PubMed Central (PMC3 - NLM DTD)
Archive of life sciences journal literature at the U.S. National Institutes of Health (NIH), developed and managed by NIH's National Center for Biotechnology Information (NCBI) in the National Library of Medicine (NLM).
Journal of Bacteriology
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Bacteria in Solitary Confinement - Mullineaux, Conrad W.
Even in clonal bacterial cultures, individual bacteria can show substantial stochastic variation, leading to pitfalls in the interpretation of data derived from millions of cells in a culture. In this issue of the Journal of Bacteriology, as part of their study on osmoadaptation in a cyanobacterium, Nanatani et al. describe employing an ingenious microfluidic device that gently cages individual cells (J Bacteriol 197:676–687, 2015, http://dx.doi.org/10.1128/JB.02276-14). The device is a welcome addition to the toolkit available to probe the responses of individual cells to environmental cues.
Mep72, a Metzincin Protease That Is Preferentially Secreted by Biofilms of Pseudomonas aeruginosa - Passmore, Ian J.; Nishikawa, Kahoko; Lilley, Kathryn S.; Bowden, Steven D.; Chung, Jade C. S.; Welch, Martin
In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain...
Functional Metabolic Map of Faecalibacterium prausnitzii, a Beneficial Human Gut Microbe - Heinken, Almut; Khan, M. Tanweer; Paglia, Giuseppe; Rodionov, Dmitry A.; Harmsen, Hermie J. M.; Thiele, Ines
The human gut microbiota plays a central role in human well-being and disease. In this study, we present an integrated, iterative approach of computational modeling, in vitro experiments, metabolomics, and genomic analysis to accelerate the identification of metabolic capabilities for poorly characterized (anaerobic) microorganisms. We demonstrate this approach for the beneficial human gut microbe Faecalibacterium prausnitzii strain A2-165. We generated an automated draft reconstruction, which we curated against the limited biochemical data. This reconstruction modeling was used to develop in silico and in vitro a chemically defined medium (CDM), which was validated experimentally. Subsequent metabolomic analysis of the spent medium...
Agrobacterium tumefaciens ExoR Controls Acid Response Genes and Impacts Exopolysaccharide Synthesis, Horizontal Gene Transfer, and Virulence Gene Expression - Heckel, Brynn C.; Tomlinson, Amelia D.; Morton, Elise R.; Choi, Jeong-Hyeon; Fuqua, Clay
Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens
exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved...
A Novel Protein, RafX, Is Important for Common Cell Wall Polysaccharide Biosynthesis in Streptococcus pneumoniae: Implications for Bacterial Virulence - Wu, Kaifeng; Huang, Jian; Zhang, Yanqing; Xu, Wenchun; Xu, Hongmei; Wang, Libin; Cao, Ju; Zhang, Xuemei; Yin, Yibing
Teichoic acid (TA), together with peptidoglycan (PG), represents a highly complex glycopolymer that ensures cell wall integrity and has several crucial physiological activities. Through an insertion-deletion mutation strategy, we show that ΔrafX mutants are impaired in cell wall covalently attached TA (WTA)-PG biosynthesis, as evidenced by their abnormal banding patterns and reduced amounts of WTA in comparison with wild-type strains. Site-directed mutagenesis revealed an essential role for external loop 4 and some highly conserved amino acid residues in the function of RafX protein. The rafX gene was highly conserved in closely related streptococcal species, suggesting an important physiological function in...
Evidence for a Hexaheteromeric Methylenetetrahydrofolate Reductase in Moorella thermoacetica - Mock, Johanna; Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Thauer, Rudolf K.
Moorella thermoacetica can grow with H2 and CO2, forming acetic acid from 2 CO2 via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4...
Emerging Roles for Anionic Non-Bilayer Phospholipids in Fortifying the Outer Membrane Permeability Barrier - Bishop, Russell E.
Lately, researchers have been actively investigating Escherichia coli
lptD mutants, which exhibit reduced transport of lipopolysaccharide to the cell surface. In this issue of the Journal of Bacteriology, Sutterlin et al. (H. A. Sutterlin, S. Zhang, and T. J. Silhavy, J. Bacteriol. 196:3214–3220, 2014) now reveal an important functional role for phosphatidic acid in fortifying the outer membrane permeability barrier in certain lptD mutant backgrounds. These findings come on the heels of the first reports of two LptD crystal structures, which now provide a structural framework for interpreting lptD genetics.
Biological Cost of Pyocin Production during the SOS Response in Pseudomonas aeruginosa - Penterman, Jon; Singh, Pradeep K.; Walker, Graham C.
LexA and two structurally related regulators, PrtR and PA0906, coordinate the Pseudomonas aeruginosa SOS response. RecA-mediated autocleavage of LexA induces the expression of a protective set of genes that increase DNA damage repair and tolerance. In contrast, RecA-mediated autocleavage of PrtR induces antimicrobial pyocin production and a program that lyses cells to release the newly synthesized pyocin. Recently, PrtR-regulated genes were shown to sensitize P. aeruginosa to quinolones, antibiotics that elicit a strong SOS response. Here, we investigated the mechanisms by which PrtR-regulated genes determine antimicrobial resistance and genotoxic stress survival. We found that induction of PrtR-regulated genes lowers resistance...
Accumulation of Phosphatidic Acid Increases Vancomycin Resistance in Escherichia coli - Sutterlin, Holly A.; Zhang, Sisi; Silhavy, Thomas J.
In Gram-negative bacteria, lipopolysaccharide (LPS) contributes to the robust permeability barrier of the outer membrane, preventing entry of toxic molecules such as antibiotics. Mutations in lptD, the beta-barrel component of the LPS transport and assembly machinery, compromise LPS assembly and result in increased antibiotic sensitivity. Here, we report rare vancomycin-resistant suppressors that improve barrier function of a subset of lptD mutations. We find that all seven suppressors analyzed mapped to the essential gene cdsA, which is responsible for the conversion of phosphatidic acid to CDP-diacylglycerol in phospholipid biosynthesis. These cdsA mutations cause a partial loss of function and, as expected,...
The Two-Component Regulators GacS and GacA Positively Regulate a Nonfluorescent Siderophore through the Gac/Rsm Signaling Cascade in High-Siderophore-Yielding Pseudomonas sp. Strain HYS - Yu, Xinyan; Chen, Min; Jiang, Zhen; Hu, Yi; Xie, Zhixiong
Siderophores, which are produced to overcome iron deficiency, are believed to be closely related to the adaptability of bacteria. The high-siderophore-yielding Pseudomonas sp. strain HYS simultaneously secretes the fluorescent siderophore pyoverdine and another nonfluorescent siderophore that is a major contributor to the high siderophore yield. Transposon mutagenesis revealed siderophore-related genes, including the two-component regulators GacS/GacA and a special cluster containing four open reading frames (the nfs cluster). Deletion mutations of these genes abolished nonfluorescent-siderophore production, and expression of the nfs cluster depended on gacA, indicating that gacS-gacA may control the nonfluorescent siderophore through regulation of the nfs cluster. Furthermore, regulation...
Structure of the RNA Polymerase Assembly Factor Crl and Identification of Its Interaction Surface with Sigma S - Banta, Amy B.; Cuff, Marianne E.; Lin, Hueylie; Myers, Angela R.; Ross, Wilma; Joachimiak, Andrzej; Gourse, Richard L.
Bacteria utilize multiple sigma factors that associate with core RNA polymerase (RNAP) to control transcription in response to changes in environmental conditions. In Escherichia coli and Salmonella enterica, Crl positively regulates the σS regulon by binding to σS to promote its association with core RNAP. We recently characterized the determinants in σS responsible for specific binding to Crl. However, little is known about the determinants in Crl required for this interaction. Here, we present the X-ray crystal structure of a Crl homolog from Proteus mirabilis in conjunction with in vivo and in vitro approaches that probe the Crl-σS interaction in...
Chemical Structures of Streptococcus pneumoniae Capsular Polysaccharide Type 39 (CPS39), CPS47F, and CPS34 Characterized by Nuclear Magnetic Resonance Spectroscopy and Their Relation to CPS10A - Bush, C. Allen; Yang, Jinghua; Yu, Bingwu; Cisar, John O.
Structural characterization of Streptococcus pneumoniae capsular polysaccharides (CPS) is a prerequisite for unraveling both antigenic and genetic relationships that exist between different serotypes. In the current study, comparative structural studies of S. pneumoniae CPS serogroup 10 (CPS10) were extended to include genetically related S. pneumoniae CPS34, CPS39, and CPS47F. High-resolution heteronuclear nuclear magnetic resonance (NMR) spectroscopy confirmed the published structure of CPS34 and, in conjunction with glycosyl composition analyses, revealed the following repeat unit structures of the other serotypes, which have not been previously characterized:
Common and unique structural features of these polysaccharides, including different positions of O-acetylation, were unambiguously...
Folding LacZ in the Periplasm of Escherichia coli - Dwyer, Robert S.; Malinverni, Juliana C.; Boyd, Dana; Beckwith, Jon; Silhavy, Thomas J.
Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been...
Pleiotropic Role of the RNA Chaperone Protein Hfq in the Human Pathogen Clostridium difficile - Boudry, P.; Gracia, C.; Monot, M.; Caillet, J.; Saujet, L.; Hajnsdorf, E.; Dupuy, B.; Martin-Verstraete, I.; Soutourina, O.
Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins...
Endogenous Synthesis of 2-Aminoacrylate Contributes to Cysteine Sensitivity in Salmonella enterica - Ernst, Dustin C.; Lambrecht, Jennifer A.; Schomer, Rebecca A.; Downs, Diana M.
RidA, the archetype member of the widely conserved RidA/YER057c/UK114 family of proteins, prevents reactive enamine/imine intermediates from accumulating in Salmonella enterica by catalyzing their hydrolysis to stable keto acid products. In the absence of RidA, endogenous 2-aminoacrylate persists in the cellular environment long enough to damage a growing list of essential metabolic enzymes. Prior studies have focused on the dehydration of serine by the pyridoxal 5′-phosphate (PLP)-dependent serine/threonine dehydratases, IlvA and TdcB, as sources of endogenous 2-aminoacrylate. The current study describes an additional source of endogenous 2-aminoacrylate derived from cysteine. The results of in vivo analysis show that the cysteine...
Identification of a Unique Radical S-Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii - Allen, Kylie D.; Xu, Huimin; White, Robert H.
Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-l-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of...
Genome-Wide Analysis of the Role of Global Transcriptional Regulator GntR1 in Corynebacterium glutamicum - Tanaka, Yuya; Takemoto, Norihiko; Ito, Terukazu; Teramoto, Haruhiko; Yukawa, Hideaki; Inui, Masayuki
The transcriptional regulator GntR1 downregulates the genes for gluconate catabolism and pentose phosphate pathway in Corynebacterium glutamicum. Gluconate lowers the DNA binding affinity of GntR1, which is probably the mechanism of gluconate-dependent induction of these genes. In addition, GntR1 positively regulates ptsG, a gene encoding a major glucose transporter, and pck, a gene encoding phosphoenolpyruvate carboxykinase. Here, we searched for the new target of GntR1 on a genome-wide scale by chromatin immunoprecipitation in conjunction with microarray (ChIP-chip) analysis. This analysis identified 56 in vivo GntR1 binding sites, of which 7 sites were previously reported. The newly identified GntR1 sites include...
Communication between Binding Sites Is Required for YqjI Regulation of Target Promoters within the yqjH-yqjI Intergenic Region - Wang, Suning; Blahut, Matthew; Wu, Yun; Philipkosky, Katherine E.; Outten, F. Wayne
The nickel-responsive transcription factor YqjI represses its own transcription and transcription of the divergent yqjH gene, which encodes a novel ferric siderophore reductase. The intergenic region between the two promoters is complex, with multiple sequence features that may impact YqjI-dependent regulation of its two target promoters. We utilized mutagenesis and DNase I footprinting to characterize YqjI regulation of the yqjH-yqjI intergenic region. The results show that YqjI binding results in an extended footprint at the yqjI promoter (site II) compared to the yqjH promoter (site I). Mutagenesis of in vivo gene reporter constructs revealed that the two YqjI binding sites,...