter discs for sulfotransferase defects.

  14. Gravitropism in roots of intermediate-starch mutants of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kiss, J. Z.; Wright, J. B.; Caspar, T.

    1996-01-01

    Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the starchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to the starchless mutant, it appears that 51-60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.

  15. Fitness of Escherichia coli mutants with reduced susceptibility to tigecycline

    PubMed Central

    Linkevicius, Marius; Anderssen, Jytte Mark; Sandegren, Linus; Andersson, Dan I.

    2016-01-01

    Objectives The objective of this study was to determine the fitness of Escherichia coli mutants with reduced susceptibility to tigecycline after exposure to adverse conditions in vitro and in vivo. Methods Survival in response to low pH, bile salts, oxidative stress and human serum was examined for E. coli mutants with reduced susceptibility to tigecycline due to single mutations that caused increased efflux (marR, lon) or impaired LPS (rfaC, rfaE, lpcA). An in vitro competition assay was used to determine growth fitness defects. Competitive fitness was assessed using mouse infection models. MICs, exponential growth rates and expression levels of efflux-related genes were measured for genetically reconstructed double and triple mutants. Results The LPS mutants were 48–85-fold more susceptible to bile salts compared with the ERN mutants and the WT. As shown by in vitro competitions, the fitness reduction was 0.3%–13% for ERN mutants and ∼24% for LPS mutants. During in vivo survival experiments, LPS mutants were outcompeted by the WT strain in the thigh infection model. Constructed double ERN and LPS mutants showed additive and synergistic increases in tigecycline MICs. Conclusions Generally, reduced susceptibility to tigecycline caused a decrease in fitness under stressful in vitro and in vivo conditions with ERN mutants being fitter than LPS mutants. When combined, ERN mutations caused a synergistic increase in the MIC of tigecycline. These findings could explain why clinical resistance to tigecycline in E. coli is mainly associated with up-regulation of the AcrAB efflux system. PMID:26851608

  16. CD147 and AGR2 expression promote cellular proliferation and metastasis of head and neck squamous cell carcinoma

    SciTech Connect

    Sweeny, Larissa; Liu, Zhiyong; Bush, Benjamin D.; Hartman, Yolanda; Zhou, Tong; Rosenthal, Eben L.

    2012-08-15

    The signaling pathways facilitating metastasis of head and neck squamous cell carcinoma (HNSCC) cells are not fully understood. CD147 is a transmembrane glycoprotein known to induce cell migration and invasion. AGR2 is a secreted peptide also known to promote cell metastasis. Here we describe their importance in the migration and invasion of HNSCC cells (FADU and OSC-19) in vitro and in vivo. In vitro, knockdown of CD147 or AGR2 decreased cellular proliferation, migration and invasion. In vivo, knockdown of CD147 or AGR2 expression decreased primary tumor growth as well as regional and distant metastasis. -- Highlights: Black-Right-Pointing-Pointer We investigated AGR2 in head and neck squamous cell carcinoma for the first time. Black-Right-Pointing-Pointer We explored the relationship between AGR2 and CD147 for the first time. Black-Right-Pointing-Pointer AGR2 and CD147 appear to co-localize in head and squamous cell carcinoma samples. Black-Right-Pointing-Pointer Knockdown of both AGR2 and CD147 reduced migration and invasion in vitro. Black-Right-Pointing-Pointer Knockdown of both AGR2 and CD147 decreased metastasis in vivo.

  17. Comparison of in vitro and in vivo agr/bgr ratios for prostate cancer

    NASA Astrophysics Data System (ADS)

    Carlson, David J.; Stewart, Robert D.; Li, X. Allen; Jennings, Kristofer; Wang, Jian Z.; Guerrero, M.

    2004-10-01

    Parallel in vitro and in vivo studies provide insight into the relationship between clinical response and intrinsic cellular radiosensitivity and may aid in the development of predictive assays. Compilations of radiosensitivity parameters from in vitro experiments can also be used to examine the potential effectiveness of alternative or new treatment plan designs until enough clinical data become available to directly estimate the requisite radiosensitivity parameters. In this work, survival data for six prostate cancer cell lines (ten datasets total) have been extracted from the literature and re-analysed using the linear-quadratic (LQ) survival model. The paired bootstrap technique for regression is used to compute 95% confidence intervals for the estimated radiosensitivity parameters. LQ radiosensitivity parameters derived from the in vitro data are then compared to radiosensitivity parameters derived from clinical data for prostate cancer. Estimates of agr range from 0.09 to 0.35 Gy-1 (all cell lines), and the agr/bgr ratio ranges from 1.09 to 6.29 Gy (all cell lines). Point estimates of the repair half-time (PPC-1, TSU-Pr1, PC-3 and DU-145 cell lines) range from 5.7 to 8.9 h (95% confidence interval from 0.26 h to 10.7 h). Differences in the radiosensitivity parameters determined from the data reported by different laboratories are as large as or larger than the differences in radiosensitivity parameters observed among the various prostate cell lines. The reported studies demonstrate that even seemingly small corrections for dose rate effects, such as those expected in high dose rate (HDR) experiments, can sometimes have a significant impact on estimates of agr and agr/bgr. By neglecting dose rate effects in the analysis of HDR experiments, estimates of the agr/bgr ratio may be too high by factors as large as 1.3 to 6.2. The half-time for repair derived from the in vitro experiments appears significantly larger (slower repair rate) than estimates derived from

  18. Uncertainty Quantification of Calculated Temperatures for the AGR 3/4 Experiment

    SciTech Connect

    Pham, Binh Thi-Cam

    2015-09-01

    A series of Advanced Gas Reactor (AGR) irradiation experiments are being conducted within the Advanced Reactor Technology (ART) Fuel Development and Qualification Program. The main objectives of the fuel experimental campaign are to provide the necessary data on fuel performance to support fuel process development, qualify a fuel design and fabrication process for normal operation and accident conditions, and support development and validation of fuel performance and fission product transport models and codes (PLN 3636, “Technical Program Plan for INL Advanced Reactor Technologies Technology Development Office/Advanced Gas Reactor Fuel Development and Qualification Program”). The AGR 3/4 test was inserted in the Northeast Flux Trap position in the Advanced Test Reactor (ATR) core at Idaho National Laboratory (INL) in December 2011 and successfully completed irradiation in mid-April 2014, resulting in irradiation of the tristructural isotropic (TRISO) fuel for 369.1 effective full-power days (EFPDs) during approximately 2.4 calendar years. The AGR 3/4 data, including the irradiation data and calculated results, were qualified and stored in the Nuclear Data Management and Analysis System (NDMAS). To support the U.S. TRISO fuel performance assessment and to provide data for validation of fuel performance and fission product transport models and codes, the daily as run thermal analysis has been performed separately on each of twelve AGR 3/4 capsules for the entire irradiation as discussed in ECAR-2807, “AGR 3/4 Daily As Run Thermal Analyses”. The ABAQUS code’s finite element-based thermal model predicts the daily average volume average (VA) fuel temperature (FT), peak FT, and graphite matrix, sleeve, and sink temperature in each capsule. The JMOCUP simulation codes were also created to perform depletion calculations for the AGR 3/4 experiment (ECAR-2753, “JMOCUP As-Run Daily Physics Depletion Calculation for the AGR 3/4 TRISO Particle Experiment in ATR

  19. Epidemiological data of staphylococcal scalded skin syndrome in France from 1997 to 2007 and microbiological characteristics of Staphylococcus aureus associated strains.

    PubMed

    Lamand, V; Dauwalder, O; Tristan, A; Casalegno, J S; Meugnier, H; Bes, M; Dumitrescu, O; Croze, M; Vandenesch, F; Etienne, J; Lina, G

    2012-12-01

    Epidemiological data on staphylococcal scalded skin syndromes (SSSS), including bullous impetigo (BI) and generalized exfoliative syndrome (GES), are scarce. To better characterize SSSS and associated Staphylococcus aureus strains, we conducted a retrospective study of 349 cases collected in France between 1997 and 2007 by the National Reference Centre of Staphylococci. Our results showed a stationary evolution of SSSS cases, with a heterogeneous distribution of cases in France. Although notification was not exhaustive, we estimated an incidence of 0.56 cases/year/million inhabitants, in accordance with previous studies conducted in France and Europe, with a median age of 2 years old and sex ratios of 1. A seasonal effect was observed, with a higher GES/BI ratio in autumn compared with other seasons, which could be explained by the impact of viral co-infection. Genetic analysis of S. aureus strains showed that accessory gene regulator (agr) 4, exfoliative toxin A (eta) and B (etb) genes, staphylococcal and enterotoxin-like O (selo) gene and agr4 etb selo profiles were predominantly associated with GES, whereas agr2 eta and agr4 eta selo were more frequently observed with BI. Only one methicillin-resistant strain was found. Protein A (spa) typing identified two main genotypes: spa clonal complex (CC) 159/sequence-type (ST) 121 (75%) and spaCC346/ST15 (18%). spaCC159 was mainly associated with agr4 eta etb selo, agr4 eta selo and agr4 etb selo, and spaCC346 was mainly associated with agr2 eta, suggesting that French SSSS cases are caused by these two main lineages. However, in a multivariate analysis, only etb was independently associated with GES. PMID:23078129

  20. Stress tolerance in doughs of Saccharomyces cerevisiae trehalase mutants derived from commercial Baker's yeast.

    PubMed

    Shima, J; Hino, A; Yamada-Iyo, C; Suzuki, Y; Nakajima, R; Watanabe, H; Mori, K; Takano, H

    1999-07-01

    Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Deltanth1), acid trehalase mutants (Deltaath1), and double mutants (Deltanth1 ath1) by using commercial baker's yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Deltanth1 and Deltaath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Deltanth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough. PMID:10388673

  1. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes. PMID:9007229

  2. Structure-Function Analyses of a Staphylococcus epidermidis Autoinducing Peptide Reveals Motifs Critical for AgrC-type Receptor Modulation.

    PubMed

    Yang, Tian; Tal-Gan, Yftah; Paharik, Alexandra E; Horswill, Alexander R; Blackwell, Helen E

    2016-07-15

    Staphylococcus epidermidis is frequently implicated in human infections associated with indwelling medical devices due to its ubiquity in the skin flora and formation of robust biofilms. The accessory gene regulator (agr) quorum sensing (QS) system plays a prominent role in the establishment of biofilms and infection by this bacterium. Agr activation is mediated by the binding of a peptide signal (or autoinducing peptide, AIP) to its cognate AgrC receptor. Many questions remain about the role of QS in S. epidermidis infections, as well as in mixed-microbial populations on a host, and chemical modulators of its agr system could provide novel insights into this signaling network. The AIP ligand provides an initial scaffold for the development of such probes; however, the structure-activity relationships (SARs) for activation of S. epidermidis AgrC receptors by AIPs are largely unknown. Herein, we report the first SAR analyses of an S. epidermidis AIP by performing systematic alanine and d-amino acid scans of the S. epidermidis AIP-I. On the basis of these results, we designed and identified potent, pan-group inhibitors of the AgrC receptors in the three S. epidermidis agr groups, as well as a set of AIP-I analogs capable of selective AgrC inhibition in either specific S. epidermidis agr groups or in another common staphylococcal species, S. aureus. In addition, we uncovered a non-native peptide agonist of AgrC-I that can strongly inhibit S. epidermidis biofilm growth. Together, these synthetic analogs represent new and readily accessible probes for investigating the roles of QS in S. epidermidis colonization and infections.

  3. Nitrate reduction mutants of Fusarium moniliforme (Gibberella fujikuroi)

    SciTech Connect

    Klittich, C.J.R.; Leslie, J.F.

    1988-03-01

    Twelve strains of Fusarium moniliforme were examined for their ability to sector spontaneously on toxic chlorate medium. All strains sectored frequently; 91% of over 1200 colonies examined formed chlorate-resistant, mutant sectors. Most of these mutants had lesions in the nitrate reduction pathway and were unable to utilize nitrate (nit mutants). nit mutations occurred in seven loci: a structural gene for nitrate reductase (nit1), a regulatory gene specific for the nitrate reduction pathway (nit3), and five genes controlling the production of a molybdenum-containing cofactors that is necessary for nitrate reductase activity (nit2, nit4, nit5, nit6, nit7). No mutations affecting nitrite reductase or a major nitrogen regulatory locus were found among over 1000 nit mutants. Mutations of nit1 were recovered most frequently (39-66%, depending on the strain) followed by nit3 mutations (23-42%). The frequency of isolation of each mutant type could be altered, however, by changing the source of nitrogen in the chlorate medium. The authors concluded that genetic control of nitrate reduction in F. moniliforme is similar to that in Aspergillus and Neurospora, but that the overall regulation of nitrogen metabolism may be different.

  4. In Vivo Studies of Temperature-Sensitive recB and recC Mutants

    PubMed Central

    Kushner, Sidney R.

    1974-01-01

    Some in vivo properties of Escherichia coli K-12 strains carrying recB270 (formerly recBts1) and recC271 (formerly recCts1) mutations have been determined. Single recB270 and recC271 mutants appear normal at 30 C with regard to ultraviolet and mitomycin C sensitivity, recombination proficiency, and viability. At 43 C these strains become sensitive to ultraviolet and mitomycin C, while showing only a slight decrease in recombination proficiency. The viable titers of the single mutants are somewhat reduced at 43 C. Double mutant strains carrying polA1 and recB270 or recC271 are inviable at 43 C. The double mutant strain (recB270 recC271) is sensitive to both UV and mitomycin C at 30 C, but shows only slightly reduced recombination proficiency. At 43 C the strain resembles absolute recB and recC mutants in all respects. In addition, the double mutant strain exhibits a temperature-induced drop in viable titer. The triple mutant polA1 recB270 recC271 is viable at 30 C. Two hypotheses are advanced to explain these results. PMID:4612007

  5. RNA Sequencing Analysis of the Broad-Host-Range Strain Sinorhizobium fredii NGR234 Identifies a Large Set of Genes Linked to Quorum Sensing-Dependent Regulation in the Background of a traI and ngrI Deletion Mutant

    PubMed Central

    Krysciak, Dagmar; Grote, Jessica; Rodriguez Orbegoso, Mariita; Utpatel, Christian; Förstner, Konrad U.; Li, Lei; Schmeisser, Christel; Krishnan, Hari B.

    2014-01-01

    The alphaproteobacterium Sinorhizobium fredii NGR234 has an exceptionally wide host range, as it forms nitrogen-fixing nodules with more legumes than any other known microsymbiont. Within its 6.9-Mbp genome, it encodes two N-acyl-homoserine-lactone synthase genes (i.e., traI and ngrI) involved in the biosynthesis of two distinct autoinducer I-type molecules. Here, we report on the construction of an NGR234-ΔtraI and an NGR234-ΔngrI mutant and their genome-wide transcriptome analysis. A high-resolution RNA sequencing (RNA-seq) analysis of early-stationary-phase cultures in the NGR234-ΔtraI background suggested that up to 316 genes were differentially expressed in the NGR234-ΔtraI mutant versus the parent strain. Similarly, in the background of NGR234-ΔngrI 466 differentially regulated genes were identified. Accordingly, a common set of 186 genes was regulated by the TraI/R and NgrI/R regulon. Coregulated genes included 42 flagellar biosynthesis genes and 22 genes linked to exopolysaccharide (EPS) biosynthesis. Among the genes and open reading frames (ORFs) that were differentially regulated in NGR234-ΔtraI were those linked to replication of the pNGR234a symbiotic plasmid and cytochrome c oxidases. Biotin and pyrroloquinoline quinone biosynthesis genes were differentially expressed in the NGR234-ΔngrI mutant as well as the entire cluster of 21 genes linked to assembly of the NGR234 type III secretion system (T3SS-II). Further, we also discovered that genes responsible for rhizopine catabolism in NGR234 were strongly repressed in the presence of high levels of N-acyl-homoserine-lactones. Together with nodulation assays, the RNA-seq-based findings suggested that quorum sensing (QS)-dependent gene regulation appears to be of higher relevance during nonsymbiotic growth rather than for life within root nodules. PMID:25002423

  6. Isolation and characterization of yeast monomorphic mutants of Candida albicans.

    PubMed Central

    Elorza, M V; Sentandreu, R; Ruiz-Herrera, J

    1994-01-01

    A method was devised for the isolation of yeast monomorphic (LEV) mutants of Candida albicans. By this procedure, about 20 stable yeast-like mutants were isolated after mutagenesis with ethyl methane sulfonate. The growth rate of the mutants in different carbon sources, both fermentable and not, was indistinguishable from that of the parental strain, but they were unable to grow as mycelial forms after application of any of the common effective inducers, i.e., heat shock, pH alterations, proline addition, or use of GlcNAc as the carbon source. Studies performed with one selected strain demonstrated that it had severe alterations in the chemical composition of the cell wall, mainly in the levels of chitin and glucans, and in specific mannoproteins, some of them recognizable by specific polyclonal and monoclonal antibodies. It is suggested that these structural alterations hinder the construction of a normal hyphal wall. Images PMID:8157600

  7. C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype.

    PubMed

    Kanteti, Rajani; Dhanasingh, Immanuel; El-Hashani, Essam; Riehm, Jacob J; Stricker, Thomas; Nagy, Stanislav; Zaborin, Alexander; Zaborina, Olga; Biron, David; Alverdy, John C; Im, Hae Kyung; Siddiqui, Shahid; Padilla, Pamela A; Salgia, Ravi

    2016-01-01

    We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.

  8. Data Compilation for AGR-1 Pre-Production Test: NUCO350-75T-Z

    SciTech Connect

    Hunn, John D; Lowden, Richard Andrew; Pappano, Peter J

    2006-03-01

    This document is a compilation of characterization data for compact lot NUCO350-75T-Z. This compact lot was fabricated using particle composite NUCO350-75T, which was a composite of three batches of TRISO-coated 350 m natural uranium oxide/uranium carbide kernels (NUCO). The compacts and coated particles were produced as part of a development effort at ORNL for the Advanced Gas Reactor Fuel Development and Qualification (AGR) program. The kernels were obtained from BWXT and were identified as composite G73B-NU-69300. The BWXT kernel lot G73B-NU-69300 was riffled into sublots for characterization and coating. The ORNL identification for these kernel sublots was NUCO350-## (where ## were a series of integers beginning with 01). NUCO350-75T-Z was produced as part of the ORNL AGR development effort and is not fully representative of a final product. This compact lot was the first run through of the entire ORNL AGR-1 irradiation test fuel production process involving coating, characterization, and compacting of TRISO-coated 350 m NUCO. The results of this exercise were used to fine tune the irradiation test fuel production process and as a basis for the decision to proceed with the production of the baseline fuel for the AGR-1 irradiation test.

  9. PIE on Safety-Tested AGR-1 Compact 5-1-1

    SciTech Connect

    Hunn, John D.; Morris, Robert Noel; Baldwin, Charles A.; Montgomery, Fred C.; Gerczak, Tyler J.

    2015-08-01

    Post-irradiation examination (PIE) is being performed in support of tristructural isotropic (TRISO) coated particle fuel development and qualification for High-Temperature Gas-cooled Reactors (HTGRs). AGR-1 was the first in a series of TRISO fuel irradiation experiments initiated in 2006 under the Advanced Gas Reactor (AGR) Fuel Development and Qualification Program; this work continues to be funded by the Department of Energy's Office of Nuclear Energy as part of the Advanced Reactor Technologies (ART) initiative. AGR-1 fuel compacts were fabricated at Oak Ridge National Laboratory (ORNL) in 2006 and irradiated for three years in the Idaho National Laboratory (INL) Advanced Test Reactor (ATR) to demonstrate and evaluate fuel performance under HTGR irradiation conditions. PIE is being performed at INL and ORNL to study how the fuel behaved during irradiation, and to examine fuel performance during exposure to elevated temperatures at or above temperatures that could occur during a depressurized conduction cooldown event. This report summarizes safety testing of irradiated AGR-1 Compact 5-1-1 in the ORNL Core Conduction Cooldown Test Facility (CCCTF) and post-safety testing PIE.

  10. Colorado's AgrAbility Project's Effects on KASA and Practice Changes with Agricultural Producers and Professionals

    ERIC Educational Resources Information Center

    Fetsch, Robert J.; Jackman, Danielle M.

    2015-01-01

    Disability rates resulting from work-related injuries remain steadily high among farmers and ranchers. To address the gap in services within this population, USDA implemented AgrAbility nationally. Using part of Bennett's hierarchical model, the current study evaluated the KASA and practice change levels of 401 farmers and ranchers and compared…

  11. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

    PubMed

    García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

    2013-12-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga.

  12. Identification of Arabidopsis rat Mutants

    PubMed Central

    Zhu, Yanmin; Nam, Jaesung; Humara, Jaime M.; Mysore, Kirankumar S.; Lee, Lan-Ying; Cao, Hongbin; Valentine, Lisa; Li, Jingling; Kaiser, Anthony D.; Kopecky, Andrea L.; Hwang, Hau-Hsuan; Bhattacharjee, Saikat; Rao, Praveen K.; Tzfira, Tzvi; Rajagopal, Jyothi; Yi, HoChul; Veena; Yadav, Badam S.; Crane, Yan M.; Lin, Kui; Larcher, Yves; Gelvin, Matthew J.K.; Knue, Marnie; Ramos, Cynthia; Zhao, Xiaowen; Davis, Susan J.; Kim, Sang-Ic; Ranjith-Kumar, C.T.; Choi, Yoo-Jin; Hallan, Vipin K.; Chattopadhyay, Sudip; Sui, Xiangzhen; Ziemienowicz, Alicja; Matthysse, Ann G.; Citovsky, Vitaly; Hohn, Barbara; Gelvin, Stanton B.

    2003-01-01

    Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants. PMID:12805582

  13. ECB deacylase mutants

    DOEpatents

    Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

    2002-01-01

    A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

  14. Advanced Gas Reactor (AGR)-5/6/7 Fuel Irradiation Experiments in the Advanced Test Reactor

    SciTech Connect

    A. Joseph Palmer; David A. Petti; S. Blaine Grover

    2014-04-01

    The United States Department of Energy’s Very High Temperature Reactor (VHTR) Advanced Gas Reactor (AGR) Fuel Development and Qualification Program will be irradiating up to seven separate low enriched uranium (LEU) tri-isotopic (TRISO) particle fuel (in compact form) experiments in the Advanced Test Reactor (ATR) located at the Idaho National Laboratory (INL). These irradiations and fuel development are being accomplished to support development of the next generation reactors in the United States. The goals of the irradiation experiments are to provide irradiation performance data to support fuel process development, to qualify fuel for normal operating conditions, to support development and validation of fuel performance and fission product transport models and codes, and to provide irradiated fuel and materials for post irradiation examination (PIE) and safety testing. The experiments, which each consist of at least five separate capsules, are being irradiated in an inert sweep gas atmosphere with individual on-line temperature monitoring and control of each capsule. The sweep gases also have on-line fission product monitoring the effluent from each capsule to track performance of the fuel during irradiation. The first two experiments (designated AGR-1 and AGR-2), have been completed. The third and fourth experiments have been combined into a single experiment designated AGR-3/4, which started its irradiation in December 2011 and is currently scheduled to be completed in April 2014. The design of the fuel qualification experiment, designated AGR-5/6/7, is well underway and incorporates lessons learned from the three previous experiments. Various design issues will be discussed with particular details related to selection of thermometry.

  15. Microstructure of TRISO Coated Particles from the AGR-1 Experiment I: SiC Grain Size and Grain Boundary Character

    SciTech Connect

    Rita Kirchhofer; John D, Hunn; Paul A. Demkowicz; James I. Cole; Brian P. Gorman

    2013-01-01

    Pre-irradiation SiC microstructures in TRISO coated fuel particles from the AGR-1 experiment were quantitatively characterized using electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM). From EBSD it was determined that only the cubic polymorph of as-deposited SiC was present and the SiC had a high fraction of CSL S3 grain boundaries. Additionally, the local area misorientation (LAM), which is a qualitative measurement of strain in the SiC lattice, was mapped for each fuel variant. The morphology of the SiC / IPyC interfaces were characterized by TEM following site-specific focused ion beam (FIB) specimen preparation. It was determined that the SiC layer had a heavily faulted microstructure typical of CVD deposited SiC and that the average grain diameter increased from the SiC/IPyC interface for all the fuel variants, except V3 that showed a constant grain size across the layer.

  16. Activation and Inhibition of the Receptor Histidine Kinase AgrC Occurs Through Opposite Helical Transduction Motions

    PubMed Central

    Wang, Boyuan; Zhao, Aishan; Novick, Richard; Muir, Tom W.

    2014-01-01

    Summary Staphylococcus aureus virulence is regulated when secreted autoinducing peptides (AIPs) are recognized by a membrane-bound receptor histidine kinase (RHK), AgrC. Some AIPs are agonists of virulence gene expression, while others are antagonists. It is unclear how AIP binding regulates AgrC activity. Here, we reconstitute an AgrC family member, AgrC-I, using nanometer-scale lipid bilayer discs. We show that AgrC-I requires membranes rich in anionic lipids to function. The agonist, AIP-I, binds AgrC-I non-cooperatively in a 2:2 stoichiometry, while an antagonist ligand, AIP-II, functions as an inverse agonist of the kinase activity. We also demonstrate the kinase and sensor domains in AgrC are connected by a helical linker whose conformational state exercises rheostat-like control over the kinase activity. Binding of agonist or inverse-agonist peptides results in twisting of the linker in different directions. These two observations provide a view of the molecular motions triggered by ligand binding in an intact membrane-bound RHK. PMID:24656130

  17. UV-induced Lactobacillus gasseri mutants resisting sodium chloride and sodium nitrite for meat fermentation.

    PubMed

    Arihara, K; Itoh, M

    2000-06-01

    Lactobacillus gasseri, one of the predominant lactobacilli in human intestinal tracts, is utilized for probiotics and dairy starter cultures. However, since L. gasseri is relatively sensitive to sodium chloride and sodium nitrite (essential compounds for meat products), it is difficult to utilize this species for conventional fermented meat products. In this study, efforts were directed to generate mutants of L. gasseri resisting sodium chloride and sodium nitrite. UV irradiation of the strain of L. gasseri JCM1131(T) generated several mutants resisting these compounds. A mutant strain 1131-M8 demonstrated satisfactory growth in meat containing 3.3% sodium chloride and 200 ppm sodium nitrite. Although proteins extracted from the cell surface of 1131-M8 were slightly different from those of the original strain, other biochemical characteristics of both strains were indistinguishable. These results suggest that the L. gasseri mutant obtained in this study could be utilized as a starter culture to develop probiotic meat products.

  18. Investigation into the resistance of lactoperoxidase tolerant Escherichia coli mutants to different forms of oxidative stress.

    PubMed

    De Spiegeleer, Philipp; Vanoirbeek, Kristof; Lietaert, Annelies; Sermon, Jan; Aertsen, Abram; Michiels, Chris W

    2005-11-15

    Six lactoperoxidase tolerant Escherichia coli transposon mutants isolated and characterized in an earlier study, and some newly constructed double mutants, were subjected to peroxide, superoxide and hypochlorite stress, and their inactivation was compared to that of the wild type strain MG1655. Knock out mutants of waaQ and waaO, which owed their lactoperoxidase tolerance to an impaired outer membrane permeability due to a reduced porin content, also exhibited higher resistance to hypochlorite, as did a knock-out strain of lrp, encoding a regulatory protein affecting a wide range of cellular functions. Unlike the outer membrane mutants however, the lrp strain was also more resistant to t-butyl hydroperoxide, but more susceptible to the superoxide generating compound plumbagin. Finally, a lactoperoxidase tolerant knock-out strain of ulaA, involved in ascorbic acid uptake, did not show resistance to any of the other oxidants. The possible modes of action of these different oxidants are discussed.

  19. Direct selection of Clostridium acetobutylicum fermentation mutants by a proton suicide method

    SciTech Connect

    Cueto, P.H.; Mendez, B.S. )

    1990-02-01

    Clostridium acetobutylicum ATCC 10132 mutants altered in acetic acid synthesis or in the shift to solventogenesis were directly selected by a proton suicide method after mutagenic treatment, by using bromide and bromate as selective agents. The mutants were characterized according to their solvent and acid production. On the selection plates they differed in colony phenotype from the parent strain.

  20. A Laboratory Exercise for Isolation and Characterizing Microbial Mutants with Metabolic Defects.

    ERIC Educational Resources Information Center

    Doe, Frank J.; Leslie, John F.

    1993-01-01

    Describes science experiments for undergraduate biology instruction on the concepts of mutation and characterization of the resulting mutant strains. The filamentous fungi "Fusarium moniliforme" is used to illustrate the induction of mutants (mutagenesis), identification of the mutated gene, construction of a biochemical pathway, and genetic…

  1. Isolation of Rhizobium phaseoli Tn5-induced mutants with altered expression of cytochrome terminal oxidases o and aa3.

    PubMed Central

    Soberón, M; Membrillo-Hernández, J; Aguilar, G R; Sánchez, F

    1990-01-01

    Two Rhizobium phaseoli mutants affected in cytochrome expression were obtained by Tn5-mob mutagenesis of the wild-type strain (CE3). Mutant strain CFN031 expressed sevenfold less cytochrome o in culture, expressed cytochrome aa3 under microaerophilic culture conditions, in contrast to strain CE3, and was affected in its vegetative growth properties and proliferation inside plant host cells. Mutant CFN037 expressed cytochrome aa3 under microaerophilic culture conditions, while bacteroid development and nitrogen fixation occurred earlier than in strain CE3. Images FIG. 2 PMID:2155209

  2. Foxa2 and Hif1ab regulate maturation of intestinal goblet cells by modulating agr2 expression in zebrafish embryos.

    PubMed

    Lai, Yun-Ren; Lu, Yu-Fen; Lien, Huang-Wei; Huang, Chang-Jen; Hwang, Sheng-Ping L

    2016-07-15

    Mammalian anterior gradient 2 (AGR2), an endoplasmic reticulum (ER) protein disulfide-isomerase (PDI), is involved in cancer cell growth and metastasis, asthma and inflammatory bowel disease (IBD). Mice lacking Agr2 exhibit decreased Muc2 protein in intestinal goblet cells, abnormal Paneth cell development, ileitis and colitis. Despite its importance in cancer biology and inflammatory diseases, the mechanisms regulating agr2 expression in the gastrointestinal tract remain unclear. In the present study, we investigated the mechanisms that control agr2 expression in the pharynx and intestine of zebrafish by transient/stable transgenesis, coupled with motif mutation, morpholino knockdown, mRNA rescue and ChIP. A 350 bp DNA sequence with a hypoxia-inducible response element (HRE) and forkhead-response element (FHRE) within a region -4.5 to -4.2 kbp upstream of agr2 directed EGFP expression specifically in the pharynx and intestine. No EGFP expression was detected in the intestinal goblet cells of Tg(HREM:EGFP) or Tg(FHREM:EGFP) embryos with mutated HRE or FHRE, whereas EGFP was expressed in the pharynx of Tg(HREM:EGFP), but not Tg(FHREM:EGFP), embryos. Morpholino knockdown of foxa1 (forkhead box A1) reduced agr2 levels in the pharynx, whereas knockdown of foxa2 or hif1ab decreased intestinal agr2 expression and affected the differentiation and maturation of intestinal goblet cells. These results demonstrate that Foxa1 regulates agr2 expression in the pharynx, whereas both Foxa2 and Hif1ab control agr2 expression in intestinal goblet cells to regulate maturation of these cells. PMID:27222589

  3. AGR3 in Breast Cancer: Prognostic Impact and Suitable Serum-Based Biomarker for Early Cancer Detection

    PubMed Central

    Garczyk, Stefan; von Stillfried, Saskia; Antonopoulos, Wiebke; Hartmann, Arndt; Schrauder, Michael G.; Fasching, Peter A.; Anzeneder, Tobias; Tannapfel, Andrea; Ergönenc, Yavuz; Knüchel, Ruth

    2015-01-01

    Blood-based early detection of breast cancer has recently gained novel momentum, as liquid biopsy diagnostics is a fast emerging field. In this study, we aimed to identify secreted proteins which are up-regulated both in tumour tissue and serum samples of breast cancer patients compared to normal tissue and sera. Based on two independent tissue cohorts (n = 75 and n = 229) and one serum cohort (n = 80) of human breast cancer and healthy serum samples, we characterised AGR3 as a novel potential biomarker both for breast cancer prognosis and early breast cancer detection from blood. AGR3 expression in breast tumours is significantly associated with oestrogen receptor α (P<0.001) and lower tumour grade (P<0.01). Interestingly, AGR3 protein expression correlates with unfavourable outcome in low (G1) and intermediate (G2) grade breast tumours (multivariate hazard ratio: 2.186, 95% CI: 1.008-4.740, P<0.05) indicating an independent prognostic impact. In sera analysed by ELISA technique, AGR3 protein concentration was significantly (P<0.001) elevated in samples from breast cancer patients (n = 40, mainly low stage tumours) compared to healthy controls (n = 40). To develop a suitable biomarker panel for early breast cancer detection, we measured AGR2 protein in human serum samples in parallel. The combined AGR3/AGR2 biomarker panel achieved a sensitivity of 64.5% and a specificity of 89.5% as shown by receiver operating characteristic (ROC) curve statistics. Thus our data clearly show the potential usability of AGR3 and AGR2 as biomarkers for blood-based early detection of human breast cancer. PMID:25875093

  4. Bacillus sp. mutant for improved biodegradation of Congo red: random mutagenesis approach.

    PubMed

    Gopinath, Kannappan Panchamoorthy; Murugesan, Shreela; Abraham, Joanna; Muthukumar, Karuppan

    2009-12-01

    This study presents the improved biodegradation of Congo red, a toxic azo dye, using mutant Bacillus sp. obtained by random mutagenesis of wild Bacillus sp. using UV and ethidium bromide. The mutants obtained were screened based on their decolorization performance and best mutants were selected for further studies. Better decolorization was observed in the initial Congo red concentration range 100-1000 mg/l for wild species whereas mutant strain was found to offer better decolorization up to 3000 mg/l. Mutant strain offered 12-30% reduction in time required for the complete decolorization by wild strain. The optimum pH and temperature were found to be 7.0 and 37 degrees C, respectively. Two efficient strains such as Bacillus sp. ACT 1 and Bacillus sp. ACT 2 were isolated from the various mutants obtained. Bacillus sp. ACT 2 showed improved enzymatic production and Bacillus sp. ACT 1 showed improved growth compared to wild strain. The enzyme responsible for the degradation was found to be azoreductase by SDS-PAGE and about 53% increased production of enzyme was achieved with mutant species. The experimental data were modeled using growth and substrate inhibition models.

  5. The molecular basis for the alternative stable phenotype in a behavioral mutant of Paramecium tetraurelia.

    PubMed

    Matsuda, A; Takahashi, M

    2001-10-01

    In the sexual reproduction of Paramecium tetraurelia, the somatic nucleus (macronucleus) undergoes massive genomic rearrangement, including gene amplification and excision of internal eliminated sequences (IESs), in its normal developmental process. Strain d4-662, one of the pawn mutants, is a behavioral mutant of P. tetraurelia that carries a recessive allele of pwB662. ThepwB gene in the macronucleus of the strain has an insertion of the IES because a base substitution within the IES prevents its excision during gene rearrangement. Cultures of this strain frequently contain cells reverting to the wild type in the behavioral phenotype. The mutant and revertant cells maintained stable clonal phenotypes under the various environmental conditions examined unless they underwent sexual reproduction. After sexual reproduction, both mutant and revertant produced 2.7-7.1% reverted progeny. A molecular analysis performed on the macronuclear DNA of the mutant and revertant of d4-662 showed that much less than 1% of the mutant IES was precisely excised at every sexual reproduction of the strain. Therefore, the alternative phenotype of strain d4-662 seems to be caused by an alternative excision of the mutant IES. PMID:11817644

  6. Anthracycline metabolites from Streptomyces violaceus A262. I. Isolation of antibiotic-blocked mutants from Streptomyces violaceus A262.

    PubMed

    Johdo, O; Ishikura, T; Yoshimoto, A; Takeuchi, T

    1991-10-01

    Five mutant (or variant) strains producing new anthracycline antibiotics were derived from Streptomyces violaceus A262 by mutagenesis treatment. Strain SE1-625 showed a limited production of three known beta-rhodomycinone diglycosides while the parent strain produced numerous unidentified beta-rhodomycinone glycosides. Strain SU2-730 was an antibiotic-blocked mutant which produced only epsilon-rhodomycinone glycosides (named epelmycins). Strains SC-7 and SE2-2385 were variants which produced alpha-citromycinone glycosides (named yellamycins) and beta-isorhodomycinone glycosides (named obelmycins), respectively. Strain SE2-2385-A1 produced alpha 2-rhodomycinone glycosides (named alldimycins). Glycosidation-less mutants which accumulated only aglycone were also obtained. Isolation of these mutants or variants and preliminary identification of their anthracycline products are described. PMID:1955394

  7. Enhancement of the Gibberella zeae growth inhibitory lipopeptides from a Bacillus subtilis mutant by ion beam implantation.

    PubMed

    Liu, J; Liu, M; Wang, J; Yao, J M; Pan, R R; Yu, Z L

    2005-11-01

    Bacillus subtilis JA antagonized the growth of Gibberella zeae. In order to reduce growth of this fungi pathogen to a greater extent, low-energy ion beam implantation was applied in mutant breeding. We studied the effects of different energies and different doses of nitrogen ion implantation. The mutant strain designated as JA026 was obtained showing higher inhibition activity in the screening plate. Its inhibition zone against indicator organism increased by 14.3% compared to the original strain. The electrospray ionization mass spectrometry (ESI/MS) analysis indicated that the antifungal lipopeptides produced by the mutant were identical to those produced by the wild-type strain. The mutant strain exhibited favorable properties including the high yield of antifungal lipopeptides production and faster growth over the parent strain, which suggested that this strain would be a promising biocontrol candidate in agriculture.

  8. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    PubMed

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme. PMID:25139657

  9. Characterization of a mutant glucose isomerase from Thermoanaerobacterium saccharolyticum.

    PubMed

    Xu, Heng; Shen, Dong; Wu, Xue-Qiang; Liu, Zhi-Wei; Yang, Qi-He

    2014-10-01

    A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co(2+) or Mg(2+) for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.

  10. Analysis of Escherichia coli mutants with a linear respiratory chain.

    PubMed

    Steinsiek, Sonja; Stagge, Stefan; Bettenbrock, Katja

    2014-01-01

    The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I and II, as well as of three terminal oxidases. They differ with respect to energetic efficiency (proton translocation) and their affinity to the different quinone/quinol species and oxygen. In order to analyze the advantages of the branched electron transport chain over a linear one and to assess how usage of the different terminal oxidases determines growth behavior at varying oxygen concentrations, a set of isogenic mutant strains was created, which lack NADH dehydrogenase I as well as two of the terminal oxidases, resulting in strains with a linear respiratory chain. These strains were analyzed in glucose-limited chemostat experiments with defined oxygen supply, adjusting aerobic, anaerobic and different microaerobic conditions. In contrast to the wild-type strain MG1655, the mutant strains produced acetate even under aerobic conditions. Strain TBE032, lacking NADH dehydrogenase I and expressing cytochrome bd-II as sole terminal oxidase, showed the highest acetate formation rate under aerobic conditions. This supports the idea that cytochrome bd-II terminal oxidase is not able to catalyze the efficient oxidation of the quinol pool at higher oxygen conditions, but is functioning mainly under limiting oxygen conditions. Phosphorylation of ArcA, the regulator of the two-component system ArcBA, besides Fnr the main transcription factor for the response towards different oxygen concentrations, was studied. Its phosphorylation pattern was changed in the mutant strains. Dephosphorylation and therefore inactivation of ArcA started at lower aerobiosis levels than in the wild-type strain. Notably, not only the micro- and aerobic metabolism was affected by the mutations, but also the anaerobic metabolism, where the respiratory chain should not be important. PMID:24475268

  11. Survival of Brucella abortus aqpX mutant in fresh and ripened cheeses.

    PubMed

    Santiago-Rodríguez, María Del Rosario; Díaz-Aparicio, Efrén; Arellano-Reynoso, Beatriz; García-Lobo, Juan M; Gimeno, Miquel; Palomares-Reséndiz, Erika G; Hernández-Castro, Rigoberto

    2015-02-01

    The objective of this work was to evaluate the survival of a Brucella abortus aqpX mutant during the elaboration and conservation of fresh and ripened cheeses at 4 °C and 24 °C. The pH values and water activity were monitored for each type of cheese. The fresh cheese was elaborated with raw milk inoculated with 6×10⁸ colony-forming units (CFU)/mL each of parental and mutant strain. Ripening cheeses were elaborated with both raw and pasteurized milk and inoculated with 12×10⁸ CFU/mL each of parental and mutant strains. In fresh cheese, survival was observed during elaboration and conservation for 7 days at 4 °C in mutant and parental strains. The number of survivors of the mutant strain was 10 times lower compared with the parental strain at pH 5 and a(w) of 0.930. In the cheese elaborated with raw milk and ripened at 24 °C, both strains survived until day 17 at pH 4.0 and a(w) of 0.89. However, when the cheese was elaborated with pasteurized milk, the parental strain survived until day 31 of ripening, and the mutant strain survived 24 days at pH 4 and a(w) of 0.886. The survival of the mutant strain showed a diminution of one logarithm during elaboration and ripening of cheese as compared with the parental strain. When the cheese was elaborated with raw milk and ripened at 4 °C, survival of the parental strain was 24 days, whereas the mutant strain survived only 17 days (pH 5 and a(w) 0.90). Regarding the cheese elaborated with pasteurized milk and maturated at 4 °C, both strains survived 31 days (pH 5 and a(w) 0.90), with the same survival diminution during elaboration and ripening. Our results show that in both types of cheese, the mutated aqpX strain survived 10 times less than the parental strain, which shows that the aqpX gene can be related to the survival of Brucella abortus in this type of cheese.

  12. Survival of Brucella abortus aqpX mutant in fresh and ripened cheeses.

    PubMed

    Santiago-Rodríguez, María Del Rosario; Díaz-Aparicio, Efrén; Arellano-Reynoso, Beatriz; García-Lobo, Juan M; Gimeno, Miquel; Palomares-Reséndiz, Erika G; Hernández-Castro, Rigoberto

    2015-02-01

    The objective of this work was to evaluate the survival of a Brucella abortus aqpX mutant during the elaboration and conservation of fresh and ripened cheeses at 4 °C and 24 °C. The pH values and water activity were monitored for each type of cheese. The fresh cheese was elaborated with raw milk inoculated with 6×10⁸ colony-forming units (CFU)/mL each of parental and mutant strain. Ripening cheeses were elaborated with both raw and pasteurized milk and inoculated with 12×10⁸ CFU/mL each of parental and mutant strains. In fresh cheese, survival was observed during elaboration and conservation for 7 days at 4 °C in mutant and parental strains. The number of survivors of the mutant strain was 10 times lower compared with the parental strain at pH 5 and a(w) of 0.930. In the cheese elaborated with raw milk and ripened at 24 °C, both strains survived until day 17 at pH 4.0 and a(w) of 0.89. However, when the cheese was elaborated with pasteurized milk, the parental strain survived until day 31 of ripening, and the mutant strain survived 24 days at pH 4 and a(w) of 0.886. The survival of the mutant strain showed a diminution of one logarithm during elaboration and ripening of cheese as compared with the parental strain. When the cheese was elaborated with raw milk and ripened at 4 °C, survival of the parental strain was 24 days, whereas the mutant strain survived only 17 days (pH 5 and a(w) 0.90). Regarding the cheese elaborated with pasteurized milk and maturated at 4 °C, both strains survived 31 days (pH 5 and a(w) 0.90), with the same survival diminution during elaboration and ripening. Our results show that in both types of cheese, the mutated aqpX strain survived 10 times less than the parental strain, which shows that the aqpX gene can be related to the survival of Brucella abortus in this type of cheese. PMID:25551422

  13. Factors influencing maternal behavior in the hubb/hubb mutant mouse.

    PubMed

    Alston-Mills, B; Parker, A C; Eisen, E J; Wilson, R; Fletcher, S

    We examined the maternal behavior of hubb/hubb mutant mice and normal control (+/hubb) siblings. From previous observations we noted that mutants groom their pups less, suckle less than normal, and often cannibalize the young. To date, these observations had not been quantified. Although prolactin (PRL) is linked to maternal behavior, it was difficult to measure because of the hyperirratibility of the mutant mice. Consequently, dopamine (DA) and its metabolite, dihydroxyphenylacetic acid (DOPAC), were measured in the median eminence in brains of both normal and mutant mice. Tyrosine hydroxylase, the rate-determining step in dopamine synthesis, was localized in the brain by immunohistochemistry. Five mutant and nine normal dams were observed for pup retrieval and crouching. Mean time for pup retrieval was slower (p < 0.06) for mutants (28.09 s) than for normal dams (18.49 s). Crouching was the same for both strains. Mutant pups were cold to the touch, and not well groomed. Brains from both strains were examined at Day 11 and Day 18 of gestation and Day 2 and Day 11 of lactation. Qualitatively, tyrosine hydroxylase localization in the arcuate nucleus and median eminence was the same in both strains for the gestation samples. The decrease in staining observed from gestation to lactation in the normal mice was increased in the mutants. Dopamine was similar in both strains at all stages, but DOPAC was significantly higher at early lactation in the mutants. We do not assume an absolute inverse relationship between dopaminergic activities and prolactin, but it is likely that the increase in DOPAC in the mutant reflects a decrease in prolactin, which could contribute to the diminished maternal care in the mutants. PMID:10627055

  14. Isolation and characterization of Escherichia coli mutants lacking inducible cyanase.

    PubMed

    Guilloton, M; Karst, F

    1987-03-01

    To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay between lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.

  15. Hydrocarbon assimilation and biosurfactant production in Pseudomonas aeruginosa mutants

    SciTech Connect

    Koch, A.K.; Fiechter, A.; Reiser, J. ); Kaeppeli, O. )

    1991-07-01

    The authors isolated transposon Tn5-GM-induced mutants of Pseudomonas aeruginosa PG201 that were unable to grow in minimal media containing hexadecane as a carbon source. Some of these mutants lacked extracellular rhamnolipids, as shown by measuring the surface and interfacial tensions of the cell culture supernatants. Furthermore, the concentrated culture media of the mutant strains were tested for the presence of rhamnolipids by thin-layer chromatography and for rhamnolipid activities, including hemolysis and growth inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the inhibition of Bacillus subtilis. Mutant 65E12 was unable to produce extracellular rhamnolipids under any of the conditions tested, lacked the capacity to take up {sup 14}C-labeled hexadecane, and did not grow in media containing individual alkanes with chain lengths ranging from C{sub 12} to C{sub 19}. However, growth on these alkanes and uptake of ({sup 14}C)hexadecane were restored when small amounts of purified rhamnolipids were added to the cultures. Mutant 59C7 was unable to grow in media containing hexadecane, nor was it able to take up ({sup 14}C)hexadecane uptake. The addition of small amounts of rhamnolipids restored on alkanes and ({sup 14}C)hexadecane uptake. In glucose-containing media, however, mutant 59C7 produced rhamnolipids at levels about twice as high as those of the wild-type strain. These results show that rhamnolipids play a major role in hexadecane uptake and utilization by P.aeruginosa.

  16. Synthesis of fluorescent phenylethanethiolated gold nanoclusters via pseudo-AGR method.

    PubMed

    Yao, Chuanhao; Tian, Shubo; Liao, Lingwen; Liu, Xinfeng; Xia, Nan; Yan, Nan; Gan, Zibao; Wu, Zhikun

    2015-10-21

    It is well known that the fluorescence of metal nanoclusters is strongly dependent of the protecting ligand and reports of phenylethanethiolated metal nanoclusters with distinct fluorescence are rare. Herein, a fluorescent phenylethanethiolated gold nanocluster is synthesized using an unexpected pseudo-AGR method (AGR: anti-galvanic reduction). The cluster is precisely determined to be Au24(SC2H4Ph)20 by isotope-resolved mass spectroscopy in tandem with thermogravimetric analysis (TGA). The fluorescence comparison between Au24(SC2H4Ph)20, Au25(SC2H4Ph)18, Au38(SC2H4Ph)24 and Au144(SC2H4Ph)60 is also presented. The finding of the fluorescent phenylethanethiolated gold nanocluster in this work has important implication for future study on the fluorescence of metal nanoclusters.

  17. AGR-1 Irradiation Test Final As-Run Report, Rev. 3

    SciTech Connect

    Collin, Blaise P.

    2015-01-01

    This document presents the as-run analysis of the AGR-1 irradiation experiment. AGR-1 is the first of eight planned irradiations for the Advanced Gas Reactor (AGR) Fuel Development and Qualification Program. Funding for this program is provided by the US Department of Energy (DOE) as part of the Next-Generation Nuclear Plant (NGNP) project. The objectives of the AGR-1 experiment are: 1. To gain experience with multi-capsule test train design, fabrication, and operation with the intent to reduce the probability of capsule or test train failure in subsequent irradiation tests. 2. To irradiate fuel produced in conjunction with the AGR fuel process development effort. 3. To provide data that will support the development of an understanding of the relationship between fuel fabrication processes, fuel product properties, and irradiation performance. In order to achieve the test objectives, the AGR-1 experiment was irradiated in the B-10 position of the Advanced Test Reactor (ATR) at the Idaho National Laboratory (INL) for a total duration of 620 effective full power days of irradiation. Irradiation began on December 24, 2006 and ended on November 6, 2009 spanning 13 ATR cycles and approximately three calendar years. The test contained six independently controlled and monitored capsules. Each capsule contained 12 compacts of a single type, or variant, of the AGR coated fuel. No fuel particles failed during the AGR-1 irradiation. Final burnup values on a per compact basis ranged from 11.5 to 19.6 %FIMA, while fast fluence values ranged from 2.21 to 4.39 x 1025 n/m2 (E >0.18 MeV). We’ll say something here about temperatures once thermal recalc is done. Thermocouples performed well, failing at a lower rate than expected. At the end of the irradiation, nine of the originally-planned 19 TCs were considered functional. Fission product release-to-birth (R/B) ratios were quite low. In most capsules, R/B values at the end of the irradiation were at or below

  18. AGR-2 Irradiation Test Final As-Run Report, Rev 2

    SciTech Connect

    Collin, Blaise P.

    2014-08-01

    This document presents the as-run analysis of the AGR-2 irradiation experiment. AGR-2 is the second of the planned irradiations for the Advanced Gas Reactor (AGR) Fuel Development and Qualification Program. Funding for this program is provided by the U.S. Department of Energy as part of the Very High Temperature Reactor (VHTR) Technical Development Office (TDO) program. The objectives of the AGR-2 experiment are to: (a) Irradiate UCO (uranium oxycarbide) and UO2 (uranium dioxide) fuel produced in a large coater. Fuel attributes are based on results obtained from the AGR-1 test and other project activities. (b) Provide irradiated fuel samples for post-irradiation experiment (PIE) and safety testing. (c) Support the development of an understanding of the relationship between fuel fabrication processes, fuel product properties, and irradiation performance. The primary objective of the test was to irradiate both UCO and UO2 TRISO (tri-structural isotropic) fuel produced from prototypic scale equipment to obtain normal operation and accident condition fuel performance data. The UCO compacts were subjected to a range of burnups and temperatures typical of anticipated prismatic reactor service conditions in three capsules. The test train also includes compacts containing UO2 particles produced independently by the United States, South Africa, and France in three separate capsules. The range of burnups and temperatures in these capsules were typical of anticipated pebble bed reactor service conditions. The results discussed in this report pertain only to U.S. produced fuel. In order to achieve the test objectives, the AGR-2 experiment was irradiated in the B-12 position of the Advanced Test Reactor (ATR) at Idaho National Laboratory (INL) for a total irradiation duration of 559.2 effective full power days (EFPD). Irradiation began on June 22, 2010, and ended on October 16, 2013, spanning 12 ATR power cycles and approximately three and a

  19. The Electrogenic Bacterium Shewanella Oneidensis MR-1 and its Mutants with Increased Reducing Capacity

    NASA Astrophysics Data System (ADS)

    Voeikova, T. A.; Emelyanova, L. K.; Novikova, L. M.; Mordkovich, N. N.; Shakulov, R. S.; Debabov, V. G.

    2013-02-01

    Mutants of Shewanella oneidensis MR-1 resistant to fosfomycin, a toxic analogue of phosphoenolpyruvate, were obtained. The mutants exhibited an increased reducing activity and a higher rate of lactate utilization. A correlation was shown between the rates of metabolism of oxidized substrates and the rate of reduction of methylene blue, a mediator of electron transport. The mutants of S.oneidensis MR-1 will be used in microbial fuel cells (MFC) to enhance energy production from organic compounds. The strain S. oneidensis MR-1 and its mutants with an increased electron production will be used as a good source of bioelectricity in MFC in the experiments on the International Space Station.

  20. New Blocking Antibodies against Novel AGR2-C4.4A Pathway Reduce Growth and Metastasis of Pancreatic Tumors and Increase Survival in Mice

    PubMed Central

    Arumugam, Thiruvengadam; Deng, Defeng; Bover, Laura; Wang, Huamin; Logsdon, Craig D.; Ramachandran, Vijaya

    2015-01-01

    Anterior gradient 2 (AGR2) promotes cancer growth, metastasis and resistance to therapy via unknown mechanisms. We investigated the effects of extracellular AGR2 signaling through the orphan GPI-linked receptor C4.4A in pancreatic ductal adenocarcinoma (PDAC). Proliferation, migration and invasion and apoptosis were measured using colorimetric, Boyden chamber, and fluorescence-activated cell sorting analyses. We developed blocking monoclonal antibodies against AGR2 and C4.4A and tested their effects, along with siRNAs, on cancer cell functions and on orthotopic tumors in nude mice. Extracellular AGR2 stimulated proliferation, migration, invasion and chemoresistance of PDAC cell lines. AGR2 interacted with C4.4A in cell lysates and mixtures of recombinant proteins. Knockdown of C4.4A reduced migration and resistance to gemcitabine. PDAC tissues, but not adjacent healthy pancreatic tissues, expressed high levels of AGR2 and C4.4A. AGR2 signaling through C4.4A required laminins 1 or 5 and integrin β1. Administration of antibodies against AGR2 and C4.4A reduced growth and metastasis and caused regression of aggressive xenograft tumors leading to increased survival of mice. These data support a model in which AGR2 binds and signals via C4.4A in an autocrine loop and promotes the growth of pancreas tumors in mice. Blocking monoclonal antibodies against AGR2 and C4.4A may have therapeutic potential against PDAC. PMID:25646014

  1. Status of the NGNP fuel experiment AGR-2 irradiated in the advanced test reactor

    SciTech Connect

    S. Blaine Grover; David A. Petti

    2014-05-01

    The United States Department of Energy's Next Generation Nuclear Plant (NGNP) Advanced Gas Reactor (AGR) Fuel Development and Qualification Program will be irradiating up to seven separate low enriched uranium (LEU) tri-isotopic (TRISO) particle fuel (in compact form) experiments in the Advanced Test Reactor (ATR) located at the Idaho National Laboratory (INL). These irradiations and fuel development are being accomplished to support development of the next generation reactors in the United States, and will be irradiated over the next several years to demonstrate and qualify new TRISO coated particle fuel for use in high temperature gas reactors. The goals of the irradiation experiments are to provide irradiation performance data to support fuel process development, to qualify fuel for normal operating conditions, to support development and validation of fuel performance and fission product transport models and codes, and to provide irradiated fuel and materials for post irradiation examination (PIE) and safety testing. The experiments, which will each consist of at least six separate capsules, will be irradiated in an inert sweep gas atmosphere with individual on-line temperature monitoring and control of each capsule. The sweep gas will also undergo on-line fission product monitoring to track performance of the fuel in each individual capsule during irradiation. The first experiment (designated AGR-1) started irradiation in December 2006 and was completed in November 2009. The second experiment (AGR-2), which utilized the same experiment design as well as control and monitoring systems as AGR-1, started irradiation in June 2010 and is currently scheduled to be completed in April 2013. The design of this experiment and sup

  2. National AgrAbility Project impact on farmers and ranchers with disabilities.

    PubMed

    Meyer, R H; Fetsch, R J

    2006-11-01

    The impact of AgrAbility was evaluated through a survey of farmers and ranchers with disabilities who have been served by AgrAbility. The general demographics of the client population and assistance received were evaluated. Other information gathered included client ability pre- and post-onset of a disability and implications of self-reported outlook for the future. Eight states with AgrAbility programs participated in this cooperative survey with the National AgrAbility Project, with a 58.7% response rate (N = 618). The client population was mostly male (85.2%) with an average age of 53.3 with many working full-time (42.4%), part-time (27.6%), only off-farm (3%), or both off and on the farm (27%) in predominately row-crop (58.2%), cattle (not dairy) (46.6%), and hay or forage (41.4%) operations. Nearly half (48.2%) of the clients reported that the origin of the disability was due to a chronic health condition, as opposed to an injury. The majority of clients reported receiving information referring them to a funding source (42.0%) and receiving technical assistance with modifications around the farm or ranch (41.3%). Only two areas of farm operation were reported to have increased after the onset of disability (farm office from 43.8% to 61.2% and household chores from 30.9% to 36.0%). Field machinery operation continues to be the most common activity on the farm, with 73.3% reporting operating field machinery after the onset of disability. The present sample was more optimistic than expected. From a simultaneous multiple linear regression analysis, the factors contributing to positive future outlook include: ability to manage one's chores, machinery, and farm, F (5, 387) = 34.91, p < 0.001). Implications for safety professionals are included. PMID:17131949

  3. Status of the NGNP Fuel Experiment AGR-2 Irradiated in the Advanced Test Reactor

    SciTech Connect

    Blaine Grover

    2012-10-01

    The United States Department of Energy’s Next Generation Nuclear Plant (NGNP) Advanced Gas Reactor (AGR) Fuel Development and Qualification Program will be irradiating up to seven separate low enriched uranium (LEU) tri-isotopic (TRISO) particle fuel (in compact form) experiments in the Advanced Test Reactor (ATR) located at the Idaho National Laboratory (INL). These irradiations and fuel development are being accomplished to support development of the next generation reactors in the United States, and will be irradiated over the next several years to demonstrate and qualify new TRISO coated particle fuel for use in high temperature gas reactors. The goals of the irradiation experiments are to provide irradiation performance data to support fuel process development, to qualify fuel for normal operating conditions, to support development and validation of fuel performance and fission product transport models and codes, and to provide irradiated fuel and materials for post irradiation examination (PIE) and safety testing. The experiments, which will each consist of at least six separate capsules, will be irradiated in an inert sweep gas atmosphere with individual on-line temperature monitoring and control of each capsule. The sweep gas will also have on-line fission product monitoring on its effluent to track performance of the fuel in each individual capsule during irradiation. The first experiment (designated AGR-1) started irradiation in December 2006 and was completed in November 2009. The second experiment (AGR-2), which utilized the same experiment design as well as control and monitoring systems as AGR-1, started irradiation in June 2010 and is currently scheduled to be completed in April 2013. The design of this experiment and support systems will be briefly discussed, followed by the progress and status of the experiment to date.

  4. Post-irradiation Examination and Fission Product Inventory Analysis of AGR-1 Irradiation Capsules

    SciTech Connect

    J M Harp; P D Demkowicz; S A Ploger

    2012-10-01

    The AGR-1 experiment was the first in a series of Advanced Gas Reactor (AGR) experiments designed to test TRISO fuel under High Temperature Gas Reactor irradiation conditions. This experiment was irradiated in the Advanced Test Reactor (ATR) at Idaho National Laboratory (INL) and is currently undergoing post-irradiation examination (PIE) at INL’s Materials and Fuels Complex (MFC). The inventory and distribution of fission products, especially Ag-110m, was assessed and analyzed for all the components of the AGR-1 capsules. This data should help inform the study of fission product migration in coated particle fuel. Gamma spectrometry was used to measure the activity of various different fission products in the different components of the AGR-1 test train. Each capsule contained: 12 fuel compacts, a graphite holder that kept the fuel compacts in place, graphite spacers that were above and below the graphite holders and fuel compacts, gas lines through which a helium neon gas mixture flowed in and out of each capsule, and the stainless steel shell that contained the experiment. Gamma spectrometry results and the experimental techniques used to capture these results will be presented for all the capsule components. The components were assayed to determine the total activity of different fission products present in or on them. These totals are compared to the total expected activity of a particular fission product in the capsule based on predictions from physics simulation. Based on this metric, a significant fraction of the Ag-110m was detected outside the fuel compacts, but the amount varied highly between the 6 capsules. Very small fractions of Cs-137 (<2E-5), Cs-134 (<1e-5), and Eu-154 (<4e-4) were detected outside of the fuel compacts. Additionally, the distribution of select fission products in some of the components including the fuel compacts and the graphite holders were measured and will be discussed.

  5. Vancomycin modifies the expression of the agr system in multidrug-resistant Staphylococcus aureus clinical isolates

    PubMed Central

    Cázares-Domínguez, Vicenta; Ochoa, Sara A.; Cruz-Córdova, Ariadnna; Rodea, Gerardo E.; Escalona, Gerardo; Olivares, Alma L.; Olivares-Trejo, José de Jesús; Velázquez-Guadarrama, Norma; Xicohtencatl-Cortes, Juan

    2015-01-01

    Staphylococcus aureus is an opportunistic pathogen that colonizes human hosts and causes a wide variety of diseases. Two interacting regulatory systems called agr (accessory gene regulator) and sar (staphylococcal accessory regulator) are involved in the regulation of virulence factors. The aim of this study was to evaluate the effect of vancomycin on hld and spa gene expression during the exponential and post-exponential growth phases in multidrug-resistant (MDR) S. aureus. Methods: Antibiotic susceptibility was evaluated by the standard microdilution method. The phylogenetic profile was obtained by pulsed-field gel electrophoresis (PFGE). Polymorphisms of agr and SCCmec (staphylococcal cassette chromosome mec) were analyzed by multiplex polymerase chain reaction (PCR). The expression levels of hld and spa were analyzed by reverse transcription-PCR. An enzyme-linked immunosorbent assay (ELISA) was performed to detect protein A, and biofilm formation was analyzed via crystal violet staining. Results: In total, 60.60% (20/33) of S. aureus clinical isolates were MDR. Half (10/20) of the MDR S. aureus isolates were distributed in subcluster 10, with >90% similarity among them. In the isolates of this subcluster, a high prevalence (100%) for the agrII and the cassette SCCmec II polymorphisms was found. Our data showed significant increases in hld expression during the post-exponential phase in the presence and absence of vancomycin. Significant increases in spa expression, protein A production and biofilm formation were observed during the post-exponential phase when the MDR S. aureus isolates were challenged with vancomycin. Conclusion: The polymorphism agrII, which is associated with nosocomial isolates, was the most prevalent polymorphism in MDR S. aureus. Additionally, under our study conditions, vancomycin modified hld and spa expression in these clinical isolates. Therefore, vancomycin may regulate alternative systems that jointly participate in the regulation of

  6. The isolation of Staphylococcus aureus tea tree oil-reduced susceptibility mutants.

    PubMed

    Cuaron, Jesus A; Dulal, Santosh; Cooke, Peter H; Torres, Nathanial J; Gustafson, John E

    2014-08-01

    Tea tree oil (TTO)-reduced susceptibility (TTORS) mutants of two Staphylococcus aureus laboratory strains were isolated utilizing TTO gradient plates. Attempts to isolate TTORS mutants employing agar plates containing single TTO concentrations failed. All TTORS mutants demonstrated a small colony variant (SCV) phenotype and produced cells with a smaller diameter, as determined by scanning electron microscopy. The addition of SCV auxotrophic supplements to media did not lead to an increase in TTORS mutant colony size. Revertants were also isolated from the TTORS mutants following growth in drug-free media, and all revertant strains demonstrated phenotypes similar to their respective parent strains. Transmission electron microscopy revealed that an SH1000 TTORS mutant demonstrated a thinner cell wall and novel septal invaginations compared with parent strain SH1000. In addition, comparative genomic sequencing did not reveal any mutations in an SH1000 TTORS mutant previously linked to well-characterized SCV genotypes. This study demonstrates that TTO can select for a unique SCV phenotype.

  7. Anterior Gradient 2 (AGR2) Induced Epidermal Growth Factor Receptor (EGFR) Signaling Is Essential for Murine Pancreatitis-Associated Tissue Regeneration

    PubMed Central

    Wodziak, Dariusz; Dong, Aiwen; Basin, Michael F.; Lowe, Anson W.

    2016-01-01

    A recently published study identified Anterior Gradient 2 (AGR2) as a regulator of EGFR signaling by promoting receptor presentation from the endoplasmic reticulum to the cell surface. AGR2 also promotes tissue regeneration in amphibians and fish. Whether AGR2-induced EGFR signaling is essential for tissue regeneration in higher vertebrates was evaluated using a well-characterized murine model for pancreatitis. The impact of AGR2 expression and EGFR signaling on tissue regeneration was evaluated using the caerulein-induced pancreatitis mouse model. EGFR signaling and cell proliferation were examined in the context of the AGR2-/- null mouse or with the EGFR-specific tyrosine kinase inhibitor, AG1478. In addition, the Hippo signaling coactivator YAP1 was evaluated in the context of AGR2 expression during pancreatitis. Pancreatitis-induced AGR2 expression enabled EGFR translocation to the plasma membrane, the initiation of cell signaling, and cell proliferation. EGFR signaling and tissue regeneration were partially inhibited by the tyrosine kinase inhibitor AG1478, but absent in the AGR2-/- null mouse. AG1478-treated and AGR2-/- null mice with pancreatitis died whereas all wild-type controls recovered. YAP1 activation was also dependent on pancreatitis-induced AGR2 expression. AGR2-induced EGFR signaling was essential for tissue regeneration and recovery from pancreatitis. The results establish tissue regeneration as a major function of AGR2-induced EGFR signaling in adult higher vertebrates. Enhanced AGR2 expression and EGFR signaling are also universally present in human pancreatic cancer, which support a linkage between tissue injury, regeneration, and cancer pathogenesis. PMID:27764193

  8. Characterization of epitopes on the rabies virus glycoprotein by selection and analysis of escape mutants.

    PubMed

    Fallahi, Firouzeh; Wandeler, Alexander I; Nadin-Davis, Susan A

    2016-07-15

    The glycoprotein (G) is the only surface protein of the lyssavirus particle and the only viral product known to be capable of eliciting the production of neutralizing antibodies. In this study, the isolation of escape mutants resistant to monoclonal antibody (Mab) neutralization was attempted by a selection strategy employing four distinct rabies virus strains: the extensively passaged Evelyn Rokitnicki Abelseth (ERA) strain and three field isolates representing two bat-associated variants and the Western Canada skunk variant (WSKV). No escape mutants were generated from either of the bat-associated viral variants but two neutralization mutants were derived from the WSKV isolate. Seven independent ERA mutants were recovered using Mabs directed against antigenic sites I (four mutants) and IIIa (three mutants) of the glycoprotein. The cross-neutralization patterns of these viral mutants were used to determine the precise location and nature of the G protein epitopes recognized by these Mabs. Nucleotide sequencing of the G gene indicated that those mutants derived using Mabs directed to antigenic site (AS) III all contained amino acid substitutions in this site. However, of the four mutants selected with AS I Mabs, two bore mutations within AS I as expected while the remaining two carried mutations in AS II. WSKV mutants exhibited mutations at the sites appropriate for the Mabs used in their selection. All ERA mutant preparations were more cytopathogenic than the parental virus when propagated in cell culture; when in vivo pathogenicity in mice was examined, three of these mutants exhibited reduced pathogenicity while the remaining four mutants exhibited comparable pathogenic properties to those of the parent virus. PMID:27132040

  9. Transposon Mutants of Bradyrhizobium japonicum Altered in Attachment to Host Roots †

    PubMed Central

    Vesper, Stephen J.; Malik, Nasir S. A.; Bauer, Wolfgang D.

    1987-01-01

    Transposon mutants of Bradyrhizobium japonicum 110 ARS were produced and screened for changes in attachment ability. Mutant CFK4 produced twice as many piliated cells, attached in 2.5-fold-higher numbers to soybean root segments, and colonized roots in about 2-fold-higher numbers than did the parental strain, 110 ARS. Mutants CFK35 and CFK38 were reduced in their attachment about 2-fold and 3.5-fold, respectively. This corresponded to reductions in piliated cells in their populations, reduced reaction with anti-pilus antiserum, and reduced hydrophobic attachment. Mutants CFK4 and CFK38 nodulated soybeans at about the same level as the parent strain, but CFK35 induced only pseudonodules. Two-dimensional gel analyses of the proteins from the mutants showed relatively few changes in proteins. PMID:16347421

  10. Genetic transformation assays for identification of strains of Moraxella urethralis.

    PubMed Central

    Juni, E

    1977-01-01

    Studies of 31 strains of Moraxella urethralis have shown that 20 of them are competent for genetic transformation. This finding has led to the development of transformation assays for identification of newly isolated strains of this organism. Crude deoxyribonucleic acid (DNA) samples from all strains of M. urethralis readily transform auxotrophic mutants of competent strains to prototrophy, whereas DNA samples from unrelated bacteria such as Acinetobacter, Moraxella, and Neisseria species uniformly fail to elicit positive transformation of mutant tester strains. One of the competent strains of M. urethralis investigated is a naturally occurring mutant defective in its ability to utilize citrate as a carbon and energy source. DNA samples from 29 of the 30 remaining strains of utilization; the one nonreacting strain is citrate negative and probably possesses the same genetic lesion as the citrate-negative mutant. Three organisms originally identified as strains of M. urethralis, because of their phenotypic properties, are probably incorrectly designated, since DNA samples from these strains failed to transform any of the tester mutant strains used in the present study. The transformation assay for M. urethralis is very simple and can be performed readily in a clinical laboratory. The entire procedure can be carried out in less than 24 h. Images PMID:845247

  11. Selective isolation of Fusarium graminearum mutants depressed for production of beta-glucosidase

    SciTech Connect

    Loureiro-Dias, M.C.

    1982-09-01

    A strain of Fusarium graminearum produced extracellular beta-glucosidase (beta-D-glucoside glucohydrolase (EC 3.2.1.21)) subject to carbon catabolite repression. Depressed mutants was selectively isolated by the use of 2-deoxyglucose, a nonmetabolizable catabolic repressor. On each plate of cellobiose and 2-deoxyglucose inoculated with 10 to the power of 6 spores and irradiated with UV light, a few colonies emerged. Comparative growth experiments with one of the derepressed mutants and the parent strain showed that the mutant produced beta-glucosidase in the presence of glucose. Furthermore, the same mutant produced more beta-glucosidase than did the wild-type strain on cellobiose alone. (Refs. 7).

  12. Selection of the Mutants with High Hydroquinone Degradation Ability of Serratia Marcesscen by Plasma Mutation

    NASA Astrophysics Data System (ADS)

    Yao, Risheng; You, Qidong; He, Weijing; Zhu, Huixia

    2009-06-01

    In this study, an efficient way by plasma induced mutation was applied to improve the hydroquinone degradation capacity of Serratia marcescens AB 90027 (SM27). The results showed that combined with the selection of hydroquinone tolerance, the mutant with high hydroquinone degradation ability induced by plasma could be achieved. The best dose for plasma mutation was 15 s, which showed a 47.0% higher positive mutation ratio. Besides, the aimed mutant was markedly different from the parent strain (SM27) in colonial traits while cultivated on Kings media. Finally, the hydroquinone degradation ratio reached 70.5% using the induced mutant strain with 1500 mg/L hydroquinone (HQ) after 15 days of cultivation as the selective conditions; however, it was only 46.7% for SM27. The improvement of the degradation capacity by the induced mutant with a high concentration of HQ selection was attributed to its faster growth and higher hydroquinone tolerance compared with that of the parent strain.

  13. Natural Strain

    NASA Technical Reports Server (NTRS)

    Freed, Alan D.

    1997-01-01

    Logarithmic strain is the preferred measure of strain used by materials scientists, who typically refer to it as the "true strain." It was Nadai who gave it the name "natural strain," which seems more appropriate. This strain measure was proposed by Ludwik for the one-dimensional extension of a rod with length l. It was defined via the integral of dl/l to which Ludwik gave the name "effective specific strain." Today, it is after Hencky, who extended Ludwik's measure to three-dimensional analysis by defining logarithmic strains for the three principal directions.

  14. Nonchemotactic Mutants of Escherichia coli

    PubMed Central

    Armstrong, John B.; Adler, Julius; Dahl, Margaret M.

    1967-01-01

    We have isolated 40 mutants of Escherichia coli which are nonchemotactic as judged by their failure to swarm on semisolid tryptone plates or to make bands in capillary tubes containing tryptone broth. All the mutants have normal flagella, a fact shown by their shape and reaction with antiflagella serum. All are fully motile under the microscope and all are sensitive to the phage chi. Unlike its parent, one of the mutants, studied in greater detail, failed to show chemotaxis toward oxygen, glucose, serine, threonine, or aspartic acid. The failure to exhibit chemotaxis does not result from a failure to use the chemicals. The swimming of this mutant was shown to be random. The growth rate was normal under several conditions, and the growth requirements were unchanged. Images PMID:5335897

  15. Plaque color method for rapid isolation of novel recA mutants of Escherichia coli K-12: new classes of protease-constitutive recA mutants.

    PubMed Central

    Tessman, E S; Peterson, P

    1985-01-01

    As a prerequisite to mutational analysis of functional sites on the RecA protein of Escherichia coli, a method was developed for rapid isolation of recA mutants with altered RecA protease function. The method involves plating mutagenized lambda recA+ cI ind on strains deleted for recA and containing, as indicators of RecA protease activity, Mu d(Ap lac) fusions in RecA-inducible genes. The lambda recA phages were recognized by their altered plaque colors, and the RecA protease activity of the lambda recA mutant lysogens was measured by expression of beta-galactosidase from dinD::lac. One class of recA mutants had constitutive protease activity and was designated Prtc; in these cells the RecA protein was always in the protease form without the usual need for DNA damage to activate it. Some Prtc mutants were recombinase negative and were designated Prtc Rec-. Another class of 65 recA mutants isolated as being protease defective were all also recombinase defective. Unlike the original temperature-dependent Prtc Rec+ mutant (recA441), the new Prtc Rec+ mutants showed constitutive protease activity at any growth temperature, with some having considerably greater activity than the recA441 strain. Study of these strong Prtc Rec+ mutants revealed a new SOS phenomenon, increased permeability to drugs. Use of this new SOS phenomenon as an index of protease strength clearly distinguished 5 Prtc mutants as the strongest among 150. These five strongest Prtc mutants showed the greatest increase in spontaneous mutation frequency and were not inhibited by cytidine plus guanosine, which inhibited the constitutive protease activity of the recA441 strain and of all the other new Prtc mutants. Strong Prtc Rec+ mutants were more UV resistant than recA+ strains and showed indications of having RecA proteins whose specific activity of recombinase function was higher than that of wild-type RecA. A Prt+ Rec- mutant with an anomalous response to effectors is described. PMID:3160686

  16. Uncertainty Quantification of Calculated Temperatures for the U.S. Capsules in the AGR-2 Experiment

    SciTech Connect

    Lybeck, Nancy; Einerson, Jeffrey J.; Pham, Binh T.; Hawkes, Grant L.

    2015-03-01

    A series of Advanced Gas Reactor (AGR) irradiation experiments are being conducted within the Advanced Reactor Technology (ART) Fuel Development and Qualification Program. The main objectives of the fuel experimental campaign are to provide the necessary data on fuel performance to support fuel process development, qualify a fuel design and fabrication process for normal operation and accident conditions, and support development and validation of fuel performance and fission product transport models and codes (PLN-3636). The AGR-2 test was inserted in the B-12 position in the Advanced Test Reactor (ATR) core at Idaho National Laboratory (INL) in June 2010 and successfully completed irradiation in October 2013, resulting in irradiation of the TRISO fuel for 559.2 effective full power days (EFPDs) during approximately 3.3 calendar years. The AGR-2 data, including the irradiation data and calculated results, were qualified and stored in the Nuclear Data Management and Analysis System (NDMAS) (Pham and Einerson 2014). To support the U.S. TRISO fuel performance assessment and to provide data for validation of fuel performance and fission product transport models and codes, the daily as-run thermal analysis has been performed separately on each of four AGR-2 U.S. capsules for the entire irradiation as discussed in (Hawkes 2014). The ABAQUS code’s finite element-based thermal model predicts the daily average volume-average fuel temperature and peak fuel temperature in each capsule. This thermal model involves complex physical mechanisms (e.g., graphite holder and fuel compact shrinkage) and properties (e.g., conductivity and density). Therefore, the thermal model predictions are affected by uncertainty in input parameters and by incomplete knowledge of the underlying physics leading to modeling assumptions. Therefore, alongside with the deterministic predictions from a set of input thermal conditions, information about prediction uncertainty is instrumental for the ART

  17. Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena.

    PubMed

    Rojas-Rejón, Oscar A; Poggi-Varaldo, Héctor M; Ramos-Valdivia, Ana C; Martínez-Jiménez, Alfredo; Cristiani-Urbina, Eliseo; de la Torre Martínez, Mayra; Ponce-Noyola, Teresa

    2011-01-01

    Derepressed mutant PR-22 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg(-1) and cellulolytic activity from 1.9 to 4 U mg(-1); this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20-32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l(-1) of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.

  18. Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile

    PubMed Central

    Odo, Chioma; DuPont, Herbert L.

    2016-01-01

    ABSTRACT Clostridium difficile infection (CDI) is responsible for most of the definable cases of antibiotic- and hospital-associated diarrhea worldwide and is a frequent cause of morbidity and mortality in older patients. C. difficile, a multidrug-resistant anaerobic pathogen, causes disease by producing toxins A and B, which are controlled by an accessory gene regulator (Agr) quorum signaling system. Some C. difficile strains encode two Agr loci in their genomes, designated agr1 and agr2. The agr1 locus is present in all of the C. difficile strains sequenced to date, whereas the agr2 locus is present in a few strains. The functional roles of agr1 and agr2 in C. difficile toxin regulation and pathogenesis were unknown until now. Using allelic exchange, we deleted components of both agr loci and examined the mutants for toxin production and virulence. The results showed that the agr1 mutant cannot produce toxins A and B; toxin production can be restored by complementation with wild-type agr1. Furthermore, the agr1 mutant is able to colonize but unable to cause disease in a murine CDI model. These findings have profound implications for CDI treatment because we have uncovered a promising therapeutic target for the development of nonantibiotic drugs to treat this life-threatening emerging pathogen by targeting the toxins directly responsible for disease. PMID:27531912

  19. Repressor mutant forms of the Azospirillum brasilense NtrC protein.

    PubMed

    Huergo, Luciano F; Assumpção, Marcelo C; Souza, Emanuel M; Steffens, M Berenice R; Yates, M Geoffrey; Chubatsu, Leda S; Pedrosa, Fábio O

    2004-10-01

    The Azospirillum brasilense mutant strains FP8 and FP9, after treatment with nitrosoguanidine, showed a null Nif phenotype and were unable to use nitrate as their sole nitrogen source. Sequencing of the ntrC genes revealed single nucleotide mutations in the NtrC nucleotide-binding site. The phenotypes of these strains are discussed in relation to their genotypes.

  20. Mutants of Saccharomyces cerevisiae with defective vacuolar function

    SciTech Connect

    Kitamoto, K.; Yoshizawa, K.; Ohsumi, Y.; Anraku, Y.

    1988-06-01

    Mutants of the yeast Saccharomyces cerevisiae that have a small vacuolar lysine pool were isolated and characterized. Mutant KL97 (lys1 slp1-1) and strain KL197-1A (slp1-1), a prototrophic derivative of KL97, did not grow well in synthetic medium supplemented with 10 mM lysine. Genetic studies indicated that the slp1-1mutation (for small lysine pool) is recessive and is due to a single chromosomal mutation. Mutant KL97 shows the following pleiotropic defects in vacuolar functions. (i) It has small vacuolar pools for lysine, arginine, and histidine. (ii) Its growth is sensitive to lysine, histidine, Ca/sup 2 +/, heavy metal ions, and antibiotics. (iii) It has many small vesicles but no large central vacuole. (iv) It has a normal amount of the vacuolar membrane marker ..cap alpha..-mannosidase but shows reduced activities of the vacuole sap markers proteinase A, proteinase B, and carboxypeptidase Y.

  1. Isolation of pigmentation mutants of the green filamentous photosynthetic bacterium Chloroflexus aurantiacus.

    PubMed

    Pierson, B K; Keith, L M; Leovy, J G

    1984-07-01

    Mutants deficient in the production of bacteriochlorophyll c (Bchl c) and one mutant lacking colored carotenoids were isolated from the filamentous gliding bacterium Chloroflexus aurantiacus. Mutagenesis was achieved by using UV radiation or N-methyl-N'-nitro-N-nitrosoguanidine. Several clones were isolated that were deficient in Bchl c synthesis. All reverted. One double mutant deficient both in Bchl c synthesis and in the synthesis of colored carotenoids under anaerobic conditions was isolated. Isolation of a revertant in Bchl c synthesis from this double mutant produced a mutant strain of Chloroflexus that grew photosynthetically under anaerobic conditions and lacked colored carotenoids. Analysis of pigment contents and growth rates of the mutants revealed a positive association between growth rate and content of Bchl c under light-limiting conditions.

  2. A photorespiratory mutant of Chlamydomonas reinhardtii

    SciTech Connect

    Suzuki, K.; Marek, L.F.; Spalding, M.H. )

    1990-05-01

    A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO{sub 2} concentration for photoautrophic growth in spite of the apparent induction of a functional CO{sub 2} concentrating mechanism in air-adapted cells. In 2% O{sub 2} the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O{sub 2}, photosynthetic O{sub 2} evolution is severely inhibited in the mutant by preillumination in limiting CO{sub 2}, although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O{sub 2} evolution measurements. Net CO{sub 2} uptake is also inhibited when the cells are exposed to air (21% O{sub 2}, 0.035% CO{sub 2}, balance N{sub 2}) for longer than a few minutes. ({sup 14}C)Phosphoglycolate accumulates within 5 minutes of photosynthetic {sup 14}CO{sub 2} fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO{sub 2} requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicate that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.

  3. Manifestations de la transition solide-liquide dans les agrégats

    NASA Astrophysics Data System (ADS)

    Calvo, F.

    The thermodynamics of clusters is a subject of increasing interest, both from the theoretical and experimental points of view. We present a set of theoretical and numerical methods for studying phase transitions, especially the solid-liquid transition, in atomic or molecular clusters. Several means of characterization (thermodynamical, geometrical and dynamical) are introduced, and the differences between the finite-size behaviour and the bulk behaviour are described. The phenomenon of "dynamical coexistence", which can be seen as the cluster spontaneously going back and forth between the solid and liquid states across the time, is illustrated. Its thermodynamical consequences are also emphasized. Finally, we present some typical thermodynamical phenomena due to the finite-size character, and strongly dependent on the physical chemistry of the matter at the microscopic level. We study these phenomena with Monte Carlo and molecular dynamics simulations: surface melting observed on rare gas clusters, multistage melting observed on ionic clusters, the special behaviours of molecular clusters, and the more intricate problem of free or solvated metallic clusters. La thermodynamique des agrégats est un sujet en développement croissant, tant sur le plan théorique qu'expérimental. Nous présentons un ensemble de méthodes théoriques et numériques destinées à l'étude des transitions de phase, en particulier de la transition solide-liquide, dans les agrégats atomiques et moléculaires. Divers moyens de caractérisation (thermodynamiques, géométriques et dynamiques) sont introduits, et les différences entre les comportements à taille finie et à taille macroscopique sont précisées. Le phénomène de "coexistence dynamique", qui voit l'agrégat passer spontanément de l'état solide à l'état liquide (et réciproquement) au cours du temps, est illustré, et ses conséquences thermodynamiques sont soulignées. Enfin, nous présentons un certain nombre de ph

  4. A ClpB chaperone knockout mutant of Mesorhizobium ciceri shows a delay in the root nodulation of chickpea plants.

    PubMed

    Brígido, Clarisse; Robledo, Marta; Menéndez, Esther; Mateos, Pedro F; Oliveira, Solange

    2012-12-01

    Several molecular chaperones are known to be involved in bacteria stress response. To investigate the role of chaperone ClpB in rhizobia stress tolerance as well as in the rhizobia-plant symbiosis process, the clpB gene from a chickpea microsymbiont, strain Mesorhizobium ciceri LMS-1, was identified and a knockout mutant was obtained. The ClpB knockout mutant was tested to several abiotic stresses, showing that it was unable to grow after a heat shock and it was more sensitive to acid shock than the wild-type strain. A plant-growth assay performed to evaluate the symbiotic performance of the clpB mutant showed a higher proportion of ineffective root nodules obtained with the mutant than with the wild-type strain. Nodulation kinetics analysis showed a 6- to 8-day delay in nodule appearance in plants inoculated with the ΔclpB mutant. Analysis of nodC gene expression showed lower levels of transcript in the ΔclpB mutant strain. Analysis of histological sections of nodules formed by the clpB mutant showed that most of the nodules presented a low number of bacteroids. No differences in the root infection abilities of green fluorescent protein-tagged clpB mutant and wild-type strains were detected. To our knowledge, this is the first study that presents evidence of the involvement of the chaperone ClpB from rhizobia in the symbiotic nodulation process.

  5. Autolysis of Microbial Cells: Salt Activation of Autolytic Enzymes in a Mutant of Staphylococcus aureus

    PubMed Central

    Gilpin, Richard W.; Chatterjee, Anadi N.; Young, Frank E.

    1972-01-01

    The effect of various salts on the autolysis of cell wall of a ribitol teichoic acid-deficient mutant of Staphylococcus aureus H (strain 52A5 carrying tar-1) was compared with the parent strain. In the presence of high concentrations of certain salts such as 1.0 m NaCl, the mutant undergoes autolysis with the release of osmotically sensitive spheroplasts. The parent strain is not affected by these conditions. The stimulation of lysis is related to an activation of N-acylmuramyl-l-alanine amidase. Images PMID:4591480

  6. Natural Strain

    NASA Technical Reports Server (NTRS)

    Freed, Alan D.

    1995-01-01

    The purpose of this paper is to present a consistent and thorough development of the strain and strain-rate measures affiliated with Hencky. Natural measures for strain and strain-rate, as I refer to them, are first expressed in terms of of the fundamental body-metric tensors of Lodge. These strain and strain-rate measures are mixed tensor fields. They are mapped from the body to space in both the Eulerian and Lagrangian configurations, and then transformed from general to Cartesian fields. There they are compared with the various strain and strain-rate measures found in the literature. A simple Cartesian description for Hencky strain-rate in the Lagrangian state is obtained.

  7. Draft Genome Sequence of Neurospora crassa Strain FGSC 73

    DOE PAGES

    Baker, Scott E.; Schackwitz, Wendy; Lipzen, Anna; Martin, Joel; Haridas, Sajeet; LaButti, Kurt; Grigoriev, Igor V.; Simmons, Blake A.; McCluskey, Kevin

    2015-04-02

    We report the elucidation of the complete genome of the Neurospora crassa (Shear and Dodge) strain FGSC 73, a mat-a, trp-3 mutant strain. The genome sequence around the idiotypic mating type locus represents the only publicly available sequence for a mat-a strain. 40.42 Megabases are assembled into 358 scaffolds carrying 11,978 gene models.

  8. [Effect of Mutations in Extracellular Nuclease on the Characteristics of the Pigmented and Nonpigmented Serratia marcescens Strains].

    PubMed

    Nizamutdinova, E Kh; Shirshikova, T V; Mardanova, A M; Sharipova, M R; Bogomol'naya, L M

    2016-01-01

    Comparative characterization of the pigmented and nonpigmented Serratia marcescens strains and their extracellular nuclease mutants was carried out. Biomass accumulation by the mutant strains decreased on average by 20%, while proteolytic activity of the culture liquid was 4-5 times lower than in the case of the wild type strains. The mutants with impaired extracellular nuclease genes exhibited higher sensitivity to reactive oxygen species. Comparative analysis of motility of the strains revealed the highest flagellar activity in the wild type nonpigmented strain, while the cells of its mutant completely lost this feature.

  9. Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

    NASA Astrophysics Data System (ADS)

    Wang, Hua; Zhang, Jian; Yang, Fan; Wang, Kai; Shen, Si-Le; Liu, Bing-Bing; Zou, Bo; Zou, Guang-Tian

    2011-01-01

    The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

  10. Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

    PubMed

    Klinsupa, Worawan; Phansiri, Salak; Thongpradis, Panida; Yongsmith, Busaba; Pothiratana, Chetsada

    2016-01-10

    To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph). PMID:26562446

  11. Enhancement of yellow pigment production by intraspecific protoplast fusion of Monascus spp. yellow mutant (ade(-)) and white mutant (prototroph).

    PubMed

    Klinsupa, Worawan; Phansiri, Salak; Thongpradis, Panida; Yongsmith, Busaba; Pothiratana, Chetsada

    2016-01-10

    To breed industrially useful strains of a slow-growing, yellow pigment producing strain of Monascus sp., protoplasts of Monascus purpureus yellow mutant (ade(-)) and rapid-growing M. purpureus white mutant (prototroph) were fused and fusants were selected on minimal medium (MM). Preliminary conventional protoplast fusion of the two strains was performed and the result showed that only white colonies were detected on MM. It was not able to differentiate the fusants from the white parental prototroph. To solve this problem, the white parental prototroph was thus pretreated with 20mM iodoacetamide (IOA) for cytoplasm inactivation and subsequently taken into protoplast fusion with slow-growing Monascus yellow mutant. Under this development technique, only the fusants, with viable cytoplasm from Monascus yellow mutant (ade(-)), could thus grow on MM, whereas neither IOA pretreated white parental prototroph nor yellow auxotroph (ade(-)) could survive. Fifty-three fusants isolated from yellow colonies obtained through this developed technique were subsequently inoculated on complete medium (MY agar). Fifteen distinguished yellow colonies from their parental yellow mutant were then selected for biochemical, morphological and fermentative properties in cassava starch and soybean flour (SS) broth. Finally, three most stable fusants (F7, F10 and F43) were then selected and compared in rice solid culture. Enhancement of yellow pigment production over the parental yellow auxotroph was found in F7 and F10, while enhanced glucoamylase activity was found in F43. The formation of fusants was further confirmed by monacolin K content, which was intermediate between the two parents (monacolin K-producing yellow auxotroph and non-monacolin K producing white prototroph).

  12. Bacteriophage-insensitive mutants for high quality Crescenza manufacture

    PubMed Central

    Chirico, Donatella; Gorla, Arianna; Verga, Viola; Pedersen, Per D.; Polgatti, Eliseo; Cava, Antonio; Dal Bello, Fabio

    2014-01-01

    Streptococcus thermophilus is a thermophilic lactic acid bacterium used as starter culture for the manufacture of fermented dairy products. For the production of Crescenza and other soft cheeses, Sacco has developed and provides dairies with three different defined blends of S. thermophilus strains. Each blend contains two different S. thermophilus strains. The strains were selected based on their unique technological properties as well as different phage profiles. Analysis of 133 whey samples collected in 2009–2010 from Italian dairies showed a high prevalence (about 50%) of bacteriophage attacks on the blend ST020. More specifically, the strain S. thermophilus ST1A was found to be the preferred target of the bacteriophages. A bacteriophage insensitive mutant (BIM5) of the phage-sensitive strain ST1A was successfully developed and used to substitute strain ST1A in the Crescenza starter culture ST020. The strain BIM5 showed identical technological and industrial traits as those of the phage-sensitive strain ST1A. The improved resistance of the modified Crescenza starter culture ST020R was confirmed at Italian dairies, and its effectiveness monitored on 122 whey samples collected in 2011–2012. Compared to the previous values (2009–2010), the use of the phage-hardened blend ST020R allowed reducing of frequency of phage attacks from about 50 to less than 5% of the whey samples investigated. PMID:24834065

  13. Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase

    SciTech Connect

    Mat-Jan, F.; Alam, K.Y.; Clark, D.P. )

    1989-01-01

    Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.

  14. Constitutively cellulose-producing mutant of trichoderma reesei

    SciTech Connect

    Mishra, S.; Gopalkrishnan, K.S.; Ghose, T.K.

    1982-01-01

    The single major factor limiting the economy of enzymatic hydrolysis of cellulose is the cost of the cellulase enzymes. Most of the work to date in reducing this cost has centered around selection of hyperproducing strains using the standard cellulose medium. Cellulose is an insoluble substrate with obvious design disadvantages in bioreactors. It is often indicated that another reasonable approach to the problem would be the isolation of constitutive mutants which require no inducer to form cellulase. In fact, the goal of several laboratories around the world working on the hydrolysis of cellulosic substances has been to isolate a constitutive mutant. Reported here is the isolation of a constitutively cellulase producing mutant in T. reesei that produces high levels of all of the component enzymes in the cellulase complex in the presence of glucose or sucrose. With this constitutive mutant, it is hoped that a major hurdle of working with an insoluble substrate will be overcome. The mutant will allow easier operation and control of cellulose bioreactors and can be used for continuous enzyme production, as opposed to the present batch production system. Also, a number of other unusable susbtrates like whey and molasses can be used for commercial preparations of cellulases. (Refs. 2).

  15. Identification of a Mutant PfCRT-Mediated Chloroquine Tolerance Phenotype in Plasmodium falciparum

    PubMed Central

    Valderramos, Stephanie G.; Valderramos, Juan-Carlos; Musset, Lise; Purcell, Lisa A.; Mercereau-Puijalon, Odile; Legrand, Eric; Fidock, David A.

    2010-01-01

    Mutant forms of the Plasmodium falciparum transporter PfCRT constitute the key determinant of parasite resistance to chloroquine (CQ), the former first-line antimalarial, and are ubiquitous to infections that fail CQ treatment. However, treatment can often be successful in individuals harboring mutant pfcrt alleles, raising questions about the role of host immunity or pharmacokinetics vs. the parasite genetic background in contributing to treatment outcomes. To examine whether the parasite genetic background dictates the degree of mutant pfcrt-mediated CQ resistance, we replaced the wild type pfcrt allele in three CQ-sensitive strains with mutant pfcrt of the 7G8 allelic type prevalent in South America, the Oceanic region and India. Recombinant clones exhibited strain-dependent CQ responses that ranged from high-level resistance to an incremental shift that did not meet CQ resistance criteria. Nonetheless, even in the most susceptible clones, 7G8 mutant pfcrt enabled parasites to tolerate CQ pressure and recrudesce in vitro after treatment with high concentrations of CQ. 7G8 mutant pfcrt was found to significantly impact parasite responses to other antimalarials used in artemisinin-based combination therapies, in a strain-dependent manner. We also report clinical isolates from French Guiana that harbor mutant pfcrt, identical or related to the 7G8 haplotype, and manifest a CQ tolerance phenotype. One isolate, H209, harbored a novel PfCRT C350R mutation and demonstrated reduced quinine and artemisinin susceptibility. Our data: 1) suggest that high-level CQR is a complex biological process dependent on the presence of mutant pfcrt; 2) implicate a role for variant pfcrt alleles in modulating parasite susceptibility to other clinically important antimalarials; and 3) uncover the existence of a phenotype of CQ tolerance in some strains harboring mutant pfcrt. PMID:20485514

  16. Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants

    PubMed Central

    Gomes, Cláudia; Martínez-Puchol, Sandra; Ruiz-Roldán, Lidia; Pons, Maria J.; del Valle Mendoza, Juana; Ruiz, Joaquim

    2016-01-01

    The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-β-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62–65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments. PMID:27667026

  17. Characterization of lipopolysaccharides from Escherichia coli K-12 mutants.

    PubMed Central

    Boman, H G; Monner, D A

    1975-01-01

    Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4. Images PMID:1089628

  18. AGR-3/4 Final Data Qualification Report for ATR Cycles 151A through 155B-1

    SciTech Connect

    Pham, Binh T.

    2015-03-01

    This report provides the qualification status of experimental data for the entire Advanced Gas Reactor 3/4 (AGR 3/4) fuel irradiation. AGR-3/4 is the third in a series of planned irradiation experiments conducted in the Advanced Test Reactor (ATR) at Idaho National Laboratory (INL) for the AGR Fuel Development and Qualification Program, which supports development of the advanced reactor technology under the INL ART Technology Development Office (TDO). The main objective of AGR-3/4 irradiation is to provide a known source of fission products for subsequent transport through compact matrix and structural graphite materials due to the presence of designed-to-fail fuel particles. Full power irradiation of the AGR 3/4 test began on December 14, 2011 (ATR Cycle 151A), and was completed on April 12, 2014 (end of ATR Cycle 155B) after 369.1 effective full power days of irradiation. The AGR-3/4 test was in the reactor core for eight of the ten ATR cycles between 151A and 155B. During the unplanned outage cycle, 153A, the experiment was removed from the ATR northeast flux trap (NEFT) location and stored in the ATR canal. This was to prevent overheating of fuel compacts due to higher than normal ATR power during the subsequent Powered Axial Locator Mechanism cycle, 153B. The AGR 3/4 test was inserted back into the ATR NEFT location during the outage of ATR Cycle 154A on April 26, 2013. Therefore, the AGR-3/4 irradiation data received during these 2 cycles (153A and 153B) are irrelevant and their qualification status isnot included in this report. Additionally, during ATR Cycle 152A the ATR core ran at low power for a short enough duration that the irradiation data are not used for physics and thermal calculations. However, the qualification status of irradiation data for this cycle is still covered in this report. As a result, this report includes data from 8 ATR Cycles: 151A, 151B, 152A, 152B, 154A, 154B, 155A, and 155B, as recorded in the Nuclear Data Management and

  19. Survival, growth, and localization of epiphytic fitness mutants of pseudomonas syringae on leaves

    SciTech Connect

    Beattie, G.A.; Lindow, S.E. )

    1994-10-01

    Among 82 epiphytic fitness mutants of a Pseudomonas syringae pv. syringae strain that were characterized in a previous study, 4 mutants were particularly intolerant of the stresses associated with dry leaf surfaces. These four mutants each exhibited distinctive behaviors when inoculated into and into plant leaves. For example, while non showed measurable growth on dry potato leaf surfaces, they grew to different population sizes in the intercellular space of bean leaves and on dry bean leaf surfaces, and one mutant appeared incapable of growth in both environments although it grew well on moist bean leaves. The presence of the parental strain did not influence the survival of the mutants immediately following exposure of leaves to dry, high-light incubation conditions, suggesting that the reduced survival of the mutants did not result from an inability to produce extracellular factors in planta. On moist bean leaves that were colonized by either a mutant or the wild type, the proportion of the total epiphytic population that was located in sizes protected from a surface sterilant was smaller for the mutants than for the wild type, indicating that the mutants were reduced in their ability to locate, multiply in, and/or survive in such protected sites. This reduced ability was only one of possible several factors contributing to the reduced epiphytic fitness of each mutant. Their reduced fitness was not specific to the host plant bean, since they also exhibited reduced fitness on the nonhost plant potato; the functions altered in these strains are thus of interest for their contribution to the general fitness of bacterial epiphytes. 52 refs., 6 figs., 1 tab.

  20. A DNA adenine methylase mutant of Shigella flexneri shows no significant attenuation of virulence.

    PubMed

    Honma, Yasuko; Fernández, Reinaldo E; Maurelli, Anthony T

    2004-04-01

    Mutants of Salmonella defective in DNA adenine methylase (dam) have been reported to be attenuated for virulence and to provide protective immunity when used as vaccine strains. To determine whether these observations could be extended to Shigella, a dam mutant of Shigella flexneri 2a was characterized and examined for the role of dam in pathogenesis. The Shigella dam mutant showed some unique characteristics; however, it retained virulence in vivo as well as in vitro. The mutant invaded cultured L2 monolayer cells as efficiently as the wild-type parent, but its intracellular growth was suppressed up to 7 h post-invasion. Furthermore, the invading dam mutant formed smaller plaques in cell monolayers compared to the parent strain. However, the mutant produced keratoconjunctivitis in the Sereny test in guinea pigs only slightly more slowly than the wild-type. While the effect of the dam mutation on virulence was modest, the rate of spontaneous mutation in the dam mutant was 1000-fold greater compared with the wild-type. The virulence and high mutability displayed by the dam mutant of Sh. flexneri suggest that a general anti-bacterial pathogen vaccine strategy based on mutations in dam needs to be re-evaluated.

  1. AGR-2 Data Qualification Report for ATR Cycles 147A, 148A, 148B, and 149A

    SciTech Connect

    Michael L. Abbott; Keith A. Daum

    2011-08-01

    This report presents the data qualification status of fuel irradiation data from the first four reactor cycles (147A, 148A, 148B, and 149A) of the on-going second Advanced Gas Reactor (AGR-2) experiment as recorded in the NGNP Data Management and Analysis System (NDMAS). This includes data received by NDMAS from the period June 22, 2010 through May 21, 2011. AGR-2 is the second in a series of eight planned irradiation experiments for the AGR Fuel Development and Qualification Program, which supports development of the very high temperature gas-cooled reactor (VHTR) under the Next Generation Nuclear Plant (NGNP) Project. Irradiation of the AGR-2 test train is being performed at the Advanced Test Reactor (ATR) at the Idaho National Laboratory (INL) and is planned for 600 effective full power days (approximately 2.75 calendar years) (PLN-3798). The experiment is intended to demonstrate the performance of UCO (uranium oxycarbide) and UO2 (uranium dioxide) fuel produced in a large coater. Data qualification status of the AGR-1 experiment was reported in INL/EXT-10-17943 (Abbott et al. 2010).

  2. Isolation of an R- M+ mutant of Yersinia enterocolitica serotype O:8 and its application in construction of rough mutants utilizing mini-Tn5 derivatives and lipopolysaccharide-specific phage.

    PubMed Central

    Zhang, L; Skurnik, M

    1994-01-01

    A generally applicable procedure was used to isolate a spontaneous restriction-deficient mutant of Yersinia enterocolitica serotype O:8. Transposition frequency in the mutant strain 8081-res was approximately 6.7 x 10(-6) per recipient, while it was practically zero in the wild-type strain 8081-c. Mobilization frequency into 8081-res was 10(5) times higher than that into the wild-type strain. The mutant had lost the ability to express the YenI restriction endonuclease activity present in serotype O:8 strains. This allowed the construction of a transposon library in 8081-res. Insertion mutants with transposons in the genes of the rfa region were selected from this library. Images PMID:8132471

  3. Gravitropism of inflorescence stems in starch-deficient mutants of Arabidopsis

    NASA Technical Reports Server (NTRS)

    Weise, S. E.; Kiss, J. Z.

    1999-01-01

    Previous studies have assayed the gravitropic response of roots and hypocotyls of wild type Arabidopsis thaliana, two reduced-starch strains, and a starchless strain. Because there have been few reports on inflorescence gravitropism, in this article, we use microscopic analyses and time-course studies of these mutants and their wild type to study gravitropism in these stems. Sedimentation of plastids was observed in endodermal cells of the wild type and reduced-starch mutants but not in the starchless mutant. In all of these strains, the short inflorescence stems (1.0-2.9 cm) were less responsive to the gravistimulus compared with the long stems (3.0-6.0 cm). In both long and short inflorescence stems, the wild type initially had the greatest response; the starchless mutant had the least response; and the reduced starch mutants exhibited an intermediate response. Furthermore, growth rates among all four strains were approximately equal. At about 6 h after reorientation, inflorescences of all strains returned to a position parallel to the gravity vector. Thus, in inflorescence stems, sedimentation of plastids may act as an accelerator but is not required to elicit a gravitropic response. Furthermore, the site of perception appears to be diffuse throughout the inflorescence stem. These results are consistent with both a plastid-based statolith model and the protoplast pressure hypothesis, and it is possible that multiple systems for gravity perception occur in plant cells.

  4. Isolation and characterization of nonspreading mutants of the gliding bacterium Cytophaga johnsonae.

    PubMed Central

    Chang, L E; Pate, J L; Betzig, R J

    1984-01-01

    Three approaches were taken to isolate a total of 153 nonspreading mutants derived from our laboratory strain of Cytophaga johnsonae, UW101, or from its auxotrophic derivative, UW10538. Characterization of 109 of these mutants led to their placement in five general categories: (i) motile, nonspreading (MNS) mutants whose cells are motile to various degrees but whose colonies fail to spread on agar gels under any conditions of incubation; (ii) conditional nonspreading (CNS) mutants with motile cells whose colonies require more moisture to spread on agar gels than do those of wild-type cells; (iii) filamentous conditional motility (FCM) mutants whose cells grow as nonmotile filaments or as motile cells with wild-type morphology, depending on conditions of incubation; (iv) short, tumbling, nonspreading (STN) mutants with short cells that tumble constantly; and (v) truly nonmotile (TNM) mutants whose cells never move and whose colonies never spread under any conditions tested. All TNM mutants exhibited a remarkable pleiotropy not seen in the other four classes of mutants: all were resistant to 39 phages to which wild-type cells are sensitive, and all were unable to digest chitin, which is digested by wild-type cells. The correlation between ability to move and phage sensitivity was strengthened further by showing that 150 additional TNM mutants derived from UW101 and 43 TNM mutants derived from 29 independent isolates of C. johnsonae were resistant to all phages to which their parents were sensitive. Furthermore, motile revertants of TNM mutants became phage sensitive, and temperature-sensitive mutants were motile and phage sensitive at 25 degrees C and nonmotile and phage resistant at 32 degrees C. Evidence supports the conclusion that any mutation rendering cells truly nonmotile invariably alters cell surface-associated properties such as phage sensitivity and chitin digestion merely as a consequence of changing a moving cell surface to a static surface. Images PMID

  5. Quality Assurance Program Plan for AGR Fuel Development and Qualification Program

    SciTech Connect

    W. Ken Sowder

    2004-02-01

    Quality Assurance Plan (QPP) is to document the Idaho National Engineering and Environmental Laboratory (INEEL) Management and Operating (M&O) Contractor’s quality assurance program for AGR Fuel Development and Qualification activities, which is under the control of the INEEL. The QPP is an integral part of the Gen IV Program Execution Plan (PEP) and establishes the set of management controls for those systems, structures and components (SSCs) and related quality affecting activities, necessary to provide adequate confidence that items will perform satisfactorily in service.

  6. Synthesis of fluorescent phenylethanethiolated gold nanoclusters via pseudo-AGR method

    NASA Astrophysics Data System (ADS)

    Yao, Chuanhao; Tian, Shubo; Liao, Lingwen; Liu, Xinfeng; Xia, Nan; Yan, Nan; Gan, Zibao; Wu, Zhikun

    2015-10-01

    It is well known that the fluorescence of metal nanoclusters is strongly dependent of the protecting ligand and reports of phenylethanethiolated metal nanoclusters with distinct fluorescence are rare. Herein, a fluorescent phenylethanethiolated gold nanocluster is synthesized using an unexpected pseudo-AGR method (AGR: anti-galvanic reduction). The cluster is precisely determined to be Au24(SC2H4Ph)20 by isotope-resolved mass spectroscopy in tandem with thermogravimetric analysis (TGA). The fluorescence comparison between Au24(SC2H4Ph)20, Au25(SC2H4Ph)18, Au38(SC2H4Ph)24 and Au144(SC2H4Ph)60 is also presented. The finding of the fluorescent phenylethanethiolated gold nanocluster in this work has important implication for future study on the fluorescence of metal nanoclusters.It is well known that the fluorescence of metal nanoclusters is strongly dependent of the protecting ligand and reports of phenylethanethiolated metal nanoclusters with distinct fluorescence are rare. Herein, a fluorescent phenylethanethiolated gold nanocluster is synthesized using an unexpected pseudo-AGR method (AGR: anti-galvanic reduction). The cluster is precisely determined to be Au24(SC2H4Ph)20 by isotope-resolved mass spectroscopy in tandem with thermogravimetric analysis (TGA). The fluorescence comparison between Au24(SC2H4Ph)20, Au25(SC2H4Ph)18, Au38(SC2H4Ph)24 and Au144(SC2H4Ph)60 is also presented. The finding of the fluorescent phenylethanethiolated gold nanocluster in this work has important implication for future study on the fluorescence of metal nanoclusters. Electronic supplementary information (ESI) available: TEM monitoring of the reaction process, digital photos of Au24 and Au25 under visible and UV light, MALDI-MS spectra of Au25, intermediate product, and Au24, fluorescence decay profiles of Au24, Au25, Au38 and Au144 and photobleaching curve of Au24(SC2H4Ph)20. See DOI: 10.1039/c5nr04760a

  7. Nox2 Modification of LDL Is Essential for Optimal Apolipoprotein B-mediated Control of agr Type III Staphylococcus aureus Quorum-sensing

    PubMed Central

    Hall, Pamela R.; Elmore, Bradley O.; Spang, Cynthia H.; Alexander, Susan M.; Manifold-Wheeler, Brett C.; Castleman, Moriah J.; Daly, Seth M.; Peterson, M. Michal; Sully, Erin K.; Femling, Jon K.; Otto, Michael; Horswill, Alexander R.; Timmins, Graham S.; Gresham, Hattie D.

    2013-01-01

    Staphylococcus aureus contains an autoinducing quorum-sensing system encoded within the agr operon that coordinates expression of virulence genes required for invasive infection. Allelic variation within agr has generated four agr specific groups, agr I–IV, each of which secretes a distinct autoinducing peptide pheromone (AIP1-4) that drives agr signaling. Because agr signaling mediates a phenotypic change in this pathogen from an adherent colonizing phenotype to one associated with considerable tissue injury and invasiveness, we postulated that a significant contribution to host defense against tissue damaging and invasive infections could be provided by innate immune mechanisms that antagonize agr signaling. We determined whether two host defense factors that inhibit AIP1-induced agrI signaling, Nox2 and apolipoprotein B (apoB), also contribute to innate control of AIP3-induced agrIII signaling. We hypothesized that apoB and Nox2 would function differently against AIP3, which differs from AIP1 in amino acid sequence and length. Here we show that unlike AIP1, AIP3 is resistant to direct oxidant inactivation by Nox2 characteristic ROS. Rather, the contribution of Nox2 to defense against agrIII signaling is through oxidation of LDL. ApoB in the context of oxLDL, and not LDL, provides optimal host defense against S. aureus agrIII infection by binding the secreted signaling peptide, AIP3, and preventing expression of the agr-driven virulence factors which mediate invasive infection. ApoB within the context of oxLDL also binds AIP 1-4 and oxLDL antagonizes agr signaling by all four agr alleles. Our results suggest that Nox2-mediated oxidation of LDL facilitates a conformational change in apoB to one sufficient for binding and sequestration of all four AIPs, demonstrating the interdependence of apoB and Nox2 in host defense against agr signaling. These data reveal a novel role for oxLDL in host defense against S. aureus quorum-sensing signaling. PMID:23459693

  8. Extract from a mutant Rhodobacter sphaeroides as an enriched carotenoid source

    PubMed Central

    Wang, Chih-Chiang; Ding, Shangwu; Chiu, Kuo-Hsun; Liu, Wen-Sheng; Lin, Tai-Jung; Wen, Zhi-Hong

    2016-01-01

    Background The extract Lycogen™ from the phototrophic bacterium Rhodobacter sphaeroides (WL-APD911) has attracted significant attention because of its promising potential as a bioactive mixture, attributed in part to its anti-inflammatory properties and anti-oxidative activity. Objective This study aims to investigate the components of Lycogen™ and its anti-inflammatory properties and anti-oxidative activity. Design and results The mutant strain R. sphaeroides (WL-APD911) whose carotenoid 1,2-hydratase gene has been altered by chemical mutagenesis was used for the production of a new carotenoid. The strain was grown at 30°C on Luria–Bertani (LB) agar plates. After a 4-day culture period, the mutant strain displayed a 3.5-fold increase in carotenoid content, relative to the wild type. In the DPPH test, Lycogen™ showed more potent anti-oxidative activity than lycopene from the wild-type strain. Primary skin irritation test with hamsters showed no irritation response in hamster skins after 30 days of treatment with 0.2% Lycogen™. Chemical investigations of Lycogen™ using nuclear magnetic resonance (NMR) 1H, 13C, and COSY/DQCOSY spectra have identified spheroidenone and methoxyneurosporene. Quantitative analysis of these identified compounds based on spectral intensities indicates that spheroidenone and methoxyneurosporene are major components (approximately 1:1); very small quantities of other derivatives are also present in the sample. Conclusions In this study, we identified the major carotenoid compounds contained in Lycogen™, including spheroidenone and methoxyneurosporene by high-resolution NMR spectroscopy analysis. The carotenoid content of this mutant strain of R. sphaeroides was 3.5-fold higher than that in normal strain. Furthermore, Lycogen™ from the mutant strain is more potent than lycopene from the wild-type strain and does not cause irritation in hamster skins. These findings suggest that this mutant strain has the potential to be used

  9. In vitro characterization of felid herpesvirus 1 (FHV-1) mutants generated by recombineering in a recombinant BAC vector.

    PubMed

    Tai, S-H Sheldon; Holz, Carine; Engstrom, Michael D; Cheng, Hans H; Maes, Roger K

    2016-08-01

    Felid herpesvirus 1 (FHV-1) mutants were constructed using two-step Red-mediated recombination techniques based on a virulent full-length FHV-1 BAC clone. The individual mutant viruses generated were deficient in glycoprotein C (gC), glycoprotein E (gE), US3 serine/threonine protein kinase (PK), or both gE and thymidine kinase (TK). The gC- mutant virus produced plaques that were similar in size to those resulting from infection with the C-27 parent strain. In contrast, the gE(-), PK(-), and gE(-)PK(-) deletion mutants produced plaques that were significantly smaller. Multistep in vitro growth kinetics of the gE(-), PK(-), and gE(-)PK(-) viruses were slightly delayed compared to those of the C-27 parent strain. Peak progeny titers of these three mutants were approximately 10-fold lower than those generated with the C-27 strain. There was no delay in the growth kinetics of the gC- mutant, but the progeny virus titer obtained with this mutant was at least 3 logs lower compared to the parental strain titer. Based upon their in vitro characteristics, these mutants will be useful for the development of novel immunization strategies against this important feline pathogen. PMID:27157860

  10. Isolation of a respiratory-deficient Kluyveromyces fragilis mutant for the production of ethanol from Jerusalem artichoke

    SciTech Connect

    Guiraud, J.P.; Bourgi, J.; Stervinou, M.; Claisse, M.; Galzy, P.

    1987-05-01

    A respiratory-deficient mutant of Kluyveromyces fragilis was isolated using a ethidium bromide mutagenesis. It was characterized by a loss of cytochromes a + a3 and by an improvement of its inulinase activity. Under anaerobic conditions this mutant was always better than the wild strain for ethanol production especially from Jerusalem artichoke extracts containing large amounts of high polyfructosans (early extracts).

  11. High level of antibiotic production in a double polyphosphate kinase and phosphate-binding protein mutant of Streptomyces lividans.

    PubMed

    Díaz, Margarita; Sevillano, Laura; Rico, Sergio; Lombo, Felipe; Braña, Alfredo F; Salas, Jose A; Mendez, Carmen; Santamaría, Ramón I

    2013-05-01

    Phosphate metabolism regulates most of the life processes of microorganisms. In the present work we obtained and studied a Streptomyces lividans ppk/pstS double mutant, which lacks polyphosphate kinase (PPK) and the high-affinity phosphate-binding protein (PstS), impairing at the same time the intracellular storage of polyphosphate and the intake of new inorganic phosphate from a phosphate-limited medium, respectively. In some of the aspects analyzed, the ppk/pstS double mutant was more similar to the wt strain than was the single pstS mutant. The double mutant was thus able to grow in phosphate-limited media, whereas the pstS mutant required the addition of 1 mM phosphate under the assay conditions used. The double mutant was able to incorporate more than one fourth of the inorganic phosphate incorporated by the wt strain, whereas phosphate incorporation was almost completely impaired in the pstS mutant. Noteworthy, under phosphate limitation conditions, the double ppk/pstS mutant showed a higher production of the endogenous antibiotic actinorhodin and the heterologous antitumor 8-demethyl-tetracenomycin (up to 10-fold with respect to the wt strain), opening new possibilities for the use of this strain in the heterologous expression of antibiotic pathways.

  12. Isolation and characterization of ultraviolet light-sensitive mutants of the blue-green alga Anacystis nidulans.

    NASA Technical Reports Server (NTRS)

    Asato, Y.

    1972-01-01

    Three independently isolated ultraviolet light sensitive (uvs) mutants of Anacystis nidulans were characterized. Strain uvs-1 showed the highest sensitivity to UV by its greatly reduced photoreactivation capacity following irradiation. Pretreatment with caffeine suppressed the dark-survival curve of strain uvs-1, thus indicating the presence of excision enzymes involved in dark repair. Under 'black' and 'white' illumination, strain uvs-1 shows photorecovery properties comparable with wild-type cultures. Results indicate that strains uvs-1, uvs-35, and uvs-88 are probably genetically distinct UV-sensitive mutants.

  13. Salmonella typhimurium TnphoA mutants with increased sensitivity to biological and chemical detergents.

    PubMed

    Lacroix, F J; Avoyne, C; Pinault, C; Popoff, M Y; Pardon, P

    1995-10-01

    Salmonella typhimurium is a ubiquitous pathogenic bacterium able to sustain the environmental conditions of the gastrointestinal tract, including biliary salts. To understand the mechanisms involved in bile salt resistance and, more generally, detergent resistance, we investigated S. typhimurium mutants produced with the random mutagenic TnphoA transposon. A total of 3,000 transpositional mutants were isolated. Three strains among the 1,432 first mutants lost the ability to grow in the presence of biological and chemical detergents. They were prototrophic and exhibited normal lipopolysaccharide and outer membrane protein profiles after SDS-PAGE. They did not show sensitivity to dyes but showed very different sensitivities to antibiotics. For each mutant strain, Southern blotting analysis revealed a unique TnphoA insertion at different chromosomal locations. These observations were confirmed by transduction experiments.

  14. Biodegradation of neonicotinoid insecticide, imidacloprid by restriction enzyme mediated integration (REMI) generated Trichoderma mutants.

    PubMed

    He, Xiangfeng; Wubie, Abebe Jenberie; Diao, Qingyun; Li, Wei; Xue, Fei; Guo, Zhanbo; Zhou, Ting; Xu, Shufa

    2014-10-01

    REMI (restriction enzyme-mediated integration) technique was employed to construct Trichoderma atroviride strain T23 mutants with degrading capability of neonicotinoid insecticide, imidacloprid. The plasmid pBluescript II KS-hph used for integration in REMI mutants was confirmed by PCR and Southern hybridization. Among 153 transformants, 57% of them have showed higher neonicotinoid insecticide, imidacloprid, degradation ability than the wild strain T23 (p<0.01). More specifically, seven single-copied T. atroviride T23 transformants have confirmed a 30% higher degradation rate than their parent isolate. Among all transformed mutants, a 95% imidacloprid degradation rate was identified as the highest. This study, thus, provided an effective approach for improving neonicotinoid insecticide-degrading capability using REMI transformed T. atroviride mutants.

  15. Selection and phenotypic characterization of nonhemagglutinating mutants of Porphyromonas gingivalis.

    PubMed Central

    Chandad, F; Mayrand, D; Grenier, D; Hinode, D; Mouton, C

    1996-01-01

    To further investigate the relationship between fimbriae and the hemagglutinating adhesin HA-Ag2 of Porphyromonas gingivalis, three spontaneous mutants of the type strain ATCC 33277 were selected by a hemadsorption procedure. They were characterized for hemagglutination, trypsin-like and lectin-binding activities, and hydrophobicity and for the presence of fimbriae. The presence of the 42-kDa (the fimbrilin subunit) and the 43- and 49-kDa (the HA-Ag2 components) polypeptides was investigated by immunoblotting using polyclonal and monoclonal antibodies directed to fimbriae and to the hemagglutinating adhesin HA-Ag2. Cells from two of the three mutants (M1 and M2) exhibited no or little hemagglutination activity and very low trypsin-like activity and did not show the 43- and 49-kDa polypeptides. Abnormal fimbriation in M1 was deduced from the following observations of cells grown for 18 h: absence of the 42-kDa polypeptide and of a 14-kDa polypeptide and no fimbriae visible on electron micrographs. While the cells of mutant M2, irrespective of the age of the culture, were found to lack the 43- and 49-kDa polypeptides and hemagglutination activity, the supernatants of cultures grown for 72 h had high hemagglutination and trypsin-like activities and revealed the presence of the 42-, 43-, and 49-kDa polypeptides. This suggests that M2 may be missing some molecules which anchor the components to the cell surface. Mutant M3 showed levels of activities similar to those of the parental strain but lacked the 43-kDa polypeptide. Other pleiotropic effects observed for the mutants included loss of dark pigmentation and lower hydrophobicity. The data from this study fuel an emerging consensus whereby fimbriation, hemagglutination, and proteolytic activities, as well as other functions in P. gingivalis, are intricate. PMID:8641806

  16. The Anterior Gradient Homolog 3 (AGR3) Gene Is Associated with Differentiation and Survival in Ovarian Cancer

    PubMed Central

    King, Erin R.; Tung, Celestine S.; Tsang, Yvonne T.M.; Zu, Zhifei; Lok, Gabriel T.M.; Deavers, Michael T.; Malpica, Anais; Wolf, Judith K.; Lu, Karen H.; Birrer, Michael J.; Mok, Samuel C.; Gershenson, David M.; Wong, Kwong-Kwok

    2011-01-01

    Low-grade serous ovarian carcinoma is believed to arise from serous borderline ovarian tumors, yet the progression from serous borderline tumors to low-grade serous ovarian carcinoma remains poorly understood. The purpose of this study was to identify differentially expressed genes between the two groups. Expression profiles were generated from 6 human ovarian surface epithelia (HOSE), 8 serous borderline ovarian tumors (SBOT), 13 low-grade serous ovarian carcinomas (LG), and 24 high-grade serous ovarian carcinomas (HG). The anterior gradient homolog 3 (AGR3) gene was found to be highly upregulated in serous borderline ovarian tumors; this finding was validated by real-time quantitative RT-PCR, Western blotting, and immunohistochemistry. Anti-AGR3 immunohistochemistry was performed on an additional 56 LG and 103 HG tissues and the results were correlated with clinical data. Expression profiling determined that 1254 genes were differentially expressed (P < 0.005) between SBOT, LG and HG tumors. Serous borderline ovarian tumors exhibited robust positive staining for AGR3, with a lower percentage of tumor cells stained in LG and HG. Immunofluorescence staining indicated that AGR3 expression was limited to ciliated cells. Tumor samples with a high percentage (>10%) of AGR3 positively stained tumor cells were associated with improved longer median survival in both the LG (P = 0.013) and HG (P = 0.008) serous ovarian carcinoma groups. The progression of serous borderline ovarian tumors to low-grade serous ovarian carcinoma may involve the de-differentiation of ciliated cells. AGR3 could serve as a prognostic marker for survival in patients with low-grade and high-grade serous ovarian carcinomas. PMID:21451362

  17. Construction and pilot screening of a signature-tagged mutant library of Sinorhizobium fredii.

    PubMed

    Wang, Dan; Wang, Yuan Chun; Wu, Li Juan; Liu, Jian Xin; Zhang, Pan; Jiao, Jian; Yan, Hui; Liu, Tao; Tian, Chang Fu; Chen, Wen Xin

    2016-03-01

    Sinorhizobium fredii is well known for its ability to establish symbiosis with diverse legumes such as Glycine max (soybean, determinate nodules) and Cajanus cajan (pigeon pea, indeterminate nodules). In order to make screening of S. fredii genes related to symbiosis cost-effective, we constructed a large Tn5 insertion mutant library of S. fredii CCBAU45436 using the signature-tagged mutagenesis (STM) technique. This STM library contains a total of 25,500 independent mutants distributed in 17 sublibraries tagged by corresponding distinct DNA bar-code sequences. After the pilot screening of 255 mutants in 15 batches, Tag85-4, Tag4-17, Tag4-11 and Tag10-13 were found to have attenuated competitiveness (0-30 % in nodule occupation) compared to the wild-type strain when inoculated on soybean. Further characterization of these mutants suggests that Tag4-11 (a pyrC mutant) and Tag10-13 (a nrdJ mutant) are defective in establishing symbiosis with soybean. The pyrC mutant induced uninfected pseudonodules while the nrdJ mutant formed significantly more nodules containing bacteroids with poor persistence ability. When these two mutants were tested on pigeon pea, host-specific symbiotic defects were found. These results demonstrated the STM library as a valuable resource for identifying S. fredii genes relevant to symbiosis. PMID:26472206

  18. Characterization of Staphylococcus aureus mutants expressing reduced susceptibility to common house-cleaners

    PubMed Central

    Davis, A.O.; O’Leary, J.O.; Muthaiyan, A.; Langevin, M.J.; Delgado, A.; Abalos, A.T.; Fajardo, A.R.; Marek, J.; Wilkinson, B.J.; Gustafson, J.E.

    2013-01-01

    Aims To characterize mutants of Staphylococcus aureus expressing reduced susceptibility to house cleaners (HC), assess the impact of the alternative sigma factor SigB on HC susceptibility, and determine the MIC of clinical methicillin-resistant S. aureus (MRSA) to a HC. Methods and Results Susceptibility to HC, HC components, H2O2, vancomycin and oxacillin and physiological parameters were determined for HC-reduced susceptibility (HCRS) mutants, parent strain COL and COLsigB::kan. HCRS mutants selected with three HC expressed reduced susceptibility to multiple HC, HC components, H2O2 and vancomycin. Two unique HCRS mutants also lost the methicillin resistance determinant. In addition, all HCRS mutants exhibited better growth at two temperatures, and one HCRS mutant expressed reduced carotenoid production. COLsigB::kan demonstrated increased susceptibility to all HC and many HC components. sigB operon mutations were not detected in one HCRS mutant background. Of 76 clinical MRSA, 20 exhibited reduced susceptibility to a HC. Conclusions HCRS mutants demonstrate altered susceptibility to multiple antimicrobials. While sigB is required for full HC resistance, one HCRS mechanism does not involve sigB operon mutations. Clinical MRSA expressing reduced susceptibility to a common HC were detected. Significance and Impact of the Study This study suggests that HCRS mutants are not protected against, nor selected by, practical HC concentrations. PMID:15659191

  19. Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP) and generation of a mutant library with diverse phenotypes.

    PubMed

    Fang, Mingyue; Jin, Lihua; Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

    2013-01-01

    In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th) subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae. PMID:24319517

  20. Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

    PubMed Central

    Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

    2013-01-01

    In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae. PMID:24319517

  1. Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP) and generation of a mutant library with diverse phenotypes.

    PubMed

    Fang, Mingyue; Jin, Lihua; Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

    2013-01-01

    In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th) subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.

  2. Imipenem- and meropenem-resistant mutants of Enterobacter cloacae and Proteus rettgeri lack porins.

    PubMed Central

    Raimondi, A; Traverso, A; Nikaido, H

    1991-01-01

    Carbapenems such as imipenem and meropenem are not rapidly hydrolyzed by commonly occurring beta-lactamases. Nevertheless, it was possible, by mutagenesis and selection, to isolate mutant strains of Enterobacter cloacae and Proteus rettgeri that are highly resistant to meropenem and imipenem. Two alterations were noted in the E. cloacae mutants. First, the mutant strains appeared to be strongly derepressed in the production of beta-lactamases, which reached a very high level when the strains were grown in the presence of imipenem. Second, these mutants were deficient in the production of nonspecific porins, as judged by the pattern of outer membrane proteins as well as by reconstitution assays of permeability. As with most porin-deficient mutants, their cultures were unstable, and their cultivation in the absence of carbapenems rapidly led to an overgrowth of porin-producing revertants. Analysis of the data suggests that the synergism between the lowered outer membrane permeability and the slow but significant hydrolysis of carbapenems by the overproduced enzymes can explain the resistance phenotypes quantitatively, although the possibility of alteration of the target cannot be excluded at present. With P. rettgeri mutants, there was no indication of further derepression of beta-lactamase, but the enzyme hydrolyzed imipenem much more efficiently than the E. cloacae enzyme did. In addition, the major porin was absent in one mutant strain. These results suggest that a major factor for the carbapenem resistance of these enteric bacteria is the porin deficiency, and this conclusion forms a contrast to the situation in Pseudomonas aeruginosa, in which the most prevalent class of imipenem-resistant mutants appears to lack the specific channel protein D2 yet retains the major nonspecific porin F. Images PMID:1656855

  3. Bacteriocin-resistant mutants of Erwinia chrysanthemi: possible involvement of iron acquisition in phytopathogenicity.

    PubMed

    Expert, D; Toussaint, A

    1985-07-01

    A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit soft-rot symptoms on saintpaulia plants. The loss of pathogenicity was correlated with the disappearance of one to three outer membrane polypeptides (molecular weights, about 80,000 to 90,000) whose production in wild-type strains was greatly enhanced under iron-limited growth conditions. The mutants did not exhibit altered extracellular pectinolytic or cellulolytic activities. PMID:4008442

  4. Isolation and characterization of Pichia heedii mutants defective in xylose uptake

    SciTech Connect

    Does, A.L.; Bisson, L.F. )

    1990-11-01

    To investigate the role of xylose uptake in xylose metabolism in yeasts, we isolated a series of mutated strains of the yeast Pichia heedii which are defective in xylose utilization. Four of these demonstrated defects in xylose uptake. Overlaps between the functional or regulatory mechanisms for glucose and xylose uptake may exist in this yeast since some of the mutants defective in xylose uptake were also defective in glucose transport. None of the mutants were defective in xylose reductase or xylitol dehydrogenase activities.

  5. Post-irradiation Examination of the AGR-1 Experiment: Plans and Preliminary Results

    SciTech Connect

    Paul Demkowicz

    2001-10-01

    Abstract – The AGR-1 irradiation experiment contains seventy-two individual cylindrical fuel compacts (25 mm long x 12.5 mm diameter) each containing approximately 4100 TRISO-coated uranium oxycarbide fuel particles. The experiment accumulated 620 effective full power days in the Advanced Test Reactor at the Idaho National Laboratory with peak burnups exceeding 19% FIMA. An extensive post-irradiation examination campaign will be performed on the AGR-1 fuel in order to characterize the irradiated fuel properties, assess the in-pile fuel performance in terms of coating integrity and fission metals release, and determine the fission product retention behavior during high temperature accident testing. PIE experiments will include dimensional measurements of fuel and irradiated graphite, burnup measurements, assessment of fission metals release during irradiation, evaluation of coating integrity using the leach-burn-leach technique, microscopic examination of kernel and coating microstructures, and accident testing of the fuel in helium at temperatures up to 1800°C. Activities completed to date include opening of the irradiated capsules, measurement of fuel dimensions, and gamma spectrometry of selected fuel compacts.

  6. Validation of the Physics Analysis used to Characterize the AGR-1 TRISO Fuel Irradiation Test

    SciTech Connect

    Sterbentz, James W.; Harp, Jason M.; Demkowicz, Paul A.; Hawkes, Grant L.; Chang, Gray S.

    2015-05-01

    The results of a detailed physics depletion calculation used to characterize the AGR-1 TRISO-coated particle fuel test irradiated in the Advanced Test Reactor (ATR) at the Idaho National Laboratory are compared to measured data for the purpose of validation. The particle fuel was irradiated for 13 ATR power cycles over three calendar years. The physics analysis predicts compact burnups ranging from 11.30-19.56% FIMA and cumulative neutron fast fluence from 2.21?4.39E+25 n/m2 under simulated high-temperature gas-cooled reactor conditions in the ATR. The physics depletion calculation can provide a full characterization of all 72 irradiated TRISO-coated particle compacts during and post-irradiation, so validation of this physics calculation was a top priority. The validation of the physics analysis was done through comparisons with available measured experimental data which included: 1) high-resolution gamma scans for compact activity and burnup, 2) mass spectrometry for compact burnup, 3) flux wires for cumulative fast fluence, and 4) mass spectrometry for individual actinide and fission product concentrations. The measured data are generally in very good agreement with the calculated results, and therefore provide an adequate validation of the physics analysis and the results used to characterize the irradiated AGR-1 TRISO fuel.

  7. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064.

    PubMed

    da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

    2013-12-01

    Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

  8. Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

    PubMed Central

    da Silva Vasconcelos, Eliton; de Lima, Vanderlei Aparecido; Goto, Leandro Seiji; Cruz-Hernández, Isara Lourdes; Hokka, Carlos Osamu

    2013-01-01

    Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant. PMID:24688492

  9. Erythritol production with minimum by-product using Candida magnoliae mutant.

    PubMed

    Ghezelbash, G R; Nahvi, I; Malekpour, A

    2014-01-01

    In order to enhance erythritol production, mutants of Candida magnoliae DSM70638 were generated by ultraviolet and chemical mutagenesis. Erythritol productivity of samples was analyzed by TLC and HPLC with the refractive index detector. One of the mutants named mutant 12-2 gave a 2.4-fold increase in erythritol (20.32 g/L) and a 5.5-fold decrease in glycerol production compared to the wild strain. A sequence-based map of erythrose reductase gene in this mutant showed a replacement of the A321 by G321 that did not cause any amino acid exchange in protein structure. Therefore, the reason of higher erythritol production in C. magnoliae mutant 12-2 is probably the increase in expression of the open reading frame gene. This study revealed that a mutation or minor change in the sequence of genes involved in a production pathway can lead to a significant increase in protein translation.

  10. Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC.

    PubMed

    Jensen, Rasmus O; Winzer, Klaus; Clarke, Simon R; Chan, Weng C; Williams, Paul

    2008-08-29

    Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the alpha-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.

  11. [Biofilm Formation by the Nonflagellated flhB1 Mutant of Azospirillum brasilense Sp245].

    PubMed

    Shelud'ko, A V; Filip'echeva, Yu A; Shumiliva, E M; Khlebtsov, B N; Burov, A M; Petrova, L P; Katsy, E I

    2015-01-01

    Azospirillum brasilense Sp245 with mixed flagellation are able to form biofilms on various surfaces. A nonflagellated mutant of this strain with inactivated chromosomal copy of the flhB gene (flhB1) was shown to exhibit specific traits at the later stages of biofilm formation on a hydrophilic (glass) surface. Mature biofilms of the flhB1::Omegon-Km mutant Sp245.1063 were considerably thinner than those of the parent strain Sp245. The biofilms of the mutant were more susceptible to the forces of hydrodynamic shear. A. brasilense Sp245 cells in biofilms were not found to possess lateral flagella. Cells with polar flagella were, however, revealed by atomic force microscopy of mature native biofilms of strain Sp245. Preservation of a polar flagellum (probably nonmotile) on the cells of A. brasilense Sp245 may enhance the biofilm stability.

  12. Fitness Studies of Azole-Resistant Strains of Aspergillus fumigatus

    PubMed Central

    Valsecchi, Isabel; Mellado, Emilia; Beau, Rémi; Raj, Shriya

    2015-01-01

    Isogenic bar-coded strains of Aspergillus fumigatus carrying the G54W or M220K mutation in Cyp51A were constructed. In vitro, the growth and conidiation capacities of the mutants were similar to those of the parental strain. Competition studies in the absence of azoles showed that there was no adverse fitness cost for the azole-resistant A. fumigatus strains in vitro or in vivo compared to the parental strain. PMID:26416854

  13. Identification of a Hydrophobic Cleft in the LytTR Domain of AgrA as a Locus for Small Molecule Interactions that Inhibit DNA Binding

    PubMed Central

    Leonard, Paul G.; Bezar, Ian F.; Sidote, David J.; Stock, Ann M.

    2012-01-01

    The AgrA transcription factor regulates the quorum-sensing response in Staphylococcus aureus, controlling the production of hemolysins and other virulence factors. AgrA binds to DNA via its C-terminal LytTR domain, a domain not found in humans but common in many pathogenic bacteria, making it a potential target for antimicrobial development. We have determined the crystal structure of the apo AgrA LytTR domain and screened a library of 500 fragment compounds to find inhibitors of AgrA DNA-binding activity. Using NMR, the binding site for five compounds has been mapped to a common locus at the C-terminal end of the LytTR domain, a site known to be important for DNA-binding activity. Three of these compounds inhibit AgrA DNA binding. These results provide the first evidence that LytTR domains can be targeted by small organic compounds. PMID:23181972

  14. Characterization of a spontaneous, pressure-tolerant Listeria monocytogenes Scott A ctsR deletion mutant.

    PubMed

    Joerger, Rolf D; Chen, Haiqiang; Kniel, Kalmia E

    2006-01-01

    A spontaneous, pressure-tolerant mutant of Listeria monocytogenes Scott A, designated 2-1, was isolated after several rounds of pressure treatments at 500 MPa for 10 min. Mutant 2-1 was almost 100,000-fold more resistant than the wild type to a pressure of 350 MPa, and about 100-fold more resistant to 450 MPa when pressurized in growth medium. Approximately ten times more mutant cells than wild-type cells survived a 20-min exposure to 55 degrees C, and the mutant appears also to be more resistant to 0.2% H(2)O(2), although the difference could not be confirmed statistically. About 10 times more wild-type than mutant cells survived exposure to growth medium adjusted to pH 2.5 with HCl. The mutant is about 16-fold more sensitive to nisin than the wild type. Mutant 2-1 is non-motile, produces hemolytic activity, is able to grow in fetal calf serum as well as the wild type, and exhibits a lower level of invasiveness of human ileocecal adenocarcinoma cells than the wild type. The mutation in strain 2-1 is a deletion in the ctsR gene that results in the predicted production of truncated CtsR of 20 amino acids compared to a CtsR of 152 amino acids in the wild type. With the exception of its response to pH and possibly also to H(2)O(2), mutant 2-1 shares most of the phenotypes of the previously described ctsR mutant, AK01. The isolation of another spontaneous, pressure-resistant ctsR mutant confirms the central role of this regulatory gene in pressure tolerance of L. monocytogenes. Although such mutants appear of lesser concern to human health then the wild type, current detection methods for Listeria monocytogenes are not able to distinguish between these mutants and wildtype cells. PMID:16761946

  15. Genotypic and phenotypic characteristics of Methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated on three different geography locations.

    PubMed

    Ostojić, Maja; Hukić, Mirsada

    2015-08-04

    Staphylococcus aureus is a major cause of hospital-acquired infections worldwide. Increased frequency of methicillin-resistant Staphylococcus aureus (MRSA) in hospitalized patients and possibility of vancomycin resistance requires rapid and reliable characterization of isolates and control of MRSA spread in hospitals. Typing of isolates helps to understand the route of a hospital pathogen spread. The aim of this study was to investigate and compare genotypic and phenotypic characteristics of MRSA samples on three different geography locations. In addition, our aim was to evaluate three different methods of MRSA typing: spa-typing, agr-typing and GenoType MRSA.  We included 104 samples of MRSA, isolated in 3 different geographical locations in clinical hospitals in Zagreb, Mostar, and Heidelberg, during the period of six months. Genotyping and phenotyping were done by spa-typing, agr-typing and dipstick assay GenoType MRSA. We failed to type all our samples by spa-typing.  The most common spa-type in clinical hospital Zagreb was t041, in Mostar t001, and in Heidelberg t003.We analyzed 102/104 of our samples by agr-typing method. We did not find any agr-type IV in our locations. We analyzed all our samples by the dipstick assay GenoType MRSA. All isolates in our study were MRSA strains. In Zagreb there were no positive strains to PVL gene. In Mostar we have found 5/25 positive strains to PVL gene, in Heidelberg there was 1/49. PVL positive isolates were associated with spa-type t008 and agr-type I, thus, genetically, they were community-associated MRSA (CA-MRSA). Dipstick assay GenoType MRSA has demonstrated sufficient specificity, sensibility, simple performance and low cost, so we could introduce it to work in smaller laboratories. Using this method may expedite MRSA screening, thus preventing its spread in hospitals.

  16. Genotypic and phenotypic characteristics of Methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated on three different geography locations

    PubMed Central

    Ostojić, Maja; Hukić, Mirsada

    2015-01-01

    Staphylococcus aureus is a major cause of hospital-acquired infections worldwide. Increased frequency of methicillin-resistant Staphylococcus aureus (MRSA) in hospitalized patients and possibility of vancomycin resistance requires rapid and reliable characterization of isolates and control of MRSA spread in hospitals. Typing of isolates helps to understand the route of a hospital pathogen spread. The aim of this study was to investigate and compare genotypic and phenotypic characteristics of MRSA samples on three different geography locations. In addition, our aim was to evaluate three different methods of MRSA typing: spa-typing, agr-typing and GenoType MRSA. We included 104 samples of MRSA, isolated in 3 different geographical locations in clinical hospitals in Zagreb, Mostar, and Heidelberg, during the period of six months. Genotyping and phenotyping were done by spa-typing, agr-typing and dipstick assay GenoType MRSA. We failed to type all our samples by spa-typing. The most common spa-type in clinical hospital Zagreb was t041, in Mostar t001, and in Heidelberg t003. We analyzed 102/104 of our samples by agr-typing method. We did not find any agr-type IV in our locations. We analyzed all our samples by the dipstick assay GenoType MRSA. All isolates in our study were MRSA strains. In Zagreb there were no positive strains to PVL gene. In Mostar we have found 5/25 positive strains to PVL gene, in Heidelberg there was 1/49. PVL positive isolates were associated with spa-type t008 and agr-type I, thus, genetically, they were community-associated MRSA (CA-MRSA). Dipstick assay GenoType MRSA has demonstrated sufficient specificity, sensibility, simple performance and low cost, so we could introduce it to work in smaller laboratories. Using this method may expedite MRSA screening, thus preventing its spread in hospitals. PMID:26295294

  17. Genotypic and phenotypic characteristics of Methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated on three different geography locations.

    PubMed

    Ostojić, Maja; Hukić, Mirsada

    2015-01-01

    Staphylococcus aureus is a major cause of hospital-acquired infections worldwide. Increased frequency of methicillin-resistant Staphylococcus aureus (MRSA) in hospitalized patients and possibility of vancomycin resistance requires rapid and reliable characterization of isolates and control of MRSA spread in hospitals. Typing of isolates helps to understand the route of a hospital pathogen spread. The aim of this study was to investigate and compare genotypic and phenotypic characteristics of MRSA samples on three different geography locations. In addition, our aim was to evaluate three different methods of MRSA typing: spa-typing, agr-typing and GenoType MRSA.  We included 104 samples of MRSA, isolated in 3 different geographical locations in clinical hospitals in Zagreb, Mostar, and Heidelberg, during the period of six months. Genotyping and phenotyping were done by spa-typing, agr-typing and dipstick assay GenoType MRSA. We failed to type all our samples by spa-typing.  The most common spa-type in clinical hospital Zagreb was t041, in Mostar t001, and in Heidelberg t003.We analyzed 102/104 of our samples by agr-typing method. We did not find any agr-type IV in our locations. We analyzed all our samples by the dipstick assay GenoType MRSA. All isolates in our study were MRSA strains. In Zagreb there were no positive strains to PVL gene. In Mostar we have found 5/25 positive strains to PVL gene, in Heidelberg there was 1/49. PVL positive isolates were associated with spa-type t008 and agr-type I, thus, genetically, they were community-associated MRSA (CA-MRSA). Dipstick assay GenoType MRSA has demonstrated sufficient specificity, sensibility, simple performance and low cost, so we could introduce it to work in smaller laboratories. Using this method may expedite MRSA screening, thus preventing its spread in hospitals. PMID:26295294

  18. Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida.

    PubMed

    Duque, Estrella; García, Vanina; de la Torre, Jesús; Godoy, Patricia; Bernal, Patricia; Ramos, Juan-Luis

    2004-10-01

    The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.

  19. Enhanced production of isoamyl alcohol and isoamyl acetate by ubiquitination-deficient Saccharomyces cerevisiae mutants.

    PubMed

    Abe, Fumiyoshi; Horikoshi, Koki

    2005-01-01

    Aromatic compounds are an important element in the flavor of yeast-fermented alcohol. We isolated mutants of Saccharomyces cerevisiae capable of growth at high levels of hydrostatic pressure. Among them, the HPG1 mutants, with a defect in their Rsp5 ubiquitin ligase, were found to produce high amounts of aromatics due to enhanced leucine uptake, with isoamyl alcohol production 2- to 3-fold and isoamyl acetate production 4- to 8-fold that of the wild-type strain. The result suggests that the HPG1/RSP5 mutant alleles could be new resources for producing these flavoring compounds for yeast-fermented alcoholic beverages. PMID:16217550

  20. [Role of RNA-polymerase in gene activity regulation of E. coli RNA-polymerase mutants with a pleiotropic effect. I. Physiological and biochemical studies].

    PubMed

    Kamzolova, S G; Arutiunian, A V; Ozolin', O N; Oganesian, M G

    1979-01-01

    Four Rifr-mutants of E. coli B/r (rpo B401, rpo B402, rpo B403, rpo B409) which differ from the wild strain in one or more phenotypic properties besides rifampicin resistance were obtained. Transfer of the mutant Rifr-alleles into the parent strain gives the latter all the properties of the mutant. This indicates that the new properties are due to the pleiotropic effect of Rifr-mutations. Biochemical studies of the properties of RNA-polymerases from the mutants and the parent showed that some new properties of the mutants could not be explained by the appearance of analogous properties in the mutant RNA-polymerase itself. They seem to be caused by alteration in functional activity of the mutant enzyme, particulary, alteration of its control properties during transcription. The function of the beta-subunit in genetic transcription is discussed.

  1. Characterization and protective property of Brucella abortus cydC and looP mutants.

    PubMed

    Truong, Quang Lam; Cho, Youngjae; Barate, Abhijit Kashinath; Kim, Suk; Hahn, Tae-Wook

    2014-11-01

    Brucella abortus readily multiplies in professional or nonprofessional phagocytes in vitro and is highly virulent in mice. Isogenic mutants of B. abortus biovar 1 strain IVKB9007 lacking the ATP/GDP-binding protein motif A (P-loop) (named looP; designated here the IVKB9007 looP::Tn5 mutant) and the ATP-binding/permease protein (cydC; designated here the IVKB9007 cydC::Tn5 mutant) were identified and characterized by transposon mutagenesis using the mini-Tn5Km2 transposon. Both mutants were found to be virtually incapable of intracellular replication in both murine macrophages (RAW264.7) and the HeLa cell line, and their virulence was significantly impaired in BALB/c mice. Respective complementation of the IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants restored their ability to survive in vitro and in vivo to a level comparable with that of the wild type. These findings indicate that the cydC and looP genes play important roles in the virulence of B. abortus. In addition, intraperitoneal immunization of mice with a dose of the live IVKB9007 looP::Tn5 and IVKB9007 cydC::Tn5 mutants provided a high degree of protection against challenge with pathogenic B. abortus strain 544. Both mutants should be evaluated further as a live attenuated vaccine against bovine brucellosis for their ability to stimulate a protective immune response.

  2. Carbon and energy metabolism of atp mutants of Escherichia coli.

    PubMed

    Jensen, P R; Michelsen, O

    1992-12-01

    The membrane-bound H(+)-ATPase plays a key role in free-energy transduction of biological systems. We report how the carbon and energy metabolism of Escherichia coli changes in response to deletion of the atp operon that encodes this enzyme. Compared with the isogenic wild-type strain, the growth rate and growth yield were decreased less than expected for a shift from oxidative phosphorylation to glycolysis alone as a source of ATP. Moreover, the respiration rate of a atp deletion strain was increased by 40% compared with the wild-type strain. This result is surprising, since the atp deletion strain is not able to utilize the resulting proton motive force for ATP synthesis. Indeed, the ratio of ATP concentration to ADP concentration was decreased from 19 in the wild type to 7 in the atp mutant, and the membrane potential of the atp deletion strain was increased by 20%, confirming that the respiration rate was not controlled by the magnitude of the opposing membrane potential. The level of type b cytochromes in the mutant cells was 80% higher than the level in the wild-type cells, suggesting that the increased respiration was caused by an increase in the expression of the respiratory genes. The atp deletion strain produced twice as much by-product (acetate) and exhibited increased flow through the tricarboxylic acid cycle and the glycolytic pathway. These three changes all lead to an increase in substrate level phosphorylation; the first two changes also lead to increased production of reducing equivalents. We interpret these data as indicating that E. coli makes use of its ability to respire even if it cannot directly couple this ability to ATP synthesis; by respiring away excess reducing equivalents E. coli enhances substrate level ATP synthesis.

  3. Screening of Bacillus subtilis transposon mutants with altered riboflavin production.

    PubMed

    Tännler, Simon; Zamboni, Nicola; Kiraly, Csilla; Aymerich, Stéphane; Sauer, Uwe

    2008-09-01

    To identify novel targets for metabolic engineering of riboflavin production, we generated about 10,000 random, transposon-tagged mutants of an industrial, riboflavin-producing strain of Bacillus subtilis. Process-relevant screening conditions were established by developing a 96-deep-well plate method with raffinose as the carbon source, which mimics, to some extent, carbon limitation in fed batch cultures. Screening in raffinose and complex LB medium identified more efficiently riboflavin overproducing and underproducing mutants, respectively. As expected for a "loss of function" analysis, most identified mutants were underproducers. Insertion mutants in two genes with yet unknown function, however, were found to attain significantly improved riboflavin titers and yields. These genes and possibly further ones that are related to them are promising candidates for metabolic engineering. While causal links to riboflavin production were not obvious for most underproducers, we demonstrated for the gluconeogenic glyceraldehyde-3-phosphate dehydrogenase GapB how a novel, non-obvious metabolic engineering strategy can be derived from such underproduction mutations. Specifically, we improved riboflavin production on various substrates significantly by deregulating expression of the gluconeogenic genes gapB and pckA through knockout of their genetic repressor CcpN. This improvement was also verified under the more process-relevant conditions of a glucose-limited fed-batch culture. PMID:18582593

  4. Abnormal grooming activity in Dab1(scm) (scrambler) mutant mice.

    PubMed

    Strazielle, C; Lefevre, A; Jacquelin, C; Lalonde, R

    2012-07-15

    Dab1(scm) mutant mice, characterized by cell ectopias and degeneration in cerebellum, hippocampus, and neocortex, were compared to non-ataxic controls for different facets of grooming caused by brief water immersions, as well as some non-grooming behaviors. Dab1(scm) mutants were strongly affected in their quantitative functional parameters, exhibiting higher starting latencies before grooming relative to non-ataxic littermates of the A/A strain, fewer grooming bouts, and grooming components of shorter duration, with an unequal regional distribution targeting almost totally the rostral part (head washing and forelimb licking) of the animal. Only bouts of a single grooming element were preserved. The cephalocaudal order of grooming elements appeared less disorganized, mutant and control mice initiating the grooming with head washing and forelimb licking prior to licking posterior parts. However, mutants differed from controls in that all their bouts were incomplete but uninterrupted, although intergroup difference for percentage of the incorrect transitions was not significant. In contrast to grooming, Dab1(scm) mice ambulated for a longer time. During walking episodes, they exhibited more body scratching than controls, possibly to compensate for the lack of licking different body parts. In conjunction with studies with other ataxic mice, these results indicate that the cerebellar