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Sample records for periplasmic s1-like nuclease

  1. Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island

    SciTech Connect

    Pimkin, Maxim; Miller, C. Glenn; Blakesley, Lauryn; Oleykowski, Catherine A.; Kodali, Nagendra S.; Yeung, Anthony T. . E-mail: AT_Yeung@fccc.edu

    2006-04-28

    DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the protein identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.

  2. The plant s1-like nuclease family has evolved a highly diverse range of catalytic capabilities.

    PubMed

    Lesniewicz, Krzysztof; Karlowski, Wojciech M; Pienkowska, Joanna R; Krzywkowski, Piotr; Poreba, Elzbieta

    2013-07-01

    Plant S1-like nucleases, often referred to as nuclease I enzymes, are the main class of enzymes involved in nucleic acid degradation during plant programmed cell death. The catalytically active site of these enzymes shows a significant similarity to the well-described P1 nuclease from Penicillium citrinum. Previously published studies reported that plant S1-like nucleases possess catalytic activities similar to their fungal orthologs, i.e. they hydrolyze single-stranded DNA and RNA, and less efficiently double-stranded DNA, in the presence of zinc ions. Here we describe a comprehensive study of the nucleolytic activities of all Arabidopsis S1-like paralogs. Our results revealed that different members of this family are characterized by a surprisingly large variety of catalytic properties. We found that, in addition to Zn(2+)-dependent enzymes, this family also comprises nucleases activated by Ca(2+) and Mn(2+), which implies that the apparently well-known S1 nuclease active site in plant nucleases is able to cooperate with different activatory ions. Moreover, particular members of this class differ in their optimum pH value and substrate specificity. These results shed new light on the widely accepted classification of plant nucleases which is based on the assumption that the catalytic requirements of plant nucleases reflect their phylogenetic origin. Our results imply the need to redefine the understanding of the term 'nuclease I'. Analysis of the phylogenetic relationships between S1-like enzymes shows that plant representatives of this family evolve toward an increase in catalytic diversity. The importance of this process for the biological functions of plant S1-type enzymes is discussed.

  3. Growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain.

    PubMed

    Nesbeth, Darren N; Perez-Pardo, Miguel-Angel; Ali, Shaukat; Ward, John; Keshavarz-Moore, Eli

    2012-02-01

    Host cell engineering is becoming a realistic option in whole bioprocess strategies to maximize product manufacturability. High molecular weight (MW) genomic DNA currently hinders bioprocessing of Escherichia coli by causing viscosity in homogenate feedstocks. We previously showed that co-expressing Staphylococcal nuclease and human Fab' fragment in the periplasm of E. coli enables auto-hydrolysis of genomic DNA upon cell disruption, with a consequent reduction in feedstock viscosity and improvement in clarification performance. Here we report the impact of periplasmic nuclease expression on stability of DNA and Fab' fragment in homogenates, host-strain growth kinetics, cell integrity at harvest and Fab' fragment productivity. Nuclease and Fab' plasmids were shown to exert comparable levels of growth burden on the host W3110 E. coli strain. Nuclease co-expression did not compromise either the growth performance or volumetric yield of the production strain. 0.5 g/L Fab' fragment (75 L scale) and 0.7 g/L (20 L scale) was achieved for both unmodified and cell-engineered production strains. Unexpectedly, nuclease-modified cells achieved maximum Fab' levels 8-10 h earlier than the original, unmodified production strain. Scale-down studies of homogenates showed that nuclease-mediated hydrolysis of high MW DNA progressed to completion within minutes of homogenization, even when homogenates were chilled on ice, with no loss of Fab' product and no need for additional co-factors or buffering.

  4. Spirochete periplasmic flagella and motility.

    PubMed

    Li, C; Motaleb, A; Sal, M; Goldstein, S F; Charon, N W

    2000-10-01

    Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria. They also have a special attribute: spirochetes can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria. In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space. A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell with those attached at the other end. The number of periplasmic flagella varies from species to species. These structures have been shown to be directly involved in spirochete motility, and they function by rotating within the periplasmic space. The mechanics of motility also vary among the spirochetes. In Leptospira, a motility model developed several years ago has been extensively tested, and the evidence supporting this model is convincing. Borrelia burgdorferi swims differently, and a model of its motility has been recently put forward. This model is based on analyzing the motion of swimming cells, high voltage electron microscopy of fixed cells, and mutant analysis. To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed. Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella. They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins. Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function. Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident. Future studies on spirochete motility should be

  5. Periplasmic glucans isolated from Proteobacteria.

    PubMed

    Lee, Sanghoo; Cho, Eunae; Jung, Seunho

    2009-12-31

    Periplasmic glucans (PGs) are general constituents in the periplasmic space of Proteobacteria. PGs from bacterial strains are found in larger amounts during growth on medium with low osmolarity and thus are often been specified as osmoregulated periplasmic glucans (OPGs). Furthermore, they appear to play crucial roles in pathogenesis and symbiosis. PGs have been classified into four families based on the structural features of their backbones, and they can be modified by a variety of non-sugar substituents. It has also recently been confirmed that novel PGs with various degrees of polymerization (DPs) and/or different substituents are produced under different growth conditions among Proteobacteria. In addition to their biological functions as regulators of low osmolarity, PGs have a variety of physico-chemical properties due to their inherent three-dimensional structures, hydrogen-bonding and complex-forming abilities. Thus, much attention has recently been focused on their physico-chemical applications. In this review, we provide an updated classification of PGs, as well as a description of the occurrences of novel PGs with substituents under various bacterial growth environments, the genes involved in PG biosynthesis and the various physico-chemical properties of PGs.

  6. Nitrate and periplasmic nitrate reductases

    PubMed Central

    Sparacino-Watkins, Courtney; Stolz, John F.; Basu, Partha

    2014-01-01

    The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed. PMID:24141308

  7. Engineered fluorescent proteins illuminate the bacterial periplasm.

    PubMed

    Dammeyer, Thorben; Tinnefeld, Philip

    2012-01-01

    The bacterial periplasm is of special interest whenever cell factories are designed and engineered. Recombinantely produced proteins are targeted to the periplasmic space of Gram negative bacteria to take advantage of the authentic N-termini, disulfide bridge formation and easy accessibility for purification with less contaminating cellular proteins. The oxidizing environment of the periplasm promotes disulfide bridge formation - a prerequisite for proper folding of many proteins into their active conformation. In contrast, the most popular reporter protein in all of cell biology, Green Fluorescent Protein (GFP), remains inactive if translocated to the periplasmic space prior to folding. Here, the self-catalyzed chromophore maturation is blocked by formation of covalent oligomers via interchain disulfide bonds in the oxidizing environment. However, different protein engineering approaches addressing folding and stability of GFP resulted in improved proteins with enhanced folding properties. Recent studies describe GFP variants that are not only active if translocated in their folded form via the twin-arginine translocation (Tat) pathway, but actively fold in the periplasm following general secretory pathway (Sec) and signal recognition particle (SRP) mediated secretion. This mini-review highlights the progress that enables new insights into bacterial export and periplasmic protein organization, as well as new biotechnological applications combining the advantages of the periplasmic production and the Aequorea-based fluorescent reporter proteins.

  8. Protein quality control in the bacterial periplasm

    PubMed Central

    Miot, Marika; Betton, Jean-Michel

    2004-01-01

    The proper functioning of extracytoplasmic proteins requires their export to, and productive folding in, the correct cellular compartment. All proteins in Escherichia coli are initially synthesized in the cytoplasm, then follow a pathway that depends upon their ultimate cellular destination. Many proteins destined for the periplasm are synthesized as precursors carrying an N-terminal signal sequence that directs them to the general secretion machinery at the inner membrane. After translocation and signal sequence cleavage, the newly exported mature proteins are folded and assembled in the periplasm. Maintaining quality control over these processes depends on chaperones, folding catalysts, and proteases. This article summarizes the general principles which control protein folding in the bacterial periplasm by focusing on the periplasmic maltose-binding protein. PMID:15132751

  9. Turbidimetric Assay of Staphylococcal Nuclease

    PubMed Central

    Erickson, Alan; Deibel, R. H.

    1973-01-01

    A simplified turbidimetric procedure was developed to assay staphylococcal nuclease activity. The ease of performance and sensitivity to nanogram quantities enhance the utilization of the method for the quantitative or qualitative estimation of the enzyme. Unlike plating methods, the turbidimetric procedure affords the differentiation between heat-stable and heat-labile nuclease activity. PMID:4735446

  10. Recombinant Cyclophilins Lack Nuclease Activity

    PubMed Central

    Manteca, Angel; Sanchez, Jesus

    2004-01-01

    Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins. PMID:15342605

  11. Recombinant cyclophilins lack nuclease activity.

    PubMed

    Manteca, Angel; Sanchez, Jesus

    2004-09-01

    Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.

  12. Mutation detection using Surveyor nuclease.

    PubMed

    Qiu, Peter; Shandilya, Harini; D'Alessio, James M; O'Connor, Kevin; Durocher, Jeffrey; Gerard, Gary F

    2004-04-01

    We have developed a simple and flexible mutation detection technology for the discovery and mapping of both known and unknown mutations. This technology is based on a new mismatch-specific DNA endonuclease from celery, Surveyor nuclease, which is a member of the CEL nuclease family of plant DNA endonucleases. Surveyor nuclease cleaves with high specificity at the 3' side of any mismatch site in both DNA strands, including all base substitutions and insertion/deletions up to at least 12 nucleotides. Surveyor nuclease technology involves four steps: (i) PCR to amplify target DNA from both mutant and wild-type reference DNA; (ii) hybridization to form heteroduplexes between mutant and wild-type reference DNA; (iii) treatment of annealed DNA with Surveyor nuclease to cleave heteroduplexes; and (iv) analysis of digested DNA products using the detection/separation platform of choice. The technology is highly sensitive, detecting rare mutants present at as low as 1 in 32 copies. Unlabeled Surveyor nuclease digestion products can be analyzed using conventional gel electrophoresis or high-performance liquid chromatography (HPLC), while end labeled digestion products are suitable for analysis by automated gel or capillary electrophoresis. The entire protocol can be performed in less than a day and is suitable for automated and high-throughput procedures.

  13. Periplasmic Proteins of the Extremophile Acidithiobacillus ferrooxidans

    PubMed Central

    Chi, An; Valenzuela, Lissette; Beard, Simon; Mackey, Aaron J.; Shabanowitz, Jeffrey; Hunt, Donald F.; Jerez, Carlos A.

    2015-01-01

    Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophile capable of obtaining energy by oxidizing ferrous iron or sulfur compounds such as metal sulfides. Some of the proteins involved in these oxidations have been described as forming part of the periplasm of this extremophile. The detailed study of the periplasmic components constitutes an important area to understand the physiology and environmental interactions of microorganisms. Proteomics analysis of the periplasmic fraction of A. ferrooxidans ATCC 23270 was performed by using high resolution linear ion trap-FT MS. We identified a total of 131 proteins in the periplasm of the microorganism grown in thiosulfate. When possible, functional categories were assigned to the proteins: 13.8% were transport and binding proteins, 14.6% were several kinds of cell envelope proteins, 10.8% were involved in energy metabolism, 10% were related to protein fate and folding, 10% were proteins with unknown functions, and 26.1% were proteins without homologues in databases. These last proteins are most likely characteristic of A. ferrooxidans and may have important roles yet to be assigned. The majority of the periplasmic proteins from A. ferrooxidans were very basic compared with those of neutrophilic microorganisms such as Escherichia coli, suggesting a special adaptation of the chemolithoautotrophic bacterium to its very acidic environment. The high throughput proteomics approach used here not only helps to understand the physiology of this extreme acidophile but also offers an important contribution to the functional annotation for the available genomes of biomining microorganisms such as A. ferrooxidans for which no efficient genetic systems are available to disrupt genes by procedures such as homologous recombination. PMID:17911085

  14. Sperm chromatin released by nucleases.

    PubMed

    Nazarov, Igor B; Shlyakhtenko, Luda S; Lyubchenko, Yuri L; Zalenskaya, Irina A; Zalensky, Andrei O

    2008-01-01

    In human spermatozoa, 15-20% of histones are retained in the nucleus to coexist with protamines. Hypothetically, nucleohistone regions of sperm chromatin mark DNA sequences for distinctive processing during fertilization and early embryogenesis. The structural organization and molecular composition of nucleohistones in human spermatozoa is poorly studied. Here, we isolate and characterize fractions of sperm chromatin that are solubilized by endogenous and micrococcal nucleases. Chromatin isolated by either nuclease have a nucleosomal organization with the periodicity of approximately 195 bp (endogenous nuclease digest) and approximately 189 bp (micrococcal nuclease digest), which is similar to that of somatic cells. A distinct feature of sperm nucleohistone is its specific compact supra-nucleosomal organization that was demonstrated by two-dimensional electrophoresis and by atomic force microscopy. The latter technique showed compacted fiber arrays composed of globular particles with the prevailing diameter of approximately 16 nm. A rough estimation indicates that histones may cover continuous stretches of >50 kbp of sperm DNA. This initial characterization of sperm chromatin solubilized by nucleases is important for our understanding of the bipartite structural organization of the paternal genome.

  15. Genome engineering with targetable nucleases.

    PubMed

    Carroll, Dana

    2014-01-01

    Current technology enables the production of highly specific genome modifications with excellent efficiency and specificity. Key to this capability are targetable DNA cleavage reagents and cellular DNA repair pathways. The break made by these reagents can produce localized sequence changes through inaccurate nonhomologous end joining (NHEJ), often leading to gene inactivation. Alternatively, user-provided DNA can be used as a template for repair by homologous recombination (HR), leading to the introduction of desired sequence changes. This review describes three classes of targetable cleavage reagents: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas RNA-guided nucleases (RGNs). As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. This review discusses the properties, advantages, and limitations of each system, as well as the specific considerations required for their use in different biological systems.

  16. Import of periplasmic bacteriocins targeting the murein.

    PubMed

    Braun, Volkmar; Helbig, Stephanie; Patzer, Silke I

    2012-12-01

    Colicins are the only proteins imported by Escherichia coli and thus serve as tools to study the protein import mechanism. Most of the colicins studied degrade DNA, 16S RNA or tRNA in the cytoplasm, or form pores in the cytoplasmic membrane. Two bacteriocins, Cma (colicin M) and Pst (pesticin), affect the murein structure in the periplasm. These two bacteriocins must be imported only across the outer membrane and therefore represent the simplest system for studying protein import. Cma can be reversibly translocated across the outer membrane. Cma and Pst unfold during import. The crystal structure of Pst reveals a phage T4L (T4 lysozyme) fold of the activity domain. Both bacteriocins require energy for import which is translocated from the cytoplasmic membrane into the outer membrane by the Ton system. Cma kills cells only when the periplasmic FkpA PPIase (peptidylprolyl cis-trans isomerase)/chaperone is present.

  17. Periplasmic Superoxide Dismutase in Meningococcal Pathogenicity

    PubMed Central

    Wilks, Kathryn E.; Dunn, Kate L. R.; Farrant, Jayne L.; Reddin, Karen M.; Gorringe, Andrew R.; Langford, Paul R.; Kroll, J. Simon

    1998-01-01

    Meningococcal sodC encodes periplasmic copper- and zinc-cofactored superoxide dismutase (Cu,Zn SOD) which catalyzes the conversion of the superoxide radical anion to hydrogen peroxide, preventing a sequence of reactions leading to production of toxic hydroxyl free radicals. From its periplasmic location, Cu,Zn SOD was inferred to acquire its substrate from outside the bacterial cell and was speculated to play a role in preserving meningococci from the action of microbicidal oxygen free radicals produced in the context of host defense. A sodC mutant was constructed by allelic exchange and was used to investigate the role of Cu,Zn SOD in pathogenicity. Wild-type and mutant meningococci grew at comparable rates and survived equally long in aerobic liquid culture. The mutant showed no increased sensitivity to paraquat, which generates superoxide within the cytosol, but was approximately 1,000-fold more sensitive to the toxicity of superoxide generated in solution by the xanthine/xanthine oxidase system. These data support a role for meningococcal Cu,Zn SOD in protection against exogenous superoxide. In experiments to translate this into a role in pathogenicity, wild-type and mutant organisms were used in an intraperitoneal mouse infection model. The sodC mutant was significantly less virulent. We conclude that periplasmic Cu,Zn SOD contributes to the virulence of Neisseria meningitidis, most likely by reducing the effectiveness of toxic oxygen host defenses. PMID:9423860

  18. Therapeutic genome editing with engineered nucleases.

    PubMed

    Haas, Simone A; Dettmer, Viviane; Cathomen, Toni

    2017-01-31

    Targeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies. Here, we review the development of the different classes of programmable nucleases, discuss the challenges and improvements in translating gene editing into clinical use, and give an outlook on what applications can expect to enter the clinic in the near future.

  19. Off-target effects of engineered nucleases.

    PubMed

    Yee, Jiing-Kuan

    2016-09-01

    Recent advances in gene editing with engineered nucleases have transformed our ability to manipulate the genome from diverse organisms for applications ranging from biomedical research to disease treatment. A major complication with these engineered nucleases is the binding of the nuclease to unintended genomic sites that share sequence homology with the on-target site. Cleavage of these off-target sites followed by DNA repair using normal cellular DNA repair mechanisms can cause gene mutation or gross chromosome rearrangement. Identification of nuclease-generated off-target sites is a daunting task due to the size and complexity of the mammalian genome. Five unbiased, genome-wide strategies have been developed to detect the off-target cleavage. Some of these strategies reach the sensitivity near the detection limit of directed deep sequencing and have sufficient precision and resolution to objectively assessing the off-target effect of any engineered nuclease. Significant progress has also been made recently to boost the nuclease targeting specificity by protein engineering to modify the structure of the nuclease and alter the interaction with its genomic target. In several studied cases, the off-target effect generated by the modified nuclease is completely eliminated. These modified nucleases significantly improve the overall fidelity of gene editing. These developments will enable gene editing tools to be applied more broadly and safely in basic research and disease treatment. © 2016 Federation of European Biochemical Societies.

  20. Periplasmic Structure in Saccharomyces rouxii (Boutroux), an Osmophil

    PubMed Central

    Arnold, Wilfred N.; Garrison, Robert G.; Boyd, Karen S.

    1974-01-01

    Electron micrographs of ultrathin sections of S. rouxii displayed electrondense, membrane-circumscribed structures between the protoplasmic membrane and the cell wall. These periplasmic bodies were numerous in cells from a 3-day culture and absent or rare in older cells. Periplasmic bodies were fewer and smaller (flattened) in specimens grown in a medium fortified with 10% sucrose; they were not detected in cells grown in 20% sucrose. A brief treatment with ethyl acetate caused the periplasmic bodies of young cells to become electron light. Periplasmic bodies were most prevalent in the regions of the bud scars and were often accommodated within large invaginations in the protoplasmic membrane. In general, conditions which favor the prevalence and electron density of periplasmic bodies are those which also mask the activity of β-fructofuranosidase in this species. Images PMID:4451363

  1. Periplasmic superoxide dismutase SodCI of Salmonella binds peptidoglycan to remain tethered within the periplasm.

    PubMed

    Tidhar, Avital; Rushing, Marcus D; Kim, Byoungkwan; Slauch, James M

    2015-09-01

    Salmonellae survive and propagate in macrophages to cause serious systemic disease. Periplasmic superoxide dismutase plays a critical role in this survival by combating phagocytic superoxide. Salmonella Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Although both proteins are produced during infection, only SodCI is functional in the macrophage phagosome. We have previously shown that SodCI, relative to SodCII, is both protease resistant and tethered within the periplasm and that either of these properties is sufficient to allow a SodC to protect against phagocytic superoxide. Tethering is defined as remaining cell-associated after osmotic shock or treatment with cationic antimicrobial peptides. Here we show that SodCI non-covalently binds peptidoglycan. SodCI binds to Salmonella and Bacillus peptidoglycan, but not peptidoglycan from Staphylococcus. Moreover, binding can be inhibited by a diaminopimelic acid containing tripeptide, but not a lysine containing tripeptide, showing that the protein recognizes the peptide portion of the peptidoglycan. Replacing nine amino acids in SodCII with the corresponding residues from SodCI confers tethering, partially delineating an apparently novel peptidoglycan binding domain. These changes in sequence increase the affinity of SodCII for peptidoglycan fragments to match that of SodCI and allow the now tethered SodCII to function during infection.

  2. Structure and metal loading of a soluble periplasm cuproprotein.

    PubMed

    Waldron, Kevin J; Firbank, Susan J; Dainty, Samantha J; Pérez-Rama, Mónica; Tottey, Steve; Robinson, Nigel J

    2010-10-15

    A copper-trafficking pathway was found to enable Cu(2+) occupancy of a soluble periplasm protein, CucA, even when competing Zn(2+) is abundant in the periplasm. Here, we solved the structure of CucA (a new cupin) and found that binding of Cu(2+), but not Zn(2+), quenches the fluorescence of Trp(165), which is adjacent to the metal site. Using this fluorescence probe, we established that CucA becomes partly occupied by Zn(2+) following exposure to equimolar Zn(2+) and Cu(2+). Cu(2+)-CucA is more thermodynamically stable than Zn(2+)-CucA but k((Zn→Cu)exchange) is slow, raising questions about how the periplasm contains solely the Cu(2+) form. We discovered that a copper-trafficking pathway involving two copper transporters (CtaA and PacS) and a metallochaperone (Atx1) is obligatory for Cu(2+)-CucA to accumulate in the periplasm. There was negligible CucA protein in the periplasm of ΔctaA cells, but the abundance of cucA transcripts was unaltered. Crucially, ΔctaA cells overaccumulate low M(r) copper complexes in the periplasm, and purified apoCucA can readily acquire Cu(2+) from ΔctaA periplasm extracts, but in vivo apoCucA fails to come into contact with these periplasmic copper pools. Instead, copper traffics via a cytoplasmic pathway that is coupled to CucA translocation to the periplasm.

  3. Structure and Metal Loading of a Soluble Periplasm Cuproprotein*

    PubMed Central

    Waldron, Kevin J.; Firbank, Susan J.; Dainty, Samantha J.; Pérez-Rama, Mónica; Tottey, Steve; Robinson, Nigel J.

    2010-01-01

    A copper-trafficking pathway was found to enable Cu2+ occupancy of a soluble periplasm protein, CucA, even when competing Zn2+ is abundant in the periplasm. Here, we solved the structure of CucA (a new cupin) and found that binding of Cu2+, but not Zn2+, quenches the fluorescence of Trp165, which is adjacent to the metal site. Using this fluorescence probe, we established that CucA becomes partly occupied by Zn2+ following exposure to equimolar Zn2+ and Cu2+. Cu2+-CucA is more thermodynamically stable than Zn2+-CucA but k(Zn→Cu)exchange is slow, raising questions about how the periplasm contains solely the Cu2+ form. We discovered that a copper-trafficking pathway involving two copper transporters (CtaA and PacS) and a metallochaperone (Atx1) is obligatory for Cu2+-CucA to accumulate in the periplasm. There was negligible CucA protein in the periplasm of ΔctaA cells, but the abundance of cucA transcripts was unaltered. Crucially, ΔctaA cells overaccumulate low Mr copper complexes in the periplasm, and purified apoCucA can readily acquire Cu2+ from ΔctaA periplasm extracts, but in vivo apoCucA fails to come into contact with these periplasmic copper pools. Instead, copper traffics via a cytoplasmic pathway that is coupled to CucA translocation to the periplasm. PMID:20702411

  4. Cold denaturation of staphylococcal nuclease.

    PubMed Central

    Griko, Y V; Privalov, P L; Sturtevant, J M; Venyaminov SYu

    1988-01-01

    Denaturation of staphylococcal nuclease was studied in a temperature range from -7 to 70 degrees C by scanning microcalorimetry and spectropolarimetry. It was found that the native protein is maximally stable at about 20 degrees C and is denatured upon heating and cooling from this temperature. The heat and cold denaturation processes are approximated rather well by a two-state transition showing that the molecule is composed of a single cooperative system. The main difference between these two processes is in the sign of the enthalpy and entropy of denaturation: whereas the heat denaturation proceeds with increases in the enthalpy and entropy, the cold denaturation proceeds with decreases in both quantities. The inversion of the enthalpy sign occurs at about 15 degrees C in an acetate buffer, but this temperature can be raised by addition of urea to the solvent. PMID:3368446

  5. Designed nucleases for targeted genome editing.

    PubMed

    Lee, Junwon; Chung, Jae-Hee; Kim, Ho Min; Kim, Dong-Wook; Kim, Hyongbum

    2016-02-01

    Targeted genome-editing technology using designed nucleases has been evolving rapidly, and its applications are widely expanding in research, medicine and biotechnology. Using this genome-modifying technology, researchers can precisely and efficiently insert, remove or change specific sequences in various cultured cells, micro-organisms, animals and plants. This genome editing is based on the generation of double-strand breaks (DSBs), repair of which modifies the genome through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR). In addition, designed nickase-induced generation of single-strand breaks can also lead to precise genome editing through HDR, albeit at relatively lower efficiencies than that induced by nucleases. Three kinds of designed nucleases have been used for targeted DSB formation: zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system. A growing number of researchers are using genome-editing technologies, which have become more accessible and affordable since the discovery and adaptation of CRISPR-Cas9. Here, the repair mechanism and outcomes of DSBs are reviewed and the three types of designed nucleases are discussed with the hope that such understanding will facilitate applications to genome editing.

  6. [TALE nuclease engineering and targeted genome modification].

    PubMed

    Shen, Yan; Xiao, An; Huang, Peng; Wang, Wei-Ye; Zhu, Zuo-Yan; Zhang, Bo

    2013-04-01

    Artificial designer nucleases targeting specific DNA sequences open up a new field for reverse genetics study. The rapid development of engineered endonucleases (EENs) enables targeted genome modification theoretically in any species. The construction of transcription activator-like effector nucleases (TALENs) is simpler with higher specificity and less toxicity than zinc-finger nucleases (ZFNs). Here, we summarized the recent progresses and prospects of TALEN technology, with an emphasis on its structure, function, and construction strategies, as well as a collection of species and genes that have been successfully modified by TALENs, especially the application in zebrafish.

  7. TALE nucleases: tailored genome engineering made easy.

    PubMed

    Mussolino, Claudio; Cathomen, Toni

    2012-10-01

    Custom-made designer nucleases have evolved into an indispensable platform to precisely alter complex genomes for basic research, biotechnology, synthetic biology, or human gene therapy. In this review we describe how transcription activator-like effector nucleases (TALENs) have rapidly developed into a chief technology for targeted genome editing in different model organisms as well as human stem cells. We summarize the technological background and provide an overview of the current state-of-the-art of TALENs with regard to activity and specificity of these nucleases for targeted genome engineering.

  8. Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    PubMed Central

    Langel, Ülo

    2016-01-01

    Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies. PMID:27101307

  9. Mouse genome engineering using designer nucleases.

    PubMed

    Hermann, Mario; Cermak, Tomas; Voytas, Daniel F; Pelczar, Pawel

    2014-04-02

    Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

  10. The Intimin periplasmic domain mediates dimerisation and binding to peptidoglycan.

    PubMed

    Leo, Jack C; Oberhettinger, Philipp; Chaubey, Manish; Schütz, Monika; Kühner, Daniel; Bertsche, Ute; Schwarz, Heinz; Götz, Friedrich; Autenrieth, Ingo B; Coles, Murray; Linke, Dirk

    2015-01-01

    Intimin and Invasin are prototypical inverse (Type Ve) autotransporters and important virulence factors of enteropathogenic Escherichia coli and Yersinia spp. respectively. In addition to a C-terminal extracellular domain and a β-barrel transmembrane domain, both proteins also contain a short N-terminal periplasmic domain that, in Intimin, includes a lysin motif (LysM), which is thought to mediate binding to peptidoglycan. We show that the periplasmic domain of Intimin does bind to peptidoglycan both in vitro and in vivo, but only under acidic conditions. We were able to determine a dissociation constant of 0.8 μM for this interaction, whereas the Invasin periplasmic domain, which lacks a LysM, bound only weakly in vitro and failed to bind peptidoglycan in vivo. We present the solution structure of the Intimin LysM, which has an additional α-helix conserved within inverse autotransporter LysMs but lacking in others. In contrast to previous reports, we demonstrate that the periplasmic domain of Intimin mediates dimerisation. We further show that dimerisation and peptidoglycan binding are general features of LysM-containing inverse autotransporters. Peptidoglycan binding by the periplasmic domain in the infection process may aid in resisting mechanical and chemical stress during transit through the gastrointestinal tract.

  11. Lanthanide accumulation in the periplasmic space of Escherichia coli B.

    PubMed Central

    Bayer, M E; Bayer, M H

    1991-01-01

    Treatment of growing Escherichia coli B with lanthanide ions [lanthanum(III), terbium(III), and europium(III)] and subsequent aldehyde-OsO4 fixation caused areas of high contrast to appear within the periplasm (the space between inner and outer membrane of the cell envelope). X-ray microanalysis of ultrathin sections of Epon-embedded or acrylic resin-embedded cells revealed the presence of the lanthanide and of phosphorus in the areas, whose contrast greatly exceeded that of other stained structures. Comparatively small amounts of the lanthanide were also present in the outer membrane and in the cytoplasm. The distribution of the periplasmic areas of high contrast was found to be random and not clustered at areas of current or future septum formation. Irregular cell shapes were observed after lanthanide treatment before onset of fixation. In contrast to glutaraldehyde-OsO4 fixation, glutaraldehyde used as the sole fixer caused a scattered distribution of the lanthanide. Cryofixation (slam-freezing) and freeze substitution revealed a lanthanum stain at both the periplasm and the outer part of the outer membrane. Deenergization of the cell membrane by either phage T4 or carbonyl cyanide m-chlorophenylhydrazone abolished the metal accumulation. Furthermore, addition of excess calcium, administered together with the lanthanide solution, diminished the quantity and size of areas of high contrast. Cells grown in media of high NaCl concentration revealed strongly stained areas of periplasmic precipitates, whereas cells grown under low-salt conditions showed very few high-contrast patches in the periplasm. Terbium treatment (during fixation) enhanced the visibility of the sites of inner-outer membrane contact (the membrane adhesion sites) in plasmolized cells, possibly as the result of an accumulation of the metal at the adhesion domains. The data suggest a rapid interaction of the lanthanides with components of the cell envelope, the periplasm, and the energized inner

  12. Biological and biomedical applications of engineered nucleases.

    PubMed

    Pan, Yunzhi; Xiao, Li; Li, Alice S S; Zhang, Xu; Sirois, Pierre; Zhang, Jia; Li, Kai

    2013-09-01

    The development of engineered nucleases is the fruit of a new technological approach developed in the last two decades which has led to significant benefits on genome engineering, particularly on gene therapy. These applications enable efficient and specific genetic modifications via the induction of a double-strand break (DSB) in a specific genomic target sequence, followed by the homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. In addition to the application on gene modification in cells and intact organisms, a number of recent papers have reported that this gene editing technology can be applied effectively to human diseases. With the promising data obtained using engineered endonucleases in gene therapy, it appears reasonable to expect that more diseases could be treated and even be cured in this new era of individualized medicine. This paper first brief introduces the development of engineered nucleases with a special emphasis on zinc-finger nucleases (ZFNs) and transcription activator-like effector (TALE) nucleases (TALENs), and then takes CCR5-based gene therapy as an example to discuss the therapeutic applications of engineered nucleases.

  13. The SMX DNA Repair Tri-nuclease.

    PubMed

    Wyatt, Haley D M; Laister, Rob C; Martin, Stephen R; Arrowsmith, Cheryl H; West, Stephen C

    2017-03-02

    The efficient removal of replication and recombination intermediates is essential for the maintenance of genome stability. Resolution of these potentially toxic structures requires the MUS81-EME1 endonuclease, which is activated at prometaphase by formation of the SMX tri-nuclease containing three DNA repair structure-selective endonucleases: SLX1-SLX4, MUS81-EME1, and XPF-ERCC1. Here we show that SMX tri-nuclease is more active than the three individual nucleases, efficiently cleaving replication forks and recombination intermediates. Within SMX, SLX4 co-ordinates the SLX1 and MUS81-EME1 nucleases for Holliday junction resolution, in a reaction stimulated by XPF-ERCC1. SMX formation activates MUS81-EME1 for replication fork and flap structure cleavage by relaxing substrate specificity. Activation involves MUS81's conserved N-terminal HhH domain, which mediates incision site selection and SLX4 binding. Cell cycle-dependent formation and activation of this tri-nuclease complex provides a unique mechanism by which cells ensure chromosome segregation and preserve genome integrity.

  14. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases

    PubMed Central

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-01-01

    Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9. PMID:26501275

  15. Osmoregulated periplasmic glucans synthesis gene family of Shigella flexneri

    USDA-ARS?s Scientific Manuscript database

    Osmoregulated periplasmic glucans (OPGs) of foodborne enteropathogen Shigella flexneri were characterized. OPGs were composed of 100 percent glucose with 2-linked glucose as the most abundant residue with terminal glucose, 2-linked and 2,6-linked glucose also present in high quantities. Most dominan...

  16. Targeting DNA double-strand breaks with TAL effector nucleases.

    PubMed

    Christian, Michelle; Cermak, Tomas; Doyle, Erin L; Schmidt, Clarice; Zhang, Feng; Hummel, Aaron; Bogdanove, Adam J; Voytas, Daniel F

    2010-10-01

    Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.

  17. Differentiation of staphylococci on the basis of nuclease properties.

    PubMed

    Gudding, R

    1983-11-01

    The quantity, thermostability, and serological pattern of nucleases produced by different staphylococci were studied. Staphylococcal strains were isolated from nine different species of animals or from humans. Staphylococcus aureus, Staphylococcus intermedius, and Staphylococcus hyicus subsp. hyicus were vigorous producers of nuclease, whereas the coagulase-negative staphylococci, except S. hyicus subsp. hyicus, produced significantly less nuclease. The nucleases of all strains were found to be thermostable. S. aureus, S. intermedius, and S. hyicus subsp. hyicus could be distinguished from each other and from coagulase-negative staphylococci on the basis of inhibition of nuclease activity by specific antibodies.

  18. Identification of off-target cleavage sites of zinc finger nucleases and TAL effector nucleases using predictive models.

    PubMed

    Fine, Eli J; Cradick, Thomas J; Bao, Gang

    2014-01-01

    Using engineered nucleases, such as Zinc Finger Nucleases (ZFNs) or Transcription Activator-Like Effector Nucleases (TALENs), to make targeted genomic modifications has become a common technique to create new model organisms and custom cell lines, and has shown great promise for disease treatment. However, these nucleases have the potential for off-target cleavage that could confound interpretation of experimental results and be detrimental for therapeutic use. Here, we describe a method to test for nuclease cleavage at potential off-target sites predicted by bioinformatics models.

  19. Periplasmal Physics: The Rotational Dynamics of Spirochetal Flagella

    NASA Astrophysics Data System (ADS)

    Huber, Greg

    2012-02-01

    Spirochetes are distinguished by the location of their flagella, which reside within the periplasm: the tiny space between the bacterial cell wall and the outer membrane. In Borrelia burgdorferi/ (the causative agent of Lyme Disease), rotation of the flagella leads to cellular undulations that drive swimming. Exactly how these shape changes arise due to the forces and torques acting between the flagella and the cell body is unknown. By applying low-Reynolds number hydrodynamic theory to the motion of an elastic flagellum rotating in the periplasm, we show that the flagella are most likely separated from the bacterial cell wall by a lubricating layer of fluid. We obtain analytical solutions for the force and torque on the rotating flagellum through lubrication analysis, as well as through scaling analysis, and find results are in close agreement numerical simulations. (Joint work with J. Yang and C.W. Wolgemuth.)

  20. Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

    PubMed Central

    Loftus, Steven R.; Walker, Daniel; Maté, Maria J.; Bonsor, Daniel A.; James, Richard; Moore, Geoffrey R.; Kleanthous, Colin

    2006-01-01

    The natively disordered N-terminal 83-aa translocation (T) domain of E group nuclease colicins recruits OmpF to a colicin-receptor complex in the outer membrane (OM) as well as TolB in the periplasm of Escherichia coli, the latter triggering translocation of the toxin across the OM. We have identified the 16-residue TolB binding epitope in the natively disordered T-domain of the nuclease colicin E9 (ColE9) and solved the crystal structure of the complex. ColE9 folds into a distorted hairpin within a canyon of the six-bladed β-propeller of TolB, using two tryptophans to bolt the toxin to the canyon floor and numerous intramolecular hydrogen bonds to stabilize the bound conformation. This mode of binding enables colicin side chains to hydrogen-bond TolB residues in and around the channel that runs through the β-propeller and that constitutes the binding site of peptidoglycan-associated lipoprotein (Pal). Pal is a globular binding partner of TolB, and their association is known to be important for OM integrity. The structure is therefore consistent with translocation models wherein the colicin disrupts the TolB–Pal complex causing local instability of the OM as a prelude to toxin import. Intriguingly, Ca2+ ions, which bind within the β-propeller channel and switch the surface electrostatics from negative to positive, are needed for the negatively charged T-domain to bind TolB with an affinity equivalent to that of Pal and competitively displace it. Our study demonstrates that natively disordered proteins can compete with globular proteins for binding to folded scaffolds but that this can require cofactors such as metal ions to offset unfavorable interactions. PMID:16894158

  1. Periplasmic c cytochromes and chlorate reduction in Ideonella dechloratans.

    PubMed

    Bäcklund, Anna Smedja; Bohlin, Jan; Gustavsson, Niklas; Nilsson, Thomas

    2009-04-01

    The aim of this study was to clarify the pathway of electron transfer between the inner membrane components and the periplasmic chlorate reductase. Several soluble c-type cytochromes were found in the periplasm. The optical difference spectrum of dithionite-reduced periplasmic extract shows that at least one of these components is capable of acting as an electron donor to the enzyme chlorate reductase. The cytochromes were partially separated, and the fractions were analyzed by UV/visible spectroscopy to determine the ability of donating electrons to chlorate reductase. Our results show that one of the c cytochromes (6 kDa) is able to donate electrons, both to chlorate reductase and to the membrane-bound cytochrome c oxidase, whereas the roles of the remaining c cytochromes still remain to be elucidated. Peptide extracts of the c cytochromes were obtained by tryptic in-gel digestion for matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Peptide sequences obtained indicate that the 6-kDa cytochrome c protein is similar to c cytochromes from the chlorate-reducing bacterium Dechloromonas aromatica.

  2. The Development of TALE Nucleases for Biotechnology.

    PubMed

    Ousterout, David G; Gersbach, Charles A

    2016-01-01

    The development of a facile genome engineering technology based on transcription activator-like effector nucleases (TALENs) has led to significant advances in diverse areas of science and medicine. In this review, we provide a broad overview of the development of TALENs and the use of this technology in basic science, biotechnology, and biomedical applications. This includes the discovery of DNA recognition by TALEs, engineering new TALE proteins to diverse targets, general advances in nuclease-based editing strategies, and challenges that are specific to various applications of the TALEN technology. We review examples of applying TALENs for studying gene function and regulation, generating disease models, and developing gene therapies. The current status of genome editing and future directions for other uses of these technologies are also discussed.

  3. Genome editing with engineered nucleases in plants.

    PubMed

    Osakabe, Yuriko; Osakabe, Keishi

    2015-03-01

    Numerous examples of successful 'genome editing' now exist. Genome editing uses engineered nucleases as powerful tools to target specific DNA sequences to edit genes precisely in the genomes of both model and crop plants, as well as a variety of other organisms. The DNA-binding domains of zinc finger (ZF) proteins were the first to be used as genome editing tools, in the form of designed ZF nucleases (ZFNs). More recently, transcription activator-like effector nucleases (TALENs), as well as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which utilizes RNA-DNA interactions, have proved useful. A key step in genome editing is the generation of a double-stranded DNA break that is specific to the target gene. This is achieved by custom-designed endonucleases, which enable site-directed mutagenesis via a non-homologous end-joining (NHEJ) repair pathway and/or gene targeting via homologous recombination (HR) to occur efficiently at specific sites in the genome. This review provides an overview of recent advances in genome editing technologies in plants, and discusses how these can provide insights into current plant molecular biology research and molecular breeding technology.

  4. Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila.

    PubMed

    Beumer, Kelly J; Trautman, Jonathan K; Christian, Michelle; Dahlem, Timothy J; Lake, Cathleen M; Hawley, R Scott; Grunwald, David J; Voytas, Daniel F; Carroll, Dana

    2013-10-03

    Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.

  5. Origins of Programmable Nucleases for Genome Engineering.

    PubMed

    Chandrasegaran, Srinivasan; Carroll, Dana

    2016-02-27

    Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins and the FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system-the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein-have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut

  6. A guide to genome engineering with programmable nucleases.

    PubMed

    Kim, Hyongbum; Kim, Jin-Soo

    2014-05-01

    Programmable nucleases - including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system - enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.

  7. Holo- And Apo- Structures of Bacterial Periplasmic Heme Binding Proteins

    SciTech Connect

    Ho, W.W.; Li, H.; Eakanunkul, S.; Tong, Y.; Wilks, A.; Guo, M.; Poulos, T.L.

    2009-06-01

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an 'in' position where it can coordinate the heme iron to an 'out' orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg{sup 228} in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg{sup 228}, and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B{sub 12}-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B{sub 12}, compared with ligands for FhuD, a peptide siderophore.

  8. Genome Editing in Mice Using TALE Nucleases.

    PubMed

    Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

    2016-01-01

    Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

  9. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

    PubMed Central

    Vertommen, Didier; Silhavy, Thomas J.; Collet, Jean-Francois

    2013-01-01

    β-barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the non-viable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli. PMID:22589188

  10. Dynamic periplasmic chaperone reservoir facilitates biogenesis of outer membrane proteins

    PubMed Central

    Costello, Shawn M.; Plummer, Ashlee M.; Fleming, Patrick J.; Fleming, Karen G.

    2016-01-01

    Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed “Outer Membrane Protein Biogenesis Model” (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway. PMID:27482090

  11. Alternative periplasmic copper-resistance mechanisms in Gram negative bacteria.

    PubMed

    Pontel, Lucas B; Soncini, Fernando C

    2009-07-01

    Bacteria have evolved different systems to tightly control both cytosolic and envelope copper concentration to fulfil their requirements and at the same time, avoid copper toxicity. We have previously demonstrated that, as in Escherichia coli, the Salmonella cue system protects the cytosol from copper excess. On the other hand, and even though Salmonella lacks the CusCFBA periplasmic copper efflux system, it can support higher copper concentrations than E. coli under anaerobic conditions. Here we show that the Salmonella cue regulon is also responsible for the control of copper toxicity in anaerobiosis. We establish that resistance in this condition requires a novel CueR-controlled gene named cueP. A DeltacueP mutant is highly susceptible to copper in the absence of oxygen, but shows a faint phenotype in aerobic conditions unless other copper-resistance genes are also deleted, resembling the E. coli CusCFBA behaviour. Species that contain a cueP homologue under CueR regulation have no functional CusR/CusS-dependent Cus-coding operon. Conversely, species that carry a CusR/CusS-regulated cus operon have no cueP homologues. Even more, we show that the CueR-controlled cueP expression increases copper resistance of a Deltacus E. coli. We posit that CueP can functionally replace the Cus complex for periplasmic copper resistance, in particular under anaerobic conditions.

  12. Sequential closure of the cytoplasm and then the periplasm during cell division in Escherichia coli.

    PubMed

    Skoog, Karl; Söderström, Bill; Widengren, Jerker; von Heijne, Gunnar; Daley, Daniel O

    2012-02-01

    To visualize the latter stages of cell division in live Escherichia coli, we have carried out fluorescence recovery after photobleaching (FRAP) on 121 cells expressing cytoplasmic green fluorescent protein and periplasmic mCherry. Our data show conclusively that the cytoplasm is sealed prior to the periplasm during the division event.

  13. TALE nucleases and next generation GM crops.

    PubMed

    Mahfouz, Magdy M; Li, Lixin

    2011-01-01

    Site-specific and adaptable DNA binding domains are essential modules to develop genome engineering technologies for crop improvement. Transcription activator-like effectors (TALEs) proteins are used to provide a highly specific and adaptable DNA binding modules. TALE chimeric nucleases (TALENs) were used to generate site-specific double strand breaks (DSBs) in vitro and in yeast, Caenorhabditis elegans, mammalian and plant cells. The genomic DSBs can be generated at predefined and user-selected loci and repaired by either the non-homologous end joining (NHEJ) or homology dependent repair (HDR). Thus, TALENs can be used to achieve site-specific gene addition, stacking, deletion or inactivation. TALE-based genome engineering tools should be powerful to develop new agricultural biotechnology approaches for crop improvement. Here, we discuss the recent research and the potential applications of TALENs to accelerate the generation of genomic variants through targeted mutagenesis and to produce a non-transgenic GM crops with the desired phenotype.

  14. Genetic engineering of human pluripotent cells using TALE nucleases.

    PubMed

    Hockemeyer, Dirk; Wang, Haoyi; Kiani, Samira; Lai, Christine S; Gao, Qing; Cassady, John P; Cost, Gregory J; Zhang, Lei; Santiago, Yolanda; Miller, Jeffrey C; Zeitler, Bryan; Cherone, Jennifer M; Meng, Xiangdong; Hinkley, Sarah J; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Jaenisch, Rudolf

    2011-07-07

    Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here we engineered transcription activator-like effector nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) clones carrying transgenic cassettes solely at the TALEN-specified location. Our data suggest that TALENs employing the specific architectures described here mediate site-specific genome modification in human pluripotent cells with similar efficiency and precision as do zinc-finger nucleases (ZFNs).

  15. Protein Diffusion in the Periplasm of E. coli under Osmotic Stress

    PubMed Central

    Sochacki, Kem A.; Shkel, Irina A.; Record, M. Thomas; Weisshaar, James C.

    2011-01-01

    The physical and mechanical properties of the cell envelope of Escherichia coli are poorly understood. We use fluorescence recovery after photobleaching to measure diffusion of periplasmic green fluorescent protein and probe the fluidity of the periplasm as a function of external osmotic conditions. For cells adapted to growth in complete medium at 0.14–1.02 Osm, the mean diffusion coefficient increases from 3.4 μm2 s−1 to 6.6 μm2 s−1 and the distribution of Dperi broadens as growth osmolality increases. This is consistent with a net gain of water by the periplasm, decreasing its biopolymer volume fraction. This supports a model in which the turgor pressure drops primarily across the thin peptidoglycan layer while the cell actively maintains osmotic balance between periplasm and cytoplasm, thus avoiding a substantial pressure differential across the cytoplasmic membrane. After sudden hyperosmotic shock (plasmolysis), the cytoplasm loses water as the periplasm gains water. Accordingly, increases threefold. The fluorescence recovery after photobleaching is complete and homogeneous in all cases, but in minimal medium, the periplasm is evidently thicker at the cell tips. For the relevant geometries, Brownian dynamics simulations in model cytoplasmic and periplasmic volumes provide analytical formulae for extraction of accurate diffusion coefficients from readily measurable quantities. PMID:21190653

  16. Rapid qualitative method for detecting staphylococcal nuclease in foods.

    PubMed Central

    Koupal, A; Deibel, R H

    1978-01-01

    A rapid method for the detection of heat-stable staphylococcal nuclease in foods is described. The procedure consists of an acid precipitation, boiling, and centrifugation followed by enzyme detection in an agar plate containing deoxyribonucleic acid. To test the efficacy of the procedure, purified Staphylococcus aureus nuclease was added to various foods and recovery experiments were performed. Additionally, foods were inoculated and incubated with S. aureus, and the staphylococcal counts were compared with nuclease activity. The results indicate that the procedure possesses merit for a rapid method that can be incorporated into quality control programs. The procedure requires approximately 2.5 h, and it will detect nuclease levels as low as 10 ng/g of food. Images PMID:677882

  17. Genome Editing in Human Cells Using CRISPR/Cas Nucleases.

    PubMed

    Wyvekens, Nicolas; Tsai, Shengdar Q; Joung, J Keith

    2015-10-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. This unit describes protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases.

  18. Catalytic activity of nuclease P1: Experiment and theory

    SciTech Connect

    Miller, J.H.; Falcone, J.M.; Shibata, M.; Box, H.C.

    1994-10-01

    Nuclease P1 from Penicillium citrinum is a zinc dependent glyco-enzyme that recognizes single stranded DNA and RNA as substrates and hydrolyzes the phosphate ester bond. Nuclease Pl seems to recognize particular conformations of the phosphodiester backbone and shows significant variation in the rate of hydrolytic activity depending upon which nucleosides are coupled by the phosphodiester bond. The efficiency of nuclease Pl in hydrolyzing the phosphodiester bonds of a substrate can be altered by modifications to one of the substrate bases induced by ionizing radiation or oxidative stress. Measurements have been made of the effect of several radiation induced lesions on the catalytic rate of nuclease Pl. A model of the structure of the enzyme has been constructed in order to better understand the binding and activity of this enzyme on various ssDNA substrates.

  19. Targeted mutagenesis of zebrafish: use of zinc finger nucleases.

    PubMed

    Leong, Ivone Un San; Lai, Daniel; Lan, Chuan-Ching; Johnson, Ross; Love, Donald R; Johnson, Ross; Love, Donald R

    2011-09-01

    The modeling of human disease in the zebrafish (Danio rerio) is moving away from chemical mutagensis and transient downregulation using morpholino oligomers to more targeted and stable transgenic methods. In this respect, zinc finger nucleases offer a means of introducing mutations at targeted sites at high efficiency. We describe here the development of zinc finger nucleases and their general use in model systems with a focus on the zebrafish.

  20. Disruption of the Membrane Nuclease Gene (MBOVPG45_0215) of Mycoplasma bovis Greatly Reduces Cellular Nuclease Activity

    PubMed Central

    Sharma, Shukriti; Tivendale, Kelly A.; Markham, Philip F.

    2015-01-01

    ABSTRACT Although the complete genome sequences of three strains of Mycoplasma bovis are available, few studies have examined gene function in this important pathogen. Mycoplasmas lack the biosynthetic machinery for the de novo synthesis of nucleic acid precursors, so nucleases are likely to be essential for them to acquire nucleotide precursors. Three putative membrane nucleases have been annotated in the genome of M. bovis strain PG45, MBOVPG45_0089 and MBOVPG45_0310, both of which have the thermonuclease (TNASE_3) functional domain, and MBOVPG45_0215 (mnuA), which has an exonuclease/endonuclease/phosphatase domain. While previous studies have demonstrated the function of TNASE_3 domain nucleases in several mycoplasmas, quantitative comparisons of the contributions of different nucleases to cellular nuclease activity have been lacking. Mapping of a library of 319 transposon mutants of M. bovis PG45 by direct genome sequencing identified mutants with insertions in MBOVPG45_0310 (the Δ0310 mutant) and MBOVPG45_0215 (the Δ0215 mutant). In this study, the detection of the product of MBOVPG45_0215 in the Triton X-114 fraction of M. bovis cell lysates, its cell surface exposure, and its predicted signal peptide suggested that it is a surface-exposed lipoprotein nuclease. Comparison of a ΔmnuA mutant with wild-type M. bovis on native and denatured DNA gels and in digestion assays using double-stranded phage λ DNA and closed circular plasmid DNA demonstrated that inactivation of this gene abolishes most of the cellular exonuclease and endonuclease activity of M. bovis. This activity could be fully restored by complementation with the wild-type mnuA gene, demonstrating that MnuA is the major cellular nuclease of M. bovis. IMPORTANCE Nucleases are thought to be important contributors to virulence and crucial for the maintenance of a nutritional supply of nucleotides in mycoplasmas that are pathogenic in animals. This study demonstrates for the first time that of the

  1. Artificial zinc finger nucleases for DNA cloning.

    PubMed

    Zeevi, Vardit; Tovkach, Andriy; Tzfira, Tzvi

    2010-01-01

    DNA cloning is fundamental for modern cell research and biotechnology. Various restriction enzymes have been isolated, characterized, and purified to facilitate the digestion and ligation of DNA molecules of different origins. Nevertheless, the very small numbers of enzymes capable of digesting novel and long DNA sequences and the tedious and nearly impossible task of re-engineering existing enzymes with novel specificities greatly limit the use of restriction enzymes for the construction of complex and long DNA molecules. Zinc finger nucleases (ZFNs) - hybrid restriction enzymes that can be tailor made for the digestion of both native and artificial DNA sequences - offer a unique opportunity for expanding the repertoire of restriction enzymes useful for various DNA cloning tasks. Here we present protocols for the assembly, expression, and purification of cloning-grade ZFNs and their use for DNA cloning. We focus our discussion on the assembly of a dual-cassette plant transformation vector, as an example of a task that is nearly impossible to perform using the current collection of naturally occurring and recombinant 6-8 bp long restriction enzymes.

  2. ARTEMIS nuclease facilitates apoptotic chromatin cleavage.

    PubMed

    Britton, Sébastien; Frit, Philippe; Biard, Denis; Salles, Bernard; Calsou, Patrick

    2009-10-15

    One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pan-caspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis.

  3. Biological function of a polysaccharide degrading enzyme in the periplasm.

    PubMed

    Wang, Yajie; Moradali, M Fata; Goudarztalejerdi, Ali; Sims, Ian M; Rehm, Bernd H A

    2016-11-08

    Carbohydrate polymers are industrially and medically important. For instance, a polysaccharide, alginate (from seaweed), is widely used in food, textile and pharmaceutical industries. Certain bacteria also produce alginate through membrane spanning multi-protein complexes. Using Pseudomonas aeruginosa as a model organism, we investigated the biological function of an alginate degrading enzyme, AlgL, in alginate production and biofilm formation. We showed that AlgL negatively impacts alginate production through its enzymatic activity. We also demonstrated that deletion of AlgL does not interfere with polymer length control, epimerization degree or stability of the biosynthesis complex, arguing that AlgL is a free periplasmic protein dispensable for alginate production. This was further supported by our protein-stability and interaction experiments. Interestingly, over-production of AlgL interfered with polymer length control, suggesting that AlgL could be loosely associated with the biosynthesis complex. In addition, chromosomal expression of algL enhanced alginate O-acetylation; both attachment and dispersal stages of the bacterial biofilm lifecycle were sensitive to the level of O-acetylation. Since this modification also protects the pathogen against host defences and enhances other virulence factors, chromosomal expression of algL could be important for the pathogenicity of this organism. Overall, this work improves our understanding of bacterial alginate production and provides new knowledge for alginate production and disease control.

  4. Biological function of a polysaccharide degrading enzyme in the periplasm

    PubMed Central

    Wang, Yajie; Moradali, M. Fata; Goudarztalejerdi, Ali; Sims, Ian M.; Rehm, Bernd H. A.

    2016-01-01

    Carbohydrate polymers are industrially and medically important. For instance, a polysaccharide, alginate (from seaweed), is widely used in food, textile and pharmaceutical industries. Certain bacteria also produce alginate through membrane spanning multi-protein complexes. Using Pseudomonas aeruginosa as a model organism, we investigated the biological function of an alginate degrading enzyme, AlgL, in alginate production and biofilm formation. We showed that AlgL negatively impacts alginate production through its enzymatic activity. We also demonstrated that deletion of AlgL does not interfere with polymer length control, epimerization degree or stability of the biosynthesis complex, arguing that AlgL is a free periplasmic protein dispensable for alginate production. This was further supported by our protein-stability and interaction experiments. Interestingly, over-production of AlgL interfered with polymer length control, suggesting that AlgL could be loosely associated with the biosynthesis complex. In addition, chromosomal expression of algL enhanced alginate O-acetylation; both attachment and dispersal stages of the bacterial biofilm lifecycle were sensitive to the level of O-acetylation. Since this modification also protects the pathogen against host defences and enhances other virulence factors, chromosomal expression of algL could be important for the pathogenicity of this organism. Overall, this work improves our understanding of bacterial alginate production and provides new knowledge for alginate production and disease control. PMID:27824067

  5. A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis.

    PubMed

    Bartlett, Thomas M; Bratton, Benjamin P; Duvshani, Amit; Miguel, Amanda; Sheng, Ying; Martin, Nicholas R; Nguyen, Jeffrey P; Persat, Alexandre; Desmarais, Samantha M; VanNieuwenhze, Michael S; Huang, Kerwyn Casey; Zhu, Jun; Shaevitz, Joshua W; Gitai, Zemer

    2017-01-12

    Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism and function of this curvature were previously unknown. Here, we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm and thus can be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (quantitative analysis of sacculus architecture remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertions into the outer face than the inner face. Furthermore, crvA is quorum regulated, and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium.

    PubMed Central

    Kier, L D; Weppelman, R; Ames, B N

    1977-01-01

    A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems. Images PMID:192712

  7. Metabolism of periplasmic membrane-derived oligosaccharides by the predatory bacterium Bdellovibrio bacteriovorus 109J

    SciTech Connect

    Ruby, E.G.; McCabe, J.B.

    1988-02-01

    Membrane-derived oligosaccharides (MDO), a class of osmotically active carbohydrates, are the major organic solutes present in the periplasm of Escherichia coli and many other gram-negative bacteria when cells are grown in a medium of low osmolarity. Analyses of growing cells of Bdellovibrio bacteriovorus, a gram-negative predator of other bacteria, have confirmed that they also synthesize a characteristic MDO-like class of oligosaccharides. The natural growth environment of bdellovibrios is the periplasm of other gram-negative bacteria. Because of this location, prey cell MDO constitute a potential source of organic nutrients for growing bdellovibrios. Using cells of E. coli whose MDO were /sup 3/H labeled, we examined the extent to which B. bacteriovorus 109J metabolizes these prey cell components. Interestingly, there was neither significant degradation nor incorporation of prey cell MDO by bdellovibrios during the course of their intracellular growth. In fact, bdellovibrios had little capability either to degrade extracellular MDO that was made available to them or to transport glucose, the major monomeric constituent of prey cell MDO. Instead, periplasmic MDO were irreversibly lost to the extracellular environment during the period of bdellovibrio attack and penetration. Thus, although prey cell periplasmic proteins are retained, other important periplasmic components are released early in the bdellovibrio growth cycle. The loss of these MDO may aid in the destabilization of the prey cell plasma membrane, increasing the availability of cytoplasmic constituents to the periplasmic bdellovibrio.

  8. Direct Metal Transfer Between Periplasmic Proteins Identifies a Bacterial Copper Chaperone

    SciTech Connect

    Bagai, I.; Rensing, C.; Blackburn, N.; McEvoy, M.M.

    2009-05-11

    Transition metals require exquisite handling within cells to ensure that cells are not harmed by an excess of free metal species. In Gram-negative bacteria, copper is required in only small amounts in the periplasm, not in the cytoplasm, so a key aspect of protection under excess metal conditions is to export copper from the periplasm. Additional protection could be conferred by a periplasmic chaperone to limit the free metal species prior to export. Using isothermal titration calorimetry, we have demonstrated that two periplasmic proteins, CusF and CusB, of the Escherichia coli Cu(I)/Ag(I) efflux system undergo a metal-dependent interaction. Through the development of a novel X-ray absorption spectroscopy approach using selenomethionine labeling to distinguish the metal sites of the two proteins, we have demonstrated transfer of Cu(I) occurs between CusF and CusB. The interaction between these proteins is highly specific, as a homologue of CusF with a 51% identical sequence and a similar affinity for metal, did not function in metal transfer. These experiments establish a metallochaperone activity for CusF in the periplasm of Gram-negative bacteria, serving to protect the periplasm from metal-mediated damage.

  9. Periplasmic response upon disruption of transmembrane Cu transport in Pseudomonas aeruginosa.

    PubMed

    Raimunda, Daniel; Padilla-Benavides, Teresita; Vogt, Stefan; Boutigny, Sylvain; Tomkinson, Kaleigh N; Finney, Lydia A; Argüello, José M

    2013-02-01

    Pseudomonas aeruginosa, an opportunistic pathogen, has two transmembrane Cu(+) transport ATPases, CopA1 and CopA2. Both proteins export cytoplasmic Cu(+) into the periplasm and mutation of either gene leads to attenuation of virulence. CopA1 is required for maintaining cytoplasmic copper levels, while CopA2 provides copper for cytochrome c oxidase assembly. We hypothesized that transported Cu(+) ions would be directed to their destination via specific periplasmic partners and disruption of transport should affect the periplasmic copper homeostasis. Supporting this, mutation of either ATPase gene led to large increments in periplasmic cuproprotein levels. Toward identifying the proteins participating in this cellular response the periplasmic metalloproteome was resolved in non-denaturing bidimensional gel electrophoresis, followed by X-ray fluorescence visualization and identification by mass-spectrometry. A single spot containing the electron shuttle protein azurin was responsible for the observed increments in cuproprotein contents. In agreement, lack of either Cu(+)-ATPase induced an increase in azu transcription. This is associated with an increase in the expression of anr and rpoS oxidative stress response regulators, rather than cueR, a copper sensing regulator. We propose that azurin overexpression and accumulation in the periplasm is part of the cellular response to cytoplasmic oxidative stress in P. aeruginosa.

  10. c-Type Cytochrome Assembly Is a Key Target of Copper Toxicity within the Bacterial Periplasm

    PubMed Central

    Durand, Anne; Azzouzi, Asma; Bourbon, Marie-Line; Steunou, Anne-Soisig; Liotenberg, Sylviane; Maeshima, Akinori; Astier, Chantal; Argentini, Manuela; Saito, Shingo

    2015-01-01

    ABSTRACT In the absence of a tight control of copper entrance into cells, bacteria have evolved different systems to control copper concentration within the cytoplasm and the periplasm. Central to these systems, the Cu+ ATPase CopA plays a major role in copper tolerance and translocates copper from the cytoplasm to the periplasm. The fate of copper in the periplasm varies among species. Copper can be sequestered, oxidized, or released outside the cells. Here we describe the identification of CopI, a periplasmic protein present in many proteobacteria, and show its requirement for copper tolerance in Rubrivivax gelatinosus. The ΔcopI mutant is more susceptible to copper than the Cu+ ATPase copA mutant. CopI is induced by copper, localized in the periplasm and could bind copper. Interestingly, copper affects cytochrome c membrane complexes (cbb3 oxidase and photosystem) in both ΔcopI and copA-null mutants, but the causes are different. In the copA mutant, heme and chlorophyll synthesis are affected, whereas in ΔcopI mutant, the decrease is a consequence of impaired cytochrome c assembly. This impact on c-type cytochromes would contribute also to the copper toxicity in the periplasm of the wild-type cells when they are exposed to high copper concentrations. PMID:26396241

  11. Direct metal transfer between periplasmic proteins identifies a bacterial copper chaperone.

    PubMed

    Bagai, Ireena; Rensing, Christopher; Blackburn, Ninian J; McEvoy, Megan M

    2008-11-04

    Transition metals require exquisite handling within cells to ensure that cells are not harmed by an excess of free metal species. In gram-negative bacteria, copper is required in only small amounts in the periplasm, not in the cytoplasm, so a key aspect of protection under excess metal conditions is to export copper from the periplasm. Additional protection could be conferred by a periplasmic chaperone to limit the free metal species prior to export. Using isothermal titration calorimetry, we have demonstrated that two periplasmic proteins, CusF and CusB, of the Escherichia coli Cu(I)/Ag(I) efflux system undergo a metal-dependent interaction. Through the development of a novel X-ray absorption spectroscopy approach using selenomethionine labeling to distinguish the metal sites of the two proteins, we have demonstrated transfer of Cu(I) occurs between CusF and CusB. The interaction between these proteins is highly specific, as a homologue of CusF with a 51% identical sequence and a similar affinity for metal, did not function in metal transfer. These experiments establish a metallochaperone activity for CusF in the periplasm of gram-negative bacteria, serving to protect the periplasm from metal-mediated damage.

  12. Periplasmic production of native human proinsulin as a fusion to E. coli ecotin.

    PubMed

    Malik, Ajamaluddin; Jenzsch, Marco; Lübbert, Andreas; Rudolph, Rainer; Söhling, Brigitte

    2007-09-01

    Native proinsulin belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its small size, a high proteolytic decay, and the necessity to form a native disulfide pattern. In the present study, human proinsulin was produced in the periplasm of E. coli as a fusion to ecotin, which is a small periplasmic protein of 16 kDa encoded by the host, containing one disulfide bond. The fusion protein was secreted to the periplasm and native proinsulin was determined by ELISA. Cultivation parameters were studied in parallel batch mode fermentations using E. coli BL21(DE3)Gold as a host. After improvement of fed-batch high density fermentation conditions, 153 mg fusion protein corresponding to 51.5mg native proinsulin was obtained per L. Proteins were extracted from the periplasm by osmotic shock treatment. The fusion protein was purified in one step by ecotin affinity chromatography on immobilized trypsinogen. After thrombin cleavage of the fusion protein, the products were separated by Ni-NTA chromatography. Proinsulin was quantified by ELISA and characterized by mass spectrometry. To evaluate the influence of periplasmic proteases, the amount of ecotin-proinsulin was determined in E. coli BL21(DE3)Gold and in a periplasmic protease deficient strain, E. coli SF120.

  13. Genetic correction using engineered nucleases for gene therapy applications.

    PubMed

    Li, Hongmei Lisa; Nakano, Takao; Hotta, Akitsu

    2014-01-01

    Genetic mutations in humans are associated with congenital disorders and phenotypic traits. Gene therapy holds the promise to cure such genetic disorders, although it has suffered from several technical limitations for decades. Recent progress in gene editing technology using tailor-made nucleases, such as meganucleases (MNs), zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and, more recently, CRISPR/Cas9, has significantly broadened our ability to precisely modify target sites in the human genome. In this review, we summarize recent progress in gene correction approaches of the human genome, with a particular emphasis on the clinical applications of gene therapy. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  14. NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.

    PubMed

    Kautz, R A; Fox, R O

    1993-05-01

    Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 degree C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116-Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis-trans equilibrium. The rate of trans-->cis isomerization in the native-like nuclease intermediate is approximately 100-fold faster than that observed in a Lys-Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116-Pro 117 peptide bond is comparable to that observed for other X-Pro isomerizations.

  15. A TALE nuclease architecture for efficient genome editing.

    PubMed

    Miller, Jeffrey C; Tan, Siyuan; Qiao, Guijuan; Barlow, Kyle A; Wang, Jianbin; Xia, Danny F; Meng, Xiangdong; Paschon, David E; Leung, Elo; Hinkley, Sarah J; Dulay, Gladys P; Hua, Kevin L; Ankoudinova, Irina; Cost, Gregory J; Urnov, Fyodor D; Zhang, H Steve; Holmes, Michael C; Zhang, Lei; Gregory, Philip D; Rebar, Edward J

    2011-02-01

    Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

  16. Hybrid nanosensor for colorimetric and ultrasensitive detection of nuclease contaminations

    NASA Astrophysics Data System (ADS)

    Cecere, Paola; Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Nucleases are ubiquitous enzymes that degrade DNA or RNA, thus they can prejudice the good outcome of molecular biology experiments involving nucleic acids. We propose a colorimetric test for the naked-eye detection of nuclease contaminations. The system uses an hybrid nanosensor, based on gold nanoparticles functionalized with DNA probes. Our assay is rapid, instrument-free, simple and low-cost. Moreover, it reaches sensitivity equal or better than those of commercial kits, and presents a lot of advantageous aspects. Therefore, it is very competitive, with a real market potential. This test will be relevant in routine process monitoring in scientific laboratories, and in quality control in clinical laboratories and industrial processes, allowing the simultaneous detection of nucleases with different substrate specificities and large-scale screening.

  17. In Vivo Detection of the Cyclic Osmoregulated Periplasmic Glucan of Ralstonia solanacearum by High-Resolution Magic Angle Spinning NMR

    NASA Astrophysics Data System (ADS)

    Wieruszeski, J.-M.; Bohin, A.; Bohin, J.-P.; Lippens, G.

    2001-07-01

    We investigate the mobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.

  18. Engineering nucleases for gene targeting: safety and regulatory considerations.

    PubMed

    Pauwels, Katia; Podevin, Nancy; Breyer, Didier; Carroll, Dana; Herman, Philippe

    2014-01-25

    Nuclease-based gene targeting (NBGT) represents a significant breakthrough in targeted genome editing since it is applicable from single-celled protozoa to human, including several species of economic importance. Along with the fast progress in NBGT and the increasing availability of customized nucleases, more data are available about off-target effects associated with the use of this approach. We discuss how NBGT may offer a new perspective for genetic modification, we address some aspects crucial for a safety improvement of the corresponding techniques and we also briefly relate the use of NBGT applications and products to the regulatory oversight.

  19. Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

    PubMed Central

    Jo, Byung Hoon; Kim, Im Gyu; Seo, Jeong Hyun; Kang, Dong Gyun

    2013-01-01

    Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration. PMID:23974145

  20. Cloning and Expression of Recombinant Human Endostatin in Periplasm of Escherichia coli Expression System

    PubMed Central

    Mohajeri, Abbas; Pilehvar-Soltanahmadi, Yones; Pourhassan-Moghaddam, Mohammad; Abdolalizadeh, Jalal; Karimi, Pouran; Zarghami, Nosratollah

    2016-01-01

    Purpose: Recombinant human endostatin (rhEs) is an angiogenesis inhibitor which is used as a specific drug in the treatment of non-small-cell lung cancer. In the current research, we developed an efficient method for expressing soluble form of the rhEs protein in the periplasmic space of Escherichia coli via fusing with pelB signal peptide. Methods: The human endostatin (hEs) gene was amplified using synthetic (hEs) gene as a template; then, cloned and expressed under T7 lac promoter. IPTG was used as an inducer for rhEs expression. Next, the osmotic shock was used to extraction of protein from the periplasmic space. The presence of rhEs in the periplasmic space was approved by SDS-PAGE and Western blotting. Results: The results show the applicability of pelB fusion protein system usage for secreting rhEs in the periplasm of E. coli in the laboratory scale. The rhEs represents approximately 35 % (0.83mg/l) of the total cell protein. Conclusion: The present study apparently is the first report of codon-optimized rhEs expression as a fusion with pelB signal peptide. The results presented the successful secretion of soluble rhEs to the periplasmic space. PMID:27478780

  1. Microscopic studies on Thermosipho globiformans implicate a role of the large periplasm of Thermotogales.

    PubMed

    Kuwabara, Tomohiko; Igarashi, Kensuke

    2012-11-01

    Thermosipho globiformans is a member of Thermotogales, which contains rod-shaped, Gram-negative, anaerobic (hyper)thermophiles. These bacteria are characterized by an outer sheath-like envelope, the toga, which includes the outer membrane and an amorphous layer, and forms large periplasm at the poles of each rod. The cytoplasmic membrane and its contents are called "cell", and the toga and its contents "rod", to distinguish between them. Optical cells were constructed to observe binary fission of T. globiformans. High-temperature microscopy of rods adhering to optical cells' coverslips showed that the large periplasm forms between newly divided cells in a rod, followed by rod fission at the middle of the periplasm, which was accompanied by a sideward motion of the newly generated rod pole(s). Electron microscopic observations revealed that sessile rods grown on a glass plate have nanotubes adhered to the glass, and these may be involved in the sideward motion. Epifluorescence microscopy with a membrane-staining dye suggested that formation of the septal outer membrane is distinct from cytokinesis. Transmission electron microscopy indicated that the amorphous layer forms in the periplasm between already-divided cells. These findings suggest that the large periplasm is the structure in which the septal toga forms, an event separate from cytokinesis.

  2. Engineered Escherichia coli with periplasmic carbonic anhydrase as a biocatalyst for CO2 sequestration.

    PubMed

    Jo, Byung Hoon; Kim, Im Gyu; Seo, Jeong Hyun; Kang, Dong Gyun; Cha, Hyung Joon

    2013-11-01

    Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO2). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO2, a major greenhouse gas, making this a promising alternative for chemical CO2 mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO2 sequestration by mineral carbonation, a process with the potential to store large quantities of CO2. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO2 compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO3) formation and exerted a striking impact on the maximal amount of CaCO3 produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO2 sequestration.

  3. Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12.

    PubMed Central

    Lazzaroni, J C; Portalier, R C

    1981-01-01

    Periplasmic-leaky mutants of Escherichia coli K-12 were isolated after nitrosoguanidine-induced mutagenesis. They released periplasmic enzymes into the extracellular medium. Excretion of alkaline phosphatase, which started immediately in the early exponential phase of growth, could reach up to 90% of the total enzyme production in the stationary phase. Leaky mutants were sensitive to ethylenediaminetetraacetic acid, cholic acid, and the antibiotics rifampin, chloramphenicol, mitomycin C, and ampicillin. Furthermore, they were resistant to colicin E1 and partially resistant to phage TuLa. Their genetic characterization showed that the lky mutations mapped between the suc and gal markers, near or in the tolPAB locus. A biochemical analysis of cell envelope components showed that periplasmic-leaky mutants contained reduced amounts of major outer membrane protein OmpF and increased amounts of a 16,000-dalton outer membrane protein. Images PMID:7009581

  4. Induction of double-strand breaks by S1 nuclease, mung bean nuclease and nuclease P1 in DNA containing abasic sites and nicks.

    PubMed Central

    Chaudhry, M A; Weinfeld, M

    1995-01-01

    Defined DNA substrates containing discrete abasic sites or paired abasic sites set 1, 3, 5 and 7 bases apart on opposite strands were constructed to examine the reactivity of S1, mung bean and P1 nucleases towards abasic sites. None of the enzymes acted on the substrate containing discrete abasic sites. Under conditions where little or no non-specific DNA degradation was observed, all three nucleases were able to generate double-strand breaks when the bistranded abasic sites were 1 and 3 base pairs apart. However, when the abasic sites were further apart, the enzymes again failed to cleave the DNA. These results indicate that single abasic sites do not cause sufficient denaturation of the DNA to allow incision by these single-strand specific endonucleases. The reactivity of these enzymes was also investigated on DNA substrates that were nicked by DNasel or more site-specifically by endonuclease III incision at the discrete abasic sites. The three nucleases readily induced a strand break opposite such nicks. Images PMID:7479020

  5. Adenoviral vector DNA for accurate genome editing with engineered nucleases.

    PubMed

    Holkers, Maarten; Maggio, Ignazio; Henriques, Sara F D; Janssen, Josephine M; Cathomen, Toni; Gonçalves, Manuel A F V

    2014-10-01

    Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.

  6. Purification, cloning, and characterization of the CEL I nuclease.

    PubMed

    Yang, B; Wen, X; Kodali, N S; Oleykowski, C A; Miller, C G; Kulinski, J; Besack, D; Yeung, J A; Kowalski, D; Yeung, A T

    2000-04-04

    CEL I, isolated from celery, is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. The enzyme requires Mg(2+) and Zn(2+) for activity, with a pH optimum at neutral pH. We have purified CEL I 33 000-fold to apparent homogeneity. A key improvement is the use of alpha-methyl-mannoside in the purification buffers to overcome the aggregation of glycoproteins with endogenous lectins. The SDS gel electrophoresis band for the homogeneous CEL I, with and without the removal of its carbohydrate moieties, was extracted, renatured, and shown to have mismatch cutting specificity. After determination of the amino acid sequence of 28% of the CEL I polypeptide, we cloned the CEL I cDNA. Potential orthologs are nucleases putatively encoded by the genes BFN1 of Arabidopsis, ZEN1 of Zinnia, and DSA6 of daylily. Homologies of CEL I with S1 and P1 nucleases are much lower. We propose that CEL I exemplifies a new family of neutral pH optimum, magnesium-stimulated, mismatch duplex-recognizing nucleases, within the S1 superfamily.

  7. Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

    PubMed

    Doke, M; Fukamachi, H; Morisaki, H; Arimoto, T; Kataoka, H; Kuwata, H

    2016-08-01

    Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg(2+) and Ca(2+) for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.

  8. A species-specific periplasmic flagellar protein of Serpulina (Treponema) hyodysenteriae.

    PubMed Central

    Li, Z; Dumas, F; Dubreuil, D; Jacques, M

    1993-01-01

    We have previously reported that a 46-kDa protein present in an outer membrane protein preparation seemed to be a species-specific antigen of Serpulina hyodysenteriae (Z. S. Li, N. S. Jensen, M. Bélanger, M.-C. L'Espérance, and M. Jacques, J. Clin. Microbiol. 30:2941-2947, 1992). The objective of this study was to further characterize this antigen. A Western blot (immunoblot) analysis and immunogold labeling with a monospecific antiserum against this protein confirmed that the protein was present in all S. hyodysenteriae reference strains but not in the nonpathogenic organism Serpulina innocens. The immunogold labeling results also indicated that the protein was associated with the periplasmic flagella of S. hyodysenteriae. N-terminal amino acid sequencing confirmed that the protein was in fact a periplasmic flagellar sheath protein. The molecular mass of this protein, first estimated to be 46 kDa by Western blotting, was determined to be 44 kDa when the protein was evaluated more precisely by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was glycosylated, as determined by glycoprotein staining and also by N-glycosidase F treatment. Five other periplasmic flagellar proteins of S. hyodysenteriae, which may have been the core proteins and had molecular masses of 39, 35, 32, 30, and 29 kDa, were antigenically related and cross-reacted with the periplasmic flagellar proteins of S. innocens. Finally, serum from a pig experimentally infected with S. hyodysenteriae recognized the 44-kDa periplasmic flagellar sheath protein. Our results suggest that the 44-kDa periplasmic flagellar sheath protein of S. hyodysenteriae is a species-specific glycoprotein antigen. Images PMID:8253687

  9. Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging) of Escherichia coli.

    PubMed

    Clark, Michelle W; Yie, Anna M; Eder, Elizabeth K; Dennis, Richard G; Basting, Preston J; Martinez, Keith A; Jones, Brian D; Slonczewski, Joan L

    2015-01-01

    Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH), no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5) the cells maintained cytoplasmic pH values at 7.2-7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress.

  10. Periplasmic Acid Stress Increases Cell Division Asymmetry (Polar Aging) of Escherichia coli

    PubMed Central

    Clark, Michelle W.; Yie, Anna M.; Eder, Elizabeth K.; Dennis, Richard G.; Basting, Preston J.; Martinez, Keith A.; Jones, Brian D.; Slonczewski, Joan L.

    2015-01-01

    Under certain kinds of cytoplasmic stress, Escherichia coli selectively reproduce by distributing the newer cytoplasmic components to new-pole cells while sequestering older, damaged components in cells inheriting the old pole. This phenomenon is termed polar aging or cell division asymmetry. It is unknown whether cell division asymmetry can arise from a periplasmic stress, such as the stress of extracellular acid, which is mediated by the periplasm. We tested the effect of periplasmic acid stress on growth and division of adherent single cells. We tracked individual cell lineages over five or more generations, using fluorescence microscopy with ratiometric pHluorin to measure cytoplasmic pH. Adherent colonies were perfused continually with LBK medium buffered at pH 6.00 or at pH 7.50; the external pH determines periplasmic pH. In each experiment, cell lineages were mapped to correlate division time, pole age and cell generation number. In colonies perfused at pH 6.0, the cells inheriting the oldest pole divided significantly more slowly than the cells inheriting the newest pole. In colonies perfused at pH 7.50 (near or above cytoplasmic pH), no significant cell division asymmetry was observed. Under both conditions (periplasmic pH 6.0 or pH 7.5) the cells maintained cytoplasmic pH values at 7.2–7.3. No evidence of cytoplasmic protein aggregation was seen. Thus, periplasmic acid stress leads to cell division asymmetry with minimal cytoplasmic stress. PMID:26713733

  11. Characterization of a periplasmic protein involved in iron utilization of Actinobacillus actinomycetemcomitans.

    PubMed

    Willemsen, P T; Vulto, I; Boxem, M; de Graaff, J

    1997-08-01

    The periodontopathic bacterium Actinobacillus actinomycetemcomitans possesses a 35-kDa periplasmic iron-repressible protein. Its regulation is mediated by the Fur protein, as was inferred from the Fur-binding consensus sequence at the -35 position of the gene for the 35-kDa protein and from the relaxed expression of the gene in a mutant with an altered Fur-binding sequence. The 35-kDa protein, designated AfuA, has strong homology to HitA and FbpA of Haemophilus influenzae and Neisseria meningitidis, respectively, which serve as periplasmic iron transport proteins.

  12. Osmoregulated Periplasmic Glucans (OPGs) of Salmonella enterica serovars Typhimurium are needed for optimal growth under nutrient limiting- hypoosmotic conditions

    USDA-ARS?s Scientific Manuscript database

    Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of gram negative bacteria. Synthesis of OPGs is regulated by the osmolarity of the growth medium. The role of OPGs has been postulated in plant-symbiotic as well as pathogenic microorganisms. On the other hand, opg mutant...

  13. Periplasmically-exported lupanine hydroxylase undergoes transition from soluble to functional inclusion bodies in Escherichia coli.

    PubMed

    Stampolidis, Pavlos; Kaderbhai, Naheed N; Kaderbhai, Mustak A

    2009-04-01

    Pseudomonas lupanine hydroxylase is a periplasmic-localised, two domain quinocytochrome c enzyme. It requires numerous post-translocation modifications involving signal peptide processing, disulphide bridge formation and, heme linkage in the carboxy-terminal cytochrome c domain to eventually generate a Ca(2+)-bound quino-c hemoprotein that hydroxylates the plant alkaloid, lupanine. An exported, functional recombinant enzyme was generated in Escherichia coli by co-expression with cytochrome c maturation factors. Increased growth temperatures ranging from 18 to 30 degrees C gradually raised the enzyme production to a peak together with its concomitant aggregation as red solid particles, readily activatable in a fully functional form by mild chaotropic treatment. Here, we demonstrate that the exported lupanine hydroxylase undergoes a cascade transition from a soluble to "non-classical" inclusion body form when build-up in the periplasm exceeded a basal threshold concentration. These periplasmic aggregates were distinct from the non-secreted, signal-sequenceless counterpart that occurred as misfolded, non-functional concatamers in the form of classical inclusion bodies. We discuss our findings in the light of current models of how aggregation of lupanine hydroxylase arises in the periplasmic space.

  14. Swarm and swim motilities of Salmonella enterica serovar Typhimurium and role of osmoregulated periplasmic glucans

    USDA-ARS?s Scientific Manuscript database

    Background: Salmonella enterica serovar Typhimurium strains synthesize osmoregulated periplasmic glucans (OPGs) under low osmolarity conditions (< 70 mos mol l-1). OPG synthesis is not observed when cells are grown in iso- or hyper-osmotic media (> 400 mos mol l-1). Mutation in OPG structural gene...

  15. Role of anionic charges of periplasmic glucans of Shigella flexneri in overcoming detergent stress

    USDA-ARS?s Scientific Manuscript database

    Osmoregulated periplasmic glucans (OPGs) are synthesized by the members of the family Enterobacteriaceae when grown under low osmotic growth conditions. Enteropathogens such as Shigella flexneri spend considerable time outside the host environment such as irrigation waters where low nutrient low os...

  16. Interaction studies between periplasmic cytochromes provide insights into extracellular electron transfer pathways of Geobacter sulfurreducens.

    PubMed

    Fernandes, Ana P; Nunes, Tiago C; Paquete, Catarina M; Salgueiro, Carlos A

    2017-02-20

    Geobacter bacteria usually prevail among other microorganisms in soils and sediments where Fe(III) reduction has a central role. This reduction is achieved by extracellular electron transfer (EET), where the electrons are exported from the interior of the cell to the surrounding environment. Periplasmic cytochromes play an important role in establishing an interface between inner and outer membrane electron transfer components. In addition, periplasmic cytochromes, in particular nanowire cytochromes that contain at least 12 haem groups, have been proposed to play a role in electron storage in conditions of an environmental lack of electron acceptors. Up to date, no redox partners have been identified in Geobacter sulfurreducens, and concomitantly, the EET and electron storage mechanisms remain unclear. In this work, NMR chemical shift perturbation measurements were used to probe for an interaction between the most abundant periplasmic cytochrome PpcA and the dodecahaem cytochrome GSU1996, one of the proposed nanowire cytochromes in G. sulfurreducens The perturbations on the haem methyl signals of GSU1996 and PpcA showed that the proteins form a transient redox complex in an interface that involves haem groups from two different domains located at the C-terminal of GSU1996. Overall, the present study provides for the first time a clear evidence for an interaction between periplasmic cytochromes that might be relevant for the EET and electron storage pathways in G. sulfurreducens. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  17. Identification and characterization of a heme periplasmic-binding protein in Haemophilus ducreyi.

    PubMed

    St Denis, Melissa; Sonier, Brigitte; Robinson, Renée; Scott, Fraser W; Cameron, D William; Lee, B Craig

    2011-08-01

    Haemophilus ducreyi, a gram-negative and heme-dependent bacterium, is the causative agent of chancroid, a genital ulcer sexually transmitted infection. Heme acquisition in H. ducreyi proceeds via a receptor mediated process in which the initial event involves binding of hemoglobin and heme to their cognate outer membrane proteins, HgbA and TdhA, respectively. Following this specific interaction, the fate of the periplasmic deposited heme is unclear. Using protein expression profiling of the H. ducreyi periplasmic proteome, a periplasmic-binding protein, termed hHbp, was identified whose expression was enhanced under heme-limited conditions. The gene encoding this protein was situated in a locus displaying genetic characteristics of an ABC transporter. The purified protein bound heme in a dose-dependent and saturable manner and this binding was specifically competitively inhibited by heme. The hhbp gene functionally complemented an Escherichia coli heme uptake mutant. Expression of the heme periplasmic-binding protein was detected in a limited survey of H. ducreyi and H. influenzae clinical strains. These results indicate that the passage of heme into the cytoplasm of H. ducreyi involves a heme dedicated ABC transporter.

  18. Opening the periplasmic cavity in lactose permease is the limiting step for sugar binding

    PubMed Central

    Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Kaback, H. Ronald

    2011-01-01

    The lactose permease (LacY) catalyzes galactoside/H+ symport via an alternating access mechanism in which sugar- and H+-binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by opening and closing of inward- and outward-facing cavities. The crystal structures of wild-type LacY, as well as accessibility data for the protein in the membrane, provide strong support for a conformation with a tightly closed periplasmic side and an open cytoplasmic side (an inward-facing conformation). In this study, rates of substrate binding were measured by stopped-flow with purified LacY either in detergent or in reconstituted proteoliposomes. Binding rates are compared with rates of sugar-induced opening of the periplasmic pathway obtained by using a recently developed method based on unquenching of Trp fluorescence. A linear dependence of galactoside-binding rates on sugar concentration is observed in detergent, whereas reconstituted LacY binds substrate at a slower rate that is independent of sugar concentration. Rates of opening of the periplasmic cavity with LacY in detergent are independent of substrate concentration and are essentially the same for different galactosidic sugars. The findings demonstrate clearly that reconstituted LacY is oriented physiologically with a closed periplasmic side that limits access of sugar to the binding site. Moreover, opening of the periplasmic cavity is the limiting factor for sugar binding with reconstituted LacY and may be the limiting step in the overall transport reaction. PMID:21896727

  19. Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels

    PubMed Central

    2013-01-01

    Background In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled. Results Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized. Conclusions Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment. PMID:23497240

  20. The multifunctional SNM1 gene family: not just nucleases

    PubMed Central

    Yan, Yiyi; Akhter, Shamima; Zhang, Xiaoshan; Legerski, Randy

    2010-01-01

    The archetypical member of the SNM1 gene family was discovered 30 years ago in the budding yeast Saccharomyces cerevisiae. This small but ubiquitous gene family is characterized by metallo-β-lactamase and β-CASP domains, which together have been demonstrated to comprise a nuclease activity. Three mammalian members of this family, SNM1A, SNM1B/Apollo and Artemis, have been demonstrated to play surprisingly divergent roles in cellular metabolism. These pathways include variable (diversity) joining recombination, nonhomologous end-joining of double-strand breaks, DNA damage and mitotic cell cycle checkpoints, telomere maintenance and protein ubiquitination. Not all of these functions are consistent with a model in which these proteins act only as nucleases, and indicate that the SNM1 gene family encodes multifunctional products that can act in diverse biochemical pathways. In this article we discuss the various functions of SNM1A, SNM1B/Apollo and Artemis. PMID:20528238

  1. Designing and testing the activities of TAL effector nucleases.

    PubMed

    Lin, Yanni; Cradick, Thomas J; Bao, Gang

    2014-01-01

    Transcription activator-like effector nucleases (TALENs) have rapidly developed into a powerful tool for genome editing. To avoid labor-intensive and time-consuming experimental screening for active TALENs, a scoring system can help select optimal target sites. Here we describe a procedure to design active TALENs using a scoring system named Scoring Algorithm for Predicted TALEN Activity (SAPTA) and a method to test the activity of individual and pairs of TALENs.

  2. Editing livestock genomes with site-specific nucleases.

    PubMed

    Carlson, Daniel F; Tan, Wenfang; Hackett, Perry B; Fahrenkrug, Scott C

    2013-01-01

    Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10(-9). Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene's encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%-50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.

  3. Nanoplasmonic molecular ruler for nuclease activity and DNAfootprinting

    SciTech Connect

    Chen, Fanqing Frank; Liu, Gang L.; Yin, Yadong; Gerion, Daniele; Kunchakarra, Siri; Mukherjee, Bipasha; Jett, Stephen D.; Bear, David G.; Alivisatos, Paul; Lee, Luke P.

    2006-08-15

    We have constructed a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of DNA length changes and perform DNA footprinting. The ruler was created by tethering double-stranded DNA to single Au nanoparticles. The scattering spectra of Au-DNA nanoconjugates showed red-shifted peak plasmon resonance wavelength dependent on DNA length, which can be measured with sub-nanometer axial resolution, averaging {approx}1.24 nm peak wavelength shift per DNA base pair. The spectra of individual Au-DNA nanoconjugates in the presence of nuclease showed a time-resolved dependence on the reaction dynamics, allowing quantitative, kinetic and real-time measurement of nuclease activity. The ruler was further developed into a new DNA footprinting platform. We showed the specific binding of a protein to DNA and the accurate mapping of its footprint. This work promises a very fast and convenient platform for mapping DNA-protein interactions, for nuclease activity monitoring, and for other DNA size-based methods.

  4. Safety evaluation of nuclease P1 from Penicillium citrinum.

    PubMed

    Okado, Nobuo; Hasegawa, Kazushige; Mizuhashi, Fukutaro; Lynch, Barry S; Vo, Trung D; Roberts, Ashley S

    2016-02-01

    Nuclease P1 has been widely used in the food industry to enhance or create flavor. One commercial source of this enzyme is Penicillium citrinum, an anamorphic mesophilic fungus with a long history of safe use in Europe and Asia as a fermentation organism used in the production of ribonucleases. Given the intended use in food for human consumption, and noting its potential presence at trace levels in finished products, a series of safety studies including an in vitro Ames and chromosome aberration assay, an in vivo rat erythrocyte micronucleus assay and a 90-day oral toxicity study in rats were conducted. No mutagenic activity was observed in the Ames assay. Equivocal activity in the chromosome aberration assay was not replicated in the micronucleus assay at doses of up to 1007 mg total organic solids (TOS)/kg body weight (bw)/day. Following oral administration of nuclease P1 at dosages of 10.1, 101 or 1007 mg TOS/kg bw/day to Sprague-Dawley rats, no adverse effects on any study parameter were observed. The no-observed-adverse-effect level was considered to be 1007 mg TOS/kg bw/day. The results of the genotoxicity studies and subchronic rat study support the safe use in food production of nuclease P1 produced from P. citrinum.

  5. Mechanism for nuclease regulation in RecBCD

    PubMed Central

    Wilkinson, Martin; Chaban, Yuriy; Wigley, Dale B

    2016-01-01

    In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5’-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system. DOI: http://dx.doi.org/10.7554/eLife.18227.001 PMID:27644322

  6. Phylogenomic analysis of the GIY-YIG nuclease superfamily

    PubMed Central

    Dunin-Horkawicz, Stanislaw; Feder, Marcin; Bujnicki, Janusz M

    2006-01-01

    Background The GIY-YIG domain was initially identified in homing endonucleases and later in other selfish mobile genetic elements (including restriction enzymes and non-LTR retrotransposons) and in enzymes involved in DNA repair and recombination. However, to date no systematic search for novel members of the GIY-YIG superfamily or comparative analysis of these enzymes has been reported. Results We carried out database searches to identify all members of known GIY-YIG nuclease families. Multiple sequence alignments together with predicted secondary structures of identified families were represented as Hidden Markov Models (HMM) and compared by the HHsearch method to the uncharacterized protein families gathered in the COG, KOG, and PFAM databases. This analysis allowed for extending the GIY-YIG superfamily to include members of COG3680 and a number of proteins not classified in COGs and to predict that these proteins may function as nucleases, potentially involved in DNA recombination and/or repair. Finally, all old and new members of the GIY-YIG superfamily were compared and analyzed to infer the phylogenetic tree. Conclusion An evolutionary classification of the GIY-YIG superfamily is presented for the very first time, along with the structural annotation of all (sub)families. It provides a comprehensive picture of sequence-structure-function relationships in this superfamily of nucleases, which will help to design experiments to study the mechanism of action of known members (especially the uncharacterized ones) and will facilitate the prediction of function for the newly discovered ones. PMID:16646971

  7. Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans

    DOE PAGES

    Wu, Si; Brown, Roslyn N.; Payne, Samuel H.; ...

    2013-01-01

    The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans . Our top-down analysis provided the confident identification of 55 proteins in the periplasm andmore » characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm.« less

  8. Top-Down Characterization of the Post-Translationally Modified Intact Periplasmic Proteome from the Bacterium Novosphingobium aromaticivorans

    PubMed Central

    Wu, Si; Brown, Roslyn N.; Payne, Samuel H.; Meng, Da; Zhao, Rui; Tolić, Nikola; Cao, Li; Shukla, Anil; Monroe, Matthew E.; Moore, Ronald J.; Lipton, Mary S.; Paša-Tolić, Ljiljana

    2013-01-01

    The periplasm of Gram-negative bacteria is a dynamic and physiologically important subcellular compartment where the constant exposure to potential environmental insults amplifies the need for proper protein folding and modifications. Top-down proteomics analysis of the periplasmic fraction at the intact protein level provides unrestricted characterization and annotation of the periplasmic proteome, including the post-translational modifications (PTMs) on these proteins. Here, we used single-dimension ultra-high pressure liquid chromatography coupled with the Fourier transform mass spectrometry (FTMS) to investigate the intact periplasmic proteome of Novosphingobium aromaticivorans. Our top-down analysis provided the confident identification of 55 proteins in the periplasm and characterized their PTMs including signal peptide removal, N-terminal methionine excision, acetylation, glutathionylation, pyroglutamate, and disulfide bond formation. This study provides the first experimental evidence for the expression and periplasmic localization of many hypothetical and uncharacterized proteins and the first unrestrictive, large-scale data on PTMs in the bacterial periplasm. PMID:23555055

  9. DNA nuclease activity of Rev-coupled transition metal chelates.

    PubMed

    Joyner, Jeff C; Keuper, Kevin D; Cowan, J A

    2012-06-07

    Artificial nucleases containing Rev-coupled metal chelates based on combinations of the transition metals Fe(2+), Co(2+), Ni(2+), and Cu(2+) and the chelators DOTA, DTPA, EDTA, NTA, tripeptide GGH, and tetrapeptide KGHK have been tested for DNA nuclease activity. Originally designed to target reactive transition metal chelates (M-chelates) to the HIV-1 Rev response element mRNA, attachment to the arginine-rich Rev peptide also increases DNA-binding affinity for the attached M-chelates. Apparent K(D) values ranging from 1.7 to 3.6 µM base pairs for binding of supercoiled pUC19 plasmid DNA by Ni-chelate-Rev complexes were observed, as a result of electrostatic attraction between the positively-charged Rev peptide and negatively-charged DNA. Attachment of M-chelates to the Rev peptide resulted in enhancements of DNA nuclease activity ranging from 1-fold (no enhancement) to at least 13-fold (for Cu-DTPA-Rev), for the rate of DNA nicking, with second order rate constants for conversion of DNA(supercoiled) to DNA(nicked) up to 6 × 10(6) M(-1) min(-1), and for conversion of DNA(nicked) to DNA(linear) up to 1 × 10(5) M(-1) min(-1). Freifelder-Trumbo analysis and the ratios of linearization and nicking rate constants (k(lin)/k(nick)) revealed concerted mechanisms for nicking and subsequent linearization of plasmid DNA for all of the Rev-coupled M-chelates, consistent with higher DNA residency times for the Rev-coupled M-chelates. Observed rates for Rev-coupled M-chelates were less skewed by differing DNA-binding affinities than for M-chelates lacking Rev, as a result of the narrow range of DNA-binding affinities observed, and therefore relationships between DNA nuclease activity and other catalyst properties, such as coordination unsaturation, the ability to consume ascorbic acid and generate diffusible radicals, and the identity of the metal center, are now clearly illustrated in light of the similar DNA-binding affinities of all M-chelate-Rev complexes. This work

  10. Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9.

    PubMed

    Koo, Taeyoung; Lee, Jungjoon; Kim, Jin-Soo

    2015-06-01

    Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.

  11. Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN.

    PubMed

    Ul Ain, Qurrat; Chung, Jee Young; Kim, Yong-Hee

    2015-05-10

    Gene therapy by engineered nucleases is a genetic intervention being investigated for curing the hereditary disorders by targeting selected genes with specific nucleotides for establishment, suppression, abolishment of a function or correction of mutation. Here, we review the fast developing technology of targeted genome engineering using site specific programmable nucleases zinc finger nucleases (ZFNs), transcription activator like nucleases (TALENs) and cluster regulatory interspaced short palindromic repeat/CRISPR associated proteins (CRISPR/Cas) based RNA-guided DNA endonucleases (RGENs) and their different characteristics including pros and cons of genome modifications by these nucleases. We have further discussed different types of delivery methods to induce gene editing, novel development in genetic engineering other than nucleases and future prospects.

  12. Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9

    PubMed Central

    Koo, Taeyoung; Lee, Jungjoon; Kim, Jin-Soo

    2015-01-01

    Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations. PMID:25985872

  13. Periplasmic Manganese in a Subsurface Bacterium During Anaerobic Growth on Birnessite

    NASA Astrophysics Data System (ADS)

    Langley, S.; Glasauer, S.; Beveridge, T.

    2002-12-01

    In subsurface environments, where oxygen is not metabolically available for energy production, bacteria use alternate terminal electron acceptors (TEAs) to respire and grow. Anaerobic TEAs include, but are not limited to, Fe3+ and Mn4+. These metals can be present as mineral phases (e.g., ferrihydrite and hematite in the case of iron; birnessite and pyrolusite in the case of manganese). Bacteria bind strongly to minerals and reduce the metal by a process called dissimilatory metal reduction (DMR). Shewanella putrefaciens strain CN32 is a Gram-negative bacterium capable of DMR. In previous reports, when this organism was grown on birnessite, we observed cytoplasmic granules of a Mn-rich mineral phase, and an unusual deposition of electron-dense material within the periplasm (that region of the cell located between the inner and outer membranes). In an attempt to characterize the periplasmic precipitates, CN32 was inoculated into an anaerobic defined medium (DM), supplemented with 20 mM Mn (birnessite) and incubated in an anaerobic chamber. Reduced and total Mn concentrations were monitored using atomic absorption spectrophotometry, and cell numbers determined by viable counts on trypticase soy agar. TEM, combined with energy dispersive X-ray spectroscopy (EDS), was used to localize and confirm the presence of any Mn-rich depositions. Soluble Mn concentration increased steadily after inoculation, indicating active metabolism and metal reduction by the cells. Viable counts indicated that the cells reached their maximum number on day 9. Stained thin sections from 4-day-old samples examined with TEM showed cells in close association with the mineral. Secondary mineral products derived from birnessite reduction were evident (e.g., manganese phosphate). TEM-EDS also revealed the presence of ~30 nm-thick deposits of electron-dense material in the periplasm of some cells. However, examination of similar sections which had not been previously stained with osmium tetroxide

  14. Non-viral delivery of genome-editing nucleases for gene therapy.

    PubMed

    Wang, M; Glass, Z A; Xu, Q

    2017-03-01

    Manipulating the genetic makeup of mammalian cells using programmable nuclease-based genome-editing technology has recently evolved into a powerful avenue that holds great potential for treating genetic disorders. There are four types of genome-editing nucleases, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases and clustered, regularly interspaced, short palindromic repeat-associated nucleases such as Cas9. These nucleases have been harnessed to introduce precise and specific changes of the genome sequence at virtually any genome locus of interest. The therapeutic relevance of these genome-editing technologies, however, is challenged by the safe and efficient delivery of nuclease into targeted cells. Herein, we summarize recent advances that have been made on non-viral delivery of genome-editing nucleases. In particular, we focus on non-viral delivery of Cas9/sgRNA ribonucleoproteins for genome editing. In addition, the future direction for developing non-viral delivery of programmable nucleases for genome editing is discussed.

  15. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump

    PubMed Central

    Hinchliffe, Philip; Greene, Nicholas P.; Paterson, Neil G.; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-01-01

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. PMID:24996185

  16. Escherichia coli pleiotropic mutant that reduces amounts of several periplasmic and outer membrane proteins.

    PubMed Central

    Wanner, B L; Sarthy, A; Beckwith, J

    1979-01-01

    We have isolated a mutant of Escherichia coli K-12 that is reduced from 6- to 10-fold in the amount of alkaline phosphatase found in the periplasmic space. The reduced synthesis is not due to effects at the level of transcription regulation of the phoA gene, the structural gene for the enzyme. In addition, the mutation (termed perA) responsible for this phenotype results in reduced amounts of possibly six or more other periplasmic proteins and at least three outer membrane proteins. One of the outer membrane proteins affected is protein IA (D. L. Diedrich, A. O. Summers, and C. A. Schnaitman, J. Bacteriol. 131:598-607, 1977). Although other possibilities exist, one explanation for the phenotype of the perA mutation is that it affects the cell's secretory apparatus. Images PMID:387722

  17. Assimilation of nicotinamide mononucleotide requires periplasmic AphA phosphatase in Salmonella enterica.

    PubMed

    Grose, Julianne H; Bergthorsson, Ulfar; Xu, Yaping; Sterneckert, Jared; Khodaverdian, Behzad; Roth, John R

    2005-07-01

    Salmonella enterica can obtain pyridine from exogenous nicotinamide mononucleotide (NMN) by three routes. In route 1, nicotinamide is removed from NMN in the periplasm and enters the cell as the free base. In route 2, described here, phosphate is removed from NMN in the periplasm by acid phosphatase (AphA), and the produced nicotinamide ribonucleoside (NmR) enters the cell via the PnuC transporter. Internal NmR is then converted back to NMN by the NmR kinase activity of NadR. Route 3 is seen only in pnuC* transporter mutants, which import NMN intact and can therefore grow on lower levels of NMN. Internal NMN produced by either route 2 or route 3 is deamidated to nicotinic acid mononucleotide and converted to NAD by the biosynthetic enzymes NadD and NadE.

  18. Periplasmic vestibule plays an important role for solute recruitment, selectivity, and gating in the Rh/Amt/MEP superfamily.

    PubMed

    Akgun, Ugur; Khademi, Shahram

    2011-03-08

    AmtB, a member of the Rh/Amt/MEP superfamily, is responsible for ammonia transport in Escherichia coli. The ammonia pathway in AmtB consists of a narrow hydrophobic lumen in between hydrophilic periplasmic and cytoplasmic vestibules. A series of molecular dynamics simulations (greater than 0.4 μs in total) were performed to determine the mechanism of solute recruitments and selectivity by the periplasmic vestibule. The results show that the periplasmic vestibule plays a crucial role in solute selectivity, and its solute preferences follow the order of NH4(+) > NH3 > CO2. Based on our results, NH4(+) recruitment is initiated by its interaction with either E70 or E225, highly conserved residues located at the entrance of the vestibule. Subsequently, the backbone carbonyl groups at the periplasmic vestibule direct NH4(+) to the conserved aromatic cage at the bottom of the vestibule (known as the Am1 site). The umbrella sampling simulations suggest that the conserved residue D160 is not directly involved in the ammonia conduction; rather its main function is to keep the structure of periplasmic vestibule intact. The MD simulations also revealed that two partially stacked phenyl rings of F107 and F215, separating the periplasmic vestibule from the hydrophobic lumen, flip open and closed simultaneously with a frequency of approximately 10(8) flipping events per second. These results show how the periplasmic vestibule selectively recruits NH4(+) to the Am1 site, and also that the synchronized flipping of two phenyl rings potentially facilitates the solute transition from the periplasmic vestibule to the hydrophobic lumen in the Rh/Amt/MEP superfamily.

  19. Periplasmic vestibule plays an important role for solute recruitment, selectivity, and gating in the Rh/Amt/MEP superfamily

    PubMed Central

    Akgun, Ugur; Khademi, Shahram

    2011-01-01

    AmtB, a member of the Rh/Amt/MEP superfamily, is responsible for ammonia transport in Escherichia coli. The ammonia pathway in AmtB consists of a narrow hydrophobic lumen in between hydrophilic periplasmic and cytoplasmic vestibules. A series of molecular dynamics simulations (greater than 0.4 μs in total) were performed to determine the mechanism of solute recruitments and selectivity by the periplasmic vestibule. The results show that the periplasmic vestibule plays a crucial role in solute selectivity, and its solute preferences follow the order of . Based on our results, recruitment is initiated by its interaction with either E70 or E225, highly conserved residues located at the entrance of the vestibule. Subsequently, the backbone carbonyl groups at the periplasmic vestibule direct to the conserved aromatic cage at the bottom of the vestibule (known as the Am1 site). The umbrella sampling simulations suggest that the conserved residue D160 is not directly involved in the ammonia conduction; rather its main function is to keep the structure of periplasmic vestibule intact. The MD simulations also revealed that two partially stacked phenyl rings of F107 and F215, separating the periplasmic vestibule from the hydrophobic lumen, flip open and closed simultaneously with a frequency of approximately 108 flipping events per second. These results show how the periplasmic vestibule selectively recruits to the Am1 site, and also that the synchronized flipping of two phenyl rings potentially facilitates the solute transition from the periplasmic vestibule to the hydrophobic lumen in the Rh/Amt/MEP superfamily. PMID:21368153

  20. Periplasmic production via the pET expression system of soluble, bioactive human growth hormone.

    PubMed

    Sockolosky, Jonathan T; Szoka, Francis C

    2013-02-01

    A pET based expression system for the production of recombinant human growth hormone (hGH) directed to the Escherichia coli periplasmic space was developed. The pET22b plasmid was used as a template for creating vectors that encode hGH fused to either a pelB or ompA secretion signal under control of the strong bacteriophage T7 promoter. The pelB- and ompA-hGH constructs expressed in BL21 (λDE3)-RIPL E. coli are secreted into the periplasm which facilitates isolation of soluble hGH by selective disruption of the outer membrane. A carboxy-terminal poly-histidine tag enabled purification by Ni(2+) affinity chromatography with an average yield of 1.4 mg/L culture of purified hGH, independent of secretion signal. Purified pelB- and ompA-hGH are monomeric based on size exclusion chromatography with an intact mass corresponding to mature hGH indicating proper cleavage of the signal peptide and folding in the periplasm. Both pelB- and ompA-hGH bind the hGH receptor with high affinity and potently stimulate Nb2 cell growth. These results demonstrate that the pET expression system is suitable for the rapid and simple isolation of bioactive, soluble hGH from E. coli.

  1. Cytoplasmic membrane protonmotive force energizes periplasmic interactions between ExbD and TonB.

    PubMed

    Ollis, Anne A; Manning, Marta; Held, Kiara G; Postle, Kathleen

    2009-08-01

    The TonB system of Escherichia coli (TonB/ExbB/ExbD) transduces the protonmotive force (pmf) of the cytoplasmic membrane to drive active transport by high-affinity outer membrane transporters. In this study, chromosomally encoded ExbD formed formaldehyde-linked complexes with TonB, ExbB and itself (homodimers) in vivo. Pmf was required for detectable cross-linking between TonB-ExbD periplasmic domains. Consistent with that observation, the presence of inactivating transmembrane domain mutations ExbD(D25N) or TonB(H20A) also prevented efficient formaldehyde cross-linking between ExbD and TonB. A specific site of periplasmic interaction occurred between ExbD(A92C) and TonB(A150C) and required functional transmembrane domains in both proteins. Conversely, neither TonB, ExbB nor pmf were required for ExbD dimer formation. These data suggest two possible models where either dynamic complex formation occurred through transmembrane domains or the transmembrane domains of ExbD and TonB configure their respective periplasmic domains. Analysis of T7-tagged ExbD with anti-ExbD antibodies revealed that a T7 tag was responsible both for our previous failure to detect T7-ExbD-ExbB and T7-ExbD-TonB formaldehyde-linked complexes and for the concomitant artefactual appearance of T7-ExbD trimers.

  2. The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel

    PubMed Central

    Gonen, Melissa; Sun, Ji; Delarosa, Jaclyn R

    2011-01-01

    The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many Gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related AB5 heterohexamers, composed of one A subunit and a B-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 Å structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism. PMID:21406971

  3. Properties of a novel periplasmic catalase-peroxidase from Escherichia coli O157:H7.

    PubMed

    Varnado, Cornelius L; Hertwig, Kristen M; Thomas, Robert; Roberts, J Kenneth; Goodwin, Douglas C

    2004-01-01

    A subset of catalase-peroxidases are distinguished by their periplasmic location and their expression by pathogens. Kinetic and spectral properties have not been reported for any of these enzymes. We report the cloning, expression, isolation, and characterization of KatP, a periplasmic catalase-peroxidase from Escherichia coli O157:H7. Absorption spectra indicated a mixture of heme states dominated by the pentacoordinate and hexacoordinate high-spin forms. Apparent k(cat) values for catalase (1.8x10(4) s(-1)) and peroxidase (77 s(-1)) activities were greater than those of other catalase-peroxidases. However, apparent K(M) values for H2O2 were also higher (27 mM for catalase and 3 mM for peroxidase). Ferric KatP reacted with peracetic acid to form compound I (8.8x10(3) M(-1) s(-1)) and with CN(-) to form a ferri-cyano complex (3.9x10(5) M(-1) s(-1)) consistent with other catalase-peroxidases. The isolation and characterization of KatP opens new avenues to explore mechanisms by which the periplasmic catalase-peroxidases may contribute to bacterial virulence.

  4. Gene targeting technologies in rats: zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats.

    PubMed

    Mashimo, Tomoji

    2014-01-01

    The laboratory rat has been widely used as an animal model in biomedical science for more than 150 years. Applying zinc-finger nucleases or transcription activator-like effector nucleases to rat embryos via microinjection is an efficient genome editing tool for generating targeted knockout rats. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonucleases have been used as an effective tool for precise and multiplex genome editing in mice and rats. In this review, the advantages and disadvantages of these site-specific nuclease technologies for genetic analysis and manipulation in rats are discussed. © 2013 The Author Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

  5. Nanoplasmonic molecular ruler for nuclease activity and DNA footprinting

    DOEpatents

    Chen, Fanqing Frank; Liu, Gang L; Lee, Luke P

    2013-10-29

    This invention provides a nanoplasmonic molecular ruler, which can perform label-free and real-time monitoring of nucleic acid (e.g., DNA) length changes and perform nucleic acid footprinting. In various embodiments the ruler comprises a nucleic acid attached to a nanoparticle, such that changes in the nucleic acid length are detectable using surface plasmon resonance. The nanoplasmonic ruler provides a fast and convenient platform for mapping nucleic acid-protein interactions, for nuclease activity monitoring, and for other footprinting related methods.

  6. Tudor Nuclease Genes and Programmed DNA Rearrangements in Tetrahymena thermophila▿

    PubMed Central

    Howard-Till, Rachel A.; Yao, Meng-Chao

    2007-01-01

    Proteins containing a Tudor domain and domains homologous to staphylococcal nucleases are found in a number of eukaryotes. These “Tudor nucleases” have been found to be associated with the RNA-induced silencing complex (A. A. Caudy, R. F. Ketting, S. M. Hammond, A. M. Denli, A. M. Bathoorn, B. B. Tops, J. M. Silva, M. M. Myers, G. J. Hannon, and R. H. Plasterk, Nature 425:411-414, 2003). We have identified two Tudor nuclease gene homologs, TTN1 and TTN2, in the ciliate Tetrahymena thermophila, which has two distinct small-RNA pathways. Characterization of single and double KOs of TTN1 and TTN2 shows that neither of these genes is essential for growth or sexual reproduction. Progeny of TTN2 KOs and double knockouts occasionally show minor defects in the small-RNA-guided process of DNA deletion but appear to be normal in hairpin RNA-induced gene silencing, suggesting that Tudor nucleases play only a minor role in RNA interference in Tetrahymena. Previous studies of Tetrahymena have shown that inserted copies of the neo gene from Escherichia coli are often deleted from the developing macronucleus during sexual reproduction (Y. Liu, X. Song, M. A. Gorovsky, and K. M. Karrer, Eukaryot. Cell 4:421-431, 2005; M. C. Yao, P. Fuller, and X. Xi, Science 300:1581-1584, 2003). This transgene deletion phenomenon is hypothesized to be a form of genome defense. Analysis of the Tudor nuclease mutants revealed exceptionally high rates of deletion of the neo transgene at the TTN2 locus but no deletion at the TTN1 locus. When present in the same genome, however, the neo gene is deleted at high rates even at the TTN1 locus, further supporting a role for trans-acting RNA in this process. This deletion is not affected by the presence of the same sequence in the macronucleus, thus providing a counterargument for the role of the macronuclear genome in specifying all sequences for deletion. PMID:17715366

  7. Targeted mutagenesis in the malaria mosquito using TALE nucleases.

    PubMed

    Smidler, Andrea L; Terenzi, Olivier; Soichot, Julien; Levashina, Elena A; Marois, Eric

    2013-01-01

    Anopheles gambiae, the main mosquito vector of human malaria, is a challenging organism to manipulate genetically. As a consequence, reverse genetics studies in this disease vector have been largely limited to RNA interference experiments. Here, we report the targeted disruption of the immunity gene TEP1 using transgenic expression of Transcription-Activator Like Effector Nucleases (TALENs), and the isolation of several TEP1 mutant A. gambiae lines. These mutations inhibited protein production and rendered TEP1 mutants hypersusceptible to Plasmodium berghei. The TALEN technology opens up new avenues for genetic analysis in this disease vector and may offer novel biotechnology-based approaches for malaria control.

  8. The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm.

    PubMed

    Mora, Liliana; Moncoq, Karine; England, Patrick; Oberto, Jacques; de Zamaroczy, Miklos

    2015-12-25

    LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar Kd determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from 13 additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.

  9. Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1

    PubMed Central

    Song, Qing; Zhang, Xiaobo

    2008-01-01

    Background Thermostable enzymes from thermophiles have attracted extensive studies. In this investigation, a nuclease-encoding gene (designated as GBSV1-NSN) was obtained from a thermophilic bacteriophage GBSV1 for the first time. Results After recombinant expression in Escherichia coli, the purified GBSV1-NSN exhibited non-specific nuclease activity, being able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Based on sequence analysis, the nuclease shared no homology with any known nucleases, suggesting that it was a novel nuclease. The characterization of the recombinant GBSV1-NSN showed that its optimal temperature and pH were 60°C and 7.5, respectively. The results indicated that the enzymatic activity was inhibited by enzyme inhibitors or detergents, such as ethylene diamine tetraacetic acid, citrate, dithiothreitol, β-mercaptoethanol, guanidine hydrochloride, urea and SDS. In contrast, the nuclease activity was enhanced by TritonX-100, Tween-20 or chaps to approximately 124.5% – 141.6%. The Km of GBSV1-NSN nuclease was 231, 61 and 92 μM, while its kcat was 1278, 241 and 300 s-1 for the cleavage of dsDNA, ssDNA and RNA, respectively. Conclusion Our study, therefore, presented a novel thermostable non-specific nuclease from thermophilic bacteriophage and its overexpression and purification for scientific research and applications. PMID:18439318

  10. Positive and Negative Regulation of Poly(A) Nuclease

    PubMed Central

    Mangus, David A.; Evans, Matthew C.; Agrin, Nathan S.; Smith, Mandy; Gongidi, Preetam; Jacobson, Allan

    2004-01-01

    PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo. PMID:15169912

  11. Gene targeting in rats using transcription activator-like effector nucleases.

    PubMed

    Ménoret, Séverine; Tesson, Laurent; Rémy, Séverine; Usal, Claire; Thépenier, Virginie; Thinard, Reynald; Ouisse, Laure-Hélène; De Cian, Anne; Giovannangeli, Carine; Concordet, Jean-Paul; Anegon, Ignacio

    2014-08-15

    The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

  12. Design of a colicin E7 based chimeric zinc-finger nuclease

    NASA Astrophysics Data System (ADS)

    Németh, Eszter; Schilli, Gabriella K.; Nagy, Gábor; Hasenhindl, Christoph; Gyurcsik, Béla; Oostenbrink, Chris

    2014-08-01

    Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.

  13. Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli.

    PubMed

    Rech, S; Wolin, C; Gunsalus, R P

    1996-02-02

    The modABCD operon, located at 17 min on the Escherichia coli chromosome, encodes the protein components of a high affinity molybdate uptake system. Sequence analysis of the modA gene (GenBank L34009) predicts that it encodes a periplasmic binding protein based on the presence of a leader-like sequence at its N terminus. To examine the properties of the ModA protein, the modA structural gene was overexpressed, and its product was purified. The ModA protein was localized to the periplasmic space of the cell, and it was released following a gentle osmotic shock. The N-terminal sequence of ModA confirmed that a leader region of 24 amino acids was removed upon export from the cell. The apparent size of ModA is 31.6 kDa as determined by gel sieve chromatography, whereas it is 22.5 kDa when examined by SDS-polyacrylamide gel electrophoresis. A ligand-dependent protein mobility shift assay was devised using a native polyacrylamide gel electrophoresis protocol to examine binding of molybdate and other anions to the ModA periplasmic protein. Whereas molybdate and tungstate were bound with high affinity (approximately 5 microM), sulfate, chromate, selenate, phosphate, and chlorate did not bind even when tested at 2 mM. A UV spectral assay revealed apparent Kd values of binding for molybdate and tungstate of 3 and 7 microM, respectively. Strains defective in the modA gene were unable to transport molybdate unless high levels of the anion were supplied in the medium. Therefore the modA gene product is essential for high affinity molybdate uptake by the cell. Tungstate interference of molybdate acquisition by the cell is apparently due in part to the high affinity of the ModA protein for this anion.

  14. Holo- and apo-bound structures of bacterial periplasmic heme-binding proteins.

    PubMed

    Ho, Winny W; Li, Huiying; Eakanunkul, Suntara; Tong, Yong; Wilks, Angela; Guo, Maolin; Poulos, Thomas L

    2007-12-07

    An essential component of heme transport in Gram-negative bacterial pathogens is the periplasmic protein that shuttles heme between outer and inner membranes. We have solved the first crystal structures of two such proteins, ShuT from Shigella dysenteriae and PhuT from Pseudomonas aeruginosa. Both share a common architecture typical of Class III periplasmic binding proteins. The heme binds in a narrow cleft between the N- and C-terminal binding domains and is coordinated by a Tyr residue. A comparison of the heme-free (apo) and -bound (holo) structures indicates little change in structure other than minor alterations in the heme pocket and movement of the Tyr heme ligand from an "in" position where it can coordinate the heme iron to an "out" orientation where it points away from the heme pocket. The detailed architecture of the heme pocket is quite different in ShuT and PhuT. Although Arg(228) in PhuT H-bonds with a heme propionate, in ShuT a peptide loop partially takes up the space occupied by Arg(228), and there is no Lys or Arg H-bonding with the heme propionates. A comparison of PhuT/ShuT with the vitamin B(12)-binding protein BtuF and the hydroxamic-type siderophore-binding protein FhuD, the only two other structurally characterized Class III periplasmic binding proteins, demonstrates that PhuT/ShuT more closely resembles BtuF, which reflects the closer similarity in ligands, heme and B(12), compared with ligands for FhuD, a peptide siderophore.

  15. Identification of Functionally Important TonB-ExbD Periplasmic Domain Interactions In Vivo

    PubMed Central

    Ollis, Anne A.

    2012-01-01

    In Gram-negative bacteria, the cytoplasmic membrane proton-motive force energizes the active transport of TonB-dependent ligands through outer membrane TonB-gated transporters. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD couple the proton-motive force to conformational changes in TonB, which are hypothesized to form the basis of energy transduction through direct contact with the transporters. While the role of ExbB is not well understood, contact between periplasmic domains of TonB and ExbD is required, with the conformational response of TonB to presence or absence of proton motive force being modulated through ExbD. A region (residues 92 to 121) within the ExbD periplasmic domain was previously identified as being important for TonB interaction. Here, the specific sites of periplasmic domain interactions between that region and the TonB carboxy terminus were identified by examining 270 combinations of 45 TonB and 6 ExbD individual cysteine substitutions for disulfide-linked heterodimer formation. ExbD residues A92C, K97C, and T109C interacted with multiple TonB substitutions in four regions of the TonB carboxy terminus. Two regions were on each side of the TonB residues known to interact with the TonB box of TonB-gated transporters, suggesting that ExbD positions TonB for correct interaction at that site. A third region contained a functionally important glycine residue, and the fourth region involved a highly conserved predicted amphipathic helix. Three ExbD substitutions, F103C, L115C, and T121C, were nonreactive with any TonB cysteine substitutions. ExbD D25, a candidate to be on a proton translocation pathway, was important to support efficient TonB-ExbD heterodimerization at these specific regions. PMID:22493017

  16. Evolution and diversity of periplasmic proteins involved in copper homeostasis in gamma proteobacteria

    PubMed Central

    2012-01-01

    Background Different systems contributing to copper homeostasis in bacteria have been described in recent years involving periplasmic and transport proteins that provide resistance via metal efflux to the extracellular media (CopA/Cue, Cus, Cut, and Pco). The participation of these proteins in the assembly of membrane, periplasmic and secreted cuproproteins has also been postulated. The integration and interrelation of these systems and their apparent redundancies are less clear since they have been studied in alternative systems. Based on the idea that cellular copper is not free but rather it is transferred via protein-protein interactions, we hypothesized that systems would coevolve and be constituted by set numbers of essential components. Results By the use of a phylogenomic approach we identified the distribution of 14 proteins previously characterized as members of homeostasis systems in the genomes of 268 gamma proteobacteria. Only 3% of the genomes presented the complete systems and 5% of them, all intracellular parasites, lacked the 14 genes. Surprisingly, copper homeostatic pathways did not behave as evolutionary units with particular species assembling different combinations of basic functions. The most frequent functions, and probably because of its distribution the most vital, were copper extrusion from the cytoplasm to the periplasm performed by CopA and copper export from the cytoplasm to the extracellular space performed by CusC, which along with the remaining 12 proteins, assemble in nine different functional repertoires. Conclusions These observations suggest complex evolutionary dynamics and still unexplored interactions to achieve copper homeostasis, challenging some of the molecular transport mechanism proposed for these systems. PMID:23122209

  17. Promoter engineering to optimize recombinant periplasmic Fab' fragment production in Escherichia coli.

    PubMed

    Schofield, Desmond M; Templar, Alex; Newton, Joseph; Nesbeth, Darren N

    2016-07-08

    Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840-847, 2016.

  18. Electron transfer between periplasmic formate dehydrogenase and cytochromes c in Desulfovibrio desulfuricans ATCC 27774.

    PubMed

    da Silva, Sofia Marques; Pacheco, Isabel; Pereira, Inês A Cardoso

    2012-06-01

    Desulfovibrio spp. are sulfate-reducing organisms characterized by having multiple periplasmic hydrogenases and formate dehydrogenases (FDHs). In contrast to enzymes in most bacteria, these enzymes do not reduce directly the quinone pool, but transfer electrons to soluble cytochromes c. Several studies have investigated electron transfer with hydrogenases, but comparatively less is known about FDHs. In this work we conducted experiments to assess potential electron transfer pathways resulting from formate oxidation in Desulfovibrio desulfuricans ATCC 27774. This organism can grow on sulfate and on nitrate, and contains a single soluble periplasmic FDH that includes a cytochrome c (3) like subunit (FdhABC(3)). It has also a unique cytochrome c composition, including two cytochromes c not yet isolated from other species, the split-Soret and nine-heme cytochromes, besides a tetraheme type I cytochrome c (3) (TpIc (3)). The FDH activity and cytochrome composition of cells grown with lactate or formate and nitrate or sulfate were determined, and the electron transfer between FDH and these cytochromes was investigated. We studied also the reduction of the Dsr complex and of the monoheme cytochrome c-553, previously proposed to be the physiological partner of FDH. FdhABC(3) was able to reduce the c-553, TpIc (3), and split-Soret cytochromes with a high rate. For comparison, the same experiments were performed with the [NiFe] hydrogenase from the same organism. This study shows that FdhABC(3) can directly reduce the periplasmic cytochrome c network, feeding electrons into several alternative metabolic pathways, which explains the advantage of not having an associated membrane subunit.

  19. Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo.

    PubMed

    Ollis, Anne A; Postle, Kathleen

    2012-06-01

    In gram-negative bacteria, the cytoplasmic membrane proton-motive force energizes the active transport of TonB-dependent ligands through outer membrane TonB-gated transporters. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD couple the proton-motive force to conformational changes in TonB, which are hypothesized to form the basis of energy transduction through direct contact with the transporters. While the role of ExbB is not well understood, contact between periplasmic domains of TonB and ExbD is required, with the conformational response of TonB to presence or absence of proton motive force being modulated through ExbD. A region (residues 92 to 121) within the ExbD periplasmic domain was previously identified as being important for TonB interaction. Here, the specific sites of periplasmic domain interactions between that region and the TonB carboxy terminus were identified by examining 270 combinations of 45 TonB and 6 ExbD individual cysteine substitutions for disulfide-linked heterodimer formation. ExbD residues A92C, K97C, and T109C interacted with multiple TonB substitutions in four regions of the TonB carboxy terminus. Two regions were on each side of the TonB residues known to interact with the TonB box of TonB-gated transporters, suggesting that ExbD positions TonB for correct interaction at that site. A third region contained a functionally important glycine residue, and the fourth region involved a highly conserved predicted amphipathic helix. Three ExbD substitutions, F103C, L115C, and T121C, were nonreactive with any TonB cysteine substitutions. ExbD D25, a candidate to be on a proton translocation pathway, was important to support efficient TonB-ExbD heterodimerization at these specific regions.

  20. The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli

    PubMed Central

    Latifi, Ali Mohammad; Khajeh, Khosro; Farnoosh, Gholamreza; Hassanpour, Kazem; Khodi, Samaneh

    2015-01-01

    Background: Organophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process. Objectives: In this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b). Materials and Methods: The sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd. Results: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band. Conclusions: The activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step. PMID:26870308

  1. FbpA iron storage and release are governed by periplasmic microenvironments.

    PubMed

    Sensoy, Ozge; Atilgan, Ali Rana; Atilgan, Canan

    2017-02-22

    Ferric binding protein (FbpA) is part of an elaborate iron piracy mechanism evolved in Gram-negative bacteria, shuttling iron in the periplasmic space, from the outer to the cytoplasmic membrane side. We address how the dissociation process of iron is facilitated, since the binding constant of iron is on the order of 10(18) M(-1) at 6.5 pH and 200 mM ionic strength (IS). We monitor the conformational preferences of FbpA by extensive molecular dynamics (MD) simulations under conditions where IS, charge states of iron coordinating tyrosines and pH are varied, as well as when a mutation is introduced at an allosteric site. Steered MD is utilized to predict the binding affinity of iron. After triggering lobe opening by changing the charge states of tyrosines, the conformations adopted and the iron binding affinity still depend on pH, IS and allosteric interactions. To relate the observed conformational changes to the environmental conditions that might be encountered in the periplasmic space, we offer a plausible model that couples electrostatic potential distribution to the mechanical motions invoked. Although low pH/IS and allosteric perturbations decrease the affinity of iron, it remains high for spontaneous dissociation. However, the conformational changes modulated by the environmental conditions expose iron for chelation. Our study provides a quantitative dimension and molecular details to interpret the contribution of possible environmental conditions present in the periplasmic space to iron dissociation from FbpA, opening up the opportunity of modulating function via allosteric mutations or altering environmental conditions, thus offering a new route to developing strategies towards antibiotic resistance by targeting nutritional requirements.

  2. Overexpression of Recombinant Human Beta Interferon (rhINF-β) in Periplasmic Space of Escherichia coli

    PubMed Central

    Morowvat, Mohammad Hossein; Babaeipour, Valiollah; Rajabi-Memari, Hamid; Vahidi, Hossein; Maghsoudi, Nader

    2014-01-01

    Human Interferon β (INF-β) is a member of cytokines family which different studies have shown its immunomodulatory and antiviral activities. In this study an expression vector was designed and constructed for expression of human INF-β-1b either in shake flasks or bench top bioreactor. The designed vector was constructed based upon pET-25b(+) with T7 promoter. Recombinant human beta interferon (rhINF-β) was codon optimized and overexpressed as a soluble, N-terminal pelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21 (DE3). The sugar, Isopropyl-β-D-thiogalactopyranoside (IPTG) was used as a chemical inducer for rhINF-β production in the shake flasks and bench top bioreactor. Timing of beta interferon expression was controlled by using the T7 promoter. The rhINF-β protein was extracted from periplasmic space by osmotic shock treatment and the expression of the beta interferon encoding gene in random selected transformants, was confirmed by western and dot blot methods. The maximum of product formation achieved at the OD600nm = 3.42 was found to be 35 % of the total protein content of the strain which translates to 0.32 g L-1. The constructed vector could efficiently overexpress the rhINF-β into the periplasmic space of E. coli. The obtained yield of the produced rhINF-β was more than previous reports. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable to express other recombinant proteins. PMID:24711841

  3. Periplasmic flagella in Borrelia burgdoferi function to maintain cellular integrity upon external stress

    PubMed Central

    Kumar, Bharath; Miller, Kelly; Charon, Nyles W.

    2017-01-01

    Tapping mode atomic force microscopy (AFM) in solution was used to analyze the role of the internally located periplasmic flagella (PFs) of the Lyme disease spirochete Borrelia burgdorferi in withstanding externally applied cellular stresses. By systematically imaging immobilized spirochetes with increasing tapping forces, we found that mutants that lack PFs are more readily compressed and damaged by the imaging process compared to wild-type cells. This finding suggest that the PFs, aside from being essential for motility and involved in cell shape, play a cytoskeletal role in dissipating energy and maintaining cellular integrity in the presence of external stress. PMID:28898274

  4. Generation of albino Xenopus tropicalis using zinc-finger nucleases.

    PubMed

    Nakajima, Keisuke; Nakajima, Taeko; Takase, Minoru; Yaoita, Yoshio

    2012-12-01

    To generate albino lines of Xenopus tropicalis, we injected fertilized eggs with mRNAs encoding zinc-finger nucleases (ZFNs) targeting the tyrosinase coding region. Surprisingly, vitiligo was observed on the skin of F0 frogs that had been injected with ZFN mRNAs, indicating that both tyrosinase genes in the genome were disrupted in all melanocytes within the vitiligo patches. Mutation analysis using genomic DNA from the skin revealed that two mosaic F0 frogs underwent spatially complex tyrosinase gene mutations. The data implies that the ZFN-induced tyrosinase gene ablations occurred randomly over space and time throughout the entire body, possibly until the young tadpole stage, and that melanocyte precursors lacking functional tyrosinase proliferated and formed vitiligo patches. Several albino X. tropicalis, which are compound heterozygotes for biallelic tyrosinase mutations, were obtained by mating the mosaic F0 frogs. To our knowledge, this is the first report of the albino vertebrates generated by the targeted gene knockout.

  5. The effect of micrococcal nuclease digestion on nucleosome positioning data.

    PubMed

    Chung, Ho-Ryun; Dunkel, Ilona; Heise, Franziska; Linke, Christian; Krobitsch, Sylvia; Ehrenhofer-Murray, Ann E; Sperling, Silke R; Vingron, Martin

    2010-12-29

    Eukaryotic genomes are packed into chromatin, whose basic repeating unit is the nucleosome. Nucleosome positioning is a widely researched area. A common experimental procedure to determine nucleosome positions involves the use of micrococcal nuclease (MNase). Here, we show that the cutting preference of MNase in combination with size selection generates a sequence-dependent bias in the resulting fragments. This strongly affects nucleosome positioning data and especially sequence-dependent models for nucleosome positioning. As a consequence we see a need to re-evaluate whether the DNA sequence is a major determinant of nucleosome positioning in vivo. More generally, our results show that data generated after MNase digestion of chromatin requires a matched control experiment in order to determine nucleosome positions.

  6. Targeted Mutagenesis in Zebrafish Using Customized Zinc Finger Nucleases

    PubMed Central

    Foley, Jonathan E.; Maeder, Morgan L.; Pearlberg, Joseph; Joung, J. Keith; Peterson, Randall T.; Yeh, Jing-Ruey J.

    2009-01-01

    Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months. PMID:20010934

  7. Editing the Trypanosoma cruzi genome with zinc finger nucleases.

    PubMed

    Burle-Caldas, Gabriela Assis; Grazielle-Silva, Viviane; Soares-Simões, Melissa; Schumann Burkard, Gabriela; Roditi, Isabel; DaRocha, Wanderson Duarte; Teixeira, Santuza M

    2017-03-01

    Gene function studies in Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, have been hindered by the lack of efficient genetic manipulation protocols. In most organisms, insertion and deletion of DNA fragments in the genome are dependent on the generation of double-stranded DNA break (DSB) and repair. By inducing a site-specific DSB, zinc finger nucleases (ZFNs) have proven to be useful to enhance gene editing in many cell types. Using a pair of ZFNs targeted to the T. cruzi gp72 gene, we were able to generate gp72 knockout parasites with improved efficiency compared to the conventional gene knockout protocol. We also provide evidence that, in T. cruzi, repair of DSBs generated by ZFNs occurs primarily by the homologous recombination pathway.

  8. Contribution of nuclease to the pathogenesis of Aeromonas hydrophila

    PubMed Central

    Ji, Yachan; Li, Jinquan; Qin, Zhendong; Li, Aihua; Gu, Zemao; Liu, Xiaoling; Lin, Li; Zhou, Yang

    2015-01-01

    Aeromonas hydrophila is a gram-negative bacterium that is widely distributed in aquatic environments and can cause septicemia in both fish and humans. However, the underlying mechanisms leading to severe infection are not well understood. In this study, an A. hydrophila nuclease (ahn) deletion mutant was constructed to investigate its contribution to pathogenesis. This mutant did not differ from the wild-type strain in terms of its growth or hemolytic phenotype. However, the ahn-deficient mutant was more susceptible to being killed by fish macrophages and mouse blood in vitro. Furthermore, evidence obtained using both fish and murine infection models strongly indicated that the inactivation of Ahn impaired the ability of A. hydrophila to evade innate immune clearance in vivo. More importantly, the virulence of the mutant was attenuated in both fish and mice, with reductions in dissemination capacities and mortality rates. These findings implicate Ahn in A. hydrophila virulence, with important functions in evading innate immune defenses. PMID:26039879

  9. Genome editing using artificial site-specific nucleases in zebrafish.

    PubMed

    Hisano, Yu; Ota, Satoshi; Kawahara, Atsuo

    2014-01-01

    Zebrafish is a model vertebrate suitable for genetic analysis. Forward genetic analysis via chemical mutagenesis screening has established a variety of zebrafish mutants that are defective in various types of organogenesis, and the genes responsible for the individual mutants have been identified from genome mapping. On the other hand, reverse genetic analysis via targeted gene disruption using embryonic stem (ES) cells (e.g., knockout mouse) can uncover gene functions by investigating the phenotypic effects. However, this approach is mostly limited to mice among the vertebrate models because of the difficulty in establishing ES cells. Recently, new gene targeting technologies, such as the transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have been developed: that can directly introduce genome modifications at the targeted genomic locus. Here, we summarize these new and powerful genome editing techniques for the study of zebrafish.

  10. Structure based design of protein linkers for zinc finger nuclease.

    PubMed

    Anand, Priya; Schug, Alexander; Wenzel, Wolfgang

    2013-10-01

    Zinc finger nucleases are a promising tool to edit DNA in many biological applications, in particular for gene knockout. Despite many efforts the number of genes that can be effectively targeted with ZFNs remains severely limited, as available constructs cannot address arbitrary gene sequences. Here, we develop a novel concept to significantly enhance the number of DNA sequences that can be targeted by ZFN. Using an efficient computational model, we provide an extensive library of possible linker molecules between individual zinc finger motifs in the construct that can skip up to 10 base pairs between adjacent zinc finger recognition sites in the DNA sequence, which increases the number of genes that can be efficiently targeted by more than an order of magnitude.

  11. Involvement of TatD nuclease during programmed cell death in the protozoan parasite Trypanosoma brucei.

    PubMed

    Gannavaram, Sreenivas; Debrabant, Alain

    2012-03-01

    In this report, we describe the involvement of TatD nuclease during programmed cell death (PCD) in the human protozoan parasite Trypanosoma brucei. T. brucei TatD nuclease showed intrinsic DNase activity, was localized in the cytoplasm and translocated to the nucleus when cells were treated with inducers previously demonstrated to cause PCD in T. brucei. Overexpression of TatD nuclease resulted in elevated PCD and conversely, loss of TatD expression by RNAi conferred significant resistance to the induction of PCD in T. brucei. Co-immunoprecipitation studies revealed that TatD nuclease interacts with endonucleaseG suggesting that these two nucleases could form a DNA degradation complex in the nucleus. Together, biochemical activity, RNAi and subcellular localization results demonstrate the role of TatD nuclease activity in DNA degradation during PCD in these evolutionarily ancient eukaryotic organisms. Further, in conjunction with endonucleaseG, TatD may represent a critical nuclease in a caspase-independent PCD pathway in trypanosomatid parasites since caspases have not been identified in these organisms. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

  12. Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.

    PubMed

    Cai, Yujia; Bak, Rasmus O; Mikkelsen, Jacob Giehm

    2014-04-24

    Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001.

  13. Characterization of Rv0888, a Novel Extracellular Nuclease from Mycobacterium tuberculosis

    PubMed Central

    Dang, Guanghui; Cao, Jun; Cui, Yingying; Song, Ningning; Chen, Liping; Pang, Hai; Liu, Siguo

    2016-01-01

    Bacterial extracellular nucleases play important roles in virulence, biofilm formation, utilization of extracellular DNA as a nutrient, and degradation of neutrophil DNA extracellular traps. However, there is no current data available for extracellular nucleases derived from M. tuberculosis. Herein, we have identified and characterized Rv0888, an extracellular nuclease in M. tuberculosis. The protein was overexpressed in E. coli, and the purified Rv0888 protein was found to require divalent cations for activity, with an optimal temperature and pH of 41 °C and 6.5, respectively. Further results demonstrated that Rv0888 nuclease activity could be inhibited by four Chinese medicine monomers. Based on sequence analysis, Rv0888 nuclease exhibited no homology with any known extracellular nucleases, indicating that Rv0888 is a novel nuclease. Site-directed mutagenesis studies revealed that the H353, D387, and D438 residues play catalytic roles in Rv0888. In vivo infection studies confirmed that Rv0888 is required for infection and is related to pathogenicity, as the persistent ability of recombinant Mycobacterium smegmatis (rMS) Rv0888NS/MS and Rv0888S/MS is significantly higher than pMV262/MS in the lung tissue, and the Rv0888NS/MS and Rv0888S/MS could produce pathological changes in the mice lung. These results show that Rv0888 is relevant to pathogenicity of M. tuberculosis. PMID:26742696

  14. The nuclease domain of the SPP1 packaging motor coordinates DNA cleavage and encapsidation

    PubMed Central

    Cornilleau, Charlène; Atmane, Noureddine; Jacquet, Eric; Smits, Callum; Alonso, Juan C.; Tavares, Paulo; Oliveira, Leonor

    2013-01-01

    The large terminase subunit is a central component of the genome packaging motor from tailed bacteriophages and herpes viruses. This two-domain enzyme has an N-terminal ATPase activity that fuels DNA translocation during packaging and a C-terminal nuclease activity required for initiation and termination of the packaging cycle. Here, we report that bacteriophage SPP1 large terminase (gp2) is a metal-dependent nuclease whose stability and activity are strongly and preferentially enhanced by Mn2+ ions. Mutation of conserved residues that coordinate Mn2+ ions in the nuclease catalytic site affect the metal-induced gp2 stabilization and impair both gp2-specific cleavage at the packaging initiation site pac and unspecific nuclease activity. Several of these mutations block also DNA encapsidation without affecting ATP hydrolysis or gp2 C-terminus binding to the procapsid portal vertex. The data are consistent with a mechanism in which the nuclease domain bound to the portal switches between nuclease activity and a coordinated action with the ATPase domain for DNA translocation. This switch of activities of the nuclease domain is critical to achieve the viral chromosome packaging cycle. PMID:23118480

  15. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  16. Periscope: quantitative prediction of soluble protein expression in the periplasm of Escherichia coli

    NASA Astrophysics Data System (ADS)

    Chang, Catherine Ching Han; Li, Chen; Webb, Geoffrey I.; Tey, Bengti; Song, Jiangning; Ramanan, Ramakrishnan Nagasundara

    2016-03-01

    Periplasmic expression of soluble proteins in Escherichia coli not only offers a much-simplified downstream purification process, but also enhances the probability of obtaining correctly folded and biologically active proteins. Different combinations of signal peptides and target proteins lead to different soluble protein expression levels, ranging from negligible to several grams per litre. Accurate algorithms for rational selection of promising candidates can serve as a powerful tool to complement with current trial-and-error approaches. Accordingly, proteomics studies can be conducted with greater efficiency and cost-effectiveness. Here, we developed a predictor with a two-stage architecture, to predict the real-valued expression level of target protein in the periplasm. The output of the first-stage support vector machine (SVM) classifier determines which second-stage support vector regression (SVR) classifier to be used. When tested on an independent test dataset, the predictor achieved an overall prediction accuracy of 78% and a Pearson’s correlation coefficient (PCC) of 0.77. We further illustrate the relative importance of various features with respect to different models. The results indicate that the occurrence of dipeptide glutamine and aspartic acid is the most important feature for the classification model. Finally, we provide access to the implemented predictor through the Periscope webserver, freely accessible at http://lightning.med.monash.edu/periscope/.

  17. Thermosensitive omsA mutation of Escherichia coli that causes thermoregulated release of periplasmic proteins.

    PubMed

    Tsuruoka, T; Ito, M; Tomioka, S; Hirata, A; Matsuhashi, M

    1988-11-01

    A mutant of Escherichia coli with a thermosensitive defect, possibly in the outer membrane (omsA mutant), was isolated from E. coli K-12 by mutagenization and selection for thermosensitivity and beta-lactam supersensitivity of growth. The mutant also showed very high sensitivity to other antibiotics, such as macarbomycin, midecamycin, rifampin, and bacitracin. The mutation was recessive to the wild type and was mapped at about 4 min on the E. coli chromosome between fhuA and metD. The mutation caused rapid release into the medium of periplasmic enzymes such as RTEM penicillinase but practically no cytoplasmic enzyme when cells grown at 30 degrees C were transferred to 37 or 42 degrees C. Electron microscopic observations showed many large double-layered vesicles attached to the surface of cells incubated at 42 degrees C. We conclude that the mutant had a mutation that caused a temperature-dependent defect in the outer membrane structure or its assembly (named an oms mutation). The omsA mutant may be useful for production of periplasmic proteins, which it releases into the culture medium on shift up of temperature.

  18. Periplasmic Vestibule Determines the Ligand Selectivity in E.Coli AMTB

    NASA Astrophysics Data System (ADS)

    Akgun, Ugur; Khademi, Shahram

    2010-03-01

    The transport of ammonia, fundamental to the nitrogen metabolism in all domains of life, is carried out by the Rh/Amt/MEP membrane protein superfamily. The first structure of this family, AmtB from E.Coli shows a pathway for ammonia that includes two vestibules connected by a long and narrow hydrophobic lumen. The accepted mechanism for AmtB is to recruit NH4^+ and conduct neutral NH3 by deprotonation of NH4^+ at the end of periplasmic vestibule. Here we report from various MD simulations performed using a model of trimeric AmtB embedded into POPE lipid bilayer to determine the mechanism of ligands selectivity and conduction in the ammonia channels. Our total more than 500ns simulations reveal that the AmtB periplasmic vestibule prefers NH4^+ over NH3 and CO2. And the rate of ammonia conduction is regulated by the motion of the phenyl rings at the bottom of the vestibule. We also report that the conserved D160 is essential for ligand conduction by stabilizing the NH4^+ at the recruitment site through charge interactions. Our simulations also suggest NH4^+ most likely releases its proton to the bulk of water as it enters to the hydrophobic lumen.

  19. Transition to the open state of the TolC periplasmic tunnel entrance.

    PubMed

    Andersen, Christian; Koronakis, Eva; Bokma, Evert; Eswaran, Jeyanthy; Humphreys, Daniel; Hughes, Colin; Koronakis, Vassilis

    2002-08-20

    The TolC channel-tunnel spans the bacterial outer membrane and periplasm, providing a large exit duct for protein export and multidrug efflux when recruited by substrate-engaged inner membrane complexes. The sole constriction in the single pore of the homotrimeric TolC is the periplasmic tunnel entrance, which in its resting configuration is closed by dense packing of the 12 tunnel-forming alpha-helices. Recruitment of TolC must trigger opening for substrate transit to occur, but the mechanism underlying transition from the closed to the open state is not known. The high resolution structure of TolC indicates that the tunnel helices are constrained at the entrance by a circular network of intra- and intermonomer hydrogen bonds and salt bridges. To assess how opening is achieved, we disrupted these connections and monitored changes in the aperture size by measuring the single channel conductance of TolC derivatives in black lipid bilayers. Elimination of individual connections caused incremental weakening of the circular network, accompanied by gradual relaxation from the closed state and increased flexibility of the entrance. Simultaneous abolition of the key links caused a substantial increase in conductance, generating an aperture that corresponds to the modeled open state, with the capacity to allow access and passage of diverse substrates. The results support a model in which transition to the open state of TolC is achieved by an iris-like realignment of the tunnel entrance helices.

  20. Trapping Open and Closed Forms of FitE-A Group III Periplasmic Binding Protein

    SciTech Connect

    Shi, R.; Proteau, A; Wagner, J; Cui, Q; Purisima, E; Matte, A; Cygler, M

    2009-01-01

    Periplasmic binding proteins (PBPs) are essential components of bacterial transport systems, necessary for bacterial growth and survival. The two-domain structures of PBPs are topologically classified into three groups based on the number of crossovers or hinges between the globular domains: group I PBPs have three connections, group II have two, and group III have only one. Although a large number of structures for group I or II PBPs are known, fewer group III PBPs have been structurally characterized. Group I and II PBPs exhibit significant domain motions during transition from the unbound to ligand-bound form, however, no large conformational changes have been observed to date in group III PBPs. We have solved the crystal structure of a periplasmic binding protein FitE, part of an iron transport system, fit, recently identified in a clinical E. coli isolate. The structure, determined at 1.8 {angstrom} resolution, shows that FitE is a group III PBP containing a single {alpha}-helix bridging the two domains. Among the individual FitE molecules present in two crystal forms we observed three different conformations (open, closed, intermediate). Our crystallographic and molecular dynamics results strongly support the notion that group III PBPs also adopt the same Venus flytrap mechanism as do groups I and II PBPs. Unlike other group III PBPs, FitE forms dimers both in solution and in the crystals. The putative siderophore binding pocket is lined with arginine residues, suggesting an anionic nature of the iron-containing siderophore.

  1. Periscope: quantitative prediction of soluble protein expression in the periplasm of Escherichia coli

    PubMed Central

    Chang, Catherine Ching Han; Li, Chen; Webb, Geoffrey I.; Tey, BengTi; Song, Jiangning; Ramanan, Ramakrishnan Nagasundara

    2016-01-01

    Periplasmic expression of soluble proteins in Escherichia coli not only offers a much-simplified downstream purification process, but also enhances the probability of obtaining correctly folded and biologically active proteins. Different combinations of signal peptides and target proteins lead to different soluble protein expression levels, ranging from negligible to several grams per litre. Accurate algorithms for rational selection of promising candidates can serve as a powerful tool to complement with current trial-and-error approaches. Accordingly, proteomics studies can be conducted with greater efficiency and cost-effectiveness. Here, we developed a predictor with a two-stage architecture, to predict the real-valued expression level of target protein in the periplasm. The output of the first-stage support vector machine (SVM) classifier determines which second-stage support vector regression (SVR) classifier to be used. When tested on an independent test dataset, the predictor achieved an overall prediction accuracy of 78% and a Pearson’s correlation coefficient (PCC) of 0.77. We further illustrate the relative importance of various features with respect to different models. The results indicate that the occurrence of dipeptide glutamine and aspartic acid is the most important feature for the classification model. Finally, we provide access to the implemented predictor through the Periscope webserver, freely accessible at http://lightning.med.monash.edu/periscope/. PMID:26931649

  2. Molybdate-dependent expression of the periplasmic nitrate reductase in Bradyrhizobium japonicum.

    PubMed

    Bonnard, N; Tresierra-Ayala, A; Bedmar, E J; Delgado, M J

    2005-02-01

    The napEDABC genes of Bradyrhizobium japonicum encode the periplasmic nitrate reductase, an Mo-containing enzyme which catalyses the reduction of nitrate to nitrite when oxygen concentrations are limiting. In this bacterium, another set of genes, modABC, code for a high affinity ABC-type Mo transport system. A B. japonicum modA mutant has been obtained that is not capable of growing anaerobically with nitrate and lacks nitrate reductase activity. Under nitrate respiring conditions, when Mo concentrations are limiting, the B. japonicum modA mutant lacked both the 90 kDa protein corresponding to the NapA component of the periplasmic nitrate reductase, and the membrane-bound 25 kDa c-type cytochrome NapC. Regulatory studies using a napE-lacZ fusion indicated that napE expression was highly reduced in the modA mutant background when the cells were incubated anaerobically with nitrate under Mo-deficient conditions.

  3. Compartment and signal-specific codependence in the transcriptional control of Salmonella periplasmic copper homeostasis

    PubMed Central

    Pezza, Alejandro; Pontel, Lucas B.; López, Carolina; Soncini, Fernando C.

    2016-01-01

    Copper homeostasis is essential for bacterial pathogen fitness and infection, and has been the focus of a number of recent studies. In Salmonella, envelope protection against copper overload and macrophage survival depends on CueP, a major copper-binding protein in the periplasm. This protein is also required to deliver the metal ion to the Cu/Zn superoxide dismutase SodCII. The Salmonella-specific CueP-coding gene was originally identified as part of the Cue regulon under the transcriptional control of the cytoplasmic copper sensor CueR, but its expression differs from the rest of CueR-regulated genes. Here we show that cueP expression is controlled by the concerted action of CueR, which detects the presence of copper in the cytoplasm, and by CpxR/CpxA, which monitors envelope stress. Copper-activated CueR is necessary for the appropriate spatial arrangement of the −10 and −35 elements of the cueP promoter, and CpxR is essential to recruit the RNA polymerase. The integration of two ancestral sensory systems—CueR, which provides signal specificity, and CpxR/CpxA, which detects stress in the bacterial envelope—restricts the expression of this periplasmic copper resistance protein solely to cells encountering surplus copper that disturbs envelope homeostasis, emulating the role of the CusR/CusS regulatory system present in other enteric bacteria. PMID:27679850

  4. The ExbD Periplasmic Domain Contains Distinct Functional Regions for Two Stages in TonB Energization

    PubMed Central

    Ollis, Anne A.; Kumar, Aruna

    2012-01-01

    The TonB system of Gram-negative bacteria energizes the active transport of diverse nutrients through high-affinity TonB-gated outer membrane transporters using energy derived from the cytoplasmic membrane proton motive force. Cytoplasmic membrane proteins ExbB and ExbD harness the proton gradient to energize TonB, which directly contacts and transmits this energy to ligand-loaded transporters. In Escherichia coli, the periplasmic domain of ExbD appears to transition from proton motive force-independent to proton motive force-dependent interactions with TonB, catalyzing the conformational changes of TonB. A 10-residue deletion scanning analysis showed that while all regions except the extreme amino terminus of ExbD were indispensable for function, distinct roles for the amino- and carboxy-terminal regions of the ExbD periplasmic domain were evident. Like residue D25 in the ExbD transmembrane domain, periplasmic residues 42 to 61 facilitated the conformational response of ExbD to proton motive force. This region appears to be important for transmitting signals between the ExbD transmembrane domain and carboxy terminus. The carboxy terminus, encompassing periplasmic residues 62 to 141, was required for initial assembly with the periplasmic domain of TonB, a stage of interaction required for ExbD to transmit its conformational response to proton motive force to TonB. Residues 92 to 121 were important for all three interactions previously observed for formaldehyde-cross-linked ExbD: ExbD homodimers, TonB-ExbD heterodimers, and ExbD-ExbB heterodimers. The distinct requirement of this ExbD region for interaction with ExbB raised the possibility of direct interaction with the few residues of ExbB known to occupy the periplasm. PMID:22493019

  5. The ExbD periplasmic domain contains distinct functional regions for two stages in TonB energization.

    PubMed

    Ollis, Anne A; Kumar, Aruna; Postle, Kathleen

    2012-06-01

    The TonB system of gram-negative bacteria energizes the active transport of diverse nutrients through high-affinity TonB-gated outer membrane transporters using energy derived from the cytoplasmic membrane proton motive force. Cytoplasmic membrane proteins ExbB and ExbD harness the proton gradient to energize TonB, which directly contacts and transmits this energy to ligand-loaded transporters. In Escherichia coli, the periplasmic domain of ExbD appears to transition from proton motive force-independent to proton motive force-dependent interactions with TonB, catalyzing the conformational changes of TonB. A 10-residue deletion scanning analysis showed that while all regions except the extreme amino terminus of ExbD were indispensable for function, distinct roles for the amino- and carboxy-terminal regions of the ExbD periplasmic domain were evident. Like residue D25 in the ExbD transmembrane domain, periplasmic residues 42 to 61 facilitated the conformational response of ExbD to proton motive force. This region appears to be important for transmitting signals between the ExbD transmembrane domain and carboxy terminus. The carboxy terminus, encompassing periplasmic residues 62 to 141, was required for initial assembly with the periplasmic domain of TonB, a stage of interaction required for ExbD to transmit its conformational response to proton motive force to TonB. Residues 92 to 121 were important for all three interactions previously observed for formaldehyde-cross-linked ExbD: ExbD homodimers, TonB-ExbD heterodimers, and ExbD-ExbB heterodimers. The distinct requirement of this ExbD region for interaction with ExbB raised the possibility of direct interaction with the few residues of ExbB known to occupy the periplasm.

  6. Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

    PubMed

    Ellis, Mark; Patel, Pareshkumar; Edon, Marjory; Ramage, Walter; Dickinson, Robert; Humphreys, David P

    2017-01-01

    Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.

  7. NikA binds heme: a new role for an Escherichia coli periplasmic nickel-binding protein.

    PubMed

    Shepherd, Mark; Heath, Mathew D; Poole, Robert K

    2007-05-01

    NikA is a periplasmic binding protein involved in nickel uptake in Escherichia coli. NikA was identified as a heme-binding protein in the periplasm of anaerobically grown cells overexpressing CydDC, an ABC transporter that exports reductant to the periplasm. CydDC-overexpressing cells accumulate a heme biosynthesis-derived pigment, P-574. For further biochemical and spectroscopic analysis, unliganded NikA was overexpressed and purified. NikA was found to comigrate with both hemin and protoporphyrin IX during gel filtration. Furthermore, tryptophan fluorescence quenching titrations demonstrated that both hemin and protoporphyrin IX bind to NikA with similar affinity. The binding affinity of NikA for these pigments (Kd approximately 0.5 microM) was unaltered in the presence and absence of saturating concentrations of nickel, suggesting that these tetrapyrroles bind to NikA in a manner independent of nickel. To test the hypothesis that NikA is required for periplasmic heme protein assembly, the effects of a nikA mutation (nikA::Tn5, Km(R) insertion) on accumulation of P-574 by CydDC-overexpressing cells was assessed. This mutation significantly lowered P-574 levels, implying that NikA may be involved in P-574 production. Thus, in the reducing environment of the periplasm, NikA may serve as a heme chaperone as well as a periplasmic nickel-binding protein. The docking of heme onto NikA was modeled using the published crystal structure; many of the predicted complexes exhibit a heme-binding cleft remote from the nickel-binding site, which is consistent with the independent binding of nickel and heme. This work has implications for the incorporation of heme into b- and c-type cytochromes.

  8. Biosynthesis of osmoregulated periplasmic glucans in Escherichia coli: the phosphoethanolamine transferase is encoded by opgE.

    PubMed

    Bontemps-Gallo, Sébastien; Cogez, Virginie; Robbe-Masselot, Catherine; Quintard, Kevin; Dondeyne, Jacqueline; Madec, Edwige; Lacroix, Jean-Marie

    2013-01-01

    Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity.

  9. Biosynthesis of Osmoregulated Periplasmic Glucans in Escherichia coli: The Phosphoethanolamine Transferase Is Encoded by opgE

    PubMed Central

    Bontemps-Gallo, Sébastien; Robbe-Masselot, Catherine; Quintard, Kevin; Dondeyne, Jacqueline; Madec, Edwige

    2013-01-01

    Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. In E. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of the opgE gene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of the opgB gene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity. PMID:24228245

  10. Osmoregulated periplasmic glucans are needed for competitive growth and biofilm formation by Salmonella enterica serovar Typhimurium in leafy-green vegetable wash-waters and colonization in mice

    USDA-ARS?s Scientific Manuscript database

    Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of Gram negative bacteria. The role of OPGs has been postulated in symbiotic as well as pathogenic host-microbe interactions. Here we report the role of OPGs from Salmonella enterica serovar Typhimurium during growth and b...

  11. Targeting of a Nuclease to Murine Leukemia Virus Capsids Inhibits Viral Multiplication

    NASA Astrophysics Data System (ADS)

    Natsoulis, Georges; Seshaiah, Partha; Federspiel, Mark J.; Rein, Alan; Hughes, Stephen H.; Boeke, Jef D.

    1995-01-01

    Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

  12. Theoretical studies on binding modes of copper-based nucleases with DNA.

    PubMed

    Liu, Chunmei; Zhu, Yanyan; Tang, Mingsheng

    2016-03-01

    In the present work, molecular simulations were performed for the purpose of predicting the binding modes of four types of copper nucleases (a total 33 compounds) with DNA. Our docking results accurately predicted the groove binding and electrostatic interaction for some copper nucleases with B-DNA. The intercalation modes were also reproduced by "gap DNA". The obtained results demonstrated that the ligand size, length, functional groups and chelate ring size bound to the copper center could influence the binding affinities of copper nucleases. The binding affinities obtained from the docking calculations herein also replicated results found using MM-PBSA approach. The predicted DNA binding modes of copper nucleases with DNA will ultimately help us to better understand the interaction of copper compounds with DNA.

  13. Delivery methods for site-specific nucleases: Achieving the full potential of therapeutic gene editing.

    PubMed

    Liu, Jia; Shui, Sai-Lan

    2016-12-28

    The advent of site-specific nucleases, particularly CRISPR/Cas9, provides researchers with the unprecedented ability to manipulate genomic sequences. These nucleases are used to create model cell lines, engineer metabolic pathways, produce transgenic animals and plants, perform genome-wide functional screen and, most importantly, treat human diseases that are difficult to tackle by traditional medications. Considerable efforts have been devoted to improving the efficiency and specificity of nucleases for clinical applications. However, safe and efficient delivery methods remain the major obstacle for therapeutic gene editing. In this review, we summarize the recent progress on nuclease delivery methods, highlight their impact on the outcomes of gene editing and discuss the potential of different delivery approaches for therapeutic gene editing.

  14. Generation of knockout rabbits using transcription activator-like effector nucleases.

    PubMed

    Wang, Yu; Fan, Nana; Song, Jun; Zhong, Juan; Guo, Xiaogang; Tian, Weihua; Zhang, Quanjun; Cui, Fenggong; Li, Li; Newsome, Philip N; Frampton, Jon; Esteban, Miguel A; Lai, Liangxue

    2014-01-01

    Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platforms contributing to redefine the boundaries of modern biological research. They are composed of a non-specific cleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications by inducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases have been employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively. This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies, biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbits using transcription activator-like effector nucleases, and a perspective of the field.

  15. Evaluation of Biological and Physical Protection against Nuclease Degradation of Clay-Bound Plasmid DNA

    PubMed Central

    Demanèche, Sandrine; Jocteur-Monrozier, Lucile; Quiquampoix, Hervé; Simonet, Pascal

    2001-01-01

    In order to determine the mechanisms involved in the persistence of extracellular DNA in soils and to monitor whether bacterial transformation could occur in such an environment, we developed artificial models composed of plasmid DNA adsorbed on clay particles. We determined that clay-bound DNA submitted to an increasing range of nuclease concentrations was physically protected. The protection mechanism was mainly related to the adsorption of the nuclease on the clay mineral. The biological potential of the resulting DNA was monitored by transforming the naturally competent proteobacterium Acinetobacter sp. strain BD413, allowing us to demonstrate that adsorbed DNA was only partially available for transformation. This part of the clay-bound DNA which was available for bacteria, was also accessible to nucleases, while the remaining fraction escaped both transformation and degradation. Finally, transformation efficiency was related to the perpetuation mechanism, with homologous recombination being less sensitive to nucleases than autonomous replication, which requires intact molecules. PMID:11133458

  16. Structure-Specific nuclease activities of Artemis and the Artemis: DNA-PKcs complex

    PubMed Central

    Chang, Howard H.Y.; Lieber, Michael R.

    2016-01-01

    Artemis is a vertebrate nuclease with both endo- and exonuclease activities that acts on a wide range of nucleic acid substrates. It is the main nuclease in the non-homologous DNA end-joining pathway (NHEJ). Not only is Artemis important for the repair of DNA double-strand breaks (DSBs) in NHEJ, it is essential in opening the DNA hairpin intermediates that are formed during V(D)J recombination. Thus, humans with Artemis deficiencies do not have T- or B-lymphocytes and are diagnosed with severe combined immunodeficiency (SCID). While Artemis is the only vertebrate nuclease capable of opening DNA hairpins, it has also been found to act on other DNA substrates that share common structural features. Here, we discuss the key structural features that all Artemis DNA substrates have in common, thus providing a basis for understanding how this structure-specific nuclease recognizes its DNA targets. PMID:27198222

  17. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases.

    PubMed

    Frock, Richard L; Hu, Jiazhi; Meyers, Robin M; Ho, Yu-Jui; Kii, Erina; Alt, Frederick W

    2015-02-01

    Although great progress has been made in the characterization of the off-target effects of engineered nucleases, sensitive and unbiased genome-wide methods for the detection of off-target cleavage events and potential collateral damage are still lacking. Here we describe a linear amplification-mediated modification of a previously published high-throughput, genome-wide, translocation sequencing (HTGTS) method that robustly detects DNA double-stranded breaks (DSBs) generated by engineered nucleases across the human genome based on their translocation to other endogenous or ectopic DSBs. HTGTS with different Cas9:sgRNA or TALEN nucleases revealed off-target hotspot numbers for given nucleases that ranged from a few or none to dozens or more, and extended the number of known off-targets for certain previously characterized nucleases more than tenfold. We also identified translocations between bona fide nuclease targets on homologous chromosomes, an undesired collateral effect that has not been described previously. Finally, HTGTS confirmed that the Cas9D10A paired nickase approach suppresses off-target cleavage genome-wide.

  18. Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease.

    PubMed

    Li, Si-Ming; Bai, Fu-Liang; Xu, Wen-Juan; Yang, Yong-Bi; An, Ying; Li, Tian-He; Yu, Yin-Hang; Li, De-Shan; Wang, Wen-Fei

    2014-09-01

    The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was <100 pg/ml in the final product. The residual nuclease in rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease.

  19. Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis.

    PubMed

    Lonowski, Lindsey A; Narimatsu, Yoshiki; Riaz, Anjum; Delay, Catherine E; Yang, Zhang; Niola, Francesco; Duda, Katarzyna; Ober, Elke A; Clausen, Henrik; Wandall, Hans H; Hansen, Steen H; Bennett, Eric P; Frödin, Morten

    2017-03-01

    This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.

  20. Role of the nuclease of nontypeable Haemophilus influenzae in dispersal of organisms from biofilms.

    PubMed

    Cho, Christine; Chande, Aroon; Gakhar, Lokesh; Bakaletz, Lauren O; Jurcisek, Joseph A; Ketterer, Margaret; Shao, Jian; Gotoh, Kenji; Foster, Eric; Hunt, Jason; O'Brien, Erin; Apicella, Michael A

    2015-03-01

    Nontypeable Haemophilus influenzae (NTHI) forms biofilms in the middle ear during human infection. The biofilm matrix of NTHI contains extracellular DNA. We show that NTHI possesses a potent nuclease, which is a homolog of the thermonuclease of Staphylococcus aureus. Using a biofilm dispersal assay, studies showed a biofilm dispersal pattern in the parent strain, no evidence of dispersal in the nuclease mutant, and a partial return of dispersion in the complemented mutant. Quantitative PCR of mRNA from biofilms from a 24-h continuous flow system demonstrated a significantly increased expression of the nuclease from planktonic organisms compared to those in the biofilm phase of growth (P < 0.042). Microscopic analysis of biofilms grown in vitro showed that in the nuclease mutant the nucleic acid matrix was increased compared to the wild-type and complemented strains. Organisms were typically found in large aggregates, unlike the wild-type and complement biofilms in which the organisms were evenly dispersed throughout the biofilm. At 48 h, the majority of the organisms in the mutant biofilm were dead. The nuclease mutant formed a biofilm in the chinchilla model of otitis media and demonstrated a propensity to also form similar large aggregates of organisms. These studies indicate that NTHI nuclease is involved in biofilm remodeling and organism dispersal.

  1. Enhanced gene disruption by programmable nucleases delivered by a minicircle vector.

    PubMed

    Dad, A-B K; Ramakrishna, S; Song, M; Kim, H

    2014-11-01

    Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors, which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group. Taken together, our results show that gene disruption using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases.

  2. Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    PubMed Central

    Ghosh-Kumar, Manjira; Alam, Tanfis I.; Draper, Bonnie; Stack, John D.; Rao, Venigalla B.

    2011-01-01

    In genome packaging by tailed bacteriophages and herpesviruses, a concatemeric DNA is cut and inserted into an empty procapsid. A series of cuts follow the encapsidation of each unit-length ‘headful’ genome, but the mechanisms by which cutting is coupled to packaging are not understood. Here we report the first biochemical characterization of a headful nuclease from bacteriophage T4. Our results show that the T4 nuclease, which resides in the C-terminal domain of large ‘terminase’ gp17, is a weak endonuclease and regulated by a variety of factors; Mg, NaCl, ATP, small terminase gp16 and N-terminal ATPase domain. The small terminase, which stimulates gp17-ATPase, also stimulates nuclease in the presence of ATP but inhibits in the absence of ATP suggesting interdomain crosstalk. Comparison of the ‘relaxed’ and ‘tensed’ states of the motor show that a number of basic residues lining the nuclease groove are positioned to interact with DNA in the tensed state but change their positions in the relaxed state. These results suggest that conformational changes in the ATPase center remodel the nuclease center via an interdomain ‘communication track’. This might be a common regulatory mechanism for coupling DNA cutting to DNA packaging among the headful packaging nucleases from dsDNA viruses. PMID:21109524

  3. Identification of Intracellular Carbonic Anhydrase in Chlamydomonas reinhardtii which Is Distinct from the Periplasmic Form of the Enzyme 1

    PubMed Central

    Husic, H. David; Kitayama, Masahiko; Togasaki, Robert K.; Moroney, James V.; Morris, Kristin L.; Tolbert, N. E.

    1989-01-01

    A physiologically significant level of intracellular carbonic anhydrase has been identified in Chlamydomonas reinhardtii after lysis of the cell wall-less mutant, cw15, and two intracellular polypeptides have been identified which bind to anti-carbonic anhydrase antisera. The susceptibility of the intracellular activity to sulfonamide carbonic anhydrase inhibitors is more than three orders-of-magnitude less than that of the periplasmic enzyme, indicating that the intracellular activity was distinct from the periplasmic from of the enzyme. When electrophoretically separated cell extracts or chloroplast stromal fractions were probed with either anti-C. reinhardtii periplasmic carbonic anhydrase antiserum or anti-spinach carbonic anhydrase antiserum, immunoreactive polypeptides of 45 kilodaltons and 110 kilodaltons were observed with both antisera. The strongly immunoreactive 37 kilodalton polypeptide due to the periplasmic carbonic anhydrase was also observed in lysed cells, but neither the 37 kilodalton nor the 110 kilodalton polypeptides were present in the chloroplast stromal fraction. These studies have identified intracellular carbonic anhydrase activity, and putative intracellular carbonic anhydrase polypeptides in Chlamydomonas reinhardtii represented by a 45 kilodalton polypeptide in the chloroplast and a 110 kilodalton form probably in the cytoplasm, which may be associated with an intracellular inorganic carbon concentrating system. Images Figure 2 Figure 3 PMID:16666640

  4. A rapid procedure for the in situ assay of periplasmic, PQQ-dependent methanol dehydrogenase in intact single bacterial colonies.

    PubMed

    Vemuluri, Venkata Ramana; Shaw, Shreya; Autenrieth, Caroline; Ghosh, Robin

    2017-03-23

    Mechanistic details of methanol oxidation catalyzed by the periplasmically-located pyrroloquinoline quinone-dependent methanol dehydrogenase of methylotrophs can be elucidated using site-directed mutants. Here, we present an in situ colony assay of methanol dehydrogenase, which allows robotic screening of large populations of intact small colonies, and regrowth of colonies for subsequent analysis.

  5. Versatile signal peptide of Flavobacterium-originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli.

    PubMed

    Kang, Dong Gyun; Seo, Jeong Hyun; Jo, Byung Hoon; Kim, Chang Sup; Choi, Suk Soon; Cha, Hyung Joon

    2016-07-08

    Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane-associated homodimeric metalloenzyme and has its own signal peptide in its N-terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide-containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin-arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec-avoidance sequence in the c-region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848-854, 2016. © 2016 American Institute of Chemical Engineers.

  6. Targeted genome modification in mice using zinc-finger nucleases.

    PubMed

    Carbery, Iara D; Ji, Diana; Harrington, Anne; Brown, Victoria; Weinstein, Edward J; Liaw, Lucy; Cui, Xiaoxia

    2010-10-01

    Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20-75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research.

  7. Targeted Genome Modification in Mice Using Zinc-Finger Nucleases

    PubMed Central

    Carbery, Iara D.; Ji, Diana; Harrington, Anne; Brown, Victoria; Weinstein, Edward J.; Liaw, Lucy; Cui, Xiaoxia

    2010-01-01

    Homologous recombination-based gene targeting using Mus musculus embryonic stem cells has greatly impacted biomedical research. This study presents a powerful new technology for more efficient and less time-consuming gene targeting in mice using embryonic injection of zinc-finger nucleases (ZFNs), which generate site-specific double strand breaks, leading to insertions or deletions via DNA repair by the nonhomologous end joining pathway. Three individual genes, multidrug resistant 1a (Mdr1a), jagged 1 (Jag1), and notch homolog 3 (Notch3), were targeted in FVB/N and C57BL/6 mice. Injection of ZFNs resulted in a range of specific gene deletions, from several nucleotides to >1000 bp in length, among 20–75% of live births. Modified alleles were efficiently transmitted through the germline, and animals homozygous for targeted modifications were obtained in as little as 4 months. In addition, the technology can be adapted to any genetic background, eliminating the need for generations of backcrossing to achieve congenic animals. We also validated the functional disruption of Mdr1a and demonstrated that the ZFN-mediated modifications lead to true knockouts. We conclude that ZFN technology is an efficient and convenient alternative to conventional gene targeting and will greatly facilitate the rapid creation of mouse models and functional genomics research. PMID:20628038

  8. DNA targeting specificity of RNA-guided Cas9 nucleases

    PubMed Central

    Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

    2014-01-01

    The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of singleguide RNAs (sgRNAs) to enable genome editing1–10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. PMID:23873081

  9. SAPTA: a new design tool for improving TALE nuclease activity.

    PubMed

    Lin, Yanni; Fine, Eli J; Zheng, Zhilan; Antico, Christopher J; Voit, Richard A; Porteus, Matthew H; Cradick, Thomas J; Bao, Gang

    2014-04-01

    Transcription activator-like effector nucleases (TALENs) have become a powerful tool for genome editing due to the simple code linking the amino acid sequences of their DNA-binding domains to TALEN nucleotide targets. While the initial TALEN-design guidelines are very useful, user-friendly tools defining optimal TALEN designs for robust genome editing need to be developed. Here we evaluated existing guidelines and developed new design guidelines for TALENs based on 205 TALENs tested, and established the scoring algorithm for predicting TALEN activity (SAPTA) as a new online design tool. For any input gene of interest, SAPTA gives a ranked list of potential TALEN target sites, facilitating the selection of optimal TALEN pairs based on predicted activity. SAPTA-based TALEN designs increased the average intracellular TALEN monomer activity by >3-fold, and resulted in an average endogenous gene-modification frequency of 39% for TALENs containing the repeat variable di-residue NK that favors specificity rather than activity. It is expected that SAPTA will become a useful and flexible tool for designing highly active TALENs for genome-editing applications. SAPTA can be accessed via the website at http://baolab.bme.gatech.edu/Research/BioinformaticTools/TAL_targeter.html.

  10. Myostatin gene mutated mice induced with tale nucleases.

    PubMed

    Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru

    2015-01-01

    Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection.

  11. A library of TAL effector nucleases spanning the human genome.

    PubMed

    Kim, Yongsub; Kweon, Jiyeon; Kim, Annie; Chon, Jae Kyung; Yoo, Ji Yeon; Kim, Hye Joo; Kim, Sojung; Lee, Choongil; Jeong, Euihwan; Chung, Eugene; Kim, Doyoung; Lee, Mi Seon; Go, Eun Mi; Song, Hye Jung; Kim, Hwangbeom; Cho, Namjin; Bang, Duhee; Kim, Seokjoong; Kim, Jin-Soo

    2013-03-01

    Transcription activator-like (TAL) effector nucleases (TALENs) can be readily engineered to bind specific genomic loci, enabling the introduction of precise genetic modifications such as gene knockouts and additions. Here we present a genome-scale collection of TALENs for efficient and scalable gene targeting in human cells. We chose target sites that did not have highly similar sequences elsewhere in the genome to avoid off-target mutations and assembled TALEN plasmids for 18,740 protein-coding genes using a high-throughput Golden-Gate cloning system. A pilot test involving 124 genes showed that all TALENs were active and disrupted their target genes at high frequencies, although two of these TALENs became active only after their target sites were partially demethylated using an inhibitor of DNA methyltransferase. We used our TALEN library to generate single- and double-gene-knockout cells in which NF-κB signaling pathways were disrupted. Compared with cells treated with short interfering RNAs, these cells showed unambiguous suppression of signal transduction.

  12. Increasing cloning possibilities using artificial zinc finger nucleases.

    PubMed

    Zeevi, Vardit; Tovkach, Andriy; Tzfira, Tzvi

    2008-09-02

    The ability to accurately digest and ligate DNA molecules of different origins is fundamental to modern recombinant DNA research. Only a handful of enzymes are capable of recognizing and cleaving novel and long DNA sequences, however. The slow evolution and engineering of new restriction enzymes calls for alternative strategies to design novel and unique restriction enzymes capable of binding and digesting specific long DNA sequences. Here we report on the use of zinc finger nucleases (ZFNs)-hybrid synthetic restriction enzymes that can be specifically designed to bind and cleave long DNA sequences-for the purpose of DNA recombination. We show that novel ZFNs can be designed for the digestion of specific sequences and can be expressed and used for cloning purposes. We also demonstrate the power of ZFNs in DNA cloning by custom-cloning a target DNA sequence and assembling dual-expression cassettes on a single target plasmid, a task that rarely can be achieved using type-II restriction enzymes. We demonstrate the flexibility of ZFN design and the ability to shuffle monomers of different ZFNs for the digestion of compatible recognition sites through ligation of compatible ends and their cleavage by heterodimer ZFNs. Of no less importance, we show that ZFNs can be designed to recognize and cleave existing DNA sequences for the custom-cloning of native target DNA molecules.

  13. RNA-protein analysis using a conditional CRISPR nuclease.

    PubMed

    Lee, Ho Young; Haurwitz, Rachel E; Apffel, Alex; Zhou, Kaihong; Smart, Brian; Wenger, Craig D; Laderman, Stephen; Bruhn, Laurakay; Doudna, Jennifer A

    2013-04-02

    RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA-protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5' ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.

  14. DddY, a periplasmic dimethylsulfoniopropionate lyase found in taxonomically diverse species of Proteobacteria

    PubMed Central

    Curson, Andrew R J; Sullivan, Matthew J; Todd, Jonathan D; Johnston, Andrew W B

    2011-01-01

    The abundant compatible solute dimethylsulfoniopropionate (DMSP) is made by many marine algae. Different marine bacteria catabolise DMSP by various mechanisms, some of which liberate the environmentally important gas dimethyl sulfide (DMS). We describe an enzyme, DddY, which cleaves DMSP into DMS plus acrylate and is located in the bacterial periplasm, unlike other DMSP lyases that catalyse this reaction. There are dddY-like genes in strains of Alcaligenes, Arcobacter and Shewanella, in the β-, ɛ- and γ-proteobacteria, respectively. In Alcaligenes, dddY is in a cluster of ddd and acu genes that resemble, but also have significant differences to, those in other bacteria that catabolise both DMSP and acrylate. Although production of DMS and transcription of Alcaligenes dddY are both apparently inducible by pre-growth of cells with DMSP, this substrate must be catabolised to form acrylate, the bona fide coinducer. PMID:21248856

  15. NMR studies of the interaction between inner membrane-associated and periplasmic cytochromes from Geobacter sulfurreducens.

    PubMed

    Dantas, Joana M; Brausemann, Anton; Einsle, Oliver; Salgueiro, Carlos A

    2017-06-01

    Geobacter sulfurreducens is a dissimilatory metal-reducing bacterium with notable properties and significance in biotechnological applications. Biochemical studies suggest that the inner membrane-associated diheme cytochrome MacA and the periplasmic triheme cytochrome PpcA from G. sulfurreducens can exchange electrons. In this work, NMR chemical shift perturbation measurements were used to map the interface region and to measure the binding affinity between PpcA and MacA. The results show that MacA binds to PpcA in a cleft defined by hemes I and IV, favoring the contact between PpcA heme IV and the MacA high-potential heme. The dissociation constant values indicate the formation of a low-affinity complex between the proteins, which is consistent with the transient interaction observed in electron transfer complexes. © 2017 Federation of European Biochemical Societies.

  16. Nanobiosensor design utilizing a periplasmic E. coli receptor protein immobilized within Au/polycarbonate nanopores.

    PubMed

    Tripathi, Abhinav; Wang, Jianbin; Luck, Linda A; Suni, Ian I

    2007-02-01

    A new type of nanopore sensor design is reported for a reagent-less electrochemical biosensor with no analyte "tagging" by fluorescent molecules, nanoparticles, or other species. This sensor design involves immobilization within Au-coated nanopores of bacterial periplasmic binding proteins (bPBP), which undergo a wide-amplitude, hinge-twist motion upon ligand binding. Ligand binding thus triggers a reduction in the effective thickness of the immobilized protein film, which is detected as an increase in electrolyte conductivity (decrease in impedance) through the nanopores. This new sensor design is demonstrated for glucose detection using a cysteine-tagged mutant (GGR Q26C) of the galactose/glucose receptor (GGR) protein from the bPBP family. The GGR Q26C protein is immobilized onto Au nanoislands that are deposited within the pores of commercially available nanoporous polycarbonate membranes.

  17. A ligand-induced switch in the periplasmic domain of sensor histidine kinase CitA.

    PubMed

    Sevvana, Madhumati; Vijayan, Vinesh; Zweckstetter, Markus; Reinelt, Stefan; Madden, Dean R; Herbst-Irmer, Regine; Sheldrick, George M; Bott, Michael; Griesinger, Christian; Becker, Stefan

    2008-03-21

    Sensor histidine kinases of two-component signal-transduction systems are essential for bacteria to adapt to variable environmental conditions. However, despite their prevalence, it is not well understood how extracellular signals such as ligand binding regulate the activity of these sensor kinases. CitA is the sensor histidine kinase in Klebsiella pneumoniae that regulates the transport and anaerobic metabolism of citrate in response to its extracellular concentration. We report here the X-ray structures of the periplasmic sensor domain of CitA in the citrate-free and citrate-bound states. A comparison of the two structures shows that ligand binding causes a considerable contraction of the sensor domain. This contraction may represent the molecular switch that activates transmembrane signaling in the receptor.

  18. DddY, a periplasmic dimethylsulfoniopropionate lyase found in taxonomically diverse species of Proteobacteria.

    PubMed

    Curson, Andrew R J; Sullivan, Matthew J; Todd, Jonathan D; Johnston, Andrew W B

    2011-07-01

    The abundant compatible solute dimethylsulfoniopropionate (DMSP) is made by many marine algae. Different marine bacteria catabolise DMSP by various mechanisms, some of which liberate the environmentally important gas dimethyl sulfide (DMS). We describe an enzyme, DddY, which cleaves DMSP into DMS plus acrylate and is located in the bacterial periplasm, unlike other DMSP lyases that catalyse this reaction. There are dddY-like genes in strains of Alcaligenes, Arcobacter and Shewanella, in the β-, ɛ- and γ-proteobacteria, respectively. In Alcaligenes, dddY is in a cluster of ddd and acu genes that resemble, but also have significant differences to, those in other bacteria that catabolise both DMSP and acrylate. Although production of DMS and transcription of Alcaligenes dddY are both apparently inducible by pre-growth of cells with DMSP, this substrate must be catabolised to form acrylate, the bona fide coinducer.

  19. Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone.

    PubMed

    Dow, Jennifer M; Grahl, Sabine; Ward, Richard; Evans, Rachael; Byron, Olwyn; Norman, David G; Palmer, Tracy; Sargent, Frank

    2014-01-01

    Escherichia coli is a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin-arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin-arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD. In addition, a complex comprising the full-length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1 : 1 molar ratio, and small angle X-ray scattering analysis of the complex indicated that NapA was at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken together, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor.

  20. Interactions between TonB from Escherichia coli and the periplasmic protein FhuD.

    PubMed

    Carter, David M; Miousse, Isabelle R; Gagnon, Jean-Nicolas; Martinez, Eric; Clements, Abigail; Lee, Jongchan; Hancock, Mark A; Gagnon, Hubert; Pawelek, Peter D; Coulton, James W

    2006-11-17

    For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent KD of approximately 300 nM. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore.

  1. Sucrose dependent mineral phosphate solubilization in Enterobacter asburiae PSI3 by heterologous overexpression of periplasmic invertases.

    PubMed

    Kumar, Chanchal; Wagh, Jitendra; Archana, G; Naresh Kumar, G

    2016-12-01

    Enterobacter asburiae PSI3 solubilizes mineral phosphates in the presence of glucose by the secretion of gluconic acid generated by the action of a periplasmic pyrroloquinoline quinone dependent glucose dehydrogenase. In order to achieve mineral phosphate solubilization phenotype in the presence of sucrose, plasmids pCNK4 and pCNK5 containing genes encoding the invertase enzyme of Zymomonas mobilis (invB) and of Saccharomyces cerevisiae (suc2) under constitutive promoters were constructed with malE signal sequence (in case of invB alone as the suc2 is secreted natively). When introduced into E. asburiae PSI3, E. a. (pCNK4) and E. a. (pCNK5) transformants secreted 21.65 ± 0.94 and 22 ± 1.3 mM gluconic acid, respectively, in the presence of 75 mM sucrose and they also solubilized 180 ± 4.3 and 438 ± 7.3 µM P from the rock phosphate. In the presence of a mixture of 50 mM sucrose and 25 mM glucose, E. a. (pCNK5) secreted 34 ± 2.3 mM gluconic acid and released 479 ± 8.1 µM P. Moreover, in the presence of a mixture of eight sugars (10 mM each) in the medium, E. a. (pCNK5) released 414 ± 5.3 µM P in the buffered medium. Thus, this study demonstrates incorporation of periplasmic invertase imparted P solubilization ability to E. asburiae PSI3 in the presence of sucrose and mixture of sugars.

  2. Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate.

    PubMed

    Cooley, Richard B; Smith, T Jarrod; Leung, Wilfred; Tierney, Valerie; Borlee, Bradley R; O'Toole, George A; Sondermann, Holger

    2015-06-22

    We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate

    PubMed Central

    Cooley, Richard B.; Smith, T. Jarrod; Leung, Wilfred; Tierney, Valerie; Borlee, Bradley R.; O'Toole, George A.

    2015-01-01

    ABSTRACT We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. IMPORTANCE Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger. PMID:26100041

  4. Designing Signal Peptides for Efficient Periplasmic Expression of Human Growth Hormone in Escherichia coli.

    PubMed

    Khameneh, Meisam Jeirani; Moshiri, Farzaneh; Falasafi, Soheil Keyhan; Zomorodipour, Alireza

    2017-08-31

    Secretion efficiency of a protein in a SEC-type secretion system is mainly determined by an N-terminal signal peptide and its combination with its cognate protein. Five signal peptides, including; two synthetic Sec-type and three Bacillus licheniformis alpha-amylase derived signal peptides were compared for periplasmic expression of the human growth hormone (hGH) in E. coli. Based on in silico predictions on the signal peptides' cleavage efficiencies and their corresponding mRNA secondary structures, a number of amino acid substitutions and silent mutations were considered in the modified signal sequences. The two synthetic signal peptides, specifically designed for the hGH secretion in E. coli, differ in their N-terminal positively charged residues and hydrophobic regions lengths. According to the mRNA secondary structure predictions, combinations of the protein and each of the five signal sequences could lead to different outcomes, especially when accessibility of the initiator ATG and ribosome binding sites were considered. In the experimental stage, the two synthetic signal peptides displayed complete processing and resulted in efficient secretion of the mature hGH in periplasmic regions, as it was demonstrated by protein analysis. The three alpha-amylase-derived signal peptides, however, were processed partially from their precursors. Therefore, to achieve efficient secretion of a protein in a heterologous system, designing of a specific signal peptide in which a combined approach of optimizations of the mRNA secondary structure and the signal peptide H-domain and cleavage site is recommended.

  5. Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

    PubMed Central

    Yatsunyk, Liliya A.; Easton, J. Allen; Kim, Lydia R.; Sugarbaker, Stacy A.; Bennett, Brian; Breece, Robert M.; Vorontsov, Ivan I.; Tierney, David L.

    2009-01-01

    ZnuA is the periplasmic Zn2+-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn2+-bound, and Co2+-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn2+ with Co2+ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn2+ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn2+ (estimated Kd < 20 nM), Co2+, Ni2+, Cu2+, Cu+, and Cd2+, but not Mn2+. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn2+ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer. PMID:18027003

  6. myo-Inositol and d-Ribose Ligand Discrimination in an ABC Periplasmic Binding Protein

    PubMed Central

    Herrou, Julien

    2013-01-01

    The periplasmic binding protein (PBP) IbpA mediates the uptake of myo-inositol by the IatP-IatA ATP-binding cassette transmembrane transporter. We report a crystal structure of Caulobacter crescentus IbpA bound to myo-inositol at 1.45 Å resolution. This constitutes the first structure of a PBP bound to inositol. IbpA adopts a type I PBP fold consisting of two α-β lobes that surround a central hinge. A pocket positioned between the lobes contains the myo-inositol ligand, which binds with submicromolar affinity (0.76 ± 0.08 μM). IbpA is homologous to ribose-binding proteins and binds d-ribose with low affinity (50.8 ± 3.4 μM). On the basis of IbpA and ribose-binding protein structures, we have designed variants of IbpA with inverted binding specificity for myo-inositol and d-ribose. Five mutations in the ligand-binding pocket are sufficient to increase the affinity of IbpA for d-ribose by 10-fold while completely abolishing binding to myo-inositol. Replacement of ibpA with these mutant alleles unable to bind myo-inositol abolishes C. crescentus growth in medium containing myo-inositol as the sole carbon source. Neither deletion of ibpA nor replacement of ibpA with the high-affinity ribose binding allele affected C. crescentus growth on d-ribose as a carbon source, providing evidence that the IatP-IatA transporter is specific for myo-inositol. This study outlines the evolutionary relationship between ribose- and inositol-binding proteins and provides insight into the molecular basis upon which these two related, but functionally distinct, classes of periplasmic proteins specifically bind carbohydrate ligands. PMID:23504019

  7. Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

    PubMed Central

    Zückert, Wolfram R.

    2014-01-01

    Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporterlike LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

  8. Use of glycol ethers for selective release of periplasmic proteins from Gram-negative bacteria.

    PubMed

    Allen, Jeffrey R; Patkar, Anant Y; Frank, Timothy C; Donate, Felipe A; Chiu, Yuk Chun; Shields, Jefry E; Gustafson, Mark E

    2007-01-01

    Genetic modification of Gram-negative bacteria to express a desired protein within the cell's periplasmic space, located between the inner cytoplasmic membrane and the outer cell wall, can offer an attractive strategy for commercial production of therapeutic proteins and industrial enzymes. In certain applications, the product expression rate is high, and the ability to isolate the product from the cell mass is greatly enhanced because of the product's compartmentalized location within the cell. Protein release methods that increase the permeability of the outer cell wall for primary recovery, but avoid rupturing the inner cell membrane, reduce contamination of the recovered product with other host cell components and simplify final purification. This article reports representative data for a new release method employing glycol ether solvents. The example involves the use of 2-butoxyethanol (commonly called ethylene glycol n-butyl ether or EB) for selective release of a proprietary biopharmaceutical protein produced in the periplasmic space of Pseudomonas fluorescens. In this example, glycol ether treatment yielded approximately 65% primary recovery with approximately 80% purity on a protein-only basis. Compared with other methods including heat treatment, osmotic shock, and the use of surfactants, the glycol ether treatment yielded significantly reduced concentrations of other host cell proteins, lipopolysaccharide endotoxin, and DNA in the recovered protein solution. The use of glycol ethers also allowed exploitation of temperature-change-induced phase splitting behavior to concentrate the desired product. Heating the aqueous EB extract solution to 55 degrees C formed two liquid phases: a glycol ether-rich phase and an aqueous product phase containing the great majority of the product protein. This phase-splitting step yielded an approximate 10-fold reduction in the volume of the initial product solution and a corresponding increase in the product's concentration.

  9. Characterization of the periplasmic redox network that sustains the versatile anaerobic metabolism of Shewanella oneidensis MR-1.

    PubMed

    Alves, Mónica N; Neto, Sónia E; Alves, Alexandra S; Fonseca, Bruno M; Carrêlo, Afonso; Pacheco, Isabel; Paquete, Catarina M; Soares, Cláudio M; Louro, Ricardo O

    2015-01-01

    The versatile anaerobic metabolism of the Gram-negative bacterium Shewanella oneidensis MR-1 (SOMR-1) relies on a multitude of redox proteins found in its periplasm. Most are multiheme cytochromes that carry electrons to terminal reductases of insoluble electron acceptors located at the cell surface, or bona fide terminal reductases of soluble electron acceptors. In this study, the interaction network of several multiheme cytochromes was explored by a combination of NMR spectroscopy, activity assays followed by UV-visible spectroscopy and comparison of surface electrostatic potentials. From these data the small tetraheme cytochrome (STC) emerges as the main periplasmic redox shuttle in SOMR-1. It accepts electrons from CymA and distributes them to a number of terminal oxidoreductases involved in the respiration of various compounds. STC is also involved in the electron transfer pathway to reduce nitrite by interaction with the octaheme tetrathionate reductase (OTR), but not with cytochrome c nitrite reductase (ccNiR). In the main pathway leading the metal respiration STC pairs with flavocytochrome c (FccA), the other major periplasmic cytochrome, which provides redundancy in this important pathway. The data reveals that the two proteins compete for the binding site at the surface of MtrA, the decaheme cytochrome inserted on the periplasmic side of the MtrCAB-OmcA outer-membrane complex. However, this is not observed for the MtrA homologues. Indeed, neither STC nor FccA interact with MtrD, the best replacement for MtrA, and only STC is able to interact with the decaheme cytochrome DmsE of the outer-membrane complex DmsEFABGH. Overall, these results shown that STC plays a central role in the anaerobic respiratory metabolism of SOMR-1. Nonetheless, the trans-periplasmic electron transfer chain is functionally resilient as a consequence of redundancies that arise from the presence of alternative pathways that bypass/compete with STC.

  10. Acinetobacter baumannii Extracellular OXA-58 Is Primarily and Selectively Released via Outer Membrane Vesicles after Sec-Dependent Periplasmic Translocation.

    PubMed

    Liao, Yu-Ting; Kuo, Shu-Chen; Chiang, Ming-Hsien; Lee, Yi-Tzu; Sung, Wang-Chou; Chen, You-Hsuan; Chen, Te-Li; Fung, Chang-Phone

    2015-12-01

    Carbapenem-resistant Acinetobacter baumannii (CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D β-lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated. In silico analysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Characterization of the periplasmic redox network that sustains the versatile anaerobic metabolism of Shewanella oneidensis MR-1

    PubMed Central

    Alves, Mónica N.; Neto, Sónia E.; Alves, Alexandra S.; Fonseca, Bruno M.; Carrêlo, Afonso; Pacheco, Isabel; Paquete, Catarina M.; Soares, Cláudio M.; Louro, Ricardo O.

    2015-01-01

    The versatile anaerobic metabolism of the Gram-negative bacterium Shewanella oneidensis MR-1 (SOMR-1) relies on a multitude of redox proteins found in its periplasm. Most are multiheme cytochromes that carry electrons to terminal reductases of insoluble electron acceptors located at the cell surface, or bona fide terminal reductases of soluble electron acceptors. In this study, the interaction network of several multiheme cytochromes was explored by a combination of NMR spectroscopy, activity assays followed by UV-visible spectroscopy and comparison of surface electrostatic potentials. From these data the small tetraheme cytochrome (STC) emerges as the main periplasmic redox shuttle in SOMR-1. It accepts electrons from CymA and distributes them to a number of terminal oxidoreductases involved in the respiration of various compounds. STC is also involved in the electron transfer pathway to reduce nitrite by interaction with the octaheme tetrathionate reductase (OTR), but not with cytochrome c nitrite reductase (ccNiR). In the main pathway leading the metal respiration STC pairs with flavocytochrome c (FccA), the other major periplasmic cytochrome, which provides redundancy in this important pathway. The data reveals that the two proteins compete for the binding site at the surface of MtrA, the decaheme cytochrome inserted on the periplasmic side of the MtrCAB–OmcA outer-membrane complex. However, this is not observed for the MtrA homologues. Indeed, neither STC nor FccA interact with MtrD, the best replacement for MtrA, and only STC is able to interact with the decaheme cytochrome DmsE of the outer-membrane complex DmsEFABGH. Overall, these results shown that STC plays a central role in the anaerobic respiratory metabolism of SOMR-1. Nonetheless, the trans-periplasmic electron transfer chain is functionally resilient as a consequence of redundancies that arise from the presence of alternative pathways that bypass/compete with STC. PMID:26175726

  12. The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration.

    PubMed

    Delgado, María J; Bonnard, Nathalie; Tresierra-Ayala, Alvaro; Bedmar, Eulogio J; Müller, Peter

    2003-12-01

    The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66.5 bp downstream of the centre of a putative FNR-like binding site.

  13. Protection of HIV neutralizing aptamers against rectal and vaginal nucleases: implications for RNA-based therapeutics.

    PubMed

    Moore, Michael D; Cookson, Jonathan; Coventry, Veronica K; Sproat, Brian; Rabe, Lorna; Cranston, Ross D; McGowan, Ian; James, William

    2011-01-28

    RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2'-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2'-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn(2+) cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.

  14. SplitAx: A novel method to assess the function of engineered nucleases

    PubMed Central

    Axton, Richard A.; Haideri, Sharmin S.; Lopez-Yrigoyen, Martha; Taylor, Helen A.; Forrester, Lesley M.

    2017-01-01

    Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity. The assay involves splitting the N-terminal (amino acid 1–158) coding region of GFP and an out-of-frame of C-terminal region with a nuclease binding site sequence. Following exposure to the test nuclease, cutting and repair by error prone non-homologous end joining (NHEJ) restores the reading frame resulting in the production of a full length fluorescent GFP protein. Fluorescence can also be restored by complementation between the N-terminal and C-terminal coding sequences in trans. We demonstrate successful use of the SplitAx assay to assess the function of zinc finger nucleases, CRISPR hCAS9 and TALENS. We also test the activity of multiple gRNAs in CRISPR/hCas9/D10A systems. The zinc finger nucleases and guide RNAs that showed functional activity in the SplitAx assay were then used successfully to target the endogenous AAVS1, SOX6 and Cfms loci. This simple method can be applied to other unrelated proteins such as ZsGreen1 and provides a test system that does not require complex optimization. PMID:28212417

  15. A rapid assay to quantify the cleavage efficiency of custom-designed nucleases in planta.

    PubMed

    Johnson, Ross A; Gurevich, Vyacheslav; Levy, Avraham A

    2013-06-01

    Custom-designed nucleases are a promising technology for genome editing through the catalysis of double-strand DNA breaks within target loci and subsequent repair by the host cell, which can result in targeted mutagenesis or gene replacement. Implementing this new technology requires a rapid means to determine the cleavage efficiency of these custom-designed proteins in planta. Here we present such an assay that is based on cleavage-dependent luciferase gene correction as part of a transient dual-luciferase(®) reporter (Promega) expression system. This assay consists of co-infiltrating Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains: one contains the target sequence embedded within a luciferase reporter gene and the second strain contains the custom-designed nuclease gene(s). We compared repair following site-specific nuclease digestion through non-homologous DNA end-joining, as opposed to single strand DNA annealing, as a means to restore an out-of-frame luciferase gene cleavage-reporter construct. We show, using luminometer measurements and bioluminescence imaging, that the assay for non-homologous end-joining is sensitive, quantitative, reproducible and rapid in estimating custom-designed nucleases' cleavage efficiency. We detected cleavage by two out of three transcription activator-like effector nucleases that we custom-designed for targets in the Arabidopsis CRUCIFERIN3 gene, and we compared with the well-established 'QQR' zinc-finger nuclease. The assay we report requires only standard equipment and basic plant molecular biology techniques, and it can be carried out within a few days. Different types of custom-designed nucleases can be preliminarily tested in our assay system before their downstream application in plant genome editing.

  16. Label-free fluorometric detection of S1 nuclease activity by using polycytosine oligonucleotide-templated silver nanoclusters.

    PubMed

    Wang, Lihui; Ma, Keke; Zhang, Yaodong

    2015-01-01

    S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of S1 nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC12-Ag NCs). In this assay, dC12 can function as both the template for the stabilization of Ag NCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without S1 nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5×10(-8)Uμl(-1) (signal/noise=3) can be determined with a linear range of 5×10(-7) to 1×10(-3)Uμl(-1). The promising application of the proposed method in S1 nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the S1 nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. DNA oxidation profiles of copper phenanthrene chemical nucleases

    PubMed Central

    Molphy, Zara; Slator, Creina; Chatgilialoglu, Chryssostomos; Kellett, Andrew

    2015-01-01

    The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, aging, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids—as demonstrated by metal-activated bleomycin—has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)]2+ (Cu-DPQ-Phen), [Cu(DPPZ)(phen)]2+ (Cu-DPPZ-Phen), and [{Cu(phen)2}2(μ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman's reagent [Cu(phen)2]2+ throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine; Terph = terephthalate). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilizers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals (•OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2′-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited—particularly along A-T rich chains—through oxidative damage of template strands. PMID:25954741

  18. Compact dimension of denatured states of staphylococcal nuclease.

    PubMed

    Chow, C-Y; Wu, Ming-Chya; Fang, Huey-Jen; Hu, Chin-Kun; Chen, Hueih-Min; Tsong, Tian-Yow

    2008-08-15

    Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.

  19. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

    PubMed

    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

    2015-07-23

    Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

  20. Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.

    PubMed Central

    Xie, D.; Fox, R.; Freire, E.

    1994-01-01

    High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy. PMID:7756977

  1. DNA Oxidation Profiles of Copper Phenanthrene Chemical Nucleases

    NASA Astrophysics Data System (ADS)

    Molphy, Zara; Slator, Creina; Chatgilialoglu, Chryssostomos; Kellett, Andrew

    2015-04-01

    The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids - as demonstrated by metal-activated bleomycin - has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)]2+ (Cu-DPQ-Phen), [Cu(DPPZ)(phen)]2+ (Cu-DPPZ-Phen), and [{Cu(phen)2}2(μ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman’s reagent [Cu(phen)2]2+ throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilisers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals (•OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2'-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited - particularly along A-T rich chains - through oxidative damage of the template strands.

  2. Cloning and sequencing of the genes encoding the large and small subunits of the periplasmic (NiFeSe) hydrogenase of Desulfovibrio baculatus

    SciTech Connect

    Menon, N.K.; Peck, H.D. Jr.; Le Gall, J.; Przybyla, A.E.

    1987-12-01

    The genes coding for the large and small subunits of the periplasmic hydrogenase from Desulfovibrio baculatus have been cloned and sequenced. The genes are arranged in an operon with the small subunit gene preceding the large subunit gene. The small subunit gene codes for a 32 amino acid leader sequence supporting the periplasmic localization of the protein, however no ferredoxin-like or other characteristic iron-sulfur coordination sites were observed. The periplasmic hydrogenases from D. baculatus (an NiFeSe protein) and D. vulgaris (an Fe protein) exhibit no homology suggesting that they are structurally different, unrelated entities.

  3. Cloning and sequencing of the genes encoding the large and small subunits of the periplasmic (NiFeSe) hydrogenase of Desulfovibrio baculatus.

    PubMed Central

    Menon, N K; Peck, H D; Gall, J L; Przybyla, A E

    1987-01-01

    The genes coding for the large and small subunits of the periplasmic hydrogenase from Desulfovibrio baculatus have been cloned and sequenced. The genes are arranged in an operon with the small subunit gene preceding the large subunit gene. The small subunit gene codes for a 32 amino acid leader sequence supporting the periplasmic localization of the protein, however no ferredoxin-like or other characteristic iron-sulfur coordination sites were observed. The periplasmic hydrogenases from D. baculatus (an NiFeSe protein) and D. vulgaris (an Fe protein) exhibit no homology suggesting that they are structurally different, unrelated entities. Images PMID:3316183

  4. High-frequency homologous recombination in plants mediated by zinc-finger nucleases.

    PubMed

    Wright, David A; Townsend, Jeffrey A; Winfrey, Ronnie Joe; Irwin, Phillip A; Rajagopal, Jyothi; Lonosky, Patricia M; Hall, Bradford D; Jondle, Michael D; Voytas, Daniel F

    2005-11-01

    Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.

  5. Chemical Biology Approaches to Genome Editing: Understanding, Controlling, and Delivering Programmable Nucleases.

    PubMed

    Hu, Johnny H; Davis, Kevin M; Liu, David R

    2016-01-21

    Programmable DNA nucleases have provided scientists with the unprecedented ability to probe, regulate, and manipulate the human genome. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat-Cas9 system (CRISPR-Cas9) represent a powerful array of tools that can bind to and cleave a specified DNA sequence. In their canonical forms, these nucleases induce double-strand breaks at a DNA locus of interest that can trigger cellular DNA repair processes that disrupt or replace genes. The fusion of these programmable nucleases with a variety of other protein domains has led to a rapidly growing suite of tools for activating, repressing, visualizing, and modifying loci of interest. Maximizing the usefulness and therapeutic relevance of these tools, however, requires precisely controlling their activity and specificity to minimize potentially toxic side effects arising from off-target activities. This need has motivated the application of chemical biology principles and methods to genome-editing proteins, including the engineering of variants of these proteins with improved or altered specificities, and the development of genetic, chemical, optical, and protein delivery methods that control the activity of these agents in cells. Advancing the capabilities, safety, effectiveness, and therapeutic relevance of genome-engineering proteins will continue to rely on chemical biology strategies that manipulate their activity, specificity, and localization.

  6. Sequence specific cleavage of African green monkey alpha-satellite DNA by micrococcal nuclease.

    PubMed

    Hörz, W; Fittler, F; Zachau, H G

    1983-07-11

    The sequence specificity of micrococcal nuclease complicates its use in experiments addressed to the still controversial issue of nucleosome phasing. In the case of alpha-satellite DNA containing chromatin from African green monkey (AGM) cells cleavage by micrococcal nuclease in the nucleus was reported to occur predominantly at only one location around position 126 of the satellite repeat unit (Musich et al. (1982) Proc. Natl. Acad. Sci. USA 79, 118-122). DNA control experiments conducted in the same study indicated the presence of many preferential cleavage sites for micrococcal nuclease on the 172 bp long alpha-satellite repeat unit. This difference was taken as evidence for a direct and simple phase relationship between the alpha-satellite DNA sequence and the position of the nucleosomes on the DNA. We have quantitatively analyzed the digestion products of the protein-free satellite monomer with micrococcal nuclease and found that 50% of all cuts occur at positions 123 and 132, 5% at position 79, and to a level of 1-3% at about 20 other positions. We also digested high molecular weight alpha-satellite DNA from AGM nuclei with micrococcal nuclease. Again cleavage occurred mostly at positions 123 and 132 of the satellite repeat unit. Thus digestion of free DNA yields results very similar to those reported by Musich et al. for the digestion of chromatin. Therefore no conclusions on a possible phase relationship can be drawn from the chromatin digestion experiments.

  7. Quantification of designer nuclease induced mutation rates: a direct comparison of different methods

    PubMed Central

    Ehrke-Schulz, Eric; Bergmann, Thorsten; Schiwon, Maren; Doerner, Johannes; Saydaminova, Kamola; Lieber, Andre; Ehrhardt, Anja

    2016-01-01

    Designer nucleases are broadly applied to induce site-specific DNA double-strand breaks (DSB) in genomic DNA. These are repaired by nonhomologous end joining leading to insertions or deletions (in/dels) at the respective DNA-locus. To detect in/del mutations, the heteroduplex based T7-endonuclease I -assay is widely used. However, it only provides semi-quantitative evidence regarding the number of mutated alleles. Here we compared T7-endonuclease I- and heteroduplex mobility assays, with a quantitative polymerase chain reaction mutation detection method. A zinc finger nuclease pair specific for the human adeno-associated virus integration site 1 (AAVS1), a transcription activator-like effector nuclease pair specific for the human DMD gene, and a zinc finger nuclease- and a transcription activator-like effector nuclease pair specific for the human CCR5 gene were explored. We found that the heteroduplex mobility assays and T7-endonuclease I - assays detected mutations but the relative number of mutated cells/alleles can only be estimated. In contrast, the quantitative polymerase chain reaction based method provided quantitative results which allow calculating mutation and homologous recombination rates in different eukaryotic cell types including human peripheral blood mononuclear cells. In conclusion, our quantitative polymerase chain reaction based mutation detection method expands the array of methods for in/del mutation detection and facilitates quantification of introduced in/del mutations for a genomic locus containing a mixture of mutated and unmutated DNA. PMID:27419195

  8. Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases

    PubMed Central

    Fonfara, Ines; Curth, Ute; Pingoud, Alfred; Wende, Wolfgang

    2012-01-01

    Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease. PMID:21965534

  9. Purification and identification of a nuclease activity in embryo axes from French bean.

    PubMed

    Lambert, Rocío; Quiles, Francisco Antonio; Cabello-Díaz, Juan Miguel; Piedras, Pedro

    2014-07-01

    Plant nucleases are involved in nucleic acid degradation associated to programmed cell death processes as well as in DNA restriction, repair and recombination processes. However, the knowledge about the function of plant nucleases is limited. A major nuclease activity was detected by in-gel assay with whole embryonic axes of common bean by using ssDNA or RNA as substrate, whereas this activity was minimal in cotyledons. The enzyme has been purified to electrophoretic homogeneity from embryonic axes. The main biochemical properties of the purified enzyme indicate that it belongs to the S1/P1 family of nucleases. This was corroborated when this protein, after SDS-electrophoresis, was excised from the gel and further analysis by MALDI TOF/TOF allowed identification of the gene (PVN1) that codes this protein. The gene that codes the purified protein was identified. The expression of PVN1 gene was induced at the specific moment of radicle protrusion. The inclusion of inorganic phosphate to the imbibition media reduced the level of expression of this gene and the nuclease activity suggesting a relationship with the phosphorous status in French bean seedlings.

  10. Nuclease-mediated genome editing: At the front-line of functional genomics technology.

    PubMed

    Sakuma, Tetsushi; Woltjen, Knut

    2014-01-01

    Genome editing with engineered endonucleases is rapidly becoming a staple method in developmental biology studies. Engineered nucleases permit random or designed genomic modification at precise loci through the stimulation of endogenous double-strand break repair. Homology-directed repair following targeted DNA damage is mediated by co-introduction of a custom repair template, allowing the derivation of knock-out and knock-in alleles in animal models previously refractory to classic gene targeting procedures. Currently there are three main types of customizable site-specific nucleases delineated by the source mechanism of DNA binding that guides nuclease activity to a genomic target: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR). Among these genome engineering tools, characteristics such as the ease of design and construction, mechanism of inducing DNA damage, and DNA sequence specificity all differ, making their application complementary. By understanding the advantages and disadvantages of each method, one may make the best choice for their particular purpose.

  11. Periplasmic Cytophaga hutchinsonii Endoglucanases Are Required for Use of Crystalline Cellulose as the Sole Source of Carbon and Energy

    PubMed Central

    Zhu, Yongtao; Han, Lanlan; Hefferon, Kathleen L.; Silvaggi, Nicholas R.; Wilson, David B.

    2016-01-01

    ABSTRACT The soil bacterium Cytophaga hutchinsonii actively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes: cel5A, cel5B, cel5C, cel9A, cel9B, cel9C, cel9E, and cel9F. Each mutant retained the ability to digest crystalline cellulose, although the deletion of cel9C caused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking both cel5B and cel9C were completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth of C. hutchinsonii on cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane. IMPORTANCE The bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by

  12. Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans.

    PubMed

    Hatchikian, C E; Traore, A S; Fernandez, V M; Cammack, R

    1990-02-14

    The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-1 cm-1, respectively. D. fructosovorans hydrogenase contains 11 +/- 1 iron atoms, 0.9 +/- 0.15 nickel atom and 12 +/- 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.

  13. Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond.

    PubMed

    Zückert, Wolfram R

    2014-08-01

    Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

  14. In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis.

    PubMed Central

    Nickoloff, J A; Singer, J D; Heffron, F

    1990-01-01

    HO nuclease introduces a specific double-strand break in the mating-type locus (MAT) of Saccharomyces cerevisiae, initiating mating-type interconversion. To define the sequence recognized by HO nuclease, random mutations were produced in a 30-base-pair region homologous to either MAT alpha or MATa by a chemical synthesis procedure. The mutant sites were introduced into S. cerevisiae on a shuttle vector and tested for the ability to stimulate recombination in an assay that mimics mating-type interconversion. The results suggest that a core of 8 noncontiguous bases near the Y-Z junction of MAT is essential for HO nuclease to bind and cleave its recognition site. Other contacts must be required because substrates that contain several mutations outside an intact core reduce or eliminate cleavage in vivo. The results show that HO site recognition is a complex phenomenon, similar to promoter-polymerase interactions. Images PMID:2406563

  15. Multiplexed Targeted Genome Engineering Using a Universal Nuclease-Assisted Vector Integration System.

    PubMed

    Brown, Alexander; Woods, Wendy S; Perez-Pinera, Pablo

    2016-07-15

    Engineered nucleases are capable of efficiently modifying complex genomes through introduction of targeted double-strand breaks. However, mammalian genome engineering remains limited by low efficiency of heterologous DNA integration at target sites, which is typically performed through homologous recombination, a complex, ineffective and costly process. In this study, we developed a multiplexable and universal nuclease-assisted vector integration system for rapid generation of gene knock outs using selection that does not require customized targeting vectors, thereby minimizing the cost and time frame needed for gene editing. Importantly, this system is capable of remodeling native mammalian genomes through integration of DNA, up to 50 kb, enabling rapid generation and screening of multigene knockouts from a single transfection. These results support that nuclease assisted vector integration is a robust tool for genome-scale gene editing that will facilitate diverse applications in synthetic biology and gene therapy.

  16. EssD, a Nuclease Effector of the Staphylococcus aureus ESS Pathway.

    PubMed

    Ohr, Ryan Jay; Anderson, Mark; Shi, Miaomiao; Schneewind, Olaf; Missiakas, Dominique

    2017-01-01

    Specialized secretion systems of bacteria evolved for selective advantage, either killing microbial competitors or implementing effector functions during parasitism. Earlier work characterized the ESAT-6 secretion system (ESS) of Staphylococcus aureus and demonstrated its contribution to persistent staphylococcal infection of vertebrate hosts. Here, we identify a novel secreted effector of the ESS pathway, EssD, that functions as a nuclease and cleaves DNA but not RNA. EssI, a protein of the DUF600 family, binds EssD to block its nuclease activity in the staphylococcal cytoplasm. An essD knockout mutant or a variant lacking nuclease activity, essD(L546P), elicited a diminished interleukin-12 (IL-12) cytokine response following bloodstream infection of mice, suggesting that the effector function of EssD stimulates immune signaling to support the pathogenesis of S. aureus infections.

  17. Cellular Site in Bacillus subtilis of a Nuclease Which Preferentially Degrades Single-Stranded Nucleic Acids

    PubMed Central

    Birnboim, H. C.

    1966-01-01

    Birnboim, H. C. (Albert Einstein College of Medicine, New York, N.Y.). Cellular site in Bacillus subtilis of a nuclease which preferentially degrades single-stranded nucleic acids. J. Bacteriol. 91:1004–1011. 1966.—A nuclease, identified by a marked preference for single-stranded nucleic acids, has been demonstrated in extracts of Bacillus subtilis. The enzyme was associated with the cell wall-membrane fraction of mechanically disrupted cells and was released from cells which had been converted to protoplasts by lysozyme. The nuclease activity prepared by the latter procedure was found to be activated and solubilized by treatment with trypsin. The enzyme had about 2% activity on native deoxyribonucleic acid (DNA) as compared with denatured DNA. By use of CsCl analytical density gradient ultracentrifugation, this preparation was shown to degrade denatured DNA selectively in mixtures of native and denatured DNA. PMID:4956329

  18. Siderophore production of a periplasmic transport binding protein kinase gene defective mutant of Magnetospirillum magneticum AMB-1.

    PubMed

    Calugay, Ronie J; Okamura, Yoshiko; Wahyudi, Aris Tri; Takeyama, Haruko; Matsunaga, Tadashi

    2004-10-22

    A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis. The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed. The transposon was inserted in an open reading frame (ORF) coding for a periplasmic transport binding protein kinase gene homologue. Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon. Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotriacetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore. Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type. These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M. magneticum AMB-1.

  19. Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta.

    PubMed

    Johnson, Ross A; Gurevich, Vyacheslav; Filler, Shdema; Samach, Aviva; Levy, Avraham A

    2015-01-01

    Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.

  20. Hepatopancreatic nuclease of black tiger shrimp Penaeus monodon unlikely to be involved in viral triggered apoptosis.

    PubMed

    Molthathong, Sudkhate; Rojtinnakorn, Jiraporn; Senapin, Saengchan; Flegel, Timothy W

    2007-06-01

    Nucleases are phosphodiesterases that hydrolyze DNA and/or RNA. In a search for shrimp nucleases involved in apoptosis, we discovered a nuclease from hepatopancreatic cDNA of the black tiger shrimp Penaeus monodon. The full-length nuclease gene was amplified and revealed to contain 1668bp corresponding to 381 deduced amino acid residues in the mature enzyme. Sequence analysis indicated 83% nucleic acid identity and 89% amino acid identity to a nuclease from the Kuruma shrimp Penaeus japonicus (also called Marsupenaeus japonicus). Comparative analysis of sequences, conserved motifs and phylogenetic trees indicated that P. monodon nuclease (PMN) belonged to the family of DNA/RNA non-specific endonucleases (DRNSN). RT-PCR analysis using primers specific for PMN mRNA with seven different shrimp tissues revealed that expression in normal shrimp was restricted to the hepatopancreas. Semiquantitative RT-PCR analysis of PMN using hepatopancreatic mRNA from normal shrimp and from shrimp challenged with white spot syndrome virus (WSSV) indicated significant up-regulation of PMN in the hepatopancreas (P<0.05) at the early stage of viral infection but a return to baseline levels as gross signs of disease developed. At the same time, expression was always confined to the hepatopancreas and never seen in other tissues, including those reported to be prime targets for WSSV and subject to increased levels of apoptosis after infection. The results suggested that PMN is probably a digestive enzyme that is unlikely to be involved in hallmark DNA digestion associated with apoptosis.

  1. Novel Mechanism for Scavenging of Hypochlorite Involving a Periplasmic Methionine-Rich Peptide and Methionine Sulfoxide Reductase

    PubMed Central

    Melnyk, Ryan A.; Youngblut, Matthew D.; Clark, Iain C.; Carlson, Hans K.; Wetmore, Kelly M.; Price, Morgan N.; Iavarone, Anthony T.; Deutschbauer, Adam M.; Arkin, Adam P.

    2015-01-01

    ABSTRACT Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and a putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo. PMID:25968643

  2. High yield purification of nanobodies from the periplasm of E. coli as fusions with the maltose binding protein.

    PubMed

    Salema, Valencio; Fernández, Luis Ángel

    2013-09-01

    Nanobodies (Nbs) are single domain antibodies based on the variable domains of heavy chain only antibodies (HCAbs) found in camelids, also referred to as VHHs. Their small size (ca. 12-15kDa), superior biophysical and antigen binding properties have made Nbs very attractive molecules for multiple biotechnological applications, including human therapy. The most widely used system for the purification of Nbs is their expression in the periplasm of Escherichia coli with a C-terminal hexa-histidine (His6) tag followed by immobilized metal affinity chromatography (IMAC). However, significant variability in the expression levels of different Nbs are routinely observed and a single affinity chromatography step is often not sufficient to obtain Nbs of high purity. Here, we report an alternative method for expression and purification of Nbs from the periplasm of E. coli based on their fusion to maltose binding protein (MBP) in the N-terminus and His6 tag in the C-terminus (MBP-NbHis6). Soluble MBP-NbHis6 fusions were consistently expressed at high levels (⩾12mg/L of induced culture in shake flasks) in the periplasm of E. coli HM140, a strain deficient in several periplasmic proteases. Highly pure MBP-NbHis6 fusions and free NbHis6 (after site specific proteolysis of the fusions), were recovered by amylose and metal affinity chromatography steps. The monomeric nature of the purified NbHis6 was determined by gel filtration chromatography. Lastly, we demonstrated by ELISA that both monomeric NbHis6 and MBP-NbHis6 fusions retained antigen binding activity and specificity, thus facilitating their direct use in antigen recognition assays.

  3. Repellent taxis in response to nickel ion requires neither Ni2+ transport nor the periplasmic NikA binding protein.

    PubMed

    Englert, Derek L; Adase, Christopher A; Jayaraman, Arul; Manson, Michael D

    2010-05-01

    Ni(2+) and Co(2+) are sensed as repellents by the Escherichia coli Tar chemoreceptor. The periplasmic Ni(2+) binding protein, NikA, has been suggested to sense Ni(2+). We show here that neither NikA nor the membrane-bound NikB and NikC proteins of the Ni(2+) transport system are required for repellent taxis in response to Ni(2+).

  4. Structure-Based Design of a Periplasmic Binding Protein Antagonist that Prevents Domain Closure

    SciTech Connect

    Borrok, M. Jack; Zhu, Yimin; Forest, Katrina T.; Kiessling, Laura L.

    2009-07-31

    Many receptors undergo ligand-induced conformational changes to initiate signal transduction. Periplasmic binding proteins (PBPs) are bacterial receptors that exhibit dramatic conformational changes upon ligand binding. These proteins mediate a wide variety of fundamental processes including transport, chemotaxis, and quorum sensing. Despite the importance of these receptors, no PBP antagonists have been identified and characterized. In this study, we identify 3-O-methyl-D-glucose as an antagonist of glucose/galactose-binding protein and demonstrate that it inhibits glucose chemotaxis in E. coli. Using small-angle X-ray scattering and X-ray crystallography, we show that this antagonist acts as a wedge. It prevents the large-scale domain closure that gives rise to the active signaling state. Guided by these results and the structures of open and closed glucose/galactose-binding protein, we designed and synthesized an antagonist composed of two linked glucose residues. These findings provide a blueprint for the design of new bacterial PBP inhibitors. Given the key role of PBPs in microbial physiology, we anticipate that PBP antagonists will have widespread uses as probes and antimicrobial agents.

  5. The periplasmic protein TolB as a potential drug target in Pseudomonas aeruginosa.

    PubMed

    Lo Sciuto, Alessandra; Fernández-Piñar, Regina; Bertuccini, Lucia; Iosi, Francesca; Superti, Fabiana; Imperi, Francesco

    2014-01-01

    The Gram-negative bacterium Pseudomonas aeruginosa is one of the most dreaded pathogens in the hospital setting, and represents a prototype of multi-drug resistant "superbug" for which effective therapeutic options are very limited. The identification and characterization of new cellular functions that are essential for P. aeruginosa viability and/or virulence could drive the development of anti-Pseudomonas compounds with novel mechanisms of action. In this study we investigated whether TolB, the periplasmic component of the Tol-Pal trans-envelope protein complex of Gram-negative bacteria, represents a potential drug target in P. aeruginosa. By combining conditional mutagenesis with the analysis of specific pathogenicity-related phenotypes, we demonstrated that TolB is essential for P. aeruginosa growth, both in laboratory and clinical strains, and that TolB-depleted P. aeruginosa cells are strongly defective in cell-envelope integrity, resistance to human serum and several antibiotics, as well as in the ability to cause infection and persist in an insect model of P. aeruginosa infection. The essentiality of TolB for P. aeruginosa growth, resistance and pathogenicity highlights the potential of TolB as a novel molecular target for anti-P. aeruginosa drug discovery.

  6. Architecture and roles of periplasmic adaptor proteins in tripartite efflux assemblies.

    PubMed

    Symmons, Martyn F; Marshall, Robert L; Bavro, Vassiliy N

    2015-01-01

    Recent years have seen major advances in the structural understanding of the different components of tripartite efflux assemblies, which encompass the multidrug efflux (MDR) pumps and type I secretion systems. The majority of these investigations have focused on the role played by the inner membrane transporters and the outer membrane factor (OMF), leaving the third component of the system - the Periplasmic Adaptor Proteins (PAPs) - relatively understudied. Here we review the current state of knowledge of these versatile proteins which, far from being passive linkers between the OMF and the transporter, emerge as active architects of tripartite assemblies, and play diverse roles in the transport process. Recognition between the PAPs and OMFs is essential for pump assembly and function, and targeting this interaction may provide a novel avenue for combating multidrug resistance. With the recent advances elucidating the drug efflux and energetics of the tripartite assemblies, the understanding of the interaction between the OMFs and PAPs is the last piece remaining in the complete structure of the tripartite pump assembly puzzle.

  7. Architecture and roles of periplasmic adaptor proteins in tripartite efflux assemblies

    PubMed Central

    Symmons, Martyn F.; Marshall, Robert L.

    2015-01-01

    Recent years have seen major advances in the structural understanding of the different components of tripartite efflux assemblies, which encompass the multidrug efflux (MDR) pumps and type I secretion systems. The majority of these investigations have focused on the role played by the inner membrane transporters and the outer membrane factor (OMF), leaving the third component of the system – the Periplasmic Adaptor Proteins (PAPs) – relatively understudied. Here we review the current state of knowledge of these versatile proteins which, far from being passive linkers between the OMF and the transporter, emerge as active architects of tripartite assemblies, and play diverse roles in the transport process. Recognition between the PAPs and OMFs is essential for pump assembly and function, and targeting this interaction may provide a novel avenue for combating multidrug resistance. With the recent advances elucidating the drug efflux and energetics of the tripartite assemblies, the understanding of the interaction between the OMFs and PAPs is the last piece remaining in the complete structure of the tripartite pump assembly puzzle. PMID:26074901

  8. TonB interacts with BtuF, the Escherichia coli periplasmic binding protein for cyanocobalamin.

    PubMed

    James, Karron J; Hancock, Mark A; Gagnon, Jean-Nicolas; Coulton, James W

    2009-10-06

    By its direct contact with outer membrane receptor BtuB, the cytoplasmic membrane transducer TonB delivers energy that mediates cyanocobalamin uptake in Escherichia coli. This activity has been generally proposed to be the role of TonB in cyanocobalamin uptake. We now report the discovery and characterization of interactions between TonB and periplasmic binding protein BtuF. Phage display experiments predicted interaction between TonB and BtuF, identifying potential binding residues on each protein. Dynamic light scattering experiments measured a complex of 55 kDa, consistent with a TonB-BtuF heterodimer. The hydrodynamic radius of the complex was unchanged in the presence of cyanocobalamin. Surface plasmon resonance measured TonB-BtuF interaction kinetics that were independent of cyanocobalamin and that deviated from a simple binding model. Binding isotherms from intrinsic fluorescence suggested a multifaceted interaction that was independent of cyanocobalamin. In addition, the presence of TonB did not abrogate subsequent binding of cyanocobalamin by BtuF. Taken together, these data support a previously proposed model wherein TonB serves as a scaffold to optimally position BtuF for initial binding of cyanocobalamin and for its subsequent release. These results substantiate a diverse role for TonB with its multiple protein-protein interactions in bacterial nutrient uptake systems.

  9. Periplasmic arabinogalactan glycoproteins act as a calcium capacitor that regulates plant growth and development.

    PubMed

    Lamport, Derek T A; Várnai, Péter

    2013-01-01

    Arabinogalactan glycoproteins (AGPs) are implicated in virtually all aspects of plant growth and development, yet their precise role remains unknown. Classical AGPs cover the plasma membrane and are highly glycosylated by numerous acidic arabinogalactan polysaccharides O-linked to hydroxyproline. Their heterogeneity and complexity hindered a structural approach until the recent determination of a highly conserved repetitive consensus structure for a 15-sugar residue arabinogalactan subunit with paired glucuronic carboxyls. Based on NMR data and molecular dynamics simulations, we identify these carboxyls as potential intramolecular Ca(2+)-binding sites. Using rapid ultrafiltration assays and mass spectrometry, we verified that AGPs bind Ca(2+) tightly (K(d) ~ 6.5 μM) and stoichiometrically at pH 5. Ca(2+) binding is reversible in a pH-dependent manner. As typical AGPs contain c. 30 Ca(2+)-binding subunits and are bulk components of the periplasm, they represent a Ca(2+) capacitor discharged at low pH by stretch-activated plasma membrane H(+)-ATPases, hence a substantial source of cytosolic Ca(2+). We propose that these Ca(2+) waves prime the 'calcium oscillator', a signal generator essential to the global Ca(2+) signalling pathway of green plants. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  10. Structural and functional homology between periplasmic bacterial molecular chaperones and small heat shock proteins.

    PubMed

    Zav'yalov, V P; Zav'yalova, G A; Denesyuk, A I; Gaestel, M; Korpela, T

    1995-07-01

    The periplasmic Yersinia pestis molecular chaperone Caf1M belongs to a superfamily of bacterial proteins for one of which (PapD protein of Escherichia coli) the immunoglobulin-like fold was solved by X-ray analysis. The N-terminal domain of Caf1M was found to share a 20% amino acid sequence identity with an inclusion body-associated protein IbpB of Escherichia coli. One of the regions that was compared, was 32 amino acids long, and displayed more than 40% identity, probability of random coincidence was 1.2 x 10(-4). IbpB is involved in a superfamily of small heat shock proteins which fulfil the function of molecular chaperone. On the basis of the revealed homology, an immunoglobulin-like one-domain model of IbpB three-dimensional structure was designed which could be a prototype conformation of sHsp's. The structure suggested is in good agreement with the known experimental data obtained for different members of sHsp's superfamily.

  11. Selenite reduction by Shewanella oneidensis MR-1 is mediated by fumarate reductase in periplasm

    PubMed Central

    Li, Dao-Bo; Cheng, Yuan-Yuan; Wu, Chao; Li, Wen-Wei; Li, Na; Yang, Zong-Chuang; Tong, Zhong-Hua; Yu, Han-Qing

    2014-01-01

    In situ reduction of selenite to elemental selenium (Se(0)), by microorganisms in sediments and soils is an important process and greatly affects the environmental distribution and the biological effects of selenium. However, the mechanism behind such a biological process remains unrevealed yet. Here we use Shewanella oneidensis MR-1, a widely-distributed dissimilatory metal-reducing bacterium with a powerful and diverse respiration capability, to evaluate the involvement of anaerobic respiration system in the microbial selenite reduction. With mutants analysis, we identify fumarate reductase FccA as the terminal reductase of selenite in periplasm. Moreover, we find that such a reduction is dependent on central respiration c-type cytochrome CymA. In contrast, nitrate reductase, nitrite reductase, and the Mtr electron transfer pathway do not work as selenite reductases. These findings reveal a previously unrecognized role of anaerobic respiration reductases of S. oneidensis MR-1 in selenite reduction and geochemical cycles of selenium in sediments and soils. PMID:24435070

  12. Chlamydia trachomatis YtgA is an iron-binding periplasmic protein induced by iron restriction

    PubMed Central

    Miller, J. D.; Sal, M. S.; Schell, M.; Whittimore, J. D.; Raulston, J. E.

    2009-01-01

    Chlamydia trachomatis is a Gram-negative obligate intracellular bacterium that is the causative agent of common sexually transmitted diseases and the leading cause of preventable blindness worldwide. It has been observed that YtgA (CT067) is very immunogenic in patients with chlamydial genital infections. Homology analyses suggested that YtgA is a soluble periplasmic protein and a component of an ATP-binding cassette (ABC) transport system for metals such as iron. Since little is known about iron transport in C. trachomatis, biochemical assays were used to determine the potential role of YtgA in iron acquisition. 59Fe binding and competition studies revealed that YtgA preferentially binds iron over nickel, zinc or manganese. Western blot and densitometry techniques showed that YtgA concentrations specifically increased 3–5-fold in C. trachomatis, when cultured under iron-starvation conditions rather than under general stress conditions, such as exposure to penicillin. Finally, immuno-transmission electron microscopy provided evidence that YtgA is more concentrated in C. trachomatis during iron restriction, supporting a possible role for YtgA as a component of an ABC transporter. PMID:19556290

  13. Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli.

    PubMed Central

    Silhavy, T J; Hartig-Beecken, I; Boos, W

    1976-01-01

    Two-dimensional gel electrophoresis of shock fluids of Escherichia coli K-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (GLPT) that is under the regulation of glpR, the regulatory gene of the glp regulon. Mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce GLPT or produced it in reduced amounts. Other mutations exhibited no apparent effect of GLPT. Transductions of glpT+ nalA phage P1 into these mutants and selection for growth on sn-glycerol-3-phosphate revealed a 50% cotransduction frequency to nalA. Reversion of mutants taht did not produce GLPT to growth on sn-glycerol-3-phosphate resulted in strains that produce GLPT. This suggests a close relationship of GLPT to the glpT gene and to sn-glycerol-3-phosphate transport. Attempts to demonstrate binding activity of GLPT in crude shock fluid towards sn-glycerol-3-phosphate have failed so far. However, all shock fluids, independent of their GLPT content, exhibited an enzymatic activity that hydrolyzes under the conditions of the binding assay, 30 to 60% of the sn-glycerol-3-phosphate to glycerol and inorganic orthophosphate. Images PMID:770459

  14. Domain dislocation: a change of core structure in periplasmic binding proteins in their evolutionary history.

    PubMed

    Fukami-Kobayashi, K; Tateno, Y; Nishikawa, K

    1999-02-12

    Periplasmic binding proteins (PBPs) serve as receptors for various water-soluble ligands in ATP-binding cassette (ABC) transport systems, and form one of the largest protein families in eubacterial and archaebacterial genomes. They are considered to be derived from a common ancestor, judging from their similarities of three-dimensional structure, their mechanism of ligand binding and the operon structure of their genes. Nevertheless, there are two types of topological arrangements of the central beta-sheets in their core structures. It follows that there must have been differentiation in the core structure, which we call "domain dislocation", in the course of evolution of the PBP family. To find a clue as to when the domain dislocation occurred, we constructed phylogenetic trees for PBPs based on their amino acid sequences and three-dimensional structures, respectively. The trees show that the proteins of each type clearly cluster together, strongly indicating that the change in the core structure occurred only once in the evolution of PBPs. We also constructed a phylogenetic tree for the ABC proteins that are encoded by the same operon of their partner PBP, and obtained the same result. Based on the phylogenetic relationship and comparison of the topological arrangements of PBPs, we obtained a reasonable genealogical chart of structural changes in the PBP family. The present analysis shows that the unidirectional change of protein evolution is clearly deduced at the level of protein three-dimensional structure rather than the level of amino acid sequence.

  15. Role of anionic charges of periplasmic glucans of Shigella flexneri in overcoming detergent stress.

    PubMed

    Bhagwat, Arvind A; Leow, Yi Ning; Liu, Liu; Dharne, Mahesh; Kannan, Porteen

    2012-07-01

    Osmoregulated periplasmic glucans (OPGs) are synthesized by the members of the family Enterobacteriaceae when grown under low osmotic growth conditions. Enteropathogens such as Shigella flexneri spend considerable time outside the host environment such as irrigation waters where low nutrient low osmolarity conditions normally may exist. We recently demonstrated that OPGs of S. flexneri are required for optimal growth under low osmolarity low nutrient conditions. Based on homology of the OPG biosynthesis genes to those of Escherichia coli, the presumptive function of opgC and opgB genes is to add succinate and phosphoglycerol residues respectively on OPGs, rendering them anionic. Using lambda-red recombination procedure, we constructed opgB, opgC, and opgBC mutants of S. flexneri. The mutant strain defective in opgC and opgB genes synthesized neutral OPGs. The OPGs without any anionic charges were beneficial for the organism's growth in hypo-osmotic media. However, with the loss of anionic charges from OPGs, mutants were compromised in their ability to combat stress caused by anionic detergents in hypo-osmotic growth conditions. Cloned wild-type genes opgB, opgC, and opgBC, when mobilized to respective opg mutants, simultaneously restored anionic charges to OPGs and tolerance to detergents. The data indicate that anionic charges on the OPGs contribute towards overcoming the stress caused by anionic detergents such as sodium dodecyl sulfate and sodium deoxycholate.

  16. The Treponema denticola Major Sheath Protein Is Predominantly Periplasmic and Has Only Limited Surface Exposure

    PubMed Central

    Caimano, Melissa J.; Bourell, Kenneth W.; Bannister, Teresa D.; Cox, David L.; Radolf, Justin D.

    1999-01-01

    The recent discovery that the Treponema pallidum genome encodes 12 orthologs of the Treponema denticola major sheath protein (Msp) prompted us to reexamine the cellular location and topology of the T. denticola polypeptide. Experiments initially were conducted to ascertain whether Msp forms an array on or within the T. denticola outer membrane. Transmission electron microscopy (EM) of negatively stained and ultrathin-sectioned organisms failed to identify a typical surface layer, whereas freeze-fracture EM revealed that the T. denticola outer membrane contains heterogeneous transmembrane proteins but no array. In contrast, a lattice-like structure was observed in vesicles released from mildly sonicated treponemes; combined EM and biochemical analyses demonstrated that this structure was the peptidoglycan sacculus. Immunoelectron microscopy (IEM) subsequently was performed to localize Msp in T. denticola. Examination of negatively stained whole mounts identified substantial amounts of Msp in sonicated organisms. IEM of ultrathin-sectioned, intact treponemes also demonstrated that the preponderance of antigen was unassociated with the outer membrane. Lastly, immunofluorescence analysis of treponemes embedded in agarose gel microdroplets revealed that only minor portions of Msp are surface exposed. Taken as a whole, our findings challenge the widely held belief that Msp forms an array within the T. denticola outer membrane and demonstrate, instead, that it is predominantly periplasmic with only limited surface exposure. These findings also have implications for our evolving understanding of the contribution(s) of Msp/Tpr orthologs to treponemal physiology and disease pathogenesis. PMID:10417176

  17. A novel periplasmic protein (Slr0280) tunes photomixotrophic growth of the cyanobacterium, Synechocystis sp. PCC 6803.

    PubMed

    Dong, Liang-Liang; Li, Qing-Dong; Wu, Dong; Sun, Ya-Fang; Zhou, Ming; Zhao, Kai-Hong

    2016-01-10

    Cyanobacteria are among the main contributors to global photosynthesis and show a high degree of metabolic plasticity. Synechocystis sp. PCC 6803 can grow under photoautotrophic, photomixotrophic or photoheterotrophic conditions. We have characterized a novel periplasmic protein (Slr0280) that tunes the photomixotrophic growth of Synechocystis sp. PCC 6803. Slr0280 is a multi-domain protein consisting mainly of β-sheets. Several proteins that interact with Slr0280 were identified via bacterial two-hybrid screening. Slr0280 may interact through its DUF2233 domain with partners that participate in sugar metabolism, thereby coordinating the respective regulations. When slr0280 was deleted, the mutant grew more slowly than wild-type in the presence of glucose, which is ascribed to the down-regulation of glycolysis, glycogen catabolism, oxidative pentose phosphate pathway, Calvin cycle and glucose utilization. A positive regulation of Slr0280 on these sugar catabolic enzymes was confirmed by transcript (qPCR) analyses. Based on these findings, we proposed a speculative model that Slr0280 plays a coordinating regulatory role in sugar metabolism. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria.

    PubMed

    Jen, Freda E-C; Djoko, Karrera Y; Bent, Stephen J; Day, Christopher J; McEwan, Alastair G; Jennings, Michael P

    2015-09-01

    Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrous oxide. In this process, nitrite reductase (AniA), a copper (Cu)-containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite-dependent killing. After random-deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteobacteria species. © FASEB.

  19. An ultrasensitive fluorescence method suitable for quantitative analysis of mung bean nuclease and inhibitor screening in vitro and vivo.

    PubMed

    Peng, Lan; Fan, Jialong; Tong, Chunyi; Xie, Zhenhua; Zhao, Chuan; Liu, Xuanming; Zhu, Yonghua; Liu, Bin

    2016-09-15

    Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5'-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10(-4) to 4×10(-2) with a detection limit of 1×10(-4) unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease in vitro and in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. The application of transcription activator-like effector nucleases for genome editing in C. elegans.

    PubMed

    Yi, Peishan; Li, Wei; Ou, Guangshuo

    2014-08-01

    The nematode Caenorhabditis elegans has been a powerful model system for biomedical research in the past decades, however, the efficient genetic tools are still demanding for gene knockout, knock-in or conditional gene mutations. Transcription activator-like effector nucleases (TALENs) that comprise a sequence-specific DNA-binding domain fused to a FokI nuclease domain facilitate the targeted genome editing in various cell types or organisms. Here we summarize the recent progresses and protocols using TALENs in C. elegans that generate gene mutations and knock-ins in the germ line and the conditional gene knockout in somatic tissues.

  1. GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases.

    PubMed

    Zhu, Lihua Julie; Lawrence, Michael; Gupta, Ankit; Pagès, Hervé; Kucukural, Alper; Garber, Manuel; Wolfe, Scot A

    2017-05-15

    Genome editing technologies developed around the CRISPR-Cas9 nuclease system have facilitated the investigation of a broad range of biological questions. These nucleases also hold tremendous promise for treating a variety of genetic disorders. In the context of their therapeutic application, it is important to identify the spectrum of genomic sequences that are cleaved by a candidate nuclease when programmed with a particular guide RNA, as well as the cleavage efficiency of these sites. Powerful new experimental approaches, such as GUIDE-seq, facilitate the sensitive, unbiased genome-wide detection of nuclease cleavage sites within the genome. Flexible bioinformatics analysis tools for processing GUIDE-seq data are needed. Here, we describe an open source, open development software suite, GUIDEseq, for GUIDE-seq data analysis and annotation as a Bioconductor package in R. The GUIDEseq package provides a flexible platform with more than 60 adjustable parameters for the analysis of datasets associated with custom nuclease applications. These parameters allow data analysis to be tailored to different nuclease platforms with different length and complexity in their guide and PAM recognition sequences or their DNA cleavage position. They also enable users to customize sequence aggregation criteria, and vary peak calling thresholds that can influence the number of potential off-target sites recovered. GUIDEseq also annotates potential off-target sites that overlap with genes based on genome annotation information, as these may be the most important off-target sites for further characterization. In addition, GUIDEseq enables the comparison and visualization of off-target site overlap between different datasets for a rapid comparison of different nuclease configurations or experimental conditions. For each identified off-target, the GUIDEseq package outputs mapped GUIDE-Seq read count as well as cleavage score from a user specified off-target cleavage score prediction

  2. Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G.

    PubMed

    Widlak, Piotr; Garrard, William T

    2005-04-15

    Toward the end of the 20th and beginning of the 21st centuries, clever in vitro biochemical complementation experiments and genetic screens from the laboratories of Xiaodong Wang, Shigekazu Nagata, and Ding Xue led to the discovery of two major apoptotic nucleases, termed DNA fragmentation factor (DFF) or caspase-activated DNase (CAD) and endonuclease G (Endo G). Both endonucleases attack chromatin to yield 3'-hydroxyl groups and 5'-phosphate residues, first at the level of 50-300 kb cleavage products and next at the level of internucleosomal DNA fragmentation, but these nucleases possess completely different cellular locations in normal cells and are regulated in vastly different ways. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45) [also called inhibitor of CAD (ICAD-L)] and a 40 kD latent nuclease subunit (DFF40/CAD). Apoptotic activation of caspase-3 or -7 results in the cleavage of DFF45/ICAD and release of active DFF40/CAD nuclease. DFF40's nuclease activity is further activated by specific chromosomal proteins, such as histone H1, HMGB1/2, and topoisomerase II. DFF is regulated by multiple pre- and post-activation fail-safe steps, which include the requirements for DFF45/ICAD, Hsp70, and Hsp40 proteins to mediate appropriate folding during translation to generate a potentially activatable nuclease, and the synthesis in stoichiometric excess of the inhibitors (DFF45/35; ICAD-S/L). By contrast, Endo G resides in the mitochondrial intermembrane space in normal cells, and is released into the nucleus upon apoptotic disruption of mitochondrial membrane permeability in association with co-activators such as apoptosis-inducing factor (AIF). Understanding further regulatory check-points involved in safeguarding non-apoptotic cells against accidental activation of these nucleases remain as future challenges, as well as designing ways to selectively activate these nucleases in tumor cells.

  3. Analyzing the Role of Periplasmic Folding Factors in the Biogenesis of OMPs and Members of the Type V Secretion System.

    PubMed

    Bodelón, Gustavo; Marín, Elvira; Fernández, Luis Ángel

    2015-01-01

    The outer membrane (OM) of gram-negative bacteria is highly packed with OM proteins (OMPs) and the trafficking and assembly of OMPs in gram-negative bacteria is a subject of intense research. Structurally, OMPs vary in the number of β-strands and in the size and complexity of extra-membrane domains, with extreme examples being the members of the type V protein secretion system (T5SS), such as the autotransporter (AT) and intimin/invasin families of secreted proteins, in which a large extracellular "passenger" domain is linked to a β-barrel that inserts in the OM. Despite their structural and functional diversity, OMPs interact in the periplasm with a relatively small set of protein chaperones that facilitate their transport from the inner membrane (IM) to the β-barrel assembly machinery (BAM complex), preventing aggregation and assisting their folding in various aspects including disulfide bond formation. This chapter is focused on the periplasmic folding factors involved in the biogenesis of integral OMPs and members of T5SS in E. coli, which are used as a model system in this field. Background information on these periplasmic folding factors is provided along with genetic methods to generate conditional mutants that deplete these factors from E. coli and biochemical methods to analyze the folding, surface display, disulfide formation and oligomerization state of OMPs/T5SS in these mutants.

  4. Cytoplasmic and Periplasmic Proteomic Signatures of Exponentially Growing Cells of the Psychrophilic Bacterium Pseudoalteromonas haloplanktis TAC125 ▿ †

    PubMed Central

    Wilmes, Boris; Kock, Holger; Glagla, Susanne; Albrecht, Dirk; Voigt, Birgit; Markert, Stephanie; Gardebrecht, Antje; Bode, Rüdiger; Danchin, Antoine; Feller, Georges; Hecker, Michael; Schweder, Thomas

    2011-01-01

    The psychrophilic model bacterium Pseudoalteromonas haloplanktis is characterized by remarkably fast growth rates under low-temperature conditions in a range from 5°C to 20°C. In this study the proteome of cellular compartments, the cytoplasm and periplasm, of P. haloplanktis strain TAC125 was analyzed under exponential growth conditions at a permissive temperature of 16°C. By means of two-dimensional protein gel electrophoresis and mass spectrometry, a first inventory of the most abundant cytoplasmic and periplasmic proteins expressed in a peptone-supplemented minimal medium was established. By this approach major enzymes of the amino acid catabolism of this marine bacterium could be functionally deduced. The cytoplasmic proteome showed a predominance of amino acid degradation pathways and tricarboxylic acid (TCA) cycle enzymes but also the protein synthesis machinery. Furthermore, high levels of cold acclimation and oxidative stress proteins could be detected at this moderate growth temperature. The periplasmic proteome was characterized by a significant abundance of transporters, especially of highly expressed putative TonB-dependent receptors. This high capacity for protein synthesis, efficient amino acid utilization, and substrate transport may contribute to the fast growth rates of the copiotrophic bacterium P. haloplanktis in its natural environments. PMID:21183643

  5. The crystal structure of the leptospiral hypothetical protein LIC12922 reveals homology with the periplasmic chaperone SurA.

    PubMed

    Giuseppe, Priscila O; Von Atzingen, Marina; Nascimento, Ana Lúcia T O; Zanchin, Nilson I T; Guimarães, Beatriz G

    2011-02-01

    Leptospirosis is a world spread zoonosis caused by members of the genus Leptospira. Although leptospires were identified as the causal agent of leptospirosis almost 100 years ago, little is known about their biology, which hinders the development of new treatment and prevention strategies. One of the several aspects of the leptospiral biology not yet elucidated is the process by which outer membrane proteins (OMPs) traverse the periplasm and are inserted into the outer membrane. The crystal structure determination of the conserved hypothetical protein LIC12922 from Leptospira interrogans revealed a two domain protein homologous to the Escherichia coli periplasmic chaperone SurA. The LIC12922 NC-domain is structurally related to the chaperone modules of E. coli SurA and trigger factor, whereas the parvulin domain is devoid of peptidyl prolyl cis-trans isomerase activity. Phylogenetic analyses suggest a relationship between LIC12922 and the chaperones PrsA, PpiD and SurA. Based on our structural and evolutionary analyses, we postulate that LIC12922 is a periplasmic chaperone involved in OMPs biogenesis in Leptospira spp. Since LIC12922 homologs were identified in all spirochetal genomes sequenced to date, this assumption may have implications for the OMPs biogenesis studies not only in leptospires but in the entire Phylum Spirochaetes. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. The periplasmic regulator ExoR inhibits ExoS/ChvI two-component signaling in Sinorhizobium meliloti

    PubMed Central

    Chen, Esther J.; Sabio, Erich A.; Long, Sharon R.

    2008-01-01

    Sinorhizobium meliloti requires ExoS/ChvI two-component signaling to establish a nitrogen-fixing symbiosis with legume hosts. The importance of ExoS/ChvI signaling in microbe-host interactions is underscored by the requirement of ExoS/ChvI orthologs for virulence of the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In S. meliloti, ExoS/ChvI is a key regulator of gene expression for exopolysaccharide synthesis, biofilm formation, motility, nutrient utilization, and free-living viability. Previously, we showed that the novel conserved regulator ExoR interacts genetically with both ExoS and ChvI, and localizes to the periplasm of S. meliloti. Here, we show that ExoR physically associates with ExoS and that this association is important for regulating ExoS/ChvI signaling. We have identified point mutations in the Sel1-like repeat region of ExoR that disrupt binding to ExoS and cause a dramatic increase in ExoS/ChvI-dependent gene expression. Furthermore, we have found that physical interaction with ExoS stabilizes the ExoR protein. Together, our results indicate that ExoR binds to ExoS in the periplasm of S. meliloti to inhibit ExoS/ChvI activity, and that ExoR represents a novel periplasmic inhibitor of two-component signaling. PMID:18631237

  7. A novel method for assessment of local pH in periplasmic space and of cell surface potential in yeast.

    PubMed

    Plášek, Jaromír; Babuka, David; Gášková, Dana; Jančíková, Iva; Zahumenský, Jakub; Hoefer, Milan

    2017-06-01

    Yeast cells exhibit a negative surface potential due to negative charges at the cell membrane surface. Consequently, local concentrations of cations at the periplasmic membrane surface may be significantly increased compared to their bulk environment. However, in cell suspensions only bulk concentrations of cations can be measured directly. Here we present a novel method enabling the assessment of local pH at the periplasmic membrane surface which can be directly related to the underlying cell surface potential. In this proof of concept study using Saccharomyces cerevisiae cells with episomally expressed pH reporter, pHluorin, intracellular acidification induced by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was measured using synchronously scanned fluorescence spectroscopy (SSF). The analysis of titration curves revealed that the pH at the periplasmic surface of S. cerevisiae cells was about two units lower than the pH of bulk medium. This pH difference was significantly decreased by increasing the ionic strength of the bulk medium. The cell surface potential was estimated to amount to -130 mV. Comparable results were obtained also with another protonophore, pentachlorophenol (PCP).

  8. Substrate-Linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System

    SciTech Connect

    Bagai, I.; Liu, W.; Rensing, C.; Blackburn, N.J.; McEvoy, M.M.

    2009-06-02

    Gram-negative bacteria utilize dual membrane resistance nodulation division-type efflux systems to export a variety of substrates. These systems contain an essential periplasmic component that is important for assembly of the protein complex. We show here that the periplasmic protein CusB from the Cus copper/silver efflux system has a critical role in Cu(I) and Ag(I) binding. Isothermal titration calorimetry experiments demonstrate that one Ag(I) ion is bound per CusB molecule with high affinity. X-ray absorption spectroscopy data indicate that the metal environment is an all-sulfur 3-coordinate environment. Candidates for the metal-coordinating residues were identified from sequence analysis, which showed four conserved methionine residues. Mutations of three of these methionine residues to isoleucine resulted in significant effects on CusB metal binding in vitro. Cells containing these CusB variants also show a decrease in their ability to grow on copper-containing plates, indicating an important functional role for metal binding by CusB. Gel filtration chromatography demonstrates that upon binding metal, CusB undergoes a conformational change to a more compact structure. Based on these structural and functional effects of metal binding, we propose that the periplasmic component of resistance nodulation division-type efflux systems plays an active role in export through substrate-linked conformational changes.

  9. Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

    PubMed Central

    Kurokawa, Yoichi; Yanagi, Hideki; Yura, Takashi

    2000-01-01

    Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli. PMID:10966415

  10. Holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories

    PubMed Central

    Aravind, L.; Makarova, Kira S.; Koonin, Eugene V.

    2000-01-01

    Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII–colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage λ exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and Aquifex. Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including McrA and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each

  11. Electron transport to periplasmic nitrate reductase (NapA) of Wolinella succinogenes is independent of a NapC protein.

    PubMed

    Simon, Jörg; Sänger, Monica; Schuster, Stephan C; Gross, Roland

    2003-07-01

    The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor. Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined. We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities. A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced. The napA gene was inactivated by inserting a kanamycin resistance gene cassette. The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type. An antigen was detected in wild-type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus. This antigen was absent in the W. succinogenes napA mutant. It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W. succinogenes, although a second nitrate-reducing enzyme is apparently induced in the napA mutant. The nap cluster of W. succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria. The W. succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC. Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration. A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild-type properties with respect to nitrate respiration. A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex. Inspection of the W. succinogenes genome sequence suggests that ammonia formation from

  12. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells

    PubMed Central

    Kleinstiver, Benjamin P.; Tsai, Shengdar Q.; Prew, Michelle S.; Nguyen, Nhu T.; Welch, Moira M.; Lopez, Jose M.; McCaw, Zachary R.; Aryee, Martin J.; Joung, J. Keith

    2016-01-01

    The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases1 are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9)2–5. We also report that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells. PMID:27347757

  13. Early zygote-specific nuclease in mitochondria of the true slime mold Physarum polycephalum.

    PubMed

    Moriyama, Yohsuke; Yamazaki, Tomokazu; Nomura, Hideo; Sasaki, Narie; Kawano, Shigeyuki

    2005-11-01

    The active, selective digestion of mtDNA from one parent is a possible molecular mechanism for the uniparental inheritance of mtDNA. In Physarum polycephalum, mtDNA is packed by DNA-binding protein Glom, which packs mtDNA into rod-shaped mt-nucleoids. After the mating, mtDNA from one parent is selectively digested, and the Glom began to disperse. Dispersed Glom was retained for at least 6 h after mtDNA digestion, but disappeared completely by about 12 h after mixing two strains. We identified two novel nucleases using DNA zymography with native-PAGE and SDS-PAGE. One is a Ca2+-dependent, high-molecular-weight nuclease complex (about 670 kDa), and the other is a Mn2+-dependent, high-molecular-weight nuclease complex (440-670 kDa); the activity of the latter was detected as a Mn2+-dependent, 13-kDa DNase band on SDS-PAGE. All mitochondria isolated from myxamoebae had mt-nucleoids, whereas half of the mitochondria isolated from the zygotes at 12 h after mixing had lost the mt-nucleoids. The activity of the Mn2+-dependent nuclease in the isolated mitochondria was detected at least 8 h after mixing of two strains. The timing and localization of the Mn2+-dependent DNase activity matched the selective digestion of mtDNA.

  14. PNA-based artificial nucleases as antisense and anti-miRNA oligonucleotide agents.

    PubMed

    Gaglione, M; Milano, G; Chambery, A; Moggio, L; Romanelli, A; Messere, A

    2011-08-01

    Because of its interesting chemical, physical and biological properties, Peptide Nucleic Acid (PNA) has attracted major attention in molecular biology, for diagnostics purposes and development of biosensors. PNAs have become candidates for gene therapeutic drugs in ANTISENSE (AO) strategy with favorable in vivo biochemical properties. Recently, antisense PNA oligonucleotides have been described in anti-miRNA approach (AMO). We propose PNA-based nucleases as AO and AMO agents. We report the design, synthesis and characterization of two kinds of artificial nucleases composed of a PEG-PNA-PEG domain conjugated to HGG·Cu (A) and DETA (B) as well known cleavage sites. Qualitative (MALDI-TOF) and quantitative (HTS) assays were planned to study nuclease activity of constructs A and B on RNA-3'-FAM target sequence. The results have highlighted the best performance of nuclease B and the relevance of the PEG spacer, in particular for conjugate A, in terms of efficiency of the cleavage, suggesting that conjugates A and B also act as potential antisense and anti-miRNA agents.

  15. Nuclease stability of boron-modified nucleic acids: application to label-free mismatch detection.

    PubMed

    Reverte, Maëva; Vasseur, Jean-Jacques; Smietana, Michael

    2015-11-21

    5'-End boronic acid-modified oligonucleotides were evaluated against various nucleases at single and double stranded levels. The results show that these modifications induce a high resistance to degradation by calf-spleen and snake venom phosphodiesterases. More importantly, this eventually led to the development of a new label-free enzyme-assisted fluorescence-based method for single mismatch detection.

  16. Sulfonamide-bridged nucleic acid: synthesis, high RNA selective hybridization, and high nuclease resistance.

    PubMed

    Mitsuoka, Yasunori; Fujimura, Yuko; Waki, Reiko; Kugimiya, Akira; Yamamoto, Tsuyoshi; Hari, Yoshiyuki; Obika, Satoshi

    2014-11-07

    2'-N,4'-C-(N-methylamino)sulfonylmethylene-bridged thymidine (SuNA), which has a six-membered linkage including a sulfonamide moiety, was synthesized and introduced into oligonucleotides. The oligonucleotides containing SuNA exhibited excellent nuclease resistance, a high affinity toward single-stranded RNA, and a low affinity toward single-stranded DNA compared to the natural oligonucleotide.

  17. Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs).

    PubMed

    Moore, Finola E; Reyon, Deepak; Sander, Jeffry D; Martinez, Sarah A; Blackburn, Jessica S; Khayter, Cyd; Ramirez, Cherie L; Joung, J Keith; Langenau, David M

    2012-01-01

    Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

  18. Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection

    PubMed Central

    Pimkin, Maxim; Caretti, Elena; Canutescu, Adrian; Yeung, Jeffrey B; Cohn, Heather; Chen, Yibai; Oleykowski, Catherine; Bellacosa, Alfonso; Yeung, Anthony T

    2007-01-01

    Background The detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria. Results We describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from Spinacia oleracea (spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in BRCA1 gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites. Conclusion The insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies. PMID:17543120

  19. A novel nuclease activity that is activated by Ca(2+) chelated to EGTA.

    PubMed

    Dominguez, Kenneth; Ward, W Steven

    2009-12-01

    Most nucleases require a divalent cation as a cofactor, usually Mg(2+) or Ca(2+), and are inhibited by the chelators EDTA and EGTA. We report the existence of a novel nuclease activity, initially identified in the luminal fluids of the mouse male reproductive tract but subsequently found in other tissues,that requires EGTA chelated to calcium to digest DNA. We refer to this unique enzyme as CEAN (Chelated EGTA Activated Nuclease). Using a fraction of vas deferens luminal fluid, plasmid DNA was degraded in the presence of excess Ca(2+) (Ca(2+) :EGTA = 16) or excess EGTA (Ca(2+) :EGTA = 0.25), but required the presence of both. Higher levels of EGTA (Ca(2+) :EGTA = 0.10) prevented activity, suggesting that unchelated EGTA may be a competitive inhibitor. The EGTA-Ca(2+) activation of CEAN is reversible as removing EGTA-Ca(2+) stops ongoing DNA degradation, but adding EGTA-Ca(2+) again reactivates the enzyme. This suggests the possibility that CEAN binds directly to EGTA-Ca(2+). CEAN has a greater specificity for the chelator than for the divalent cation. Two other chelators, BAPTA and sodium citrate, do not activate CEAN in the presence of cation, but chelated EDTA does. EGTA chelated to other divalent cations such as Mn(2+), Zn(2+) , and Cu(2+) activate CEAN, but not Mg(2+) . The activity is lost upon boiling suggesting that it is a protein. These data suggest that EGTA and EDTA may not always protect DNA from nuclease damage.

  20. Multicomponent synthesis of artificial nucleases and their RNase and DNase activity

    PubMed Central

    Gulevich, Anton V; Koroleva, Lyudmila S; Morozova, Olga V; Bakhvalova, Valentina N

    2011-01-01

    Summary The synthesis of new, artificial ribonucleases containing two amino acid residues connected by an aliphatic linker has been developed. Target molecules were synthesized via a catalytic three-component Ugi reaction from aliphatic diisocyanides. Preliminary investigations proved unspecific nuclease activity of the new compounds towards single-stranded RNA and double-stranded circular DNA. PMID:21915218

  1. MegaTevs: single-chain dual nucleases for efficient gene disruption.

    PubMed

    Wolfs, Jason M; DaSilva, Matthew; Meister, Sarah E; Wang, Xu; Schild-Poulter, Caroline; Edgell, David R

    2014-07-01

    Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, the MegaTev enzyme generates two double-strand breaks to excise an intervening 30-bp fragment. In HEK 293 cells, we observe a high frequency of gene disruption without co-expression of DNA end-processing enzymes. Deep sequencing of disrupted target sites revealed minimal processing, consistent with the MegaTev sequestering the double-strand breaks from the DNA repair machinery. Off-target profiling revealed no detectable cleavage at sites where the I-TevI CNNNG cleavage motif is not appropriately spaced from the I-OnuI binding site. The MegaTev enzyme represents a small, programmable nuclease platform for extremely specific genome-engineering applications. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. MegaTevs: single-chain dual nucleases for efficient gene disruption

    PubMed Central

    Wolfs, Jason M.; DaSilva, Matthew; Meister, Sarah E.; Wang, Xu; Schild-Poulter, Caroline; Edgell, David R.

    2014-01-01

    Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, the MegaTev enzyme generates two double-strand breaks to excise an intervening 30-bp fragment. In HEK 293 cells, we observe a high frequency of gene disruption without co-expression of DNA end-processing enzymes. Deep sequencing of disrupted target sites revealed minimal processing, consistent with the MegaTev sequestering the double-strand breaks from the DNA repair machinery. Off-target profiling revealed no detectable cleavage at sites where the I-TevI CNNNG cleavage motif is not appropriately spaced from the I-OnuI binding site. The MegaTev enzyme represents a small, programmable nuclease platform for extremely specific genome-engineering applications. PMID:25013171

  3. Escherichia coli DNA photolyase stimulates uvrABC excision nuclease in vitro.

    PubMed Central

    Sancar, A; Franklin, K A; Sancar, G B

    1984-01-01

    Pyrimidine dimers are the major photoproducts produced in cellular DNA upon UV irradiation. In Escherichia coli there are dark and photorepair mechanisms that eliminate the dimers from DNA and prevent their lethal and mutagenic effects. To determine whether these repair mechanisms act cooperatively or competitively in repairing DNA, we investigated the effects upon one another of DNA photolyase, which mediates photorepair, and uvrABC excision nuclease, an enzyme complex of the uvrABC gene products, which catalyzes nucleotide excision repair. We found that photolyase stimulates the removal of pyrimidine dimers but not other DNA adducts by uvrABC excision nuclease. The two subunits of uvrABC excision nuclease, the uvrA and uvrB proteins which together bind to the dimer region of DNA, had no effect on the activity of photolyase. T4 endonuclease V, which like photolyase is specific for pyrimidine dimers, was inhibited by photolyase, suggesting that these two proteins recognize the same or similar chemical structures in UV-irradiated DNA that are different from those recognized by uvrABC excision nuclease. Images PMID:6390436

  4. Donor plasmid design for codon and single base genome editing using zinc finger nucleases.

    PubMed

    Pruett-Miller, Shondra M; Davis, Gregory D

    2015-01-01

    In recent years, CompoZr zinc finger nuclease (ZFN) technology has matured to the point that a user-defined double strand break (DSB) can be placed at virtually any location in the human genome within 50 bp of a desired site. Such high resolution ZFN engineering is well within the conversion tract limitations demarcated by the mammalian DNA repair machinery, resulting in a nearly universal ability to create point mutations throughout the human genome. Additionally, new architectures for targeted nuclease engineering have been rapidly developed, namely transcription activator like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems, further expanding options for placement of DSBs. This new capability has created a need to explore the practical limitations of delivering plasmid-based information to the sites of chromosomal double strand breaks so that nuclease-donor methods can be widely deployed in fundamental and therapeutic research. In this chapter, we explore a ZFN-compatible donor design in the context of codon changes at an endogenous locus encoding the human RSK2 kinase.

  5. Tudor domain proteins in protozoan parasites and characterization of Plasmodium falciparum tudor staphylococcal nuclease.

    PubMed

    Hossain, Manzar J; Korde, Reshma; Singh, Shivani; Mohmmed, Asif; Dasaradhi, P V N; Chauhan, V S; Malhotra, Pawan

    2008-04-01

    RNA-binding proteins play key roles in post-transcriptional regulation of gene expression. In eukaryotic cells, a multitude of RNA-binding proteins with several RNA-binding domains/motifs have been described. Here, we show the existence of two Tudor domain containing proteins, a survival of motor neuron (SMN)-like protein and a Staphylococcus aureus nuclease homologue referred to as TSN, in Plasmodium and other protozoan parasites. Activity analysis shows that Plasmodium falciparum TSN (PfTSN) possesses nuclease activity and Tudor domain is the RNA-binding domain. A specific inhibitor of micrococcal nucleases, 3',5'-deoxythymidine bisphosphate (pdTp) inhibits the nuclease as well as RNA-binding activities of the protein. PfTSN shows a predominant nuclear localization. Treatment of P. falciparum with pdTp, inhibited in vitro growth of both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, while a four fold concentration of pdTp did not have any significant effect on the mammalian cell line, Huh-7D12. Altogether, these results suggest that PfTSN is an essential enzyme in the parasite's life cycle.

  6. Structure-Specific nuclease activities of Artemis and the Artemis: DNA-PKcs complex.

    PubMed

    Chang, Howard H Y; Lieber, Michael R

    2016-06-20

    Artemis is a vertebrate nuclease with both endo- and exonuclease activities that acts on a wide range of nucleic acid substrates. It is the main nuclease in the non-homologous DNA end-joining pathway (NHEJ). Not only is Artemis important for the repair of DNA double-strand breaks (DSBs) in NHEJ, it is essential in opening the DNA hairpin intermediates that are formed during V(D)J recombination. Thus, humans with Artemis deficiencies do not have T- or B-lymphocytes and are diagnosed with severe combined immunodeficiency (SCID). While Artemis is the only vertebrate nuclease capable of opening DNA hairpins, it has also been found to act on other DNA substrates that share common structural features. Here, we discuss the key structural features that all Artemis DNA substrates have in common, thus providing a basis for understanding how this structure-specific nuclease recognizes its DNA targets. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Cold adaptation of the mononuclear molybdoenzyme periplasmic nitrate reductase from the Antarctic bacterium Shewanella gelidimarina

    SciTech Connect

    Simpson, Philippa J.L.; Codd, Rachel

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Cold-adapted phenotype of NapA from the Antarctic bacterium Shewanella gelidimarina. Black-Right-Pointing-Pointer Protein homology model of NapA from S. gelidimarina and mesophilic homologue. Black-Right-Pointing-Pointer Six amino acid residues identified as lead candidates governing NapA cold adaptation. Black-Right-Pointing-Pointer Molecular-level understanding of designing cool-temperature in situ oxyanion sensors. -- Abstract: The reduction of nitrate to nitrite is catalysed in bacteria by periplasmic nitrate reductase (Nap) which describes a system of variable protein subunits encoded by the nap operon. Nitrate reduction occurs in the NapA subunit, which contains a bis-molybdopterin guanine dinucleotide (Mo-MGD) cofactor and one [4Fe-4S] iron-sulfur cluster. The activity of periplasmic nitrate reductase (Nap) isolated as native protein from the cold-adapted (psychrophilic) Antarctic bacterium Shewanella gelidimarina (Nap{sub Sgel}) and middle-temperature adapted (mesophilic) Shewanella putrefaciens (Nap{sub Sput}) was examined at varied temperature. Irreversible deactivation of Nap{sub Sgel} and Nap{sub Sput} occurred at 54.5 and 65 Degree-Sign C, respectively. When Nap{sub Sgel} was preincubated at 21-70 Degree-Sign C for 30 min, the room-temperature nitrate reductase activity was maximal and invariant between 21 and 54 Degree-Sign C, which suggested that Nap{sub Sgel} was poised for optimal catalysis at modest temperatures and, unlike Nap{sub Sput}, did not benefit from thermally-induced refolding. At 20 Degree-Sign C, Nap{sub Sgel} reduced selenate at 16% of the rate of nitrate reduction. Nap{sub Sput} did not reduce selenate. Sequence alignment showed 46 amino acid residue substitutions in Nap{sub Sgel} that were conserved in NapA from mesophilic Shewanella, Rhodobacter and Escherichia species and could be associated with the Nap{sub Sgel} cold-adapted phenotype. Protein homology modeling of Nap{sub Sgel} using a

  8. Identification of the znuA-Encoded Periplasmic Zinc Transport Protein of Haemophilus ducreyi

    PubMed Central

    Lewis, David A.; Klesney-Tait, Julia; Lumbley, Sheryl R.; Ward, Christine K.; Latimer, Jo L.; Ison, Catherine A.; Hansen, Eric J.

    1999-01-01

    The znuA gene of Haemophilus ducreyi encodes a 32-kDa (mature) protein that has homology to both the ZnuA protein of Escherichia coli and the Pzp1 protein of H. influenzae; both of these latter proteins are members of a growing family of prokaryotic zinc transporters. Inactivation of the H. ducreyi 35000 znuA gene by insertional mutagenesis resulted in a mutant that grew more slowly than the wild-type parent strain in vitro unless ZnCl2 was provided at a final concentration of 100 μM. Other cations tested did not restore growth of this H. ducreyi mutant to wild-type levels. The H. ducreyi ZnuA protein was localized to the periplasm, where it is believed to function as the binding component of a zinc transport system. Complementation of the znuA mutation with the wild-type H. ducreyi znuA gene provided in trans restored the ability of this H. ducreyi mutant to grow normally in the absence of exogenously added ZnCl2. The wild-type H. ducreyi znuA gene was also able to complement a H. influenzae pzp1 mutation. The H. ducreyi znuA isogenic mutant exhibited significantly decreased virulence (P = 0.0001) when tested in the temperature-dependent rabbit model for experimental chancroid. This decreased virulence was not observed when the znuA mutant was complemented with the wild-type H. ducreyi znuA gene provided in trans. PMID:10496878

  9. Structure of the Periplasmic Stress Response Protein CpxP▿†

    PubMed Central

    Thede, Gina L.; Arthur, David C.; Edwards, Ross A.; Buelow, Daelynn R.; Wong, Julia L.; Raivio, Tracy L.; Glover, J. N. Mark

    2011-01-01

    CpxP is a novel bacterial periplasmic protein with no homologues of known function. In Gram-negative enteric bacteria, CpxP is thought to interact with the two-component sensor kinase, CpxA, to inhibit induction of the Cpx envelope stress response in the absence of protein misfolding. CpxP has also been shown to facilitate DegP-mediated proteolysis of misfolded proteins. Six mutations that negate the ability of CpxP to function as a signaling protein are localized in or near two conserved LTXXQ motifs that define a class of proteins with similarity to CpxP, Pfam PF07813. To gain insight into how these mutations might affect CpxP signaling and/or proteolytic adaptor functions, the crystal structure of CpxP from Escherichia coli was determined to 2.85-Å resolution. The structure revealed an antiparallel dimer of intertwined α-helices with a highly basic concave surface. Each protomer consists of a long, hooked and bent hairpin fold, with the conserved LTXXQ motifs forming two diverging turns at one end. Biochemical studies demonstrated that CpxP maintains a dimeric state but may undergo a slight structural adjustment in response to the inducing cue, alkaline pH. Three of the six previously characterized cpxP loss-of-function mutations, M59T, Q55P, and Q128H, likely result from a destabilization of the protein fold, whereas the R60Q, D61E, and D61V mutations may alter intermolecular interactions. PMID:21317318

  10. Function of periplasmic hydrogenases in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

    PubMed

    Caffrey, Sean M; Park, Hyung-Soo; Voordouw, Johanna K; He, Zhili; Zhou, Jizhong; Voordouw, Gerrit

    2007-09-01

    The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.

  11. TupA: a tungstate binding protein in the periplasm of Desulfovibrio alaskensis G20.

    PubMed

    Otrelo-Cardoso, Ana Rita; Nair, Rashmi R; Correia, Márcia A S; Rivas, Maria G; Santos-Silva, Teresa

    2014-07-02

    The TupABC system is involved in the cellular uptake of tungsten and belongs to the ABC (ATP binding cassette)-type transporter systems. The TupA component is a periplasmic protein that binds tungstate anions, which are then transported through the membrane by the TupB component using ATP hydrolysis as the energy source (the reaction catalyzed by the ModC component). We report the heterologous expression, purification, determination of affinity binding constants and crystallization of the Desulfovibrio alaskensis G20 TupA. The tupA gene (locus tag Dde_0234) was cloned in the pET46 Enterokinase/Ligation-Independent Cloning (LIC) expression vector, and the construct was used to transform BL21 (DE3) cells. TupA expression and purification were optimized to a final yield of 10 mg of soluble pure protein per liter of culture medium. Native polyacrylamide gel electrophoresis was carried out showing that TupA binds both tungstate and molybdate ions and has no significant interaction with sulfate, phosphate or perchlorate. Quantitative analysis of metal binding by isothermal titration calorimetry was in agreement with these results, but in addition, shows that TupA has higher affinity to tungstate than molybdate. The protein crystallizes in the presence of 30% (w/v) polyethylene glycol 3350 using the hanging-drop vapor diffusion method. The crystals diffract X-rays beyond 1.4 Å resolution and belong to the P21 space group, with cell parameters a = 52.25 Å, b = 42.50 Å, c = 54.71 Å, β = 95.43°. A molecular replacement solution was found, and the structure is currently under refinement.

  12. Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

    PubMed Central

    Hall Sedlak, Ruth; Hnilova, Marketa; Grosh, Carolynn; Fong, Hanson; Baneyx, Francois; Schwartz, Dan; Sarikaya, Mehmet; Tamerler, Candan

    2012-01-01

    Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo. PMID:22286990

  13. Periplasmic Cleavage and Modification of the 1-Phosphate Group of Helicobacter pylori Lipid A*

    PubMed Central

    Tran, An X.; Karbarz, Mark J.; Wang, Xiaoyuan; Raetz, Christian R. H.; McGrath, Sara C.; Cotter, Robert J.; Trent, M. Stephen

    2008-01-01

    Pathogenic bacteria modify the lipid A portion of their lipopolysaccharide to help evade the host innate immune response. Modification of the negatively charged phosphate groups of lipid A aids in resistance to cationic antimicrobial peptides targeting the bacterial cell surface. The lipid A of Helicobacter pylori contains a phosphoethanolamine (pEtN) unit directly linked to the 1-position of the disaccharide backbone. This is in contrast to the pEtN units found in other pathogenic Gram-negative bacteria, which are attached to the lipid A phosphate group to form a pyrophosphate linkage. This study describes two enzymes involved in the periplasmic modification of the 1-phosphate group of H. pylori lipid A. By using an in vitro assay system, we demonstrate the presence of lipid A 1-phosphatase activity in membranes of H. pylori. In an attempt to identify genes encoding possible lipid A phosphatases, we cloned four putative orthologs of Escherichia coli pgpB, the phosphatidylglycerol-phosphate phosphatase, from H. pylori 26695. One of these orthologs, Hp0021, is the structural gene for the lipid A 1-phosphatase and is required for removal of the 1-phosphate group from mature lipid A in an in vitro assay system. Heterologous expression of Hp0021 in E. coli resulted in the highly selective removal of the 1-phosphate group from E. coli lipid A, as demonstrated by mass spectrometry. We also identified the structural gene for the H. pylori lipid A pEtN transferase (Hp0022). Mass spectrometric analysis of the lipid A isolated from E. coli expressing Hp0021 and Hp0022 shows the addition of a single pEtN group at the 1-position, confirming that Hp0022 is responsible for the addition of a pEtN unit at the 1-position in H. pylori lipid A. In summary, we demonstrate that modification of the 1-phosphate group of H. pylori lipid A requires two enzymatic steps. PMID:15489235

  14. Catalytic Mechanism and Mode of Action of the Periplasmic Alginate Epimerase AlgG*

    PubMed Central

    Wolfram, Francis; Kitova, Elena N.; Robinson, Howard; Walvoort, Marthe T. C.; Codée, Jeroen D. C.; Klassen, John S.; Howell, P. Lynne

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen that forms chronic biofilm infections in the lungs of cystic fibrosis patients. A major component of the biofilm during these infections is the exopolysaccharide alginate, which is synthesized at the inner membrane as a homopolymer of 1–4-linked β-d-mannuronate. As the polymer passages through the periplasm, 22–44% of the mannuronate residues are converted to α-l-guluronate by the C5-epimerase AlgG to produce a polymer of alternating β-d-mannuronate and α-l-guluronate blocks and stretches of polymannuronate. To understand the molecular basis of alginate epimerization, the structure of Pseudomonas syringae AlgG has been determined at 2.1-Å resolution, and the protein was functionally characterized. The structure reveals that AlgG is a long right-handed parallel β-helix with an elaborate lid structure. Functional analysis of AlgG mutants suggests that His319 acts as the catalytic base and that Arg345 neutralizes the acidic group during the epimerase reaction. Water is the likely catalytic acid. Electrostatic surface potential and residue conservation analyses in conjunction with activity and substrate docking studies suggest that a conserved electropositive groove facilitates polymannuronate binding and contains at least nine substrate binding subsites. These subsites likely align the polymer in the correct register for catalysis to occur. The presence of multiple subsites, the electropositive groove, and the non-random distribution of guluronate in the alginate polymer suggest that AlgG is a processive enzyme. Moreover, comparison of AlgG and the extracellular alginate epimerase AlgE4 of Azotobacter vinelandii provides a structural rationale for the differences in their Ca2+ dependence. PMID:24398681

  15. The periplasmic chaperone Skp is required for successful Salmonella Typhimurium infection in a murine typhoid model.

    PubMed

    Rowley, Gary; Skovierova, Henrieta; Stevenson, Andrew; Rezuchova, Bronislava; Homerova, Dagmar; Lewis, Claire; Sherry, Aileen; Kormanec, Jan; Roberts, Mark

    2011-03-01

    The alternative sigma factor σ(E) (rpoE) is essential for survival in vivo of Salmonella Typhimurium but is dispensable during growth in the laboratory. We have been identifying σ(E)-regulated genes and studying their regulation and function to elucidate their potential role in the severe attenuation of S. Typhimurium rpoE mutants. In this study we identify five promoters that control the rseP, yaeT (bamA), skp region. A confirmed σ(E)-dependent promoter, yaeTp1, and a second downstream promoter, yaeTp2, are located within the upstream gene rseP and direct expression of the downstream genes. The only known function of RseP is σ(E) activation, and it is therefore not expected to be essential for S. Typhimurium in vitro. However, it proved impossible to delete the entire rseP gene due to the presence of internal promoters that regulate the essential gene yaeT. We could inactivate rseP by deleting the first third of the gene, leaving the yaeT promoters intact. Like the rpoE mutant, the rseP mutant exhibited severe attenuation in vivo. We were able to delete the entire coding sequence of skp, which encodes a periplasmic chaperone involved in targeting misfolded outer-membrane proteins to the β-barrel assembly machinery. The skp mutant was attenuated in mice after oral and parenteral infection. Virulence could be complemented by providing skp in trans but only by linking it to a heterologous σ(E)-regulated promoter. The reason the skp mutant is attenuated is currently enigmatic, but we know it is not through increased sensitivity to a variety of RpoE-activating host stresses, such as H(2)O(2), polymyxin B and high temperature, or through altered secretion of effector proteins by either the Salmonella pathogenicity island (SPI)-1 or the SPI-2 type III secretion system.

  16. Large scale, in situ isolation of periplasmic IGF-I from E. coli.

    PubMed

    Hart, R A; Lester, P M; Reifsnyder, D H; Ogez, J R; Builder, S E

    1994-11-01

    Human insulin-like growth factor I (IGF-I) accumulates in both folded and aggregated forms in the fermentation medium and cellular periplasmic space when expressed in E. coli with an endogenous secretory signal sequence. Due to its heterogeneity in form and location, low yield of IGF-I was obtained using a typical refractile body recovery strategy. To enhance recovery yield, a new procedure was developed to solubilize and extract IGF-I from cells while in fermentation broth. This method, called in situ solubilization, involves addition of chaotrope and reductant to alkaline fermentation broth and provides recovery of about 90% of all IGF-I in an isolated supernatant. To further enhance recovery, a new aqueous two-phase extraction procedure was developed which partitions soluble non-native IGF-I and biomass solids into separate liquid phases. This two-phase extraction procedure involves addition of polymer and salt to the solubilization mixture and provides about 90% recovery of solubilized IGF-I in the light phase. The performance of the solubilization and aqueous extraction procedures is reproducible at scales ranging from 10 to 1000 liters and provides a 70% cumulative recovery yield of IGF-I in the isolated light phase. The procedure provides significant initial IGF-I purification since most host proteins remain cell associated during solubilization and are enriched in heavy phase. ELISA analysis for E. coli proteins indicates that 97% of the protein in the light phase is IGF-I. Together, the techniques of in situ solubilization and aqueous two-phase extraction provide a new, high yield approach for isolating recombinant protein which is accumulated in more than one form during fermentation.

  17. Catalytic mechanism and mode of action of the periplasmic alginate epimerase AlgG.

    PubMed

    Wolfram, Francis; Kitova, Elena N; Robinson, Howard; Walvoort, Marthe T C; Codée, Jeroen D C; Klassen, John S; Howell, P Lynne

    2014-02-28

    Pseudomonas aeruginosa is an opportunistic pathogen that forms chronic biofilm infections in the lungs of cystic fibrosis patients. A major component of the biofilm during these infections is the exopolysaccharide alginate, which is synthesized at the inner membrane as a homopolymer of 1-4-linked β-D-mannuronate. As the polymer passages through the periplasm, 22-44% of the mannuronate residues are converted to α-L-guluronate by the C5-epimerase AlgG to produce a polymer of alternating β-D-mannuronate and α-L-guluronate blocks and stretches of polymannuronate. To understand the molecular basis of alginate epimerization, the structure of Pseudomonas syringae AlgG has been determined at 2.1-Å resolution, and the protein was functionally characterized. The structure reveals that AlgG is a long right-handed parallel β-helix with an elaborate lid structure. Functional analysis of AlgG mutants suggests that His(319) acts as the catalytic base and that Arg(345) neutralizes the acidic group during the epimerase reaction. Water is the likely catalytic acid. Electrostatic surface potential and residue conservation analyses in conjunction with activity and substrate docking studies suggest that a conserved electropositive groove facilitates polymannuronate binding and contains at least nine substrate binding subsites. These subsites likely align the polymer in the correct register for catalysis to occur. The presence of multiple subsites, the electropositive groove, and the non-random distribution of guluronate in the alginate polymer suggest that AlgG is a processive enzyme. Moreover, comparison of AlgG and the extracellular alginate epimerase AlgE4 of Azotobacter vinelandii provides a structural rationale for the differences in their Ca(2+) dependence.

  18. Function of Periplasmic Hydrogenases in the Sulfate-ReducingBacterium Desulfovibrio vulgaris Hildenborough

    SciTech Connect

    Caffrey, Sean M.; Park, Hyung-Soo; Voordouw, Johanna K.; He,Zhili; Zhou, Jizhong; Voordouw, Gerrit

    2007-09-24

    The sulfate-reducing bacterium Desulfovibrio vulgarisHildenborough possesses four periplasmic hydrogenases to facilitate theoxidation of molecular hydrogen. These include an [Fe]hydrogenase, an[NiFeSe]hydrogenase, and two [NiFe]hydrogenases encoded by the hyd,hys, hyn1, and hyn2 genes, respectively. In order to understand theircellular functions, we have compared the growth rates of existing (hydand hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those ofthe wild type in defined media in which lactate or hydrogen at either 5or 50 percent (vol/vol) was used as the sole electron donor for sulfatereduction. Only strains missing the [Fe]hydrogenase were significantlyaffected during growth with lactate or with 50 percent (vol/vol) hydrogenas the sole electron donor. When the cells were grown at low (5 percent[vol/vol]) hydrogen concentrations, those missing the [NiFeSe]hydrogenase suffered the greatest impairment. The growth rate datacorrelated strongly with gene expression results obtained from microarrayhybridizations and real-time PCR using mRNA extracted from cells grownunder the three conditions. Expression of the hys genes followed theorder 5 percent hydrogen>50 percent hydrogen>lactate, whereasexpression of the hyd genes followed the reverse order. These resultssuggest that growth with lactate and 50 percent hydrogen is associatedwith high intracellular hydrogen concentrations, which are best capturedby the higher activity, lower affinity [Fe]hydrogenase. In contrast,growth with 5 percent hydrogen is associated with a low intracellularhydrogen concentration, requiring the lower activity, higher affinity[NiFeSe]hydrogenase.

  19. Function of Periplasmic Hydrogenases in the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough▿ †

    PubMed Central

    Caffrey, Sean M.; Park, Hyung-Soo; Voordouw, Johanna K.; He, Zhili; Zhou, Jizhong; Voordouw, Gerrit

    2007-01-01

    The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen > 50% hydrogen > lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase. PMID:17601789

  20. Nucleases activities during French bean leaf aging and dark-induced senescence.

    PubMed

    Lambert, Rocío; Quiles, Francisco Antonio; Gálvez-Valdivieso, Gregorio; Piedras, Pedro

    2017-09-07

    During leaf senescence resources are managed, with nutrients mobilized from older leaves to new sink tissues. The latter implies a dilemma in terms of resource utilization, the leaf senescence should increase seed quality whereas delay in senescence should improve the seed yield. Increased knowledge about nutrient recycling during leaf senescence could lead to advances in agriculture and improved seed quality. Macromolecules mobilized during leaf senescence include proteins and nucleic acids. Although nucleic acids have been less well studied than protein degradation, they are possible reservoirs of nitrogen and phosphorous. The present study investigated nuclease activities and gene expression patterns of five members of the S1/P1 family in French bean (Phaseolus vulgaris L. cv.)Page: 2 during leaf senescence. An in-gel assay was used to detect nuclease activity during natural and dark-induced senescence, with single-stranded DNA (ssDNA) used as a substrate. The results revealed two nucleases (glycoproteins), with molecular masses of 34 and 39kDa in the senescent leaves. The nuclease activities were higher at a neutral than at an acidic pH. EDTA treatment inhibited the activities of the nucleases, and the addition of zinc resulted in the recovery of these activities. Both the 34 and 39kDa nucleases were able to use RNA and double-stranded DNA (dsDNA) as substrates, although their activities were low when dsDNA was used as a substrate. In addition, two ribonucleases with molecular masses of 14 and 16kDa, both of which could only utilize RNA as a substrate, were detected in the senescent leaves. Two members of the S1/P1 family, PVN2 and PVN5, were expressed under the experimental conditions, suggesting that these two genes were involved in senescence. The nuclease activity of the glycoproteins and gene expression were similar under both natural senescence and dark-induced senescence conditions. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights

  1. Kinetic analysis of the cleavage of natural and synthetic substrates by the Serratia nuclease.

    PubMed

    Friedhoff, P; Meiss, G; Kolmes, B; Pieper, U; Gimadutdinow, O; Urbanke, C; Pingoud, A

    1996-10-15

    The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serratia nuclease. Most of the natural substrates are cleaved with similar Kcat and K(m) values, the Kcat/K(m) ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA).poly(dT) or poly(dG).poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K(m), respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-Rp-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the Rp-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the Rp-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally

  2. Mouse spermatozoa contain a nuclease that is activated by pretreatment with EGTA and subsequent calcium incubation.

    PubMed

    Boaz, Segal M; Dominguez, Kenneth; Shaman, Jeffrey A; Ward, W Steven

    2008-04-01

    We demonstrated that mouse spermatozoa cleave their DNA into approximately 50 kb loop-sized fragments with topoisomerase IIB when treated with MnCl(2) and CaCl(2) in a process we term sperm chromatin fragmentation (SCF). SCF can be reversed by EDTA. A nuclease then further degrades the DNA in a process we term sperm DNA degradation (SDD). MnCl(2) alone could elicit this activity, but CaCl(2) had no effect. Here, we demonstrate the existence of a nuclease in the vas deferens that can be activated by ethylene glycol tetraacetic acid (EGTA) to digest the sperm DNA by SDD. Spermatozoa were extracted with salt and dithiothreitol to remove protamines and then incubated with EGTA. Next, the EGTA was removed and divalent cations were added. We found that Mn(2+), Ca(2+), or Zn(2+) could each activate SDD in spermatozoa but Mg(2+) could not. When the reaction was slowed by incubation on ice, EGTA pretreatment followed by incubation in Ca(2+) elicited the reversible fragmentation of sperm DNA evident in SCF. When the reactions were then incubated at 37 degrees C they progressed to the more complete degradation of DNA by SDD. EDTA could also be used to activate the nuclease, but required a higher concentration than EGTA. This EGTA-activatable nuclease activity was found in each fraction of the vas deferens plasma: in the spermatozoa, in the surrounding fluid, and in the insoluble components in the fluid. These results suggest that this sperm nuclease is regulated by a mechanism that is sensitive to EGTA, possibly by removing inhibition of a calcium binding protein.

  3. The Extracellular Nuclease Dns and Its Role in Natural Transformation of Vibrio cholerae▿

    PubMed Central

    Blokesch, Melanie; Schoolnik, Gary K.

    2008-01-01

    Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification. PMID:18757542

  4. The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae.

    PubMed

    Blokesch, Melanie; Schoolnik, Gary K

    2008-11-01

    Free extracellular DNA is abundant in many aquatic environments. While much of this DNA will be degraded by nucleases secreted by the surrounding microbial community, some is available as transforming material that can be taken up by naturally competent bacteria. One such species is Vibrio cholerae, an autochthonous member of estuarine, riverine, and marine habitats and the causative agent of cholera, whose competence program is induced after colonization of chitin surfaces. In this study, we investigate how Vibrio cholerae's two extracellular nucleases, Xds and Dns, influence its natural transformability. We show that in the absence of Dns, transformation frequencies are significantly higher than in its presence. During growth on a chitin surface, an increase in transformation efficiency was found to correspond in time with increasing cell density and the repression of dns expression by the quorum-sensing regulator HapR. In contrast, at low cell density, the absence of HapR relieves dns repression, leading to the degradation of free DNA and to the abrogation of the transformation phenotype. Thus, as cell density increases, Vibrio cholerae undergoes a switch from nuclease-mediated degradation of extracellular DNA to the uptake of DNA by bacteria induced to a state of competence by chitin. Taken together, these results suggest the following model: nuclease production by low-density populations of V. cholerae might foster rapid growth by providing a source of nucleotides for the repletion of nucleotide pools. In contrast, the termination of nuclease production by static, high-density populations allows the uptake of intact DNA and coincides with a phase of potential genome diversification.

  5. Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases

    PubMed Central

    2014-01-01

    Background The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. Results In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases. PMID:24997498

  6. Mouse Spermatozoa Contain a Nuclease that Is Activated by Pretreatment with EGTA and Subsequent Calcium Incubation

    PubMed Central

    Boaz, Segal M.; Dominguez, Kenneth; Shaman, Jeffrey A.; Ward, W. Steven

    2009-01-01

    We demonstrated that mouse spermatozoa cleave their DNA into ~50 kb loop-sized fragments with topoisomerase IIB when treated with MnCl2 and CaCl2 in a process we term sperm chromatin fragmentation (SCF). SCF can be reversed by EDTA. A nuclease then further degrades the DNA in a process we term sperm DNA degradation (SDD). MnCl2 alone could elicit this activity, but CaCl2 had no effect. Here, we demonstrate the existence of a nuclease in the vas deferens that can be activated by EGTA to digest the sperm DNA by SDD. Spermatozoa were extracted with salt and dithiothreitol to remove protamines and then incubated with EGTA. Next, the EGTA was removed and divalent cations were added. We found that Mn+2, Ca+2, or Zn+2 could each activate SDD in spermatozoa but Mg+2 could not. When the reaction was slowed by incubation on ice, EGTA pretreatment followed by incubation in Ca+2 elicited the reversible fragmentation of sperm DNA evident in SCF. When the reactions were then incubated at 37°C they progressed to the more complete degradation of DNA by SDD. EDTA could also be used to activate the nuclease, but required a higher concentration than EGTA. This EGTA-activatable nuclease activity was found in each fraction of the vas deferens plasma: in the spermatozoa, in the surrounding fluid, and in the insoluble components in the fluid. These results suggest that this sperm nuclease is regulated by a mechanism that is sensitive to EGTA, possibly by removing inhibition of a calcium binding protein. PMID:17879959

  7. Vibrio cholerae evades neutrophil extracellular traps by the activity of two extracellular nucleases.

    PubMed

    Seper, Andrea; Hosseinzadeh, Ava; Gorkiewicz, Gregor; Lichtenegger, Sabine; Roier, Sandro; Leitner, Deborah R; Röhm, Marc; Grutsch, Andreas; Reidl, Joachim; Urban, Constantin F; Schild, Stefan

    2013-01-01

    The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.

  8. Vibrio cholerae Evades Neutrophil Extracellular Traps by the Activity of Two Extracellular Nucleases

    PubMed Central

    Seper, Andrea; Hosseinzadeh, Ava; Gorkiewicz, Gregor; Lichtenegger, Sabine; Roier, Sandro; Leitner, Deborah R.; Röhm, Marc; Grutsch, Andreas; Reidl, Joachim; Urban, Constantin F.; Schild, Stefan

    2013-01-01

    The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation. PMID:24039581

  9. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases.

    PubMed

    Watanabe, Takahito; Ochiai, Hiroshi; Sakuma, Tetsushi; Horch, Hadley W; Hamaguchi, Naoya; Nakamura, Taro; Bando, Tetsuya; Ohuchi, Hideyo; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro

    2012-01-01

    Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

  10. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases

    PubMed Central

    Watanabe, Takahito; Ochiai, Hiroshi; Sakuma, Tetsushi; Horch, Hadley W.; Hamaguchi, Naoya; Nakamura, Taro; Bando, Tetsuya; Ohuchi, Hideyo; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro

    2012-01-01

    Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy. PMID:22910363

  11. Effects of thyrotropin on the phosphorylation of histones and nonhistone phosphoproteins in micrococcal nuclease-sensitive and resistant thyroid chromatin

    SciTech Connect

    Cooper, E.; Spaulding, S.W.

    1983-05-01

    Actively transcribed regions of chromatin are more susceptible than bulk chromatin to digestion by nucleases, and useful information about the composition and structure of active chromatin may be obtained by studying the chromatin fragments released from nuclei by limited nuclease digestion. In the present study, we have used micrococcal nuclease to investigate the effects of TSH on protein phosphorylation in nuclease-sensitive fractions of calf thyroid chromatin. Batches of calf thyroid slices were incubated for 2 h with /sup 32/Pi, with or without 50 mU/ml TSH. Nuclei were then prepared and the distribution of /sup 32/P-labeled histones, high mobility group (HMG) proteins, and other acid-soluble phosphoproteins between micrococcal nuclease-sensitive and resistant fractions of chromatin was examined. TSH increased the amount of /sup 32/P incorporated into HMG 14 and the histones H1 and H3. Hormone-dependent increases in the /sup 32/P-labeling of H1 and H3 were not selectively associated with micrococcal nuclease-sensitive chromatin. In contrast, (/sup 32/P) HMG-14 was preferentially solubilized from nuclei by micrococcal nuclease. This lends support to the view that TSH-induced effects on the structure and function of transcriptionally active chromatin may be mediated in part by phosphorylation of HMG 14.

  12. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

    PubMed Central

    Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

    2017-01-01

    Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

  13. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.

    PubMed

    Vouillot, Léna; Thélie, Aurore; Pollet, Nicolas

    2015-01-07

    Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases.

  14. Comparison of T7E1 and Surveyor Mismatch Cleavage Assays to Detect Mutations Triggered by Engineered Nucleases

    PubMed Central

    Vouillot, Léna; Thélie, Aurore; Pollet, Nicolas

    2015-01-01

    Genome editing using engineered nucleases is used for targeted mutagenesis. But because genome editing does not target all loci with similar efficiencies, the mutation hit-rate at a given locus needs to be evaluated. The analysis of mutants obtained using engineered nucleases requires specific methods for mutation detection, and the enzyme mismatch cleavage method is used commonly for this purpose. This method uses enzymes that cleave heteroduplex DNA at mismatches and extrahelical loops formed by single or multiple nucleotides. Bacteriophage resolvases and single-stranded nucleases are used commonly in the assay but have not been compared side-by-side on mutations obtained by engineered nucleases. We present the first comparison of the sensitivity of T7E1 and Surveyor EMC assays on deletions and point mutations obtained by zinc finger nuclease targeting in frog embryos. We report the mutation detection limits and efficiencies of T7E1 and Surveyor. In addition, we find that T7E1 outperforms the Surveyor nuclease in terms of sensitivity with deletion substrates, whereas Surveyor is better for detecting single nucleotide changes. We conclude that T7E1 is the preferred enzyme to scan mutations triggered by engineered nucleases. PMID:25566793

  15. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain.

    PubMed

    Hilbert, Brendan J; Hayes, Janelle A; Stone, Nicholas P; Xu, Rui-Gang; Kelch, Brian A

    2017-01-12

    Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA.

  16. A novel nuclease-ATPase (Nar71) from archaea is part of a proposed thermophilic DNA repair system.

    PubMed

    Guy, Colin P; Majerník, Alan I; Chong, James P J; Bolt, Edward L

    2004-01-01

    We have identified a novel structure-specific nuclease in highly fractionated extracts of the thermophilic archaeon Methanothermobacter thermautotrophicus (Mth). The 71 kDa protein product of open reading frame mth1090 is a nuclease with ATPase activity, which we call Nar71 (Nuclease-ATPase in Repair, 71 kDa). The nar71 gene is located in a gene neighbourhood proposed by genomics to encode a novel DNA repair system conserved in thermophiles. The biochemical characterization of Nar71 presented here is the first analysis from within this neighbourhood, and it supports the insight from genomics. Nuclease activity of Nar71 is specific for 3' flaps and flayed duplexes, targeting single-stranded DNA (ssDNA) regions. This activity requires Mg2+ or Mn2+ and is greatly reduced in ATP. In ATP, Nar71 displaces ssDNA, also with high specificity for 3' flap and flayed duplex DNA. Strand displacement is weak compared with nuclease activity, but in ATPS it is abolished, suggesting that Nar71 couples ATP hydrolysis to DNA strand separation. ATPase assays confirmed that Nar71 is stimulated by ssDNA, though not double-stranded DNA. Mutation of Lys-117 in Nar71 abolished ATPase and nuclease activity, and we describe a separation-of-function mutant (K68A) that has lost ATPase activity but retains nuclease activity. A model of possible Nar71 function in DNA repair is presented.

  17. Selective and efficient extraction of recombinant proteins from the periplasm of Escherichia coli using low concentrations of chemicals.

    PubMed

    Jalalirad, Reza

    2013-10-01

    Experiments were conducted to determine chemicals at low concentrations, which can be utilized for selective release of periplasmic proteins. It was revealed that 80-100 % of the activity of alpha-amylase, beta-lactamase, and Fab D1.3 was retained in the presence of 0.05 and 0.1 % Triton X-100, 0.1 % Tween 20, 0.1 % DOC, 0.01 % BAC, 0.01 % CTAB, 10 mM EDTA, 1 mM and 10 mM DEA, 10 mM NTA, 0.1 and 1 % SHMP, 200 mM urea, 100-500 mM GndCl, and 1 % solvents (hexane, xylene, toluene, benzene, pyridine and isoamyl alcohol). Performance of these chemicals, recognized as generally safe, for selective release of proteins from the periplasm of Escherichia coli was investigated. DOC was a general and very efficient agent, and at concentrations as low as 0.05, 0.1, and 0.025 %, released beta-lactamase, alpha-amylase, and Fab D1.3 selectively with yield factors of 2.7, 2.3, and 3.6 times greater than osmotic shock procedure, respectively. EDTA (1 and 10 mM) discharged Fab D1.3 with efficiency more than osmotic shock (target protein yield of 110 and 138 %, correspondingly). Isoamyl alcohol (10 % v/v) was effective for periplasmic release of alpha-amylase and particularly Fab D1.3, with target protein yields of 75 and 168 %, respectively.

  18. Novel mechanism for scavenging of hypochlorite involving a periplasmic methionine-rich peptide and methionine sulfoxide reductase

    DOE PAGES

    Melnyk, Ryan A.; Youngblut, Matthew D.; Clark, Iain C.; ...

    2015-05-12

    Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and amore » putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo. In addition, bacteria are often stressed in the environment by reactive chlorine species (RCS) of

  19. Novel mechanism for scavenging of hypochlorite involving a periplasmic methionine-rich peptide and methionine sulfoxide reductase

    SciTech Connect

    Melnyk, Ryan A.; Youngblut, Matthew D.; Clark, Iain C.; Carlson, Hans K.; Wetmore, Kelly M.; Price, Morgan N.; Lavarone, Anthony T.; Deutschbauer, Adam M.; Arkin, Adam P.; Coates, John D.

    2015-05-12

    Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and a putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo. In addition, bacteria are often

  20. Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes.

    PubMed

    Quiocho, F A; Ledvina, P S

    1996-04-01

    Crystallographic structure refinement at very high resolutions of a dozen periplasmic receptors has revealed that, though they have different sizes (26 to 60 kDa) and little sequence homology, they have high tertiary structure similarity. They consist of two distinct globular domains bisected by a cleft or groove wherein the ligand binds and is buried by a hinge-bending motion between the two domains. Structural analysis also reveals how hydrogen-bonding interactions can be tailored to a wide spectrum of specificity, ranging from the stringent specificity for phosphate and sulphate to the more loose specificity for peptides.

  1. Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm.

    PubMed

    Levy, Raphael; Ahluwalia, Kiran; Bohmann, David J; Giang, Hoa M; Schwimmer, Lauren J; Issafras, Hassan; Reddy, Nithin B; Chan, Chung; Horwitz, Arnold H; Takeuchi, Toshihiko

    2013-08-30

    Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.

  2. The superoxide dismutase SodA is targeted to the periplasm in a SecA-dependent manner by a novel mechanism.

    PubMed

    Krehenbrink, Martin; Edwards, Anne; Downie, J Allan

    2011-10-01

    The manganese/iron-type superoxide dismutase (SodA) of Rhizobium leguminosarum bv. viciae 3841 is exported to the periplasm of R. l. bv. viciae and Escherichia coli. However, it does not possess a hydrophobic cleaved N-terminal signal peptide typically present in soluble proteins exported by the Sec-dependent (Sec) pathway or the twin-arginine translocation (TAT) pathway. A tatC mutant of R. l. bv. viciae exported SodA to the periplasm, ruling out export of SodA as a complex with a TAT substrate as a chaperone. The export of SodA was unaffected in a secB mutant of E. coli, but its export from R. l. bv. viciae was inhibited by azide, an inhibitor of SecA ATPase activity. A temperature-sensitive secA mutant of E. coli was strongly reduced for SodA export. The 10 N-terminal amino acid residues of SodA were sufficient to target the reporter protein alkaline phosphatase to the periplasm. Our results demonstrate the export of a protein lacking a classical signal peptide to the periplasm by a SecA-dependent, but SecB-independent targeting mechanism. Export of the R. l. bv. viciae SodA to the periplasm was not limited to the genus Rhizobium, but was also observed in other proteobacteria.

  3. Dynamic interplay between the periplasmic and transmembrane domains of GspL and GspM in the type II secretion system.

    PubMed

    Lallemand, Mathilde; Login, Frédéric H; Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E

    2013-01-01

    The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine.

  4. Dynamic Interplay between the Periplasmic and Transmembrane Domains of GspL and GspM in the Type II Secretion System

    PubMed Central

    Guschinskaya, Natalia; Pineau, Camille; Effantin, Géraldine; Robert, Xavier; Shevchik, Vladimir E.

    2013-01-01

    The type II secretion system (T2SS) is a multiprotein nanomachine that transports folded proteins across the outer membrane of gram-negative bacteria. The molecular mechanisms that govern the secretion process remain poorly understood. The inner membrane components GspC, GspL and GspM possess a single transmembrane segment (TMS) and a large periplasmic region and they are thought to form a platform of unknown function. Here, using two-hybrid and pull-down assays we performed a systematic mapping of the GspC/GspL/GspM interaction regions in the plant pathogen Dickeya dadantii. We found that the TMS of these components interact with each other, implying a complex interaction network within the inner membrane. We also showed that the periplasmic, ferredoxin-like, domains of GspL and GspM drive homo- and heterodimerizations of these proteins. Disulfide bonding analyses revealed that the respective domain interfaces include the equivalent secondary-structure elements, suggesting alternating interactions of the periplasmic domains, L/L and M/M versus L/M. Finally, we found that displacements of the periplasmic GspM domain mediate coordinated shifts or rotations of the cognate TMS. These data suggest a plausible mechanism for signal transmission between the periplasmic and the cytoplasmic portions of the T2SS machine. PMID:24223969

  5. Spectropotentiometric and structural analysis of the periplasmic nitrate reductase from Escherichia coli.

    PubMed

    Jepson, Brian J N; Mohan, Sudesh; Clarke, Thomas A; Gates, Andrew J; Cole, Jeffrey A; Butler, Clive S; Butt, Julea N; Hemmings, Andrew M; Richardson, David J

    2007-03-02

    The Escherichia coli NapA (periplasmic nitrate reductase) contains a [4Fe-4S] cluster and a Mo-bis-molybdopterin guanine dinucleotide cofactor. The NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). These proteins have been purified and studied by spectropotentiometry, and the structure of NapA has been determined. In contrast to the well characterized heterodimeric NapAB systems ofalpha-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the gamma-proteobacterial E. coli NapA and NapB proteins purify independently and not as a tight heterodimeric complex. This relatively weak interaction is reflected in dissociation constants of 15 and 32 mum determined for oxidized and reduced NapAB complexes, respectively. The surface electrostatic potential of E. coli NapA in the apparent NapB binding region is markedly less polar and anionic than that of the alpha-proteobacterial NapA, which may underlie the weaker binding of NapB. The molybdenum ion coordination sphere of E. coli NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 A, which is indicative of a water ligand. The potential range over which the Mo(6+) state is reduced to the Mo(5+) state in either NapA (between +100 and -100 mV) or the NapAB complex (-150 to -350 mV) is much lower than that reported for R. sphaeroides NapA (midpoint potential Mo(6+/5+) > +350 mV), and the form of the Mo(5+) EPR signal is quite distinct. In E. coli NapA or NapAB, the Mo(5+) state could not be further reduced to Mo(4+). We then propose a catalytic cycle for E. coli NapA in which nitrate binds to the Mo(5+) ion and where a stable des-oxo Mo(6+) species may participate.

  6. A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1

    PubMed Central

    Wang, Yingfei; An, Ran; Umanah, George K.; Park, Hyejin; Nambiar, Kalyani; Eacker, Stephen M.; Kim, BongWoo; Bao, Lei; Harraz, Maged M.; Chang, Calvin; Chen, Rong; Wang, Jennifer E.; Kam, Tae-In; Jeong, Jun Seop; Xie, Zhi; Neifert, Stewart; Qian, Jiang; Andrabi, Shaida A.; Blackshaw, Seth; Zhu, Heng; Song, Hongjun; Ming, Guo-li; Dawson, Valina L.; Dawson, Ted M.

    2016-01-01

    Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF's nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation. PMID:27846469

  7. A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1.

    PubMed

    Wang, Yingfei; An, Ran; Umanah, George K; Park, Hyejin; Nambiar, Kalyani; Eacker, Stephen M; Kim, BongWoo; Bao, Lei; Harraz, Maged M; Chang, Calvin; Chen, Rong; Wang, Jennifer E; Kam, Tae-In; Jeong, Jun Seop; Xie, Zhi; Neifert, Stewart; Qian, Jiang; Andrabi, Shaida A; Blackshaw, Seth; Zhu, Heng; Song, Hongjun; Ming, Guo-Li; Dawson, Valina L; Dawson, Ted M

    2016-10-07

    Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1-dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1-dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF's nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation. Copyright © 2016, American Association for the Advancement of Science.

  8. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Fine, Eli J; Bao, Gang

    2016-01-01

    The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications. PMID:26750397

  9. Cleavage of the HIV replication primer tRNALys,3 in human cells expressing bacterial anticodon nuclease.

    PubMed Central

    Shterman, N; Elroy-Stein, O; Morad, I; Amitsur, M; Kaufmann, G

    1995-01-01

    Anticodon nuclease is a bacterial restriction enzyme directed against tRNA(Lys). We report that anticodon nuclease also cleaves mammalian tRNA(Lys) molecules, with preference and site specificity shown towards the natural substrate. Expression of the anticodon nuclease core polypeptide PrrC in HeLa cells from a recombinant vaccinia virus elicited cleavage of intracellular tRNA(Lys),3. The data justify an inquiry into the possible application of anticodon nuclease as an inhibitor of tRNA(Lys),3-primed HIV replication. They also indicate that the anticodon region of tRNA(Lys) is a substrate recognition site and suggest that PrrC harbors the enzymatic activity. Images PMID:7784179

  10. Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos

    PubMed Central

    2013-01-01

    Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embryos. We also provide details for detection of somatic and germ line transmitted mutations. And finally, we briefly describe establishment of knockout Xenopus lines. This protocol will facilitate broader applications of TALENs in studies of Xenopus biology. PMID:23663889

  11. Studies of interaction between a new synthesized minor-groove targeting artificial nuclease and DNA

    NASA Astrophysics Data System (ADS)

    Yin, Qiang; Zhang, Zhen; Zhao, Yu-Fen

    2007-04-01

    Nuclease plays an important role in molecular biology, such as DNA sequencing. Synthetic polyamide conjugates can be considered as new tool in the selective inhibition of gene expression and as potential drugs in anticancer or antiviral chemotherapy. In this paper, a new synthesized minor-groove targeting artificial nuclease, oligopyrrol-containing peptide, was reported. It was found that this new compound can bind DNA in AT-riched minor groove with high affinity and site specificity. DNA binding behavior was determined by UV-vis and circular dichroism (CD) methods. It was indicated that compound 6 can enhance the Tm of oligomer DNA from 51.8 to 63.5 °C and possesses large binding constant ( Kb = 8.83 × 10 4 L/mol).

  12. An improved zinc-finger nuclease architecture for highly specific genome editing.

    PubMed

    Miller, Jeffrey C; Holmes, Michael C; Wang, Jianbin; Guschin, Dmitry Y; Lee, Ya-Li; Rupniewski, Igor; Beausejour, Christian M; Waite, Adam J; Wang, Nathaniel S; Kim, Kenneth A; Gregory, Philip D; Pabo, Carl O; Rebar, Edward J

    2007-07-01

    Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

  13. Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases.

    PubMed

    Yoshida, Keita; Treen, Nicholas; Hozumi, Akiko; Sakuma, Tetsushi; Yamamoto, Takashi; Sasakura, Yasunori

    2014-05-01

    Targeted mutagenesis of genes-of-interest, or gene-knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis.

  14. Detection of Non-B-DNA Secondary Structures by S1 Nuclease Digestion

    NASA Astrophysics Data System (ADS)

    Del Olmo, Marcel. Li; Aranda, Agustin; Perez-Ortin, Jose E.; Tordera, Vicente

    1998-06-01

    In nature, almost all DNA strands are supercoiled in both prokaryotic and eukaryotic cells. Here, we present two cheap and simple laboratory experiments to analyze the different topological states of DNA and, simultaneously, to detect denatured regions and cruciforms in vitro, using the single-strand specific S1 nuclease. A natural (A+T)-rich region of the 3' region of Saccharomyces cerevisiae FBP1 gene and a DNA (A+T)-rich region in pUC plasmids around the terminator of the ampicillin resistance gene (both capable of undergoing supercoiling-dependent denaturation and therefore sensitive to S1 nuclease) have been used in the experiments. Experimental costs are low, and the small amounts of chemicals and the laboratory equipment used are available in every laboratory.

  15. Generation of gene disruptions by transcription activator-like effector nucleases (TALENs) in Xenopus tropicalis embryos.

    PubMed

    Lei, Yong; Guo, Xiaogang; Deng, Yi; Chen, Yonglong; Zhao, Hui

    2013-05-10

    Transcription activator-like effector nucleases (TALENs) are novel engineered DNA nucleases, and have been proven to be effective for gene specific targeting in various species. Recently we reported gene disruptions in Xenopus embryos by using TALENs. Here we summarize the protocol that is used in our studies for gene disruption. This protocol covers selection of TALEN targeting sites, TALEN assembly with a modified Golden Gate method, and injection of TALEN mRNAs into Xenopus tropicalis embryos. We also provide details for detection of somatic and germ line transmitted mutations. And finally, we briefly describe establishment of knockout Xenopus lines. This protocol will facilitate broader applications of TALENs in studies of Xenopus biology.

  16. Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases.

    PubMed

    Kasparek, Petr; Krausova, Michaela; Haneckova, Radka; Kriz, Vitezslav; Zbodakova, Olga; Korinek, Vladimir; Sedlacek, Radislav

    2014-11-03

    Gene targeting in mice mainly employs homologous recombination (HR) in embryonic stem (ES) cells. Although it is a standard way for production of genetically modified mice, the procedure is laborious and time-consuming. This study describes targeting of the mouse Rosa26 locus by transcription activator-like effector nucleases (TALENs). We employed TALEN-assisted HR in zygotes to introduce constructs encoding TurboRFP and TagBFP fluorescent proteins into the first intron of the Rosa26 gene, and in this way generated two transgenic mice. We also demonstrated that these Rosa26-specific TALENs exhibit high targeting efficiency superior to that of zinc-finger nucleases (ZFNs) specific for the same targeting sequence. Moreover, we devised a reporter assay to assess TALENs activity and specificity to improve the quality of TALEN-assisted targeting.

  17. Processing of meiotic DNA double strand breaks requires cyclin-dependent kinase and multiple nucleases.

    PubMed

    Manfrini, Nicola; Guerini, Ilaria; Citterio, Andrea; Lucchini, Giovanna; Longhese, Maria Pia

    2010-04-09

    Meiotic recombination requires the formation of programmed Spo11-dependent DNA double strand breaks (DSBs). In Saccharomyces cerevisiae, the Sae2 protein and the Mre11-Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends, which are then resected by so far unknown nucleases. Here, we demonstrate that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5'-3' resection tracts during meiosis by controlling a step subsequent to Spo11 removal.

  18. Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm

    PubMed Central

    El Khatib, Mariam; Martins, Alexandre; Bourgeois, Dominique; Colletier, Jacques-Philippe; Adam, Virgile

    2016-01-01

    Phototransformable fluorescent proteins are central to several nanoscopy approaches. As yet however, there is no available variant allowing super-resolution imaging in cell compartments that maintain oxidative conditions. Here, we report the rational design of two reversibly switchable fluorescent proteins able to fold and photoswitch in the bacterial periplasm, rsFolder and rsFolder2. rsFolder was designed by hybridisation of Superfolder-GFP with rsEGFP2, and inherited the fast folding properties of the former together with the rapid switching of the latter, but at the cost of a reduced switching contrast. Structural characterisation of the switching mechanisms of rsFolder and rsEGFP2 revealed different scenarios for chromophore cis-trans isomerisation and allowed designing rsFolder2, a variant of rsFolder that exhibits improved switching contrast and is amenable to RESOLFT nanoscopy. The rsFolders can be efficiently expressed in the E. coli periplasm, opening the door to the nanoscale investigation of proteins localised in hitherto non-observable cellular compartments. PMID:26732634

  19. NMR assignments of the GacS histidine-kinase periplasmic detection domain from Pseudomonas aeruginosa PAO1.

    PubMed

    Ali-Ahmad, Ahmad; Bornet, Olivier; Fadel, Firas; Bourne, Yves; Vincent, Florence; Bordi, Christophe; Guerlesquin, Françoise; Sebban-Kreuzer, Corinne

    2017-04-01

    Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen. It can infect vulnerable patients such as those with cystic fibrosis or hospitalized in intensive care units where it is responsible for both acute and chronic infection. The switch between these infections is controlled by a complex regulatory system involving the central GacS/GacA two-component system that activates the production of two small non-coding RNAs. GacS is a histidine kinase harboring one periplasmic detection domain, two inner-membrane helices and three H1/D1/H2 cytoplasmic domains. By detecting a yet unknown signal, the GacS histidine-kinase periplasmic detection domain (GacSp) is predicted to play a key role in activating the GacS/GacA pathway. Here, we present the chemical shift assignment of 96 % of backbone atoms (HN, N, C, Cα, Cβ and Hα), 88 % aliphatic hydrogen atoms and 90 % of aliphatic carbon atoms of this domain. The NMR-chemical shift data, on the basis of Talos server secondary structure predictions, reveal that GacSp consists of 3 β-strands, 3 α-helices and a major loop devoid of secondary structures.

  20. Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries

    PubMed Central

    Harvey, Barrett R.; Georgiou, George; Hayhurst, Andrew; Jeong, Ki Jun; Iverson, Brent L.; Rogers, Geoffrey K.

    2004-01-01

    Anchored periplasmic expression (APEx) is a technology for the isolation of ligand-binding proteins from combinatorial libraries anchored on the periplasmic face of the inner membrane of Escherichia coli. After disruption of the outer membrane by Tris-EDTA-lysozyme, the inner-membrane-anchored proteins readily bind fluorescently labeled ligands as large as 240 kDa. Fluorescently labeled cells are isolated by flow cytometry, and the DNA of isolated clones is rescued by PCR. By using two rounds of APEx, the affinity of a neutralizing antibody to the Bacillus anthracis protective antigen was improved >200-fold, exhibiting a final KD of 21 pM. This approach has several technical advantages compared with previous library screening technologies, including the unique ability to screen for ligand-binding proteins that bind endogenously expressed ligands fused to a short-lived GFP. Further, APEx is able to display proteins either as an N-terminal fusion to a six-residue sequence derived from the native E. coli lipoprotein NlpA, or as a C-terminal fusion to the phage gene three minor coat protein of M13. The latter fusions allow hybrid phage display/APEx strategies without the need for further subcloning. PMID:15197275

  1. Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli.

    PubMed

    Khairnar, Nivedita P; Kamble, Vidya A; Mangoli, Suhas H; Apte, Shree K; Misra, Hari S

    2007-07-01

    The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli.

  2. Pectocin M1 (PcaM1) Inhibits Escherichia coli Cell Growth and Peptidoglycan Biosynthesis through Periplasmic Expression.

    PubMed

    Chérier, Dimitri; Giacomucci, Sean; Patin, Delphine; Bouhss, Ahmed; Touzé, Thierry; Blanot, Didier; Mengin-Lecreulx, Dominique; Barreteau, Hélène

    2016-10-08

    Colicins are bacterial toxins produced by some Escherichia coli strains. They exhibit either enzymatic or pore-forming activity towards a very limited number of bacterial species, due to the high specificity of their reception and translocation systems. Yet, we succeeded in making the colicin M homologue from Pectobacterium carotovorum, pectocin M1 (PcaM1), capable of inhibiting E. coli cell growth by bypassing these reception and translocation steps. This goal was achieved through periplasmic expression of this pectocin. Indeed, when appropriately addressed to the periplasm of E. coli, this pectocin could exert its deleterious effects, i.e., the enzymatic degradation of the peptidoglycan lipid II precursor, which resulted in the arrest of the biosynthesis of this essential cell wall polymer, dramatic morphological changes and, ultimately, cell lysis. This result leads to the conclusion that colicin M and its various orthologues constitute powerful antibacterial molecules able to kill any kind of bacterium, once they can reach their lipid II target. They thus have to be seriously considered as promising alternatives to antibiotics.

  3. The periplasmic TorT protein is required for trimethylamine N-oxide reductase gene induction in Escherichia coli.

    PubMed Central

    Jourlin, C; Simon, G; Pommier, J; Chippaux, M; Méjean, V

    1996-01-01

    Expression of the Escherichia coli torCAD operon, which encodes the trimethylamine N-oxide reductase system, is regulated by the presence of trimethylamine N-oxide through the action of the TorR response regulator. We have identified an additional gene, torT, located just downstream from the torR gene, which is necessary for torCAD structural operon expression. Insertion within the torT gene dramatically reduced the expression of a torA'-'lacZ fusion, while presence of the gene in trans restored the wild-type phenotype. Overproduction of TorR in a torT strain resulted in partial constitutive expression of the torA'-'lacZ fusion, suggesting that TorR acts downstream from TorT. The torT gene codes for a 35.7-kDa periplasmic protein which presents some homology with the periplasmic ribose-binding protein of E. coli. We discuss the possible role of TorT as an inducer-binding protein involved in signal transduction of the tor regulatory pathway. PMID:8576063

  4. Genetic and mechanistic analyses of the periplasmic domain of the enterohemorrhagic E. coli (EHEC) QseC histidine sensor kinase.

    PubMed

    Parker, Christopher T; Russell, Regan; Njoroge, Jacqueline W; Jimenez, Angel G; Taussig, Ron; Sperandio, Vanessa

    2017-01-30

    The histidine sensor kinase (HK) QseC, senses autoinducer-3 (AI-3), and the adrenergic hormones epinephrine and norepinephrine. Upon sensing these signals, QseC acts through three response regulators (RRs) to regulate expression of virulence genes in enterohemorrhagic E. coli (EHEC). The QseB, QseF and KdpE RRs that are phosphorylated by QseC constitute a tripartite signaling cascade having different and overlapping targets, including flagella and motility, the type three secretion system encoded by the locus of enterocyte effacement (LEE), and Shiga toxin. We modeled the tertiary structure of QseC's periplasmic sensing domain, and also aligned these sequences from 12 different species to identify the most conserved amino acids. We selected eight conserved aminoacids in all of these QseC homologs. These QseC site directed mutants were expressed and still able to autophosphorylate, albeit four mutants depicted increased basal level of phosphorylation. These mutants have differential flagella and motility, LEE and Shiga toxin expression phenotypes. We selected four mutants for more in depth analyses and found that they differed in their ability to phosphorylate QseB, KdpE and QseF. This suggests that these mutations in the periplasmic sensing domain affected the downstream of the QseC signaling cascade, and therefore, can influence which pathway QseC regulates.

  5. Pectocin M1 (PcaM1) Inhibits Escherichia coli Cell Growth and Peptidoglycan Biosynthesis through Periplasmic Expression

    PubMed Central

    Chérier, Dimitri; Giacomucci, Sean; Patin, Delphine; Bouhss, Ahmed; Touzé, Thierry; Blanot, Didier; Mengin-Lecreulx, Dominique; Barreteau, Hélène

    2016-01-01

    Colicins are bacterial toxins produced by some Escherichia coli strains. They exhibit either enzymatic or pore-forming activity towards a very limited number of bacterial species, due to the high specificity of their reception and translocation systems. Yet, we succeeded in making the colicin M homologue from Pectobacterium carotovorum, pectocin M1 (PcaM1), capable of inhibiting E. coli cell growth by bypassing these reception and translocation steps. This goal was achieved through periplasmic expression of this pectocin. Indeed, when appropriately addressed to the periplasm of E. coli, this pectocin could exert its deleterious effects, i.e., the enzymatic degradation of the peptidoglycan lipid II precursor, which resulted in the arrest of the biosynthesis of this essential cell wall polymer, dramatic morphological changes and, ultimately, cell lysis. This result leads to the conclusion that colicin M and its various orthologues constitute powerful antibacterial molecules able to kill any kind of bacterium, once they can reach their lipid II target. They thus have to be seriously considered as promising alternatives to antibiotics. PMID:27740593

  6. Topological plasticity of enzymes involved in disulfide bond formation allows catalysis in either the periplasm or the cytoplasm.

    PubMed

    Hatahet, Feras; Ruddock, Lloyd W

    2013-09-23

    The transmembrane enzymes disulfide bond forming enzyme B (DsbB) and vitamin K epoxide reductase (VKOR) are central to oxidative protein folding in the periplasm of prokaryotes. Catalyzed formation of structural disulfide bonds in proteins also occurs in the cytoplasm of some hyperthermophilic prokaryotes through currently, poorly defined mechanisms. We aimed to determine whether DsbB and VKOR can be inverted in the membrane with retention of activity. By rational design of inversion of membrane topology, we engineered DsbB mutants that catalyze disulfide bond formation in the cytoplasm of Escherichia coli. This represents the first engineered inversion of a transmembrane protein with demonstrated conservation of activity and substrate specificity. This successful designed engineering led us to identify two naturally occurring and oppositely oriented VKOR homologues from the hyperthermophile Aeropyrum pernix that promote oxidative protein folding in the periplasm or cytoplasm, respectively, and hence defines the probable route for disulfide bond formation in the cytoplasm of hyperthermophiles. Our findings demonstrate how knowledge on the determinants of membrane protein topology can be used to de novo engineer a metabolic pathway and to unravel an intriguingly simple evolutionary scenario where a new "adaptive" cellular process is constructed by means of membrane protein topology inversion.

  7. The history and market impact of CRISPR RNA-guided nucleases.

    PubMed

    van Erp, Paul Bg; Bloomer, Gary; Wilkinson, Royce; Wiedenheft, Blake

    2015-06-01

    The interface between viruses and their hosts' are hot spots for biological and biotechnological innovation. Bacteria use restriction endonucleases to destroy invading DNA, and industry has exploited these enzymes for molecular cut-and-paste reactions that are central to many recombinant DNA technologies. Today, another class of nucleases central to adaptive immune systems that protect bacteria and archaea from invading viruses and plasmids are blazing a similar path from basic science to profound biomedical and industrial applications.

  8. Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects.

    PubMed

    Soucek, J; Skvor, J; Poucková, P; Matousek, J; Slavík, T; Matousek, J

    2006-01-01

    Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease (RNase A), is known to display special biological activities namely cytotoxicity for human tumor cells. Because some plant ribonucleases have a similar mass weight and structure as the animal ribonuclease, effects of a commercial product of Mung bean (Phaseolus aureus) nuclease (PhA) were studied on proliferation of ML-2 human tumor cells, as well as it's aspermatogenic, embryotoxic, immunogenic, and immunosuppressive activity, and therapeutic efficiency in athymic mice bearing human melanoma tumor. Concerning the antiproliferative activity, PhA nuclease was almost non-effective in vitro on ML-2 cells and also immunosuppressive activity on human lymphocyte in mixed culture was very low compared to that of BS RNase. However, significant antitumor activity was detected on human melanoma tumor after intratumoral or intraperitoneal administration into the mice. Furthermore conjugate of PhA nuclease with polyethylene glycol (PEG) injected seven times at the dose of 10 microg intraperitoneally showed identical antitumor activity as that of bovine seminal ribonuclease (BS RNase) injected by the same way at ten times higher dose. Both PhA and BS RNases exerted strong aspermatogenic effect on the width of spermatogenic layers while RNase A administration at ten times higher concentration was ineffective. PhA nuclease when compared by means of antibody cross reaction with RNase A, BS RNase and wheat leaf neutral RNase (WLN-RNase) was found to be immunologically similar to RNase A and WLN-RNase, meanwhile BS RNase showed much higher antigenicity in comparison with them.

  9. Orthogonal gene knock out and activation with a catalytically active Cas9 nuclease

    PubMed Central

    Dahlman, James E.; Abudayyeh, Omar O.; Joung, Julia; Gootenberg, Jonathan S.; Zhang, Feng; Konermann, Silvana

    2015-01-01

    We have developed a CRISPR-based method that uses catalytically active Cas9 and distinct sgRNA constructs to knock out and activate different genes in the same cell. These sgRNAs, with 14 15 bp target sequences and MS2 binding loops, can activate gene expression using an active Cas9 nuclease, without inducing DSBs. We use these ‘dead RNAs’ to perform orthogonal gene knockout and transcriptional activation in human cells. PMID:26436575

  10. A Novel Nuclease Activity that is Activated by Ca2+ Chelated to EGTA

    PubMed Central

    Dominguez, Kenneth; Ward, W. Steven

    2010-01-01

    Most nucleases require a divalent cation as a cofactor, usually Mg2+ or Ca2+, and are inhibited by the chelators EDTA and EGTA. We report the existence of a novel nuclease activity, initially identified in the luminal fluids of the mouse male reproductive tract but subsequently found in other tissues, that requires EGTA chelated to calcium to digest DNA. We refer to this unique enzyme as CEAN (Chelated EGTA Activated Nuclease). Using a fraction of vas deferens luminal fluid, plasmid DNA was degraded in the presence of excess Ca2+ (Ca2+:EGTA = 16) or excess EGTA (Ca2+:EGTA = 0.25), but required the presence of both. Higher levels of EGTA (Ca2+:EGTA = 0.10) prevented activity, suggesting that unchelated EGTA may be a competitive inhibitor. The EGTA-Ca2+ activation of CEAN is reversible as removing EGTA-Ca2+ stops ongoing DNA degradation, but adding EGTA-Ca2+ again reactivates the enzyme. This suggests the possibility that CEAN binds directly to EGTA-Ca2+. CEAN has a greater specificity for the chelator than for the divalent cation. Two other chelators, BAPTA and sodium citrate, do not activate CEAN in the presence of cation, but chelated EDTA does. EGTA chelated to other divalent cations such as Mn2+, Zn2+, and Cu2+ activate CEAN, but not Mg2+. The activity is lost upon boiling suggesting that it is a protein. These data suggest that EGTA and EDTA may not always prevent DNA from nuclease damage. PMID:19938954

  11. The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis.

    PubMed

    Ipsaro, Jonathan J; Haase, Astrid D; Knott, Simon R; Joshua-Tor, Leemor; Hannon, Gregory J

    2012-11-08

    PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism to provide essential protection for germ-cell genomes against the activity of mobile genetic elements. piRNA populations comprise a molecular definition of transposons, which permits them to distinguish transposons from host genes and selectively silence them. piRNAs can be generated in two distinct ways, forming either primary or secondary piRNAs. Primary piRNAs come from discrete genomic loci, termed piRNA clusters, and seem to be derived from long, single-stranded precursors. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are probably formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner. Secondary piRNAs arise during the adaptive 'ping-pong' cycle, with their 5' termini being formed by the activity of PIWIs themselves. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Drosophila melanogaster Zucchini, is a member of the phospholipase-D family of phosphodiesterases, which includes both phospholipases and nucleases. Here we produced a dimeric, soluble fragment of the mouse Zucchini homologue (mZuc; also known as PLD6) and show that it possesses single-strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to phospholipase-D family nucleases than to phospholipases. Together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs.

  12. Enhancement of nuclease P1 production by Penicillium citrinum YL104 immobilized on activated carbon filter sponge.

    PubMed

    Zhao, Nan; Ren, Hengfei; Li, Zhenjian; Zhao, Ting; Shi, Xinchi; Cheng, Hao; Zhuang, Wei; Chen, Yong; Ying, Hanjie

    2015-02-01

    The efficiency of current methods for industrial production of the enzyme nuclease P1 is limited. In this study, we sought to improve fermentation methods for the production of nuclease P1. An immobilized fermentation system using an activated carbon filter sponge as a carrier was used for the production of nuclease P1. In an airlift internal loop reactor (ALR), the fermentation performance of three different fermentation modes, including free-cell fermentation, repeated-batch fermentation, and semi-continuous immobilized fermentation, were compared. The fermentation kinetics in the fermentation broth of the three fermentation modes, including dissolved oxygen (DO), pH value, cell concentration, residual sugar concentration, and enzyme activity, were tested. The productivity of semi-continuous immobilized fermentation reached 8.76 U/mL/h, which was 33.3 and 80.2% higher than that of repeated-batch fermentation and free-cell fermentation, respectively. The sugar consumption of free-cell, repeated-batch, and semi-continuous immobilized fermentations was 41.2, 30.8, and 25.9 g/L, respectively. These results showed that immobilized-cell fermentation by using Penicillium citrinum with activated carbon filter sponge in an ALR was advantageous for nuclease P1 production, especially in the semi-continuous immobilized fermentation mode. In spite of the significant improvement in nuclease P1 production in semi-continuous immobilized fermentation mode, the specific activity of nuclease P1 was almost equal among the three fermentation modes.

  13. Crystal Structure of the First Eubacterial Mre11 Nuclease Reveals Novel Features that may Discriminate Substrates During DNA Repair

    PubMed Central

    Das, Debanu; Moiani, Davide; Axelrod, Herbert L.; Miller, Mitchell D.; McMullan, Daniel; Jin, Kevin K.; Abdubek, Polat; Astakhova, Tamara; Burra, Prasad; Carlton, Dennis; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Ernst, Dustin; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Grzechnik, Slawomir K.; Han, Gye Won; Jaroszewski, Lukasz; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Kumar, Abhinav; Marciano, David; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Paulsen, Jessica; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; van den Bedem, Henry; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Tainer, John A.; Wilson, Ian A.

    2010-01-01

    Mre11 nuclease plays a central role in the repair of cytotoxic and mutagenic DNA double-strand breaks (DSBs). As x-ray structural information has only been available for the Pyrococcus furiosus enzyme (PfMre11), the conserved and variable features of this nuclease across the domains of life have not been experimentally defined. Our crystal structure and biochemical studies demonstrate that TM1635 from Thermotoga maritima, originally annotated as a putative nuclease, is the Mre11 endo/exonuclease from T. maritima (TmMre11) and the first such structure from eubacteria. TmMre11 and PfMre11 display similar overall structures, despite sequence identity in the twilight zone of only ∼20%. However, they differ substantially in their DNA specificity domains and in their dimeric organization. Residues in the nuclease domain are highly conserved, but those in the DNA specificity domain are not. The structural differences likely affect how Mre11s from different organisms recognize and interact with single-stranded DNA, double-stranded DNA and DNA hairpin structures during DNA repair. The TmMre11 nuclease active site has no bound metal ions, but is conserved in sequence and structure with exception of a histidine that is important in PfMre11 nuclease activity. Nevertheless, biochemical characterization confirms that TmMre11 possesses both endonuclease and exonuclease activities on ssDNA and dsDNA substrates, respectively. PMID:20122942

  14. New insights into the assembly of bacterial secretins: structural studies of the periplasmic domain of XcpQ from Pseudomonas aeruginosa.

    PubMed

    Van der Meeren, Ruben; Wen, Yurong; Van Gelder, Patrick; Tommassen, Jan; Devreese, Bart; Savvides, Savvas N

    2013-01-11

    The type II secretion system is a multiprotein assembly spanning the inner and outer membranes in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization, and infection. The exoproteins are secreted across the outer membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration-dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of type II secretion system secretins and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.

  15. Virulence regulation with Venus flytrap domains: structure and function of the periplasmic moiety of the sensor-kinase BvgS.

    PubMed

    Dupré, Elian; Herrou, Julien; Lensink, Marc F; Wintjens, René; Vagin, Alexey; Lebedev, Andrey; Crosson, Sean; Villeret, Vincent; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2015-03-01

    Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions.

  16. Virulence Regulation with Venus Flytrap Domains: Structure and Function of the Periplasmic Moiety of the Sensor-Kinase BvgS

    PubMed Central

    Lensink, Marc F.; Wintjens, René; Vagin, Alexey; Lebedev, Andrey; Crosson, Sean; Villeret, Vincent; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2015-01-01

    Two-component systems (TCS) represent major signal-transduction pathways for adaptation to environmental conditions, and regulate many aspects of bacterial physiology. In the whooping cough agent Bordetella pertussis, the TCS BvgAS controls the virulence regulon, and is therefore critical for pathogenicity. BvgS is a prototypical TCS sensor-kinase with tandem periplasmic Venus flytrap (VFT) domains. VFT are bi-lobed domains that typically close around specific ligands using clamshell motions. We report the X-ray structure of the periplasmic moiety of BvgS, an intricate homodimer with a novel architecture. By combining site-directed mutagenesis, functional analyses and molecular modeling, we show that the conformation of the periplasmic moiety determines the state of BvgS activity. The intertwined structure of the periplasmic portion and the different conformation and dynamics of its mobile, membrane-distal VFT1 domains, and closed, membrane-proximal VFT2 domains, exert a conformational strain onto the transmembrane helices, which sets the cytoplasmic moiety in a kinase-on state by default corresponding to the virulent phase of the bacterium. Signaling the presence of negative signals perceived by the periplasmic domains implies a shift of BvgS to a distinct state of conformation and activity, corresponding to the avirulent phase. The response to negative modulation depends on the integrity of the periplasmic dimer, indicating that the shift to the kinase-off state implies a concerted conformational transition. This work lays the bases to understand virulence regulation in Bordetella. As homologous sensor-kinases control virulence features of diverse bacterial pathogens, the BvgS structure and mechanism may pave the way for new modes of targeted therapeutic interventions. PMID:25738876

  17. Bioinformatics analysis of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica.

    PubMed

    Li, Zhen-Hua; Tang, Zhen-Xing; Fang, Xiu-Juan; Zhang, Zhi-Liang; Shi, Lu-E

    2013-12-01

    In this paper, the physical and chemical characteristics, biological structure and function of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica (Y. NSN) found in our group were studied using multiple bioinformatics approaches. The results showed that Y. NSN had 283 amino acids, a weight of 30,692.5 ku and a certain hydrophilic property. Y. NSN had a signal peptide, no transmembrane domains and disulphide bonds. Cleavage site in Y. NSN was between pos. 23 and 24. The prediction result of the secondary structure showed Y. NSN was a coil structure-based protein. The ratio of α-helix, β-folded and random coil were 18.73%, 16.96% and 64.31%, respectively. Active sites were pos. 124, 125, 127, 157, 165 and 169. Mg(2+) binding site was pos. 157. Substrate binding sites were pos. 124, 125 and 169. The analysis of multisequencing alignment and phylogenetic tree indicated that Y. NSN shared high similarity with the nuclease from Y. enterocolitica subsp. enterocolitica 8081. The enzyme activity results showed that Y. NSN was a nuclease with good thermostability.

  18. Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae

    SciTech Connect

    Moon, Andrea F.; Gaudu, Philippe; Pedersen, Lars C.

    2014-11-01

    The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae , facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. Lastly, these structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.

  19. Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae

    DOE PAGES

    Moon, Andrea F.; Gaudu, Philippe; Pedersen, Lars C.

    2014-11-01

    The group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor for S. agalactiae , facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structuremore » of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. Lastly, these structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted to S. agalactiae.« less

  20. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.

    PubMed

    Mock, Ulrike; Hauber, Ilona; Fehse, Boris

    2016-03-01

    Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d.

  1. Applications of genome editing by programmable nucleases to the metabolic engineering of secondary metabolites.

    PubMed

    Leitão, Ana Lúcia; Costa, Marina C; Enguita, Francisco J

    2017-01-10

    Genome engineering is a branch of modern biotechnology composed of a cohort of protocols designed to construct and modify a genotype with the main objective of giving rise to a desired phenotype. Conceptually, genome engineering is based on the so called genome editing technologies, a group of genetic techniques that allow either to delete or to insert genetic information in a particular genomic locus. Ten years ago, genome editing tools were limited to virus-driven integration and homologous DNA recombination. However, nowadays the uprising of programmable nucleases is rapidly changing this paradigm. There are two main families of modern tools for genome editing depending on the molecule that controls the specificity of the system and drives the editor machinery to its place of action. Enzymes such as Zn-finger and TALEN nucleases are protein-driven genome editors; while CRISPR system is a nucleic acid-guided editing system. Genome editing techniques are still not widely applied for the design of new compounds with pharmacological activity, but they are starting to be considered as promising tools for rational genome manipulation in biotechnology applications. In this review we will discuss the potential applications of programmable nucleases for the metabolic engineering of secondary metabolites with biological activity.

  2. Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases.

    PubMed

    Butler, Nathaniel M; Baltes, Nicholas J; Voytas, Daniel F; Douches, David S

    2016-01-01

    Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species.

  3. Comprehensive analysis of the specificity of transcription activator-like effector nucleases.

    PubMed

    Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien; Thomas, Séverine; Stella, Stefano; Maréchal, Alan; Langevin, Stéphanie; Benomari, Nassima; Bertonati, Claudia; Silva, George H; Daboussi, Fayza; Epinat, Jean-Charles; Montoya, Guillermo; Duclert, Aymeric; Duchateau, Philippe

    2014-04-01

    A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.

  4. Recent developments and clinical studies utilizing engineered zinc finger nuclease technology.

    PubMed

    Jo, Young-Il; Kim, Hyongbum; Ramakrishna, Suresh

    2015-10-01

    Efficient methods for creating targeted genetic modifications have long been sought for the investigation of gene function and the development of therapeutic modalities for various diseases, including genetic disorders. Although such modifications are possible using homologous recombination, the efficiency is extremely low. Zinc finger nucleases (ZFNs) are custom-designed artificial nucleases that make double-strand breaks at specific sequences, enabling efficient targeted genetic modifications such as corrections, additions, gene knockouts and structural variations. ZFNs are composed of two domains: (i) a DNA-binding domain comprised of zinc finger modules and (ii) the FokI nuclease domain that cleaves the DNA strand. Over 17 years after ZFNs were initially developed, a number of improvements have been made. Here, we will review the developments and future perspectives of ZFN technology. For example, ZFN activity and specificity have been significantly enhanced by modifying the DNA-binding domain and FokI cleavage domain. Advances in culture methods, such as the application of a cold shock and the use of small molecules that affect ZFN stability, have also increased ZFN activity. Furthermore, ZFN-induced mutant cells can be enriched using episomal surrogate reporters. Additionally, we discuss several ongoing clinical studies that are based on ZFN-mediated genome editing in humans. These breakthroughs have substantially facilitated the use of ZFNs in research, medicine and biotechnology.

  5. Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia.

    PubMed

    Crossan, Gerry P; van der Weyden, Louise; Rosado, Ivan V; Langevin, Frederic; Gaillard, Pierre-Henri L; McIntyre, Rebecca E; Gallagher, Ferdia; Kettunen, Mikko I; Lewis, David Y; Brindle, Kevin; Arends, Mark J; Adams, David J; Patel, Ketan J

    2011-02-01

    The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway.

  6. Geminivirus-Mediated Genome Editing in Potato (Solanum tuberosum L.) Using Sequence-Specific Nucleases

    PubMed Central

    Butler, Nathaniel M.; Baltes, Nicholas J.; Voytas, Daniel F.; Douches, David S.

    2016-01-01

    Genome editing using sequence-specific nucleases (SSNs) is rapidly being developed for genetic engineering in crop species. The utilization of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated systems (CRISPR/Cas) for inducing double-strand breaks facilitates targeting of virtually any sequence for modification. Targeted mutagenesis via non-homologous end-joining (NHEJ) has been demonstrated extensively as being the preferred DNA repair pathway in plants. However, gene targeting via homologous recombination (HR) remains more elusive but could be a powerful tool for directed DNA repair. To overcome barriers associated with gene targeting, a geminivirus replicon (GVR) was used to deliver SSNs targeting the potato ACETOLACTATE SYNTHASE1 (ALS1) gene and repair templates designed to incorporate herbicide-inhibiting point mutations within the ALS1 locus. Transformed events modified with GVRs held point mutations that were capable of supporting a reduced herbicide susceptibility phenotype, while events transformed with conventional T-DNAs held no detectable mutations and were similar to wild-type. Regeneration of transformed events improved detection of point mutations that supported a stronger reduced herbicide susceptibility phenotype. These results demonstrate the use of geminiviruses for delivering genome editing reagents in plant species, and a novel approach to gene targeting in a vegetatively propagated species. PMID:27493650

  7. Purification and characterization of a novel human acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase.

    PubMed

    Famulski, K S; Liuzzi, M; Bashir, S; Mirzayans, R; Paterson, M C

    2000-02-01

    A novel N-glycosylated, mannose-rich protein has been purified approx. 4000-fold from human liver in a seven-step procedure including ion-exchange chromatography and fractionation on concanavalin A-Sepharose, Sephadex G-75 and oligo(dT)-cellulose matrices. The molecular mass of the protein is 46 kDa when measured by gel filtration (i.e. under non-denaturing conditions) and 60 kDa by SDS/PAGE (i.e. under denaturing conditions). The protein possesses two DNA backbone-incising activities, namely, the random introduction of single-strand breaks in native DNA and the rupture of the phosphodiester linkage internal to cyclobutyl pyrimidine dimers, the major class of DNA lesions induced by solar UV rays. Both activities are optimal at pH 5.0 in vitro, although the non-specific nuclease displays appreciable activity at neutral pH, depending on the buffer composition. The protein has been named acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase (AN/IDP). As a nuclease, the protein 'prefers' a linear DNA structure over a covalently closed circular molecule and is more proficient at digesting single-stranded than double-stranded DNA. The polynucleotide cleavage products of the nuclease contain 5'-OH and 3'-PO(4) termini, which are refractory to direct rejoining by DNA ligases. Depending on the substrate, the nuclease activity exhibits a temperature optimum of 50 degrees C or greater, and is neither stimulated by Mg(2+) or Ca(2+) nor inhibited by Zn(2+). AN/IDP is present in human liver and in cultured human cells of both fibroblastic and lymphocytic origins. Intracellularly, the protein can be readily detected in both the cytosolic and nuclear fractions, although much more (approx. 3-fold) is found in the latter fraction. We propose that this bifunctional enzyme may be involved in both apoptotic DNA digestion and metabolism of cyclobutyl pyrimidine dimers in UV-irradiated human cells.

  8. Purification and characterization of a novel human acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase.

    PubMed Central

    Famulski, K S; Liuzzi, M; Bashir, S; Mirzayans, R; Paterson, M C

    2000-01-01

    A novel N-glycosylated, mannose-rich protein has been purified approx. 4000-fold from human liver in a seven-step procedure including ion-exchange chromatography and fractionation on concanavalin A-Sepharose, Sephadex G-75 and oligo(dT)-cellulose matrices. The molecular mass of the protein is 46 kDa when measured by gel filtration (i.e. under non-denaturing conditions) and 60 kDa by SDS/PAGE (i.e. under denaturing conditions). The protein possesses two DNA backbone-incising activities, namely, the random introduction of single-strand breaks in native DNA and the rupture of the phosphodiester linkage internal to cyclobutyl pyrimidine dimers, the major class of DNA lesions induced by solar UV rays. Both activities are optimal at pH 5.0 in vitro, although the non-specific nuclease displays appreciable activity at neutral pH, depending on the buffer composition. The protein has been named acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase (AN/IDP). As a nuclease, the protein 'prefers' a linear DNA structure over a covalently closed circular molecule and is more proficient at digesting single-stranded than double-stranded DNA. The polynucleotide cleavage products of the nuclease contain 5'-OH and 3'-PO(4) termini, which are refractory to direct rejoining by DNA ligases. Depending on the substrate, the nuclease activity exhibits a temperature optimum of 50 degrees C or greater, and is neither stimulated by Mg(2+) or Ca(2+) nor inhibited by Zn(2+). AN/IDP is present in human liver and in cultured human cells of both fibroblastic and lymphocytic origins. Intracellularly, the protein can be readily detected in both the cytosolic and nuclear fractions, although much more (approx. 3-fold) is found in the latter fraction. We propose that this bifunctional enzyme may be involved in both apoptotic DNA digestion and metabolism of cyclobutyl pyrimidine dimers in UV-irradiated human cells. PMID:10642517

  9. Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex

    SciTech Connect

    Sampaleanu, L.M.; Bonanno, J.B.; Ayers, M.; Koo, J.; Tammam, S.; Burley, S.K.; Almo, S.C.; Burrows, L.L.; Howell, P.L.

    2010-01-12

    Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 {angstrom} resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.

  10. The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor.

    PubMed

    Kaspar, S; Perozzo, R; Reinelt, S; Meyer, M; Pfister, K; Scapozza, L; Bott, M

    1999-08-01

    The two-component regulatory system CitA/CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain flanked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE-CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327-547) showed constitutive autokinase activity and transferred the gamma-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB-D56N formed a stable complex with MalE-CitAC. To analyse the sensory properties of CitA, the periplasmic domain (amino acids 45-176) was overproduced as a soluble, cytoplasmic protein with a C-terminally attached histidine tag (CitAPHis). Purified CitAPHis bound citrate, but none of the other tri- and dicarboxylates tested, with high affinity (KD approximately 5 microM at pH 7) in a 1:1 stoichiometry. As shown by isothermal titration calorimetry, the binding reaction was driven by the enthalpy change (DeltaH = -76.3 kJ mol-1), whereas the entropy change was opposed (-TDeltaS = + 46.3 kJ mol-1). The pH dependency of the binding reaction indicated that the dianionic form H-citrate2- is the citrate species recognized by CitAPHis. In the presence of Mg2+ ions, the dissociation constant increased significantly, suggesting that the Mg-citrate complex is not bound by CitAPHis. This work defines the periplasmic domain of CitA as a highly specific citrate receptor and elucidates the binding characteristics of CitAPHis.

  11. Periplasmic Electron Transfer via the c-Type Cytochromes MtrA and FccA of Shewanella oneidensis MR-1 ▿

    PubMed Central

    Schuetz, Bjoern; Schicklberger, Marcus; Kuermann, Johannes; Spormann, Alfred M.; Gescher, Johannes

    2009-01-01

    Dissimilatory microbial reduction of insoluble Fe(III) oxides is a geochemically and ecologically important process which involves the transfer of cellular, respiratory electrons from the cytoplasmic membrane to insoluble, extracellular, mineral-phase electron acceptors. In this paper evidence is provided for the function of the periplasmic fumarate reductase FccA and the decaheme c-type cytochrome MtrA in periplasmic electron transfer reactions in the gammaproteobacterium Shewanella oneidensis. Both proteins are abundant in the periplasm of ferric citrate-reducing S. oneidensis cells. In vitro fumarate reductase FccA and c-type cytochrome MtrA were reduced by the cytoplasmic membrane-bound protein CymA. Electron transfer between CymA and MtrA was 1.4-fold faster than the CymA-catalyzed reduction of FccA. Further experiments showing a bidirectional electron transfer between FccA and MtrA provided evidence for an electron transfer network in the periplasmic space of S. oneidensis. Hence, FccA could function in both the electron transport to fumarate and via MtrA to mineral-phase Fe(III). Growth experiments with a ΔfccA deletion mutant suggest a role of FccA as a transient electron storage protein. PMID:19837833

  12. Motor Rotation Is Essential for the Formation of the Periplasmic Flagellar Ribbon, Cellular Morphology, and Borrelia burgdorferi Persistence within Ixodes scapularis Tick and Murine Hosts

    PubMed Central

    Sultan, Syed Z.; Sekar, Padmapriya; Zhao, Xiaowei; Manne, Akarsh; Liu, Jun; Wooten, R. Mark

    2015-01-01

    Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ΔmotB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ΔmotB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ΔmotB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts. PMID:25690096

  13. Characterization of a c-type heme-containing PAS sensor domain from Geobacter sulfurreducens representing a novel family of periplasmic sensors in Geobacteraceae and other bacteria.

    PubMed

    Londer, Yuri Y; Dementieva, Irina S; D'Ausilio, Cori A; Pokkuluri, P Raj; Schiffer, Marianne

    2006-05-01

    Geobacter sulfurreducens encodes one of the largest numbers of proteins annotated as parts of the two-component signal transduction and/or chemotaxis pathways. Ten of these signal transducers have homologous periplasmic sensor domains that contain the sequence signature for c-type hemes. One such sensor domain encoded by gene GSU0303 was isolated and characterized. The protein was expressed in Escherichia coli and was isolated as two colored species (green and red). The green species is a monomer of the sensor domain with a five-coordinated high-spin heme and the red species is probably a noncovalent dimer of the sensor domain which might have an uncharacterized ligand bound to the dimer. The UV-VIS spectrum of the green species indicates that it has a c'-type heme, but its structure is predicted to be homologous to CitA, a periplasmic PAS domain that does not contain heme. The GSU0303 sensor domain represents a previously unreported family of PAS-type periplasmic sensor domains that contain c-type hemes; these proteins could be part of an important mechanism for sensing redox potential or small ligands in the periplasm. Homologs to the sensor domains we identified in G. sulfurreducens are observed in various bacteria although they occur in larger numbers in the Geobacteraceae.

  14. MxaJ structure reveals a periplasmic binding protein-like architecture with unique secondary structural elements.

    PubMed

    Myung Choi, Jin; Cao, Thinh-Phat; Wouk Kim, Si; Ho Lee, Kun; Haeng Lee, Sung

    2017-07-01

    MxaJ is a component of type II methanol dehydrogenase (MDH) that mediates electron transfer during methanol oxidation in methanotrophic bacteria. However, little is known about how MxaJ structurally cooperates with MDH and Cytochrome cL . Here, we report for the first time the crystal structure of MxaJ. MxaJ consists of eight α-helices and six β-strands, and resembles the "bi-lobate" folding architecture found in periplasmic binding proteins. Distinctive features of MxaJ include prominent loops and a β-strand around the hinge region supporting the ligand-binding cavity, which might provide a more favorable framework for interacting with proteins rather than small molecules. Proteins 2017; 85:1379-1386. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. 3D structure of AcrB: the archetypal multidrug efflux transporter of Escherichia coli likely captures substrates from periplasm.

    PubMed

    Elkins, Christopher A; Nikaido, Hiroshi

    2003-02-01

    Recent advances in structural biology have extended our understanding of the multiple drug efflux complex, AcrAB-TolC, of Escherichia coli. This tripartite complex and its homologs are the major mechanisms that give most Gram-negative bacteria their characteristic intrinsic resistance to a variety of lipophilic drugs, dyes, and detergents. Most recently, the structure of the transporter AcrB was elucidated at high resolution [Nature 419(2002)587]. It is a particularly significant achievement since integral membrane proteins are notoriously elusive structures for crystallography. The striking features of this trimeric pump, such as the presence of potential substrate-binding sites in the periplasmic domain and the possibility of direct interaction with the end of TolC tunnel, refine our understanding of the mode of action of this tripartite efflux transport complex.

  16. Histidine 416 of the periplasmic binding protein NikA is essential for nickel uptake in Escherichia coli.

    PubMed

    Cavazza, Christine; Martin, Lydie; Laffly, Emmanuelle; Lebrette, Hugo; Cherrier, Mickaël V; Zeppieri, Laura; Richaud, Pierre; Carrière, Marie; Fontecilla-Camps, Juan C

    2011-02-18

    Escherichia coli require nickel for the synthesis of [NiFe] hydrogenases under anaerobic growth conditions. Nickel import depends on the specific ABC-transporter NikABCDE encoded by the nik operon, which deletion causes the complete abolition of hydrogenase activity. We have previously postulated that the periplasmic binding protein NikA binds a natural metallophore containing three carboxylate functions that coordinate a Ni(II) ion, the fourth ligand being His416, the only direct metal-protein contact, completing a square-planar coordination for the metal. The crystal structure of the H416I mutant showed no electron density corresponding to a metal-chelator complex. In vivo experiments indicate that the mutation causes a significant decrease in nickel uptake and hydrogenase activity. These results confirm the essential role of His416 in nickel transport by NikA.

  17. Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB.

    PubMed

    Javelle, Arnaud; Lupo, Domenico; Ripoche, Pierre; Fulford, Tim; Merrick, Mike; Winkler, Fritz K

    2008-04-01

    The conduction mechanism of Escherichia coli AmtB, the structurally and functionally best characterized representative of the ubiquitous Amt/Rh family, has remained controversial in several aspects. The predominant view has been that it facilitates the movement of ammonium in its uncharged form as indicated by the hydrophobic nature of a pore located in the center of each subunit of the homotrimer. Using site-directed mutagenesis and a combination of biochemical and crystallographic methods, we have investigated mechanistic questions concerning the putative periplasmic ammonium ion binding site S1 and the adjacent periplasmic "gate" formed by two highly conserved phenylalanine residues, F107 and F215. Our results challenge models that propose that NH(4)(+) deprotonation takes place at S1 before NH(3) conduction through the pore. The presence of S1 confers two critical features on AmtB, both essential for its function: ammonium scavenging efficiency at very low ammonium concentration and selectivity against water and physiologically important cations. We show that AmtB activity absolutely requires F215 but not F107 and that removal or obstruction of the phenylalanine gate produces an open but inactive channel. The phenyl ring of F215 must thus play a very specific role in promoting transfer and deprotonation of substrate from S1 to the central pore. We discuss these results with respect to three distinct mechanisms of conduction that have been considered so far. We conclude that substrate deprotonation is an essential part of the conduction mechanism, but we do not rule out net electrogenic transport.

  18. Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB

    PubMed Central

    Javelle, Arnaud; Lupo, Domenico; Ripoche, Pierre; Fulford, Tim; Merrick, Mike; Winkler, Fritz K.

    2008-01-01

    The conduction mechanism of Escherichia coli AmtB, the structurally and functionally best characterized representative of the ubiquitous Amt/Rh family, has remained controversial in several aspects. The predominant view has been that it facilitates the movement of ammonium in its uncharged form as indicated by the hydrophobic nature of a pore located in the center of each subunit of the homotrimer. Using site-directed mutagenesis and a combination of biochemical and crystallographic methods, we have investigated mechanistic questions concerning the putative periplasmic ammonium ion binding site S1 and the adjacent periplasmic “gate” formed by two highly conserved phenylalanine residues, F107 and F215. Our results challenge models that propose that NH4+ deprotonation takes place at S1 before NH3 conduction through the pore. The presence of S1 confers two critical features on AmtB, both essential for its function: ammonium scavenging efficiency at very low ammonium concentration and selectivity against water and physiologically important cations. We show that AmtB activity absolutely requires F215 but not F107 and that removal or obstruction of the phenylalanine gate produces an open but inactive channel. The phenyl ring of F215 must thus play a very specific role in promoting transfer and deprotonation of substrate from S1 to the central pore. We discuss these results with respect to three distinct mechanisms of conduction that have been considered so far. We conclude that substrate deprotonation is an essential part of the conduction mechanism, but we do not rule out net electrogenic transport. PMID:18362341

  19. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

    SciTech Connect

    Chacon, Kelly N.; Mealman, Tiffany D.; McEvoy, Megan M.; Blackburn, Ninian J.

    2014-10-13

    Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host–pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. In this paper, we use selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a “switch” role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Finally, our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.

  20. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

    DOE PAGES

    Chacon, Kelly N.; Mealman, Tiffany D.; McEvoy, Megan M.; ...

    2014-10-13

    Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host–pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. In this paper, we use selenomethionine (SeM) active site labelsmore » in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a “switch” role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Finally, our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.« less

  1. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

    PubMed Central

    Mealman, Tiffany D.; McEvoy, Megan M.; Blackburn, Ninian J.

    2014-01-01

    Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host–pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. Here, we use selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a “switch” role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm. PMID:25313055

  2. Involvement of Agrobacterium tumefaciens Galacturonate Tripartite ATP-Independent Periplasmic (TRAP) Transporter GaaPQM in Virulence Gene Expression

    PubMed Central

    Zhao, Jinlei

    2015-01-01

    Monosaccharides capable of serving as nutrients for the soil bacterium Agrobacterium tumefaciens are also inducers of the vir regulon present in the tumor-inducing (Ti) plasmid of this plant pathogen. One such monosaccharide is galacturonate, the predominant monomer of pectin found in plant cell walls. This ligand is recognized by the periplasmic sugar binding protein ChvE, which interacts with the VirA histidine kinase that controls vir gene expression. Although ChvE is also a member of the ChvE-MmsAB ABC transporter involved in the utilization of many neutral sugars, it is not involved in galacturonate utilization. In this study, a putative tripartite ATP-independent periplasmic (TRAP) transporter, GaaPQM, is shown to be essential for the utilization of galacturonic acid; we show that residue R169 in the predicted sugar binding site of the GaaP is required for activity. The gene upstream of gaaPQM (gaaR) encodes a member of the GntR family of regulators. GaaR is shown to repress the expression of gaaPQM, and the repression is relieved in the presence of the substrate for GaaPQM. Moreover, GaaR is shown to bind putative promoter regions in the sequences required for galacturonic acid utilization. Finally, A. tumefaciens strains carrying a deletion of gaaPQM are more sensitive to galacturonate as an inducer of vir gene expression, while the overexpression of gaaPQM results in strains being less sensitive to this vir inducer. This supports a model in which transporter activity is crucial in ensuring that vir gene expression occurs only at sites of high ligand concentration, such as those at a plant wound site. PMID:26637603

  3. Role of the two structural domains from the periplasmic Escherichia coli histidine-binding protein HisJ.

    PubMed

    Chu, Byron C H; DeWolf, Timothy; Vogel, Hans J

    2013-11-01

    Escherichia coli HisJ is a type II periplasmic binding protein that functions to reversibly capture histidine and transfer it to its cognate inner membrane ABC permease. Here, we used NMR spectroscopy to determine the structure of apo-HisJ (26.5 kDa) in solution. HisJ is a bilobal protein in which domain 1 (D1) is made up of two noncontiguous subdomains, and domain 2 (D2) is expressed as the inner domain. To better understand the roles of D1 and D2, we have isolated and characterized each domain separately. Structurally, D1 closely resembles its homologous domain in apo- and holo-HisJ, whereas D2 is more similar to the holo-form. NMR relaxation experiments reveal that HisJ becomes more ordered upon ligand binding, whereas isolated D2 experiences a significant reduction in slower (millisecond to microsecond) motions compared with the homologous domain in apo-HisJ. NMR titrations reveal that D1 is able to bind histidine in a similar manner as full-length HisJ, albeit with lower affinity. Unexpectedly, isolated D1 and D2 do not interact with each other in the presence or absence of histidine, which indicates the importance of intact interdomain-connecting elements (i.e. hinge regions) for HisJ functioning. Our results shed light on the binding mechanism of type II periplasmic binding proteins where ligand is initially bound by D1, and D2 plays a supporting role in this dynamic process.

  4. Structural and functional significance of the FGL sequence of the periplasmic chaperone Caf1M of Yersinia pestis.

    PubMed

    Chapman, D A; Zavialov, A V; Chernovskaya, T V; Karlyshev, A V; Zav'yalova, G A; Vasiliev, A M; Dudich, I V; Abramov, V M; Zav'yalov, V P; MacIntyre, S

    1999-04-01

    The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 beta-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 beta-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.

  5. Structural and Functional Significance of the FGL Sequence of the Periplasmic Chaperone Caf1M of Yersinia pestis

    PubMed Central

    Chapman, David A. G.; Zavialov, Anton V.; Chernovskaya, Tatiana V.; Karlyshev, Andrey V.; Zav’yalova, Galina A.; Vasiliev, Anatoly M.; Dudich, Igor V.; Abramov, Vyacheslav M.; Zav’yalov, Vladimir P.; MacIntyre, Sheila

    1999-01-01

    The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 β-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 β-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones. PMID:10198004

  6. Crystal structure of LptH, the periplasmic component of the lipopolysaccharide transport machinery from Pseudomonas aeruginosa.

    PubMed

    Bollati, Michela; Villa, Riccardo; Gourlay, Louise J; Benedet, Mattia; Dehò, Gianni; Polissi, Alessandra; Barbiroli, Alberto; Martorana, Alessandra M; Sperandeo, Paola; Bolognesi, Martino; Nardini, Marco

    2015-05-01

    Lipopolysaccharide (LPS) is the main glycolipid present in the outer leaflet of the outer membrane (OM) of Gram-negative bacteria, where it modulates OM permeability, therefore preventing many toxic compounds from entering the cell. LPS biogenesis is an essential process in Gram-negative bacteria and thus is an ideal target pathway for the development of novel specific antimicrobials. The lipopolysaccharide transport (Lpt) system is responsible for transporting LPS from the periplasmic surface of the inner membrane, where it is assembled, to the cell surface where it is then inserted in the OM. The Lpt system has been widely studied in Escherichia coli, where it consists of seven essential proteins located in the inner membrane (LptBCFG), in the periplasm (LptA) and in the OM (LptDE). In the present study, we focus our attention on the Pseudomonas aeruginosa PAO1 Lpt system. We identified an LptA orthologue, named LptH, and solved its crystal structure at a resolution of 2.75 Å. Using interspecies complementation and site-directed mutagenesis of a conserved glycine residue, we demonstrate that P. aeruginosa LptH is the genetic and functional homologue of E. coli LptA, with whom it shares the β-jellyroll fold identified also in other members of the canonical E. coli Lpt model system. Furthermore, we modeled the N-terminal β-jellyroll domain of P. aeruginosa LptD, based on the crystal structure of its homologue from Shigella flexneri, aiming to provide more general insight into the mechanism of LPS binding and transport in P. aeruginosa. Both LptH and LptD may represent new targets for the discovery of next generation antibacterial drugs, targeting specific opportunistic pathogens such as P. aeruginosa. Coordinates and structure factors have been deposited in the Protein Data Bank under accession number PDB 4uu4. © 2015 FEBS.

  7. The Same Periplasmic ExbD Residues Mediate In Vivo Interactions between ExbD Homodimers and ExbD-TonB Heterodimers ▿ †

    PubMed Central

    Ollis, Anne A.; Postle, Kathleen

    2011-01-01

    The TonB system couples cytoplasmic membrane proton motive force to TonB-gated outer membrane transporters for active transport of nutrients into the periplasm. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD promote conformational changes in TonB, which transmits this energy to the transporters. The only known energy-dependent interaction occurs between the periplasmic domains of TonB and ExbD. This study identified sites of in vivo homodimeric interactions within ExbD periplasmic domain residues 92 to 121. ExbD was active as a homodimer (ExbD2) but not through all Cys substitution sites, suggesting the existence of conformationally dynamic regions in the ExbD periplasmic domain. A subset of homodimeric interactions could not be modeled on the nuclear magnetic resonance (NMR) structure without significant distortion. Most importantly, the majority of ExbD Cys substitutions that mediated homodimer formation also mediated ExbD-TonB heterodimer formation with TonB A150C. Consistent with the implied competition, ExbD homodimer formation increased in the absence of TonB. Although ExbD D25 was not required for their formation, ExbD dimers interacted in vivo with ExbB. ExbD-TonB interactions required ExbD transmembrane domain residue D25. These results suggested a model where ExbD2 assembled with ExbB undergoes a transmembrane domain-dependent transition and exchanges partners in localized homodimeric interfaces to form an ExbD2-TonB heterotrimer. The findings here were also consistent with our previous hypothesis that ExbD guides the conformation of the TonB periplasmic domain, which itself is conformationally dynamic. PMID:21984795

  8. The same periplasmic ExbD residues mediate in vivo interactions between ExbD homodimers and ExbD-TonB heterodimers.

    PubMed

    Ollis, Anne A; Postle, Kathleen

    2011-12-01

    The TonB system couples cytoplasmic membrane proton motive force to TonB-gated outer membrane transporters for active transport of nutrients into the periplasm. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD promote conformational changes in TonB, which transmits this energy to the transporters. The only known energy-dependent interaction occurs between the periplasmic domains of TonB and ExbD. This study identified sites of in vivo homodimeric interactions within ExbD periplasmic domain residues 92 to 121. ExbD was active as a homodimer (ExbD(2)) but not through all Cys substitution sites, suggesting the existence of conformationally dynamic regions in the ExbD periplasmic domain. A subset of homodimeric interactions could not be modeled on the nuclear magnetic resonance (NMR) structure without significant distortion. Most importantly, the majority of ExbD Cys substitutions that mediated homodimer formation also mediated ExbD-TonB heterodimer formation with TonB A150C. Consistent with the implied competition, ExbD homodimer formation increased in the absence of TonB. Although ExbD D25 was not required for their formation, ExbD dimers interacted in vivo with ExbB. ExbD-TonB interactions required ExbD transmembrane domain residue D25. These results suggested a model where ExbD(2) assembled with ExbB undergoes a transmembrane domain-dependent transition and exchanges partners in localized homodimeric interfaces to form an ExbD(2)-TonB heterotrimer. The findings here were also consistent with our previous hypothesis that ExbD guides the conformation of the TonB periplasmic domain, which itself is conformationally dynamic.

  9. Defective export in Escherichia coli caused by DsbA'-PhoA hybrid proteins whose DsbA' domain cannot fold into a conformation resistant to periplasmic proteases.

    PubMed

    Guigueno, A; Belin, P; Boquet, P L

    1997-05-01

    The disulfide bond-forming factor DsbA and the alkaline phosphatase are stable in the Escherichia coli periplasmic space and can be overproduced without significant perturbation of the cell's physiology. By contrast, DsbA'-PhoA hybrid proteins resulting from TnphoA insertions into different regions of a plasmid-borne dsbA gene could become toxic (lethal) to bacteria. Toxicity was concomitant with an impairment of some step of the export mechanism and depended on at least three parameters, i.e., (i) the rate of expression of the hybrid protein, (ii) the ability of the amino-terminal DsbA' domain of the hybrid protein to fold into a protease-resistant conformation in the periplasmic space, and (iii) the activity of the DegP periplasmic protease. Even under viable conditions of low expression, DsbA' folding-deficient hybrid proteins accumulated more than the folding-proficient ones in the insoluble material and this was aggravated in a strain lacking the DegP protease. When production was more elevated, the folding-deficient hybrid proteins became lethal, but only in strains lacking the DegP activity, while the folding-proficient ones were not. Under conditions of very high production by degP+ or degP strains, both types of hybrid proteins accumulated as insoluble preproteins. Meanwhile, the export machinery was dramatically handicapped and the cells lost viability. However, the folding-deficient hybrid proteins had a higher killing efficiency than the folding-proficient ones. Free DsbA'-truncated polypeptides, although not toxic, were processed more slowly when they could not fold into a protease-resistant form in the periplasmic space. This provides indications in E. coli for a direct or indirect influence of the folding of a protein in the periplasmic environment on export efficiency.

  10. Defective export in Escherichia coli caused by DsbA'-PhoA hybrid proteins whose DsbA' domain cannot fold into a conformation resistant to periplasmic proteases.

    PubMed Central

    Guigueno, A; Belin, P; Boquet, P L

    1997-01-01

    The disulfide bond-forming factor DsbA and the alkaline phosphatase are stable in the Escherichia coli periplasmic space and can be overproduced without significant perturbation of the cell's physiology. By contrast, DsbA'-PhoA hybrid proteins resulting from TnphoA insertions into different regions of a plasmid-borne dsbA gene could become toxic (lethal) to bacteria. Toxicity was concomitant with an impairment of some step of the export mechanism and depended on at least three parameters, i.e., (i) the rate of expression of the hybrid protein, (ii) the ability of the amino-terminal DsbA' domain of the hybrid protein to fold into a protease-resistant conformation in the periplasmic space, and (iii) the activity of the DegP periplasmic protease. Even under viable conditions of low expression, DsbA' folding-deficient hybrid proteins accumulated more than the folding-proficient ones in the insoluble material and this was aggravated in a strain lacking the DegP protease. When production was more elevated, the folding-deficient hybrid proteins became lethal, but only in strains lacking the DegP activity, while the folding-proficient ones were not. Under conditions of very high production by degP+ or degP strains, both types of hybrid proteins accumulated as insoluble preproteins. Meanwhile, the export machinery was dramatically handicapped and the cells lost viability. However, the folding-deficient hybrid proteins had a higher killing efficiency than the folding-proficient ones. Free DsbA'-truncated polypeptides, although not toxic, were processed more slowly when they could not fold into a protease-resistant form in the periplasmic space. This provides indications in E. coli for a direct or indirect influence of the folding of a protein in the periplasmic environment on export efficiency. PMID:9150222

  11. A Novel Periplasmic Protein, VrpA, Contributes to Efficient Protein Secretion by the Type III Secretion System in Xanthomonas spp.

    PubMed

    Zhou, Xiaofeng; Hu, Xiufang; Li, Jinyun; Wang, Nian

    2015-02-01

    Efficient secretion of type III effector proteins from the bacterial cytoplasm to host cell cytosol via a type III secretion system (T3SS) is crucial for virulence of plant-pathogenic bacterium. Our previous study revealed a conserved hypothetical protein, virulence-related periplasm protein A (VrpA), which was identified as a critical virulence factor for Xanthomonas citri subsp. citri. In this study, we demonstrate that mutation of vrpA compromises X. citri subsp. citri virulence and hypersensitive response induction. This deficiency is also observed in the X. campestris pv. campestris strain, suggesting a functional conservation of VrpA in Xanthomonas spp. Our study indicates that VrpA is required for efficient protein secretion via T3SS, which is supported by multiple lines of evidence. A CyaA reporter assay shows that VrpA is involved in type III effector secretion; quantitative reverse-transcription polymerase chain reaction analysis suggests that the vrpA mutant fails to activate citrus-canker-susceptible gene CsLOB1, which is transcriptionally activated by transcription activator-like effector PthA4; in vitro secretion study reveals that VrpA plays an important role in secretion of T3SS pilus, translocon, and effector proteins. Our data also indicate that VrpA in X. citri subsp. citri localizes to bacterial periplasmic space and the periplasmic localization is required for full function of VrpA and X. citri subsp. citri virulence. Protein-protein interaction studies show that VrpA physically interacts with periplasmic T3SS components HrcJ and HrcC. However, the mutation of VrpA does not affect T3SS gene expression. Additionally, VrpA is involved in X. citri subsp. citri tolerance of oxidative stress. Our data contribute to the mechanical understanding of an important periplasmic protein VrpA in Xanthomonas spp.

  12. A conditional suicide system for Saccharomyces cerevisiae relying on the intracellular production of the Serratia marcescens nuclease.

    PubMed

    Balan, Andrea; Schenberg, Ana Clara G

    2005-02-01

    A conditional lethal system for biological containment of genetically modified strains of Saccharomyces cerevisiae is described. This suicide system is based on the intracellular production of the Serratia marcescens nuclease in the yeast cell, aiming at the destruction of the host genetic material. The S. marcescens nuclease, encoded by the nucA gene, is normally secreted by the bacterium into the medium. In the present work, the nucA gene, devoid of its signal peptide coding sequence, was cloned in a yeast expression vector, under control of the glucose-repressed S. cerevisiae alcohol dehydrogenase 2 gene (ADH2) promoter. When transformed into S. cerevisiae, the recombinant plasmid proved to be effective in killing the host cells upon glucose depletion from the medium, and the nuclease activity was found in lysates prepared from the transformants. In addition, the nuclease degrading effect was shown to reach chromosomal DNA in the yeast host. The killing effect of the nucA plasmid was also demonstrated in soil microcosm assays, indicating that whenever the GMM escapes into the environment where glucose is scarce, the nucA gene will be expressed and the resulting nuclease will destroy the genetic material and kill the cells. In contrast to other suicide systems that target the cell envelope, the advantage of the one described here is that it disfavours horizontal gene transfer from recombinant yeast cells to other microorganisms found in the environment.

  13. Development and application of dual-fluorescence reporter systems for measuring specific nuclease activity based on SSA repair mechanism.

    PubMed

    Furong, Han; Ling, Wang; Kun, Xu; Zhiying, Zhang; Xin, Wang

    2015-10-01

    Reporter vector system has become an important method for measuring activity of specific nucleases because of its fast construction, simple modification, easy operation, economic effectiveness as well as its role in enriching positive cells with genomic modification through mediating screen of specific nucleases positive cells. After introducing double strand breaks (DSBs), a reporter system based on non-homologous end joining (NHEJ)-mediated repair can only repair maximally two thirds of reporter genes after optimization, while single strand annealing (SSA)-mediated repair can repair all reporter genes theoretically which has higher sensitivity and facilitates the detection of specific nuclease with low activity and provides an effective way to detect specific nuclease activity in genome modification studies. In this study, we designed and constructed three sets of dual-fluorescence reporter systems based on SSA repair mechanism and applied the mRFP-eGFP system in measuring the effective activity of three pairs of ZFNs, which was 8.9%, 9.3% and 5.0%, respectively. Our study provides an effective way to detect the activity of nucleases.

  14. Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing

    PubMed Central

    Oakes, Benjamin L.; Xia, Danny F.; Rowland, Elizabeth F.; Xu, Denise J.; Ankoudinova, Irina; Borchardt, Jennifer S.; Zhang, Lei; Li, Patrick; Miller, Jeffrey C.; Rebar, Edward J.; Noyes, Marcus B.

    2016-01-01

    Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity. PMID:26738816

  15. Selection-Independent Generation of Gene Knockout Mouse Embryonic Stem Cells Using Zinc-Finger Nucleases

    PubMed Central

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10−6. In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells. PMID:22194948

  16. Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

    PubMed

    Osiak, Anna; Radecke, Frank; Guhl, Eva; Radecke, Sarah; Dannemann, Nadine; Lütge, Fabienne; Glage, Silke; Rudolph, Cornelia; Cantz, Tobias; Schwarz, Klaus; Heilbronn, Regine; Cathomen, Toni

    2011-01-01

    Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

  17. Use of designer nucleases for targeted gene and genome editing in plants.

    PubMed

    Weeks, Donald P; Spalding, Martin H; Yang, Bing

    2016-02-01

    The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single-celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double-stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production.

  18. Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

    PubMed Central

    Hodel, A.; Rice, L. M.; Simonson, T.; Fox, R. O.; Brünger, A. T.

    1995-01-01

    Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation. PMID:7613463

  19. Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases.

    PubMed

    Sizova, Irina; Greiner, Andre; Awasthi, Mayanka; Kateriya, Suneel; Hegemann, Peter

    2013-03-01

    The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc-finger nucleases (ZFNs). Our approach includes (i) design of gene-specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light-activated ion channel channelrhodopsin-1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non-functional aminoglycoside 3'-phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co-transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin-resistant (Pm-R) clones with expressing nucleases. Of these Pm-R clones, 1% also contained a modified COP3 locus. In cases where cells were co-transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.

  20. Nuclease Resistant DNA via High-Density Packing in Polymeric Micellar Nanoparticle Coronas

    PubMed Central

    Rush, Anthony M.; Thompson, Matthew P.; Tatro, Erick T.

    2013-01-01

    Herein, we describe a polymeric micellar nanoparticle capable of rendering nucleic acids resistant to nuclease digestion. This approach relies on utilizing DNA as the polar head group of a DNA-polymer amphiphile in order to assemble well-defined, discrete nanoparticles. Dense packing of DNA in the micelle corona allows for hybridization of complementary oligonucleotides while prohibiting enzymatic degradation. We demonstrate the preparation, purification and characterization of the nanoparticles, then describe their resistance to treatment with endo- and exonucleases including snake-venom phosphodiesterase (SVP) a common, general DNA digestion enzyme. PMID:23379679

  1. Zinc finger nuclease technology: advances and obstacles in modelling and treating genetic disorders.

    PubMed

    Jabalameli, Hamid Reza; Zahednasab, Hamid; Karimi-Moghaddam, Amin; Jabalameli, Mohammad Reza

    2015-03-01

    Zinc finger nucleases (ZFNs) are engineered restriction enzymes designed to target specific DNA sequences within the genome. Assembly of zinc finger DNA-binding domain to a DNA-cleavage domain enables the enzyme machinery to target unique locus in the genome and invoke endogenous DNA repair mechanisms. This machinery offers a versatile approach in allele editing and gene therapy. Here we discuss the architecture of ZFNs and strategies for generating targeted modifications within the genome. We review advances in gene therapy and modelling of the disease using these enzymes and finally, discuss the practical obstacles in using this technology. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. A new thio-Schiff base fluorophore with copper ion sensing, DNA binding and nuclease activity.

    PubMed

    Vikneswaran, R; Syafiq, Muhamad Syamir; Eltayeb, Naser Eltaher; Kamaruddin, Mohd Naqiuddin; Ramesh, S; Yahya, R

    2015-01-01

    Copper ion recognition and DNA interaction of a newly synthesized fluorescent Schiff base (HPyETSC) were investigated using UV-vis and fluorescent spectroscopy. Examination using these two techniques revealed that the detection of copper by HPyETSC is highly sensitive and selective, with a detection limit of 0.39 μm and the mode of interaction between HPyETSC and DNA is electrostatic, with a binding constant of 8.97×10(4) M(-1). Furthermore, gel electrophoresis studies showed that HPyETSC exhibited nuclease activity through oxidative pathway.

  3. Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs).

    PubMed

    Taheri-Ghahfarokhi, Amir; Malaver-Ortega, Luis F; Sumer, Huseyin

    2015-01-01

    Interest is increasing in transcription activator-like effector nucleases (TALENs) as a tool to introduce targeted double-strand breaks into the large genomes of human and animal cell lines. The produced DNA lesions stimulate DNA repair pathways, error-prone but dominant non-homologous end joining (NHEJ) and accurate but less occurring homology-directed repair (HDR), and as a result targeted genes can be modified. Here, we describe a modified Golden-Gate cloning method for generating TALENs and also details for targeting genes in mouse embryonic stem cells. The protocol described here can be used for modifying the genome of a broad range of pluripotent cell lines.

  4. Transcription activator-like effector nucleases (TALENs): a highly efficient and versatile tool for genome editing.

    PubMed

    Sun, Ning; Zhao, Huimin

    2013-07-01

    Transcription activator-like effector (TALE) nucleases (TALENs) have recently emerged as a revolutionary genome editing tool in many different organisms and cell types. The site-specific chromosomal double-strand breaks introduced by TALENs significantly increase the efficiency of genomic modification. The modular nature of the TALE central repeat domains enables researchers to tailor DNA recognition specificity with ease and target essentially any desired DNA sequence. Here, we comprehensively review the development of TALEN technology in terms of scaffold optimization, DNA recognition, and repeat array assembly. In addition, we provide some perspectives on the future development of this technology.

  5. Zinc finger nuclease technology: A stable tool for high efficiency transformation in bloodstream form T. brucei.

    PubMed

    Schumann, Gabriela; Kangussu-Marcolino, Monica M; Doiron, Nicholas; Käser, Sandro; de Assis Burle-Caldas, Gabriela; DaRocha, Wanderson D; Teixeira, Santuza M; Roditi, Isabel

    2017-04-01

    In Trypanosoma brucei, the generation of knockout mutants is relatively easy compared to other organisms as transfection methods are well established. These methods have their limitations, however, when it comes to the generation of genome-wide libraries that require a minimum of several hundred thousand transformants. Double-strand breaks with the meganuclease ISce-I dramatically increase transformation efficiency, but are not widely in use as cell lines need to be generated de novo before each transfection. Here we show that zinc finger nucleases are a robust and stable tool that can enhance transformation in bloodstream forms by more than an order of magnitude.

  6. A simple strategy for heritable chromosomal deletions in zebrafish via the combinatorial action of targeting nucleases.

    PubMed

    Lim, Shimin; Wang, Yin; Yu, Xueyao; Huang, Yian; Featherstone, Mark S; Sampath, Karuna

    2013-07-01

    Precise and effective genome-editing tools are essential for functional genomics and gene therapy. Targeting nucleases have been successfully used to edit genomes. However, whole-locus or element-specific deletions abolishing transcript expression have not previously been reported. Here, we show heritable targeting of locus-specific deletions in the zebrafish nodal-related genes squint (sqt) and cyclops (cyc). Our strategy of heritable chromosomal editing can be used for disease modeling, analyzing gene clusters, regulatory regions, and determining the functions of non-coding RNAs in genomes.

  7. TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants.

    PubMed

    Wendt, Toni; Holm, Preben Bach; Starker, Colby G; Christian, Michelle; Voytas, Daniel F; Brinch-Pedersen, Henrik; Holme, Inger Bæksted

    2013-10-01

    Transcription activator-like effector nucleases (TALENs) enable targeted mutagenesis in a variety of organisms. The primary advantage of TALENs over other sequence-specific nucleases, namely zinc finger nucleases and meganucleases, lies in their ease of assembly, reliability of function, and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation. Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently in different cells.

  8. Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin.

    PubMed Central

    Cognetti, G; Shaw, B R

    1981-01-01

    The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease. In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates. The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases. The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development. Images PMID:7312627

  9. [Transgenic Expression of Serratia marcescens Native and Mutant Nucleases Modulates Tobacco Mosaic Virus Resistance in Nicotiana tabacum L].

    PubMed

    Trifonova, E A; Saveleva, A V; Romanova, A V; Filipenko, E A; Sapotsky, M V; Malinovsky, V I; Kochetov, A V; Shumny, V K

    2015-07-01

    Extracellular Serratia marcescens nuclease is an extremely active enzyme which non-specifically degrades RNA and DNA. Its antiviral activity was previously shown both in animals and in plants when applied exogenously. Transgenic tobacco plants (Nicotiana tabacum L cv. SR1) expressing S. marcescens chimeric, mutant, and intracellular mutant nuclease gene variants were regenerated and challenged with tobacco mosaic virus. The transgenic plants exhibited a higher level of resistance to the virus infection than the control non-transgenic plants. The resistance was evidenced by the delay of the appearance of mosaic symptoms and the retarded accumulation of viral antigen. Thus, these results reveal that modulations of both extracellular nuclease activity and intracellular RNA/DNA binding can protect plants against viral diseases.

  10. Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair

    PubMed Central

    Meineke, Birthe; Shuman, Stewart

    2012-01-01

    Breakage of tRNALys(UUU) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNALys(UUU) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 5′-kinase and ligase functions. PMID:22101242

  11. Determinants of the cytotoxicity of PrrC anticodon nuclease and its amelioration by tRNA repair.

    PubMed

    Meineke, Birthe; Shuman, Stewart

    2012-01-01

    Breakage of tRNA(Lys(UUU)) by the Escherichia coli anticodon nuclease PrrC (EcoPrrC) underlies a host antiviral response to phage T4 infection that is ultimately thwarted by a virus-encoded RNA repair system. PrrC homologs are prevalent in other bacteria, but their activities and substrates are not defined. We find that induced expression of EcoPrrC is toxic in Saccharomyces cerevisiae and E. coli, whereas the Neisseria meningitidis PrrC (NmePrrC) is not. PrrCs consist of an N-terminal NTPase module and a C-terminal nuclease module. Domain swaps identified the EcoPrrC nuclease domain as decisive for toxicity when linked to either the Eco or Nme NTPase. Indeed, a single arginine-to-tryptophan change in the NmePrrC nuclease domain (R316W) educed a gain-of-function and rendered NmePrrC toxic to yeast, with genetic evidence for tRNA(Lys(UUU)) being the relevant target. The reciprocal Trp-to-Arg change in EcoPrrC (W335R) abolished its toxicity. Further mutagenesis of the EcoPrrC nuclease domain highlighted an ensemble of 15 essential residues and distinguished between hypomorphic alleles and potential nuclease-nulls. We report that the RNA repair phase of the bacterial virus-host dynamic is also portable to yeast, where coexpression of the T4 enzymes Pnkp and Rnl1 ameliorated the toxicity of NmePrrC-R316W. Plant tRNA ligase AtRNL also countered NmePrrC-R316W toxicity, in a manner that depended on AtRNL's 5'-kinase and ligase functions.

  12. A Biochemical Approach to Understanding the Fanconi Anemia Pathway-Regulated Nucleases in Genome Maintenance for Preventing Bone Marrow Failure and Cancer

    DTIC Science & Technology

    2014-04-01

    the Fanconi Anemia Pathway- Regulated Nucleases in Genome Maintenance for Preventing Bone Marrow Failure and Cancer PRINCIPAL INVESTIGATOR...GRANT NUMBER 4. TITLE AND SUBTITLE A Biochemical Approach to Understanding the Fanconi Anemia Pathway-Regulated Nucleases in Genome Maintenance for...Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Fanconi anemia is the most prevalent inherited BMF syndromes, caused by mutations in

  13. Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases

    PubMed Central

    Muller, Mandy

    2017-01-01

    During lytic Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, the viral endonu- clease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs, including the transcript encoding interleukin-6 (IL-6), escape SOX-induced cleavage. IL-6 escape is mediated through a 3’ UTR RNA regulatory element that overrides the SOX targeting mechanism. Here, we reveal that this protective RNA element functions to broadly restrict cleavage by a range of homologous and non-homologous viral endonucleases. However, it does not impede cleavage by cellular endonucleases. The IL-6 protective sequence may be representative of a larger class of nuclease escape elements, as we identified a similar protective element in the GADD45B mRNA. The IL-6 and GADD45B-derived elements display similarities in their sequence, putative structure, and several associated RNA binding proteins. However, the overall composition of their ribonucleoprotein complexes appears distinct, leading to differences in the breadth of nucleases restricted. These findings highlight how RNA elements can selectively control transcript abundance in the background of widespread virus-induced mRNA degradation. PMID:28841715

  14. Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation.

    PubMed

    Seper, Andrea; Fengler, Vera H I; Roier, Sandro; Wolinski, Heimo; Kohlwein, Sepp D; Bishop, Anne L; Camilli, Andrew; Reidl, Joachim; Schild, Stefan

    2011-11-01

    Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae. © 2011 Blackwell Publishing Ltd.

  15. Crystallization and preliminary X-ray characterization of two thermostable DNA nucleases

    SciTech Connect

    Kuettner, E. Bartholomeus; Pfeifer, Sven; Keim, Antje; Greiner-Stöffele, Thomas; Sträter, Norbert

    2006-12-01

    Two thermostable DNA nucleases from archaea were crystallized in different space groups; the crystals were suitable for X-ray analysis. Temperature-tolerant organisms are an important source to enhance the stability of enzymes used in biotechnological processes. The DNA-cleaving enzyme exonuclease III from Escherichia coli is used in several applications in gene technology. A thermostable variant could expand the applicability of the enzyme in these methods. Two homologous nucleases from Archaeoglobus fulgidus (ExoAf) and Methanothermobacter thermoautrophicus (ExoMt) were studied for this purpose. Both enzymes were crystallized in different space groups using (poly)ethylene glycols, 2,4-methyl pentandiol, dioxane, ethanol or 2-propanol as precipitants. The addition of a 10-mer DNA oligonucleotide was important to obtain monoclinic crystals of ExoAf and ExoMt that diffracted to resolutions better than 2 Å using synchrotron radiation. The crystal structures of the homologous proteins can serve as templates for genetic engineering of the E. coli exonuclease III and will aid in understanding the different catalytic properties of the enzymes.

  16. S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

    PubMed Central

    Hubbell, H R; Robberson, D L; Hsu, T C

    1979-01-01

    Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA. Images PMID:523322

  17. Rh D blood group conversion using transcription activator-like effector nucleases

    PubMed Central

    Kim, Young-Hoon; Kim, Hyun O.; Baek, Eun J.; Kurita, Ryo; Cha, Hyuk-Jin; Nakamura, Yukio; Kim, Hyongbum

    2015-01-01

    Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine. PMID:26078220

  18. Rh D blood group conversion using transcription activator-like effector nucleases.

    PubMed

    Kim, Young-Hoon; Kim, Hyun O; Baek, Eun J; Kurita, Ryo; Cha, Hyuk-Jin; Nakamura, Yukio; Kim, Hyongbum

    2015-06-16

    Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

  19. Leishmania infantum EndoG Is an Endo/Exo-Nuclease Essential for Parasite Survival

    PubMed Central

    Gutierrez, Kilian Jesús; Alzate, Juan Fernando; Genes, Carlos Mario; Moreno, David; Casanova, Elena; Gigante, Alba; Pérez-Pérez, María-Jesús; Camarasa, María-José; Clos, Joachim; Gago, Federico; Jiménez-Ruiz, Antonio

    2014-01-01

    EndoG, a member of the DNA/RNA non-specific ββα-metal family of nucleases, has been demonstrated to be present in many organisms, including Trypanosomatids. This nuclease participates in the apoptotic program in these parasites by migrating from the mitochondrion to the nucleus, where it takes part in the degradation of genomic DNA that characterizes this process. We now demonstrate that Leishmania infantum EndoG (LiEndoG) is an endo-exonuclease that has a preferential 5′ exonuclease activity on linear DNA. Regardless of its role during apoptotic cell death, this enzyme seems to be necessary during normal development of the parasites as indicated by the reduced growth rates observed in LiEndoG hemi-knockouts and their poor infectivity in differentiated THP-1 cells. The pro-life role of this protein is also corroborated by the higher survival rates of parasites that over-express this protein after treatment with the LiEndoG inhibitor Lei49. Taken together, our results demonstrate that this enzyme plays essential roles in both survival and death of Leishmania parasites. PMID:24651293

  20. Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants

    PubMed Central

    Forsyth, Adrienne; Weeks, Troy; Richael, Craig; Duan, Hui

    2016-01-01

    Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described. PMID:27826306

  1. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.

    PubMed

    Tsai, Shengdar Q; Wyvekens, Nicolas; Khayter, Cyd; Foden, Jennifer A; Thapar, Vishal; Reyon, Deepak; Goodwin, Mathew J; Aryee, Martin J; Joung, J Keith

    2014-06-01

    Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

  2. Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

    PubMed Central

    Ponnuswamy, Nandhini; Bastings, Maartje M. C.; Nathwani, Bhavik; Ryu, Ju Hee; Chou, Leo Y. T.; Vinther, Mathias; Li, Weiwei Aileen; Anastassacos, Frances M.; Mooney, David J.; Shih, William M.

    2017-01-01

    DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications. PMID:28561045

  3. Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation

    PubMed Central

    Seper, Andrea; Fengler, Vera H I; Roier, Sandro; Wolinski, Heimo; Kohlwein, Sepp D; Bishop, Anne L; Camilli, Andrew; Reidl, Joachim; Schild, Stefan

    2011-01-01

    Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae. PMID:22032623

  4. Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

    NASA Astrophysics Data System (ADS)

    Ponnuswamy, Nandhini; Bastings, Maartje M. C.; Nathwani, Bhavik; Ryu, Ju Hee; Chou, Leo Y. T.; Vinther, Mathias; Li, Weiwei Aileen; Anastassacos, Frances M.; Mooney, David J.; Shih, William M.

    2017-05-01

    DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.

  5. Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

    PubMed Central

    Flowers, G. Parker; Timberlake, Andrew T.; Mclean, Kaitlin C.; Monaghan, James R.; Crews, Craig M.

    2014-01-01

    Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability. PMID:24764077

  6. Engineered zinc finger nuclease-mediated homologous recombination of the human rhodopsin gene.

    PubMed

    Greenwald, David L; Cashman, Siobhan M; Kumar-Singh, Rajendra

    2010-12-01

    Novel zinc finger nucleases (ZFNs) were designed to target the human rhodopsin gene and induce homologous recombination of a donor DNA fragment. Three-finger zinc finger nucleases were designed based on previously published guidelines. To assay for ZFN specificity, the authors generated human embryonic retinoblast cell lines stably expressing a Pro23His rhodopsin, the most common mutation associated with autosomal dominant retinitis pigmentosa in North America. They report quantification of these rhodopsin-specific ZFNs to induce a targeted double-strand break in the human genome, demonstrate their ability to induce homologous recombination of a donor DNA fragment, and report the quantification of the frequency of ZFN-mediated homologous recombination. Compared with endogenous homologous recombination, the authors observed a 12-fold increase in homologous recombination and an absolute frequency of ZFN-directed homologous recombination as high as 17% in the human rhodopsin gene. ZFNs are chimeric proteins with significant potential for the treatment of inherited diseases. In this study, the authors report the design of novel ZFNs targeting the human rhodopsin gene. These ZFNs may be useful for the treatment of retinal diseases such as retinitis pigmentosa, one of the most common causes of inherited blindness in the developed world. Herein, they also report on several aspects of donor fragment design and in vitro conditions that facilitate ZFN-mediated homologous recombination.

  7. Nuclease activity of Saccharomyces cerevisiae Mre11 functions in targeted nucleotide alteration.

    PubMed

    Liu, Li; Usher, Michael; Hu, Yiling; Kmiec, Eric B

    2003-10-01

    Oligonucleotides can be used to direct site-specific changes in genomic DNA through a process in which mismatched base pairs in the oligonucleotide and the target DNA are created. The mechanism by which these complexes are developed and resolved is being studied by using Saccharomyces cerevisiae as a model system. Genetic analyses have revealed that in all likelihood the reaction occurs in two phases: DNA pairing and DNA repair. While the former phase involves strand assimilation, the latter phase likely involves an endonucleolytic processing step that leads to joint resolution. In this study, we established the importance of a functioning MRE11 gene in the overall reaction, as yeast strains deficient in MRE11 exhibited severely reduced activity. The activity could be rescued by complementation with wild-type MRE11 genes but not with MRE11 alleles lacking the nuclease function. Taken together, the data suggest that Mre11 provides nuclease activity for targeted nucleotide exchange, a process that could be used to reengineer yeast genes.

  8. The adnAB locus, encoding a putative helicase-nuclease activity, is essential in Streptomyces.

    PubMed

    Zhang, Lingli; Nguyen, Hoang Chuong; Chipot, Ludovic; Piotrowski, Emilie; Bertrand, Claire; Thibessard, Annabelle; Leblond, Pierre

    2014-07-01

    Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance.

  9. Butyrate-induced changes in nuclease sensitivity of chromatin cannot be correlated with transcriptional activation.

    PubMed Central

    Birren, B W; Taplitz, S J; Herschman, H R

    1987-01-01

    We examined in the H4IIE rat hepatoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequences to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation. Images PMID:3431545

  10. Improved Cell-Penetrating Zinc-Finger Nuclease Proteins for Precision Genome Engineering

    PubMed Central

    Liu, Jia; Gaj, Thomas; Wallen, Mark C; Barbas, Carlos F

    2015-01-01

    Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4+ T cells and 17% in CD34+ hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20–30% in CD4+ T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering. PMID:25756962

  11. Generation of myostatin B knockout yellow catfish (Tachysurus fulvidraco) using transcription activator-like effector nucleases.

    PubMed

    Dong, Zhangji; Ge, Jiachun; Xu, Zhiqiang; Dong, Xiaohua; Cao, Shasha; Pan, Jianlin; Zhao, Qingshun

    2014-06-01

    Myostatin (Mstn), a member of the transforming growth factor β superfamily, plays an inhibiting role in mammalian muscle growth. Mammals like human, cattle, mouse, sheep, and dog carrying null alleles of Mstn display a double-muscle phenotype. Mstn is conserved in fish; however, little is known whether the fish with mutated mstn display a similar phenotype to mammals because of the lack of mutant fish with mstn null alleles. Previously, we knocked out one of the duplicated copies of myostatin gene (mstna) in yellow catfish using zinc-finger nucleases. In this study, we report the identification of the second myostatin gene (mstnb) and knockout of mstnb in yellow catfish. The gene comprises three exons. It is predicted to encode 373 amino acid residues. The predicted protein exhibits 59.3% identity with yellow catfish Mstna and 57.3% identity with human MSTN. Employing TALEN (transcription activator-like effector nucleases) technology, we obtained two founders (from four randomly selected founders) of yellow catfish carrying the mutated mstnb gene in their germ cells. Totally, six mutated alleles of mstnb were obtained from the founders. Among the six alleles, four are nonframeshift and two are frameshift mutation. The frameshift mutated alleles include mstnb(nju22), an 8 bp deletion, and mstnb(nju24), a complex type of mutation comprising a 7 bp deletion and a 12 bp insertion. They are predicted to encode function null Mstnb. Our results will help to understand the roles of mstn genes in fish growth.

  12. Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation.

    PubMed

    Ponnuswamy, Nandhini; Bastings, Maartje M C; Nathwani, Bhavik; Ryu, Ju Hee; Chou, Leo Y T; Vinther, Mathias; Li, Weiwei Aileen; Anastassacos, Frances M; Mooney, David J; Shih, William M

    2017-05-31

    DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.

  13. Transcription Activator-Like Effector Nucleases (TALEN)-Mediated Targeted DNA Insertion in Potato Plants.

    PubMed

    Forsyth, Adrienne; Weeks, Troy; Richael, Craig; Duan, Hui

    2016-01-01

    Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. Specifically integrated transgenes are guaranteed to co-segregate, and expression level is more predictable, which makes downstream characterization and line selection more manageable. Because the site of DNA integration is known, the steps to deregulation of transgenic crops may be simplified. Here we describe a method that combines transcription activator-like effector nuclease (TALEN)-mediated induction of double strand breaks (DSBs) and non-autonomous marker selection to insert a transgene into a pre-selected, transcriptionally active region in the potato genome. In our experiment, TALEN was designed to create a DSB in the genome sequence following an endogenous constitutive promoter. A cytokinin vector was utilized for TALENs expression and prevention of stable integration of the nucleases. The donor vector contained a gene of interest cassette and a promoter-less plant-derived herbicide resistant gene positioned near the T-DNA left border which was used to select desired transgenic events. Our results indicated that TALEN induced T-DNA integration occurred with high frequency and resulting events have consistent expression of the gene of interest. Interestingly, it was found that, in most lines integration took place through one sided homology directed repair despite the minimal homologous sequence at the right border. An efficient transient assay for TALEN activity verification is also described.

  14. Editing of the heavy chain gene of Bombyx mori using transcription activator like effector nucleases.

    PubMed

    Wang, Yujun; Nakagaki, Masao

    2014-07-18

    The silk gland of Bombyx mori represents an established in vivo system for producing recombinant proteins. However, low yields of recombinant proteins have limited the system's further development because endogenous silk proteins were present. Transcription activator-like effector nucleases (TALENs) tool which work in pairs to bind and cleave DNA at specific sites, have recently been shown to be effective for genome editing in various organisms, including silkworms. To improve the yield of recombinant proteins synthesized in the silkworm by eliminated competition with endogenous fibroin synthesis, the heavy chain (H-chain) gene was knocked out using transcription activator-like effector nucleases (TALENs). A pair of TALENs that targets the 1st exon in the H-chain gene was synthesized and microinjected into silkworm embryos; the injected silkworms were screened for H-chain gene knock out (H-KO) based on their sericin cocoon-making characteristics. Sequence analysis revealed that the H-chain of the mutation was successfully edited. The TALENs was very efficient in editing the genome DNA of silkworm. By being eliminated competition with the H-chain, the production of recombinant proteins would be expected to increase markedly if this H-KO system is used.

  15. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes.

    PubMed

    Li, Ting; Huang, Sheng; Zhao, Xuefeng; Wright, David A; Carpenter, Susan; Spalding, Martin H; Weeks, Donald P; Yang, Bing

    2011-08-01

    Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

  16. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites

    PubMed Central

    Skene, Peter J; Henikoff, Steven

    2017-01-01

    We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contact sites at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq. DOI: http://dx.doi.org/10.7554/eLife.21856.001 PMID:28079019

  17. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

    PubMed Central

    Santiago, Yolanda; Chan, Edmond; Liu, Pei-Qi; Orlando, Salvatore; Zhang, Lin; Urnov, Fyodor D.; Holmes, Michael C.; Guschin, Dmitry; Waite, Adam; Miller, Jeffrey C.; Rebar, Edward J.; Gregory, Philip D.; Klug, Aaron; Collingwood, Trevor N.

    2008-01-01

    Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural—but imperfect—DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR−/− cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2–3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR−/− cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production. PMID:18359850

  18. The adnAB Locus, Encoding a Putative Helicase-Nuclease Activity, Is Essential in Streptomyces

    PubMed Central

    Zhang, Lingli; Nguyen, Hoang Chuong; Chipot, Ludovic; Piotrowski, Emilie; Bertrand, Claire

    2014-01-01

    Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance. PMID:24837284

  19. Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases.

    PubMed

    Merlin, Christine; Beaver, Lauren E; Taylor, Orley R; Wolfe, Scot A; Reppert, Steven M

    2013-01-01

    The development of reverse-genetic tools in "nonmodel" insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into "one nucleus" stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and "nonmodel" insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes.

  20. Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

    PubMed Central

    Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

    2013-01-01

    The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

  1. Degradation of nuclease-stabilized RNA oligonucleotides in Mycoplasma-contaminated cell culture media.

    PubMed

    Hernandez, Frank J; Stockdale, Katie R; Huang, Lingyan; Horswill, Alexander R; Behlke, Mark A; McNamara, James O

    2012-02-01

    Artificial RNA reagents such as small interfering RNAs (siRNAs) and aptamers often must be chemically modified for optimal effectiveness in environments that include ribonucleases. Mycoplasmas are common bacterial contaminants of mammalian cell cultures that are known to produce ribonucleases. Here we describe the rapid degradation of nuclease-stabilized RNA oligonucleotides in a human embryonic kidney 293 (HEK) cell culture contaminated with Mycoplasma fermentans, a common species of mycoplasma. RNA with 2'-fluoro- or 2'-O-methyl- modified pyrimidines was readily degraded in conditioned media from this culture, but was stable in conditioned media from uncontaminated HEK cells. RNA completely modified with 2'-O-methyls was not degraded in the mycoplasma-contaminated media. RNA zymogram analysis of conditioned culture media and material centrifuged from the media revealed several distinct protein bands (ranging from 30 to 68 kDa) capable of degrading RNA with 2'-fluoro- or 2'-O-methyl-modified pyrimidines. Finally, the mycoplasma-associated nuclease was detected in material centrifuged from the contaminated culture supernatants in as little as 15 minutes with an RNA oligo-containing 2'-O-methyl-modified pyrimidines and labeled with a 5'-fluorescein amidite (FAM) and 3'-quencher. These results suggest that mycoplasma contamination may be a critical confounding variable for cell culture experiments involving RNA-based reagents, with particular relevance for applications involving naked RNA (e.g., aptamer-siRNA chimeras).

  2. Surveyor nuclease detection of mutations and polymorphisms of mtDNA in children.

    PubMed

    Pilch, Jacek; Asman, Marek; Jamroz, Ewa; Kajor, Maciej; Kotrys-Puchalska, Elżbieta; Goss, Małgorzata; Krzak, Maria; Witecka, Joanna; Gmiński, Jan; Sieroń, Aleksander L

    2010-11-01

    Mitochondrial encephalomyopathies are complex disorders with wide range of clinical manifestations. Particularly time-consuming is the identification of mutations in mitochondrial DNA. A group of 20 children with clinical manifestations of mitochondrial encephalomyopathies was selected for molecular studies. The aims were (a) to identify mutations in mtDNA isolated from muscle and (b) to verify detected mutations in DNA isolated from blood, in order to assess the utility of a Surveyor nuclease assay kit for patient screening. The most common changes found were polymorphisms, including a few missense mutations altering the amino acid sequence of mitochondrial proteins. In two boys with MELAS (i.e., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), a mutation A→G3243 was detected in the tRNALeu gene of mtDNA isolated from muscle and blood. In one boy, the carrier status of his mother was confirmed, based on molecular analysis of DNA isolated from blood. A method using Surveyor nuclease allows systematic screening for small mutations in mtDNA, using as its source blood of the patients and asymptomatic carriers. The method still requires confirmation studying a larger group. In some patients, the use of this method should precede and might limit indications for traumatic muscle and skin biopsy.

  3. Multinuclear magnetic resonance and kinetic studies of single amino acid replacements in staphylococcal nuclease

    SciTech Connect

    Grissom, C.B.; Alexandrescu, A.T.; Ulrich, E.L.; Mills, D.A.; Markley, J.L.

    1986-05-01

    Staphylococcal nuclease hydrolyzes the 5'-phosphate bond of deoxythymidine 5'-p-nitrophenylphosphate to yield p-nitrophenyl phosphate (PNPP) and deoxythymidine in the presence of Ca/sup 2 +/. The PNPP produced can be hydrolyzed to p-nitrophenol and inorganic phosphate by alkaline phosphatase in a coupled assay to provide a chromophore suitable for kinetic studies. By using this assay, the following single amino acid replacements have been characterized at 20 mM Ca/sup 2 +/ and pH 9.25 with the following results (kinetic parameters are expressed relative to those for SNase). SNase, Km = 2.5 mM, V = 1, V/K = 1; substitution of Tyr for Phe at position 85 (Y85F), Km = 61 mM, V/K = 1; H124R, Km = 3.8 mM, V = 3, V/K = 6; H46Y, Km = 2.5 mM, V = 0.78, V/K = 0.66; F76V, Km = 2.4 mM, V = 2.1, and V/K = 2. Only a small perturbation in the kinetic constants is seen for H124R, H46Y, and F76V. Removal of the hydroxyl from tyrosine 85 diminishes the affinity for substrate. Interactions of the wild-type and variant nucleases with metal ions and inhibitors are being investigated by /sup 1/H, /sup 13/C, and /sup 113/Cd NMR.

  4. 3'nucleotidase/nuclease in protozoan parasites: Molecular and biochemical properties and physiological roles.

    PubMed

    Freitas-Mesquita, Anita Leocadio; Meyer-Fernandes, José Roberto

    2017-08-01

    3'-nucleotidase/nuclease (3'NT/NU) is a bi-functional enzyme that is able to hydrolyze 3'-monophosphorylated nucleotides and nucleic acids. This review summarizes the major molecular and biochemical properties of this enzyme in different trypanosomatid species. Sequence analysis of the gene encoding 3'NT/NU in Leishmania and Crithidia species showed that the protein possesses five highly conserved regions that are characteristic of members of the class I nuclease family. 3'NT/NU presents a molecular weight of approximately 40 kDa, which is conserved among the studied species. Throughout the review, we discuss inhibitors and substrate specificity, relating them to the putative structure of the enzyme. Finally, we present the major biological roles performed by 3'NT/NU. The involvement of 3'NT/NU in the purine salvage pathway was confirmed by the increase of activity and expression of the enzyme when the parasites were submitted to purine starvation. The generation of extracellular adenosine is also important to the modulation of the host immune response. Interaction assays involving Leishmania parasites and macrophages indicated that 3'-nucleotidase activity increases the association index between them. Recently, it was shown that 3'NT/NU plays a role in parasite escape from neutrophil extracellular traps, one of the first mechanisms of the host immune system for preventing infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Genome Editing in Mouse Spermatogonial Stem/Progenitor Cells Using Engineered Nucleases

    PubMed Central

    Fanslow, Danielle A.; Wirt, Stacey E.; Barker, Jenny C.; Connelly, Jon P.; Porteus, Matthew H.; Dann, Christina Tenenhaus

    2014-01-01

    Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse “GS” (germline stem) cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro. PMID:25409432

  6. Cloning, sequencing, and expression of Bacillus subtilis genes involved in ATP-dependent nuclease synthesis.

    PubMed Central

    Kooistra, J; Venema, G

    1991-01-01

    The genes encoding the subunits of the Bacillus subtilis ATP-dependent nuclease (add genes) have been cloned. The genes were located on an 8.8-kb SalI-SmaI chromosomal DNA fragment. Transformants of a recBCD deletion mutant of Escherichia coli with plasmid pGV1 carrying this DNA fragment showed ATP-dependent nuclease activity. Three open reading frames were identified on the 8.8-kb SalI-SmaI fragment, which could encode three proteins with molecular masses of 135 (AddB protein), 141 (AddA protein), and 28 kDa. Only the AddB and AddA proteins are required for ATP-dependent exonuclease activity. Both the AddB and AddA proteins contained a conserved amino acid sequence for ATP binding. In the AddA protein, a number of small regions were present showing a high degree of sequence similarity with regions in the E. coli RecB protein. The AddA protein contained six conserved motifs which were also present in the E. coli helicase II (UvrD protein) and the Rep helicase, suggesting that these motifs are involved in the DNA unwinding activity of the enzyme. When linked to the T7 promoter, a high level of expression was obtained in E. coli. Images PMID:1646786

  7. Simultaneous Screening and Validation of Effective Zinc Finger Nucleases in Yeast

    PubMed Central

    Wang, Ling; Lin, Juan; Zhang, Tingting; Xu, Kun; Ren, Chonghua; Zhang, Zhiying

    2013-01-01

    Zinc finger nucleases (ZFNs) have been successfully used for genome modification in various cell types and species. However, construction of an effective ZFN remained challenging. Previous studies all focused on obtaining specific zinc finger proteins (ZFPs) first via bacterial 2-hybrid approach, and then fusing selected ZFPs to FokI nuclease domain. These assembled ZFNs have high rate of failing to cleave target sites in vivo. In this study, we developed a simultaneous screening and validation system to obtain effective ZFNs directly in yeast AH109. This system is based on Gal4 reporter system carrying a unique intermediate reporter plasmid with two 30-bp Gal4 homology arms and a ZFN target site. DNA double strand breaks introduced on target sequence by ZFNs were repaired by single strand annealing (SSA) mechanism, and the restored Gal4 drove reporter genes expression. Taking the advantage of OPEN (Oligomerized Pool ENgineering) selection, we constructed 3 randomized ZFNs libraries and 9 reporter strains for each target gene. We tested this system by taking goat α s1-casein as target gene following three-step selection. Consequently, 3 efficient pairs of ZFNs were obtained from positive colonies on selective medium. The ZFNs achieved a 15.9% disruption frequency in goat mammary epithelial cells. In conclusion, we created a novel system to obtain effective ZFNs directly with simultaneous screening and validation. PMID:23741369

  8. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes

    SciTech Connect

    Li, T; Huang, S; Zhao, XF; Wright, DA; Carpenter, S; Spalding, MH; Weeks, DP; Yang, B

    2011-08-08

    Recent studies indicate that the DNA recognition domain of transcription activator-like (TAL) effectors can be combined with the nuclease domain of FokI restriction enzyme to produce TAL effector nucleases (TALENs) that, in pairs, bind adjacent DNA target sites and produce double-strand breaks between the target sequences, stimulating non-homologous end-joining and homologous recombination. Here, we exploit the four prevalent TAL repeats and their DNA recognition cipher to develop a 'modular assembly' method for rapid production of designer TALENs (dTALENs) that recognize unique DNA sequence up to 23 bases in any gene. We have used this approach to engineer 10 dTALENs to target specific loci in native yeast chromosomal genes. All dTALENs produced high rates of site-specific gene disruptions and created strains with expected mutant phenotypes. Moreover, dTALENs stimulated high rates (up to 34%) of gene replacement by homologous recombination. Finally, dTALENs caused no detectable cytotoxicity and minimal levels of undesired genetic mutations in the treated yeast strains. These studies expand the realm of verified TALEN activity from cultured human cells to an intact eukaryotic organism and suggest that low-cost, highly dependable dTALENs can assume a significant role for gene modifications of value in human and animal health, agriculture and industry.

  9. Fractionation of Sulfur Isotopes by Desulfovibrio vulgaris Mutants Lacking Periplasmic Hydrogenases or the Type I Tetraheme Cytochrome c3

    NASA Astrophysics Data System (ADS)

    Sim, M.; Ono, S.; Bosak, T.

    2012-12-01

    A large fraction of anaerobic mineralization of organic compounds relies on microbial sulfate reduction. Sulfur isotope fractionation by these microbes has been widely used to trace the biogeochemical cycling of sulfur and carbon, but intracellular mechanisms behind the wide range of fractionations observed in nature and cultures are not fully understood. In this study, we investigated the influence of electron transport chain components on the fractionation of sulfur isotopes by culturing Desulfovibrio vulgaris Hildenborough mutants lacking hydrogenases or type I tetraheme cytochrome c3 (Tp1-c3). The mutants were grown both in batch and continuous cultures. All tested mutants grew on lactate or pyruvate as the sole carbon and energy sources, generating sulfide. Mutants lacking cytoplasmic and periplasmic hydrogenases exhibited similar growth physiologies and sulfur isotope fractionations to their parent strains. On the other hand, a mutant lacking Tp1-c3 (ΔcycA) fractionated the 34S/32S ratio more than the wild type, evolving H2 in the headspace and exhibiting a lower specific respiration rate. In the presence of high concentrations of pyruvate, the growth of ΔcycA relied largely on fermentation rather than sulfate reduction, even when sulfate was abundant, producing the largest sulfur isotope effect observed in this study. Differences between sulfur isotope fractionation by ΔcycA and the wild type highlight the effect of electron transfer chains on the magnitude of sulfur isotope fractionation. Because Tp1-c3 is known to exclusively shuttle electrons from periplasmic hydrogenases to transmembrane complexes, electron transfers in the absence of Tp1-c3 should bypass the periplasmic hydrogen cycling, and the loss of reducing equivalents in the form of H2 can impair the flow of electrons from organic acids to sulfur, increasing isotope fractionation. Larger fractionation by ΔcycA can inform interpretations of sulfur isotope data at an environmental scale as well

  10. Modification and periplasmic translocation of the biofilm exopolysaccharide poly-β-1,6-N-acetyl-d-glucosamine

    PubMed Central

    Little, Dustin J.; Li, Grace; Ing, Christopher; DiFrancesco, Benjamin R.; Bamford, Natalie C.; Robinson, Howard; Nitz, Mark; Pomès, Régis; Howell, P. Lynne

    2014-01-01

    Poly-β-1,6-N-acetyl-d-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Polyglucosamine subunit B (PgaB) is responsible for the de–N-acetylation of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB’s N- and C-terminal domains forms a cleft required for the binding and de–N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C-terminal domain that would allow for a processive mechanism. PgaB’s C-terminal domain (PgaB310–672) directly binds PNAG oligomers with dissociation constants of ∼1–3 mM, and the structures of PgaB310–672 in complex with β-1,6-(GlcNAc)6, GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de–N-acetylated PNAG (dPNAG). Furthermore, PgaB310–672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB42–655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB310–672 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release. PMID:24994902

  11. An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

    DOE PAGES

    Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan; ...

    2015-01-01

    Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essentialmore » for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.« less

  12. An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

    SciTech Connect

    Dai, Jingcheng; Wei, Hehong; Tian, Chunyuan; Damron, Fredrick; Zhou, Jizhong; Qiu, Dongru

    2015-01-01

    Background: Bacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR. Results: RpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains. Conclusion: In S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.

  13. Motor rotation is essential for the formation of the periplasmic flagellar ribbon, cellular morphology, and Borrelia burgdorferi persistence within Ixodes scapularis tick and murine hosts.

    PubMed

    Sultan, Syed Z; Sekar, Padmapriya; Zhao, Xiaowei; Manne, Akarsh; Liu, Jun; Wooten, R Mark; Motaleb, M A

    2015-05-01

    Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ΔmotB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ΔmotB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ΔmotB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Efficient export of prefolded, disulfide-bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains.

    PubMed

    Matos, Cristina F R O; Robinson, Colin; Alanen, Heli I; Prus, Piotr; Uchida, Yuko; Ruddock, Lloyd W; Freedman, Robert B; Keshavarz-Moore, Eli

    2014-01-01

    Numerous high-value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide-containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild-type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide-bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm.

  15. Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: Structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein

    DOE PAGES

    Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.; ...

    2015-05-05

    The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redoxmore » system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg²⁺-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg²⁺-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg²⁺ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.« less

  16. Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein

    PubMed Central

    Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.

    2015-01-01

    ABSTRACT The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg2+-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg2+-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg2+ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm. PMID:25944861

  17. Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement.

    PubMed

    Sun, Yongwei; Li, Jingying; Xia, Lanqin

    2016-01-01

    Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes' encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired "safe" harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in plant

  18. Precise Genome Modification via Sequence-Specific Nucleases-Mediated Gene Targeting for Crop Improvement

    PubMed Central

    Sun, Yongwei; Li, Jingying; Xia, Lanqin

    2016-01-01

    Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired “safe” harbor in a predefined manner. The emergence of three programmable SSNs, such as zinc finger nucleases, transcriptional activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in

  19. Bacteriophage T4-encoded Stp can be replaced as activator of anticodon nuclease by a normal host cell metabolite.

    PubMed

    Amitsur, Michal; Benjamin, Sima; Rosner, Rachel; Chapman-Shimshoni, Daphne; Meidler, Roberto; Blanga, Shani; Kaufmann, Gabriel

    2003-10-01

    The bacterial tRNALys-specific anticodon nuclease is known as a phage T4 exclusion system. In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction-modification endonuclease EcoprrI. Stp, the T4-encoded peptide inhibitor of EcoprrI activates the latent enzyme. Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides. Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate. Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC. The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP. The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex. Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC. The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection.

  20. The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II.

    PubMed

    Lyon, C J; Evans, C J; Bill, B R; Otsuka, A J; Aguilera, R J

    2000-07-11

    The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.

  1. Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

    PubMed Central

    Nakata, Masanobu; Okahashi, Nobuo; Wada, Satoshi; Yamashiro, Takashi; Hayashi, Mikako; Hamada, Shigeyuki; Sumitomo, Tomoko; Kawabata, Shigetada

    2014-01-01

    Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg2+ and Ca2+ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression. PMID:25084357

  2. Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient nuclease active in protists

    PubMed Central

    Chen, Kuan-Ming; Campbell, Edgar; Pandey, Radha Raman; Yang, Zhaolin; McCarthy, Andrew A.; Pillai, Ramesh S.

    2015-01-01

    Piwi-interacting RNAs (piRNAs) guide Piwi argonautes to their transposon targets for silencing. The highly conserved protein Maelstrom is linked to both piRNA biogenesis and effector roles in this pathway. One defining feature of Maelstrom is the predicted MAEL domain of unknown molecular function. Here, we present the first crystal structure of the MAEL domain from Bombyx Maelstrom, which reveals a nuclease fold. The overall architecture resembles that found in Mg2+- or Mn2+-dependent DEDD nucleases, but a clear distinguishing feature is the presence of a structural Zn2+ ion coordinated by the conserved ECHC residues. Strikingly, metazoan Maelstrom orthologs across the animal kingdom lack the catalytic DEDD residues, and as we show for Bombyx Maelstrom are inactive as nucleases. However, a MAEL domain-containing protein from amoeba having both sequence motifs (DEDD and ECHC) is robustly active as an exoribonuclease. Finally, we show that the MAEL domain of Bombyx Maelstrom displays a strong affinity for single-stranded RNAs. Our studies suggest that the ancient MAEL nuclease domain evolved to function as an RNA-binding module in metazoan Maelstrom. PMID:25778731

  3. Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient nuclease active in protists.

    PubMed

    Chen, Kuan-Ming; Campbell, Edgar; Pandey, Radha Raman; Yang, Zhaolin; McCarthy, Andrew A; Pillai, Ramesh S

    2015-05-01

    Piwi-interacting RNAs (piRNAs) guide Piwi argonautes to their transposon targets for silencing. The highly conserved protein Maelstrom is linked to both piRNA biogenesis and effector roles in this pathway. One defining feature of Maelstrom is the predicted MAEL domain of unknown molecular function. Here, we present the first crystal structure of the MAEL domain from Bombyx Maelstrom, which reveals a nuclease fold. The overall architecture resembles that found in Mg(2+)- or Mn(2+)-dependent DEDD nucleases, but a clear distinguishing feature is the presence of a structural Zn(2+) ion coordinated by the conserved ECHC residues. Strikingly, metazoan Maelstrom orthologs across the animal kingdom lack the catalytic DEDD residues, and as we show for Bombyx Maelstrom are inactive as nucleases. However, a MAEL domain-containing protein from amoeba having both sequence motifs (DEDD and ECHC) is robustly active as an exoribonuclease. Finally, we show that the MAEL domain of Bombyx Maelstrom displays a strong affinity for single-stranded RNAs. Our studies suggest that the ancient MAEL nuclease domain evolved to function as an RNA-binding module in metazoan Maelstrom. © 2015 Chen et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  4. A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis.

    PubMed

    Huang, Mo Chao; Cheong, Wai Chye; Lim, Li Shi; Li, Mo-Huang

    2012-03-01

    Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real-time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.

  5. The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    PubMed Central

    Liao, Shuren; Tammaro, Margaret; Yan, Hong

    2016-01-01

    The key event in the choice of repair pathways for DNA double-strand breaks (DSBs) is the initial processing of ends. Non-homologous end joining (NHEJ) involves limited processing, but homology-dependent repair (HDR) requires extensive resection of the 5′ strand. How cells decide if an end is channeled to resection or NHEJ is not well understood. We hypothesize that the structure of ends is a major determinant and tested this hypothesis with model DNA substrates in Xenopus egg extracts. While ends with normal nucleotides are efficiently channeled to NHEJ, ends with damaged nucleotides or bulky adducts are channeled to resection. Resection is dependent on Mre11, but its nuclease activity is critical only for ends with 5′ bulky adducts. CtIP is absolutely required for activating the nuclease-dependent mechanism of Mre11 but not the nuclease-independent mechanism. Together, these findings suggest that the structure of ends is a major determinant for the pathway choice of DSB repair and the Mre11 nuclease dependency of resection. PMID:27084932

  6. AlgK is a TPR-containing protein and the periplasmic component of a novel exopolysaccharide secretin

    PubMed Central

    Keiski, Carrie-Lynn; Harwich, Michael; Jain, Sumita; Neculai, Ana Mirela; Yip, Patrick; Robinson, Howard; Whitney, John C.; Riley, Laura; Burrows, Lori L.; Ohman, Dennis E.; Howell, P. Lynne

    2010-01-01

    The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin, AlgE. Our 2.5Å structure shows AlgK is composed of 9.5 tetratricopeptide (TPR)-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA and PelB, involved in the production and export of cellulose, poly-β-1,6-N-Acetyl-D-glucosamine and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems. PMID:20159471

  7. Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

    PubMed Central

    Pirbadian, Sahand; Barchinger, Sarah E.; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A.; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad A.; Shi, Liang; Gorby, Yuri A.; Golbeck, John H.; El-Naggar, Mohamed Y.

    2014-01-01

    Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic–abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution. PMID:25143589

  8. Molybdate binding by ModA, the periplasmic component of the Escherichia coli mod molybdate transport system.

    PubMed

    Imperial, J; Hadi, M; Amy, N K

    1998-03-13

    ModA, the periplasmic-binding protein of the Escherichia coli mod transport system was overexpressed and purified. Binding of molybdate and tungstate to ModA was found to modify the UV absorption and fluorescence emission spectra of the protein. Titration of these changes showed that ModA binds molybdate and tungstate in a 1:1 molar ratio. ModA showed an intrinsic fluorescence emission spectrum attributable to its three tryptophanyl residues. Molybdate binding caused a conformational change in the protein characterized by: (i) a shift of tryptophanyl groups to a more hydrophobic environment; (ii) a quenching (at pH 5.0) or enhancement (at pH 7.8) of fluorescence; and (iii) a higher availability of tryptophanyl groups to the polar quencher acrylamide. The tight binding of molybdate did not allow an accurate estimation of the binding constants by these indirect methods. An isotopic binding method with 99MoO42- was used for accurate determination of KD (20 nM) and stoichiometry (1:1 molar ratio). ModA bound tungstate with approximately the same affinity, but did not bind sulfate or phosphate. These KDs are 150- to 250-fold lower than those previously reported, and compatible with the high molybdate transport affinity of the mod system. The affinity of ModA for molybdate was also determined in vivo and found to be similar to that determined in vitro.

  9. Crystal Structure of the Metallo-β-Lactamase GOB in the Periplasmic Dizinc Form Reveals an Unusual Metal Site

    PubMed Central

    Morán-Barrio, Jorgelina; Lisa, María-Natalia; Larrieux, Nicole; Drusin, Salvador I.; Viale, Alejandro M.; Moreno, Diego M.

    2016-01-01

    Metallo-beta-lactamases (MBLs) are broad-spectrum, Zn(II)-dependent lactamases able to confer resistance to virtually every β-lactam antibiotic currently available. The large diversity of active-site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3 that is expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica. This MBL is atypical, since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here, we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18. This enzyme displays a unique active-site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono- and di-Zn(II) forms of GOB-18 are active against penicillins, cephalosporins, and carbapenems. In silico docking and molecular dynamics simulations indicate that residue Met221 is not involved in substrate binding, in contrast to Ser221, which otherwise is conserved in most B3 enzymes. These distinctive features are conserved in recently reported GOB orthologues in environmental bacteria. These findings provide valuable information for inhibitor design and also posit that GOB enzymes have alternative functions. PMID:27458232

  10. Iron- and 4-hydroxy-2-alkylquinoline-containing periplasmic inclusion bodies of Pseudomonas aeruginosa: A chemical analysis

    USGS Publications Warehouse

    Royt, P.W.; Honeychuck, R.V.; Pant, R.R.; Rogers, M.L.; Asher, L.V.; Lloyd, J.R.; Carlos, W.E.; Belkin, H.E.; Patwardhan, S.

    2007-01-01

    Dark aggregated particles were seen on pellets of iron-rich, mid-logarithmic phase Pseudomonas aeruginosa. Transmission electron microscopy of these cells showed inclusion bodies in periplasmic vacuoles. Aggregated particles isolated from the spent medium of these cells contained iron as indicated by atomic absorption spectroscopy and by electron paramagnetic resonance spectroscopy that revealed Fe3+. Scanning electron microscopy/energy dispersive X-ray analysis of whole cells revealed the presence of iron-containing particles beneath the surface of the cell, indicating that the isolated aggregates were the intracellular inclusion bodies. Collectively, mass spectroscopy and nuclear magnetic resonance spectroscopy of the isolated inclusion bodies revealed the presence of 3,4-dihydroxy-2-heptylquinoline which is the Pseudomonas quinolone signaling compound (PQS) and an iron chelator; 4-hydroxy-2-heptylquinoline (pseudan VII), which is an iron chelator, antibacterial compound and precursor of PQS; 4-hydroxy-2-nonylquinoline (pseudan IX) which is an iron chelator and antibacterial compound; 4-hydroxy-2-methylquinoline (pseudan I), and 4-hydroxy-2-nonylquinoline N-oxide. ?? 2006 Elsevier Inc. All rights reserved.

  11. ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

    PubMed Central

    Seitz, Patrick; Pezeshgi Modarres, Hassan; Borgeaud, Sandrine; Bulushev, Roman D.; Steinbock, Lorenz J.; Radenovic, Aleksandra; Dal Peraro, Matteo; Blokesch, Melanie

    2014-01-01

    The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria. PMID:24391524

  12. Extracellular amylases of starch-fermenting yeast: pH effect on export and residence time in the periplasm

    SciTech Connect

    Calleja, G.B.; Levy-Rick, S.R.; Nasim, A.; Lusena, C.V.

    1987-01-01

    Aerobic cultures of S. alluvius in Wickerham's yeast-nitrogen-base medium with starch as sole carbon source become strongly acidic and contain no detectable extra-cellular amylolytic activity during stationary phase, when the activity in buffered cultures is maximal. The extracellular amylases are irreversibly inactivated at the low pH value (less than 3.5) attained by the cultures. When adequately buffered, the medium yields maximal extracellular amylolytic activity. About 0.2 M phosphate buffer is adequate for substrate concentrations of up to 0.5% starch; higher starch concentrations require more buffer. Unbuffered cultures that are adjusted once with alkali to pH 5.5 also allow maximal extracellular amylolytic activity, provided the adjustment is made prior to the end of exponential growth. Automatic pH control allows use of high starch concentrations of up to 4%. Export is optimal at pH values higher than the optima for enzyme activity and stability and for population growth. The need for pH adjustment prior to the appearance of amylolytic activity in the medium suggests pH dependence of the export process itself and/or acid inactivation of enzymes transiently resident in the periplasm. (Refs. 23).

  13. Mechanistic insight into the conserved allosteric regulation of periplasmic proteolysis by the signaling molecule cyclic-di-GMP

    PubMed Central

    Chatterjee, Debashree; Cooley, Richard B; Boyd, Chelsea D; Mehl, Ryan A; O'Toole, George A; Sondermann, Holger

    2014-01-01

    Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation, a prevalent adaptation strategy in response to changing environments. In Pseudomonas fluorescens, this process is regulated by the Lap system and the second messenger cyclic-di-GMP. High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG, preventing it from cleaving a cell surface-bound adhesin, thereby promoting cell adhesion. In this study, we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapD's HAMP domain. LapD appears to act as a coincidence detector, whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase. Given the conservation of key elements of this receptor system in many bacterial species, the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling. DOI: http://dx.doi.org/10.7554/eLife.03650.001 PMID:25182848

  14. Structural flexibility of the periplasmic protein, FlgA, regulates flagellar P-ring assembly in Salmonella enterica

    PubMed Central

    Matsunami, Hideyuki; Yoon, Young-Ho; Meshcheryakov, Vladimir A.; Namba, Keiichi; Samatey, Fadel A.

    2016-01-01

    A periplasmic flagellar chaperone protein, FlgA, is required for P-ring assembly in bacterial flagella of taxa such as Salmonella enterica or Escherichia coli. The mechanism of chaperone-mediated P-ring formation is poorly understood. Here we present the open and closed crystal structures of FlgA from Salmonella enterica serovar Typhimurium, grown under different crystallization conditions. An intramolecular disulfide cross-linked form of FlgA caused a dominant negative effect on motility of the wild-type strain. Pull-down experiments support a specific protein-protein interaction between FlgI, the P-ring component protein, and the C-terminal domain of FlgA. Surface plasmon resonance and limited-proteolysis indicate that flexibility of the domain is reduced in the covalently closed form. These results show that the structural flexibility of the C-terminal domain of FlgA, which is related to the structural difference between the two crystal forms, is intrinsically associated with its molecular chaperone function in P-ring assembly. PMID:27273476

  15. Characterization of periplasmic protein BP26 epitopes of Brucella melitensis reacting with murine monoclonal and sheep antibodies.

    PubMed

    Qiu, Jinlang; Wang, Wenjing; Wu, Jingbo; Zhang, Hui; Wang, Yuanzhi; Qiao, Jun; Chen, Chuangfu; Gao, Goege F; Allain, Jean-Pierre; Li, Chengyao

    2012-01-01

    More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues ⁹³DRDLQTGGI¹⁰¹ (position 93 to 101) or residues ¹⁰⁴QPIYVYPD¹¹¹, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65-70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90.

  16. Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors.

    PubMed

    Veith, Paul D; Chen, Yu-Yen; Gorasia, Dhana G; Chen, Dina; Glew, Michelle D; O'Brien-Simpson, Neil M; Cecil, Jessica D; Holden, James A; Reynolds, Eric C

    2014-05-02

    Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC-MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs' volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs.

  17. SilE is an intrinsically disordered periplasmic “molecular sponge” involved in bacterial silver resistance

    PubMed Central

    Asiani, Karishma R.; Williams, Huw; Bird, Louise; Jenner, Matthew; Searle, Mark S.

    2016-01-01

    Summary Ag+ resistance was initially found on the Salmonella enetrica serovar Typhimurium multi‐resistance plasmid pMG101 from burns patients in 1975. The putative model of Ag+ resistance, encoded by the sil operon from pMG101, involves export of Ag+ via an ATPase (SilP), an effluxer complex (SilCFBA) and a periplasmic chaperon of Ag+ (SilE). SilE is predicted to be intrinsically disordered. We tested this hypothesis using structural and biophysical studies and show that SilE is an intrinsically disordered protein in its free apo‐form but folds to a compact structure upon optimal binding to six Ag+ ions in its holo‐form. Sequence analyses and site‐directed mutagenesis established the importance of histidine and methionine containing motifs for Ag+‐binding, and identified a nucleation core that initiates Ag+‐mediated folding of SilE. We conclude that SilE is a molecular sponge for absorbing metal ions. PMID:27085056

  18. Transcriptomic Analysis of the Swarm Motility Phenotype of Salmonella enterica Serovar Typhimurium Mutant Defective in Periplasmic Glucan Synthesis.

    PubMed

    Bhagwat, Arvind A; Young, Lynn; Smith, Allen D; Bhagwat, Medha

    2017-09-01

    Movement of food-borne pathogens on moist surfaces enables them to migrate towards more favorable niches and facilitate their survival for extended periods of time. Salmonella enterica serovar Typhimurium mutants defective in Osmoregulated periplasmic glucans (OPG) synthesis are unable to exhibit motility on moist surfaces (swarming); however, their mobility in liquid (swim