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John D Helmann - One of the best experts on this subject based on the ideXlab platform.
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depletion of undecaprenyl pyrophosphate phosphatases disrupts Cell Envelope biogenesis in bacillus subtilis
Journal of Bacteriology, 2016Co-Authors: Heng Zhao, Yingjie Sun, Jason M Peters, Carol A Gross, Ethan C Garner, John D HelmannAbstract:ABSTRACT The integrity of the bacterial Cell Envelope is essential to sustain life by countering the high turgor pressure of the Cell and providing a barrier against chemical insults. In Bacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C 55 lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimized clustered regularly interspaced short palindromic repeat (CRISPR) system with catalytically inactive (“dead”) CRISPR-associated protein 9 (dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate that B. subtilis requires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of Cell Envelope synthesis and strongly activates the σ M -dependent Cell Envelope stress response, including bcrC , which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of the B. subtilis UPP-Pase enzymes, and provide further evidence linking the σ M regulon to Cell Envelope homeostasis pathways. IMPORTANCE The emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.
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bacillus subtilis extracytoplasmic function ecf sigma factors and defense of the Cell Envelope
Current Opinion in Microbiology, 2016Co-Authors: John D HelmannAbstract:Bacillus subtilis provides a model for investigation of the bacterial Cell Envelope, the first line of defense against environmental threats. Extracytoplasmic function (ECF) sigma factors activate genes that confer resistance to agents that threaten the integrity of the Envelope. Although their individual regulons overlap, σW is most closely associated with membrane-active agents, σX with cationic antimicrobial peptide resistance, and σV with resistance to lysozyme. Here, I highlight the role of the σM regulon, which is strongly induced by conditions that impair peptidoglycan synthesis and includes the core pathways of Envelope synthesis and Cell division, as well as stress-inducible alternative enzymes. Studies of these Cell Envelope stress responses provide insights into how bacteria acclimate to the presence of antibiotics.
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bacillus subtilis extracytoplasmic function ecf sigma factors and defense of the Cell Envelope
Current Opinion in Microbiology, 2016Co-Authors: John D HelmannAbstract:Bacillus subtilis provides a model for investigation of the bacterial Cell Envelope, the first line of defense against environmental threats. Extracytoplasmic function (ECF) sigma factors activate genes that confer resistance to agents that threaten the integrity of the Envelope. Although their individual regulons overlap, σW is most closely associated with membrane-active agents, σX with cationic antimicrobial peptide resistance, and σV with resistance to lysozyme. Here, I highlight the role of the σM regulon, which is strongly induced by conditions that impair peptidoglycan synthesis and includes the core pathways of Envelope synthesis and Cell division, as well as stress-inducible alternative enzymes. Studies of these Cell Envelope stress responses provide insights into how bacteria acclimate to the presence of antibiotics.
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the bacillus subtilis σm regulon and its contribution to Cell Envelope stress responses
Molecular Microbiology, 2008Co-Authors: Warawan Eiamphungporn, John D HelmannAbstract:Summary The Bacillus subtilis extracytoplasmic function (ECF) σM factor is activated by Cell Envelope stress elicited by antibiotics, and by acid, heat, ethanol and superoxide stresses. Here, we have used several complementary approaches to identify genes controlled by σM. In many cases, expression is only partially dependent on σM because of both overlapping promoter recognition with other ECF σ factors and the presence of additional promoter elements. Genes regulated by σM have a characteristic pattern of induction in response to Cell Envelope-acting antibiotics as evidenced by hierarchical clustering analysis. σM also contributes to the expression of the Spx transcription factor and thereby indirectly regulates genes of the Spx regulon. Cell Envelope stress responses also include regulons controlled by σW, σB and several two-component regulatory systems (e.g. LiaRS, YycFG, BceRS). Activation of the σM regulon increases expression of proteins functioning in transcriptional control, Cell wall synthesis and shape determination, Cell division, DNA damage monitoring, recombinational repair and detoxification.
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the bacillus subtilis extracytoplasmic function σx factor regulates modification of the Cell Envelope and resistance to cationic antimicrobial peptides
Journal of Bacteriology, 2004Co-Authors: John D HelmannAbstract:Bacillus subtilis contains seven extracytoplasmic-function σ factors that activate partially overlapping regulons. We here identify four additional members of the σX regulon, pbpX (penicillin-binding protein), ywnJ, the dlt operon (d-alanylation of teichoic acids), and the pss ybfM psd operon (phosphatidylethanolamine biosynthesis). Modification of teichoic acids by esterification with d-alanine and incorporation of phosphatidylethanolamine into the Cell membrane have a common consequence: in both cases positively charged amino groups are introduced into the Cell Envelope. The resulting reduction in the net negative charge of the Cell Envelope has been previously implicated as a resistance mechanism specific for cationic antimicrobial peptides. Consistent with this notion, we find that both sigX and dltA mutants are more sensitive to nisin than wild-type Cells. We conclude that activation of the σX regulon serves to alter Cell surface properties to provide protection against antimicrobial peptides.
Brigid M Davis - One of the best experts on this subject based on the ideXlab platform.
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a vibrio cholerae bola like protein is required for proper Cell shape and Cell Envelope integrity
bioRxiv, 2019Co-Authors: Brigid M Davis, Aurore Fleurie, Abdelrahim Zoued, Laura Alvarez, Kelly M Hines, Felipe Cava, Matthew K WaldorAbstract:BolA family proteins are conserved in gram-negative bacteria and many eukaryotes. While diverse Cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well-described. Here, we investigated the role of BolA family proteins in Vibrio cholerae , the cholera pathogen. Like Escherichia coli , V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli , where bolA is linked to Cell shape and ibaG is not, in V. cholerae , bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen9s morphology. Notably, the bizarre-shaped, multi-polar, elongated and wide Cells that predominated in exponential phase ΔibaG V. cholerae cultures were not observed in stationary phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to Cell Envelope stressors, including Cell wall acting antibiotics and bile, and was defective in intestinal colonization. ΔibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant9s morphological defects and sensitivity to Envelope stressors. Transposon-insertion sequencing analysis of ibaG9s genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the Cell Envelope. Furthermore, co-purification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls Cell morphology and Cell Envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.
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a vibrio cholerae bola like protein is required for proper Cell shape and Cell Envelope integrity
bioRxiv, 2019Co-Authors: Brigid M Davis, Aurore Fleurie, Abdelrahim Zoued, Laura Alvarez, Kelly M Hines, Felipe Cava, Matthew K WaldorAbstract:Abstract BolA family proteins are conserved in gram-negative bacteria and many eukaryotes. While diverse Cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well-described. Here, we investigated the role of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to Cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen’s morphology. Notably, the bizarre-shaped, multi-polar, elongated and wide Cells that predominated in exponential phase ΔibaG V. cholerae cultures were not observed in stationary phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to Cell Envelope stressors, including Cell wall acting antibiotics and bile, and was defective in intestinal colonization. ΔibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant’s morphological defects and sensitivity to Envelope stressors. Transposon-insertion sequencing analysis of ibaG’s genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the Cell Envelope. Furthermore, co-purification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls Cell morphology and Cell Envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins. Importance BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae. The absence of IbaG was associated with dramatic changes in Cell morphology, sensitivity to Envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae Cell Envelope and to interact with numerous iron-sulfur cluster containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae Cell shape and Cell Envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.
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a cytosine methytransferase modulates the Cell Envelope stress response in the cholera pathogen
PLOS Genetics, 2015Co-Authors: Michael C Chao, Satoshi Kimura, Brigid M Davis, Eric E Schadt, Gang Fang, Matthew K WaldorAbstract:DNA methylation is a key epigenetic regulator in all domains of life, yet the effects of most bacterial DNA methyltransferases on Cellular processes are largely undefined. Here, we used diverse techniques, including bisulfite sequencing, transcriptomics, and transposon insertion site sequencing to extensively characterize a 5-methylcytosine (5mC) methyltransferase, VchM, in the cholera pathogen, Vibrio cholerae. We have comprehensively defined VchM’s DNA targets, its genetic interactions and the gene networks that it regulates. Although VchM is a relatively new component of the V. cholerae genome, it is required for optimal V. cholerae growth in vitro and during infection. Unexpectedly, the usually essential σE Cell Envelope stress pathway is dispensable in ∆vchM V. cholerae, likely due to its lower activation in this mutant and the capacity for VchM methylation to limit expression of some Cell Envelope modifying genes. Our work illuminates how an acquired DNA methyltransferase can become integrated within complex Cell circuits to control critical housekeeping processes.
Thomas J Silhavy - One of the best experts on this subject based on the ideXlab platform.
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disruption of lipid homeostasis in the gram negative Cell Envelope activates a novel Cell death pathway
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Holly A Sutterlin, Handuo Shi, Kerrie L May, Amanda Miguel, Somya Khare, Kerwyn Casey Huang, Thomas J SilhavyAbstract:Gram-negative bacteria balance synthesis of the outer membrane (OM), Cell wall, and cytoplasmic contents during growth via unknown mechanisms. Here, we show that a dominant mutation (designated mlaA*, maintenance of lipid asymmetry) that alters MlaA, a lipoprotein that removes phospholipids from the outer leaflet of the OM of Escherichia coli, increases OM permeability, lipopolysaccharide levels, drug sensitivity, and Cell death in stationary phase. Surprisingly, single-Cell imaging revealed that death occurs after protracted loss of OM material through vesiculation and blebbing at Cell-division sites and compensatory shrinkage of the inner membrane, eventually resulting in rupture and slow leakage of cytoplasmic contents. The death of mlaA* Cells was linked to fatty acid depletion and was not affected by membrane depolarization, suggesting that lipids flow from the inner membrane to the OM in an energy-independent manner. Suppressor analysis suggested that the dominant mlaA* mutation activates phospholipase A, resulting in increased levels of lipopolysaccharide and OM vesiculation that ultimately undermine the integrity of the Cell Envelope by depleting the inner membrane of phospholipids. This novel Cell-death pathway suggests that balanced synthesis across both membranes is key to the mechanical integrity of the Gram-negative Cell Envelope.
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sensing external stress watchdogs of the escherichia coli Cell Envelope
Current Opinion in Microbiology, 2005Co-Authors: Natividad Ruiz, Thomas J SilhavyAbstract:The Cpx and σE signaling systems monitor the Cell Envelope in Escherichia coli. When induced, each system triggers a signaling cascade that leads to the upregulation of factors needed to combat Envelope damage. Although each system is distinct and can be uniquely induced by certain cues, they also share striking similarities. In this review, we discuss the recent progress in our understanding of the Cpx and σE systems and compare how both function to maintain the integrity of the Cell Envelope.
Matthew K Waldor - One of the best experts on this subject based on the ideXlab platform.
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a vibrio cholerae bola like protein is required for proper Cell shape and Cell Envelope integrity
bioRxiv, 2019Co-Authors: Brigid M Davis, Aurore Fleurie, Abdelrahim Zoued, Laura Alvarez, Kelly M Hines, Felipe Cava, Matthew K WaldorAbstract:BolA family proteins are conserved in gram-negative bacteria and many eukaryotes. While diverse Cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well-described. Here, we investigated the role of BolA family proteins in Vibrio cholerae , the cholera pathogen. Like Escherichia coli , V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli , where bolA is linked to Cell shape and ibaG is not, in V. cholerae , bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen9s morphology. Notably, the bizarre-shaped, multi-polar, elongated and wide Cells that predominated in exponential phase ΔibaG V. cholerae cultures were not observed in stationary phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to Cell Envelope stressors, including Cell wall acting antibiotics and bile, and was defective in intestinal colonization. ΔibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant9s morphological defects and sensitivity to Envelope stressors. Transposon-insertion sequencing analysis of ibaG9s genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the Cell Envelope. Furthermore, co-purification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls Cell morphology and Cell Envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.
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a vibrio cholerae bola like protein is required for proper Cell shape and Cell Envelope integrity
bioRxiv, 2019Co-Authors: Brigid M Davis, Aurore Fleurie, Abdelrahim Zoued, Laura Alvarez, Kelly M Hines, Felipe Cava, Matthew K WaldorAbstract:Abstract BolA family proteins are conserved in gram-negative bacteria and many eukaryotes. While diverse Cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well-described. Here, we investigated the role of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to Cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen’s morphology. Notably, the bizarre-shaped, multi-polar, elongated and wide Cells that predominated in exponential phase ΔibaG V. cholerae cultures were not observed in stationary phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to Cell Envelope stressors, including Cell wall acting antibiotics and bile, and was defective in intestinal colonization. ΔibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant’s morphological defects and sensitivity to Envelope stressors. Transposon-insertion sequencing analysis of ibaG’s genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the Cell Envelope. Furthermore, co-purification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls Cell morphology and Cell Envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins. Importance BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae. The absence of IbaG was associated with dramatic changes in Cell morphology, sensitivity to Envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae Cell Envelope and to interact with numerous iron-sulfur cluster containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae Cell shape and Cell Envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.
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a cytosine methytransferase modulates the Cell Envelope stress response in the cholera pathogen
PLOS Genetics, 2015Co-Authors: Michael C Chao, Satoshi Kimura, Brigid M Davis, Eric E Schadt, Gang Fang, Matthew K WaldorAbstract:DNA methylation is a key epigenetic regulator in all domains of life, yet the effects of most bacterial DNA methyltransferases on Cellular processes are largely undefined. Here, we used diverse techniques, including bisulfite sequencing, transcriptomics, and transposon insertion site sequencing to extensively characterize a 5-methylcytosine (5mC) methyltransferase, VchM, in the cholera pathogen, Vibrio cholerae. We have comprehensively defined VchM’s DNA targets, its genetic interactions and the gene networks that it regulates. Although VchM is a relatively new component of the V. cholerae genome, it is required for optimal V. cholerae growth in vitro and during infection. Unexpectedly, the usually essential σE Cell Envelope stress pathway is dispensable in ∆vchM V. cholerae, likely due to its lower activation in this mutant and the capacity for VchM methylation to limit expression of some Cell Envelope modifying genes. Our work illuminates how an acquired DNA methyltransferase can become integrated within complex Cell circuits to control critical housekeeping processes.
Min Cao - One of the best experts on this subject based on the ideXlab platform.
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the bacillus subtilis extracytoplasmic function sigmax factor regulates modification of the Cell Envelope and resistance to cationic antimicrobial peptides
Journal of Bacteriology, 2004Co-Authors: Min Cao, John D HelmannAbstract:Bacillus subtilis contains seven extracytoplasmic-function sigma factors that activate partially overlapping regulons. We here identify four additional members of the sigma(X) regulon, pbpX (penicillin-binding protein), ywnJ, the dlt operon (D-alanylation of teichoic acids), and the pss ybfM psd operon (phosphatidylethanolamine biosynthesis). Modification of teichoic acids by esterification with D-alanine and incorporation of phosphatidylethanolamine into the Cell membrane have a common consequence: in both cases positively charged amino groups are introduced into the Cell Envelope. The resulting reduction in the net negative charge of the Cell Envelope has been previously implicated as a resistance mechanism specific for cationic antimicrobial peptides. Consistent with this notion, we find that both sigX and dltA mutants are more sensitive to nisin than wild-type Cells. We conclude that activation of the sigma(X) regulon serves to alter Cell surface properties to provide protection against antimicrobial peptides.