Bryan Davies - Academia.edu (original) (raw)

Papers by Bryan Davies

PLoS pathogens, 2016

The virulence regulator ToxR initiates and coordinates gene expression needed by Vibrio cholerae ... more The virulence regulator ToxR initiates and coordinates gene expression needed by Vibrio cholerae to colonize the small intestine and cause disease. Despite its prominence in V. cholerae virulence, our understanding of the direct ToxR regulon is limited to four genes: toxT, ompT, ompU and ctxA. Here, we determine ToxR's genome-wide DNA-binding profile and demonstrate that ToxR is a global regulator of both progenitor genome-encoded genes and horizontally acquired islands that encode V. cholerae's major virulence factors and define pandemic lineages. We show that ToxR shares more than a third of its regulon with the histone-like nucleoid structuring protein H-NS, and antagonizes H-NS binding at shared binding locations. Importantly, we demonstrate that this regulatory interaction is the critical function of ToxR in V. cholerae colonization and biofilm formation. In the absence of H-NS, ToxR is no longer required for V. cholerae to colonize the infant mouse intestine or for rob...

Mol Cell, 2008

MutS homologs function in several cellular pathways including mismatch repair (MMR), the process ... more MutS homologs function in several cellular pathways including mismatch repair (MMR), the process by which mismatches introduced during DNA replication are corrected. We demonstrate that the C terminus of Bacillus subtilis MutS is necessary for an interaction with β clamp. This interaction is required for MutS-GFP focus formation in response to mismatches. Reciprocally, we show that a mutant of the β clamp causes elevated mutation frequencies and is reduced for MutS-GFP focus formation. MutS mutants defective for interaction with β clamp failed to support the next step of MMR, MutL-GFP focus formation. We conclude that the interaction between MutS and β is the major molecular interaction facilitating focus formation and that β clamp aids in the stabilization of MutS at a mismatch in vivo. The striking ability of the MutS C terminus to direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses MutS's interaction with β clamp.

Molecular Cell, Feb 15, 2008

MutS homologs function in several cellular pathways including mismatch repair (MMR), the process ... more MutS homologs function in several cellular pathways including mismatch repair (MMR), the process by which mismatches introduced during DNA replication are corrected. We demonstrate that the Cterminus of Bacillus subtilis MutS is necessary for an interaction with β-clamp. This interaction is required for MutS-GFP focus formation in response to mismatches. Reciprocally, we show a mutant of the β-clamp causes elevated mutation frequencies and is reduced for MutS-GFP focus formation. MutS mutants defective for interaction with β-clamp failed to support the next step of MMR, MutL-GFP focus formation. We conclude that the interaction between MutS and β is the major molecular interaction facilitating focus formation and that β-clamp aids in the stabilization of MutS at a mismatch in vivo. The striking ability of the MutS C-terminus to direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses MutS's interaction with β-clamp.

Mbio, Aug 28, 2012

The Harvard community has made this article openly available. Please share how this access benefi... more The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters. Citation Bogard, Ryan W., Bryan W. Davies, and John J. Mekalanos. 2012. MetR-regulated Vibrio cholerae metabolism is required for virulence. mBio 3(5): e00236-12.

Molecular Cell, Nov 12, 2009

Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP poo... more Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP pools and leading to replication fork arrest. While HU inhibition of RNR has been recognized for decades, the mechanism by which it leads to cell death remains unknown. To investigate the mechanism of HU-induced cell death we used a systems-level approach to determine the genomic and physiological responses of E. coli to HU treatment. Our results suggest a model by which HU treatment rapidly induces a set of protective responses to manage genomic instability in the majority of the cell population. Continued HU stress activates iron uptake as well as the toxins MazF and RelE whose activity causes the synthesis of incompletely translated proteins and stimulation of the envelope stress response system. These effects alter the properties of one of the cell's two terminal cytochrome oxidases in the electron transport chain, causing an increase in the production of superoxide. The increased superoxide production from the respiratory chain together with the increased iron uptake fuels the formation of hydroxyl radicals that contribute to HUinduced cell death. This work significantly expands our understanding of HU-mediated cell death and more broadly suggests a pathway whereby replication fork arrest leads to cell death.

mBio, 2015

Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive ca... more Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments, A. baumannii withstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. However, many Gram-negative pathogenic bacteria, including A. baumannii, fortify their outer membrane with hepta-acylated lipid A to protect the cell from CAMP-dependent cell lysis. Whereas in Escherichia coli and Salmonella, increased production of the outer membrane acyltransferase PagP results in formation of protective hepta-acylated lipid A, which reinforces the lipopolysaccharide portion of the outer membrane barrier, A. baumannii does not carry a gene that encodes a PagP homolog. Instead, A. baumannii has evolved a PagP-independent mechanism to synthesize protective hepta-acylated lipid A. Taking advantage of a recently adapted A. baumannii genetic recombineering system, we characterized two putative acyltransferases in A. baumannii designated LpxL Ab (A. baumannii LpxL) and LpxM Ab (A. baumannii LpxM), which transfer one and two lauroyl (C 12:0 ) acyl chains, respectively, during lipid A biosynthesis. Hepta-acylation of A. baumannii lipid A promoted resistance to vertebrate and polymyxin CAMPs, which are prescribed as last-resort treatment options. Intriguingly, our analysis also showed that LpxM Ab -

PLoS Pathogens, 2014

YbeY, a highly conserved protein, is an RNase in E. coli and plays key roles in both processing o... more YbeY, a highly conserved protein, is an RNase in E. coli and plays key roles in both processing of the critical 39 end of 16 S rRNA and in 70 S ribosome quality control under stress. These central roles account for YbeY's inclusion in the postulated minimal bacterial genome. However, YbeY is not essential in E. coli although loss of ybeY severely sensitizes it to multiple physiological stresses. Here, we show that YbeY is an essential endoribonuclease in Vibrio cholerae and is crucial for virulence, stress regulation, RNA processing and ribosome quality control, and is part of a core set of RNases essential in most representative pathogens. To understand its function, we analyzed the rRNA and ribosome profiles of a V. cholerae strain partially depleted for YbeY and other RNase mutants associated with 16 S rRNA processing; our results demonstrate that YbeY is also crucial for 16 S rRNA 39 end maturation in V. cholerae and that its depletion impedes subunit assembly into 70 S ribosomes. YbeY's importance to V. cholerae pathogenesis was demonstrated by the complete loss of mice colonization and biofilm formation, reduced cholera toxin production, and altered expression levels of virulence-associated small RNAs of a V. cholerae strain partially depleted for YbeY. Notably, the ybeY genes of several distantly related pathogens can fully complement an E. coli DybeY strain under various stress conditions, demonstrating the high conservation of YbeY's activity in stress regulation. Taken together, this work provides the first comprehensive exploration of YbeY's physiological role in a human pathogen, showing its conserved function across species in essential cellular processes.

Proceedings of the National Academy of Sciences, 2011

ChIP coupled with next-generation sequencing (ChIP-seq) has revolutionized whole-genome mapping o... more ChIP coupled with next-generation sequencing (ChIP-seq) has revolutionized whole-genome mapping of DNA-binding protein sites. Although ChIP-seq rapidly gained support in eukaryotic systems, it remains underused in the mapping of bacterial transcriptional regulator-binding sites. Using the virulence-required iron-responsive ferric uptake regulator (Fur), we report a simple, broadly applicable ChIP-seq method in the pathogen Vibrio cholerae. Combining our ChIP-seq results with available microarray data, we clarify direct and indirect Fur regulation of known iron-responsive genes. We validate a subset of Fur-binding sites in vivo and show a common motif present in all Fur ChIP-seq peaks that has enhanced binding affinity for purified V. cholerae Fur. Further analysis shows that V. cholerae Fur directly regulates several additional genes associated with Fur-binding sites, expanding the role of this transcription factor into the regulation of ribosome formation, additional transport functions, and unique sRNAs.

PLoS Pathogens, 2011

Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. ... more Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.

Nature Reviews Microbiology, 2007

Molecular Microbiology, 2010

The UPF0054 protein family is highly conserved with homologs present in nearly every sequenced ba... more The UPF0054 protein family is highly conserved with homologs present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homolog, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5′-and 3′-ends of 16S rRNA as well as maturation of the 5′-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.

Molecular Cell, 2008

MutS homologs function in several cellular pathways including mismatch repair (MMR), the process ... more MutS homologs function in several cellular pathways including mismatch repair (MMR), the process by which mismatches introduced during DNA replication are corrected. We demonstrate that the Cterminus of Bacillus subtilis MutS is necessary for an interaction with β-clamp. This interaction is required for MutS-GFP focus formation in response to mismatches. Reciprocally, we show a mutant of the β-clamp causes elevated mutation frequencies and is reduced for MutS-GFP focus formation. MutS mutants defective for interaction with β-clamp failed to support the next step of MMR, MutL-GFP focus formation. We conclude that the interaction between MutS and β is the major molecular interaction facilitating focus formation and that β-clamp aids in the stabilization of MutS at a mismatch in vivo. The striking ability of the MutS C-terminus to direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses MutS's interaction with β-clamp.

Molecular Cell, 2013

Quality control of ribosomes is critical for cellular function since protein mistranslation leads... more Quality control of ribosomes is critical for cellular function since protein mistranslation leads to severe physiological consequences. We report evidence of a previously unrecognized ribosome quality control system in bacteria that operates at the level of 70S to remove defective ribosomes. YbeY, a previously unidentified endoribonuclease, and the exonuclease RNase R act together by a process mediated specifically by the 30S ribosomal subunit, to degrade defective 70S ribosomes but not properly matured 70S ribosomes or individual subunits. Furthermore, there is essentially no fully matured 16S rRNA in a DybeY mutant at 45 C, making YbeY the only endoribonuclease to be implicated in the critically important processing of the 16S rRNA 3 0 terminus. These key roles in ribosome quality control and maturation indicate why YbeY is a member of the minimal bacterial gene set and suggest that it could be a potential target for antibacterial drugs.

Molecular Cell, 2009

Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP poo... more Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP pools and leading to replication fork arrest. While HU inhibition of RNR has been recognized for decades, the mechanism by which it leads to cell death remains unknown. To investigate the mechanism of HU-induced cell death we used a systems-level approach to determine the genomic and physiological responses of E. coli to HU treatment. Our results suggest a model by which HU treatment rapidly induces a set of protective responses to manage genomic instability in the majority of the cell population. Continued HU stress activates iron uptake as well as the toxins MazF and RelE whose activity causes the synthesis of incompletely translated proteins and stimulation of the envelope stress response system. These effects alter the properties of one of the cell's two terminal cytochrome oxidases in the electron transport chain, causing an increase in the production of superoxide. The increased superoxide production from the respiratory chain together with the increased iron uptake fuels the formation of hydroxyl radicals that contribute to HUinduced cell death. This work significantly expands our understanding of HU-mediated cell death and more broadly suggests a pathway whereby replication fork arrest leads to cell death.

Journal of Bacteriology, 2007

During the initial stages of symbiosis with the host plant Medicago sativa, Sinorhizobium melilot... more During the initial stages of symbiosis with the host plant Medicago sativa, Sinorhizobium meliloti must overcome an oxidative burst produced by the plant in order for proper symbiotic development to continue. While identifying mutants defective in symbiosis and oxidative stress defense, we isolated a mutant with a transposon insertion mutation of sitA, which encodes the periplasmic binding protein of the putative iron/ manganese ABC transporter SitABCD. Disruption of sitA causes elevated sensitivity to the reactive oxygen species hydrogen peroxide and superoxide. Disruption of sitA leads to elevated catalase activity and a severe decrease in superoxide dismutase B (SodB) activity and protein level. The decrease in SodB level strongly correlates with the superoxide sensitivity of the sitA mutant. We demonstrate that all free-living phenotypes of the sitA mutant can be rescued by the addition of exogenous manganese but not iron, a result that strongly implies that SitABCD plays an important role in manganese uptake in S. meliloti.

Journal of Bacteriology, 2009

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instabilit... more DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell.

Journal of Bacteriology, 2008

We report here the first characterization of the Sinorhizobium meliloti open reading frame SMc011... more We report here the first characterization of the Sinorhizobium meliloti open reading frame SMc01113. The SMc01113 protein is a member of a highly conserved protein family, universal among bacteria. We demonstrate that the SMc01113 gene is absolutely required for S. meliloti symbiosis with alfalfa and also for the protection of the bacterium from a wide range of environmental stresses.

Journal of Bacteriology, 2007

Employing a novel two-part screen, we identified Sinorhizobium meliloti mutants that were both se... more Employing a novel two-part screen, we identified Sinorhizobium meliloti mutants that were both sensitive to hydrogen peroxide and symbiotically defective on the host plant Medicago sativa. The mutations affect a wide variety of cellular processes and represent both novel and previously identified genes important in symbiosis.

PLoS pathogens, 2016

The virulence regulator ToxR initiates and coordinates gene expression needed by Vibrio cholerae ... more The virulence regulator ToxR initiates and coordinates gene expression needed by Vibrio cholerae to colonize the small intestine and cause disease. Despite its prominence in V. cholerae virulence, our understanding of the direct ToxR regulon is limited to four genes: toxT, ompT, ompU and ctxA. Here, we determine ToxR's genome-wide DNA-binding profile and demonstrate that ToxR is a global regulator of both progenitor genome-encoded genes and horizontally acquired islands that encode V. cholerae's major virulence factors and define pandemic lineages. We show that ToxR shares more than a third of its regulon with the histone-like nucleoid structuring protein H-NS, and antagonizes H-NS binding at shared binding locations. Importantly, we demonstrate that this regulatory interaction is the critical function of ToxR in V. cholerae colonization and biofilm formation. In the absence of H-NS, ToxR is no longer required for V. cholerae to colonize the infant mouse intestine or for rob...

Mol Cell, 2008

MutS homologs function in several cellular pathways including mismatch repair (MMR), the process ... more MutS homologs function in several cellular pathways including mismatch repair (MMR), the process by which mismatches introduced during DNA replication are corrected. We demonstrate that the C terminus of Bacillus subtilis MutS is necessary for an interaction with β clamp. This interaction is required for MutS-GFP focus formation in response to mismatches. Reciprocally, we show that a mutant of the β clamp causes elevated mutation frequencies and is reduced for MutS-GFP focus formation. MutS mutants defective for interaction with β clamp failed to support the next step of MMR, MutL-GFP focus formation. We conclude that the interaction between MutS and β is the major molecular interaction facilitating focus formation and that β clamp aids in the stabilization of MutS at a mismatch in vivo. The striking ability of the MutS C terminus to direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses MutS's interaction with β clamp.

Molecular Cell, Feb 15, 2008

MutS homologs function in several cellular pathways including mismatch repair (MMR), the process ... more MutS homologs function in several cellular pathways including mismatch repair (MMR), the process by which mismatches introduced during DNA replication are corrected. We demonstrate that the Cterminus of Bacillus subtilis MutS is necessary for an interaction with β-clamp. This interaction is required for MutS-GFP focus formation in response to mismatches. Reciprocally, we show a mutant of the β-clamp causes elevated mutation frequencies and is reduced for MutS-GFP focus formation. MutS mutants defective for interaction with β-clamp failed to support the next step of MMR, MutL-GFP focus formation. We conclude that the interaction between MutS and β is the major molecular interaction facilitating focus formation and that β-clamp aids in the stabilization of MutS at a mismatch in vivo. The striking ability of the MutS C-terminus to direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses MutS's interaction with β-clamp.

Mbio, Aug 28, 2012

The Harvard community has made this article openly available. Please share how this access benefi... more The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters. Citation Bogard, Ryan W., Bryan W. Davies, and John J. Mekalanos. 2012. MetR-regulated Vibrio cholerae metabolism is required for virulence. mBio 3(5): e00236-12.

Molecular Cell, Nov 12, 2009

Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP poo... more Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP pools and leading to replication fork arrest. While HU inhibition of RNR has been recognized for decades, the mechanism by which it leads to cell death remains unknown. To investigate the mechanism of HU-induced cell death we used a systems-level approach to determine the genomic and physiological responses of E. coli to HU treatment. Our results suggest a model by which HU treatment rapidly induces a set of protective responses to manage genomic instability in the majority of the cell population. Continued HU stress activates iron uptake as well as the toxins MazF and RelE whose activity causes the synthesis of incompletely translated proteins and stimulation of the envelope stress response system. These effects alter the properties of one of the cell's two terminal cytochrome oxidases in the electron transport chain, causing an increase in the production of superoxide. The increased superoxide production from the respiratory chain together with the increased iron uptake fuels the formation of hydroxyl radicals that contribute to HUinduced cell death. This work significantly expands our understanding of HU-mediated cell death and more broadly suggests a pathway whereby replication fork arrest leads to cell death.

mBio, 2015

Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive ca... more Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments, A. baumannii withstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the conserved lipid A component of the Gram-negative outer membrane to lyse the bacterial cell. However, many Gram-negative pathogenic bacteria, including A. baumannii, fortify their outer membrane with hepta-acylated lipid A to protect the cell from CAMP-dependent cell lysis. Whereas in Escherichia coli and Salmonella, increased production of the outer membrane acyltransferase PagP results in formation of protective hepta-acylated lipid A, which reinforces the lipopolysaccharide portion of the outer membrane barrier, A. baumannii does not carry a gene that encodes a PagP homolog. Instead, A. baumannii has evolved a PagP-independent mechanism to synthesize protective hepta-acylated lipid A. Taking advantage of a recently adapted A. baumannii genetic recombineering system, we characterized two putative acyltransferases in A. baumannii designated LpxL Ab (A. baumannii LpxL) and LpxM Ab (A. baumannii LpxM), which transfer one and two lauroyl (C 12:0 ) acyl chains, respectively, during lipid A biosynthesis. Hepta-acylation of A. baumannii lipid A promoted resistance to vertebrate and polymyxin CAMPs, which are prescribed as last-resort treatment options. Intriguingly, our analysis also showed that LpxM Ab -

PLoS Pathogens, 2014

YbeY, a highly conserved protein, is an RNase in E. coli and plays key roles in both processing o... more YbeY, a highly conserved protein, is an RNase in E. coli and plays key roles in both processing of the critical 39 end of 16 S rRNA and in 70 S ribosome quality control under stress. These central roles account for YbeY's inclusion in the postulated minimal bacterial genome. However, YbeY is not essential in E. coli although loss of ybeY severely sensitizes it to multiple physiological stresses. Here, we show that YbeY is an essential endoribonuclease in Vibrio cholerae and is crucial for virulence, stress regulation, RNA processing and ribosome quality control, and is part of a core set of RNases essential in most representative pathogens. To understand its function, we analyzed the rRNA and ribosome profiles of a V. cholerae strain partially depleted for YbeY and other RNase mutants associated with 16 S rRNA processing; our results demonstrate that YbeY is also crucial for 16 S rRNA 39 end maturation in V. cholerae and that its depletion impedes subunit assembly into 70 S ribosomes. YbeY's importance to V. cholerae pathogenesis was demonstrated by the complete loss of mice colonization and biofilm formation, reduced cholera toxin production, and altered expression levels of virulence-associated small RNAs of a V. cholerae strain partially depleted for YbeY. Notably, the ybeY genes of several distantly related pathogens can fully complement an E. coli DybeY strain under various stress conditions, demonstrating the high conservation of YbeY's activity in stress regulation. Taken together, this work provides the first comprehensive exploration of YbeY's physiological role in a human pathogen, showing its conserved function across species in essential cellular processes.

Proceedings of the National Academy of Sciences, 2011

ChIP coupled with next-generation sequencing (ChIP-seq) has revolutionized whole-genome mapping o... more ChIP coupled with next-generation sequencing (ChIP-seq) has revolutionized whole-genome mapping of DNA-binding protein sites. Although ChIP-seq rapidly gained support in eukaryotic systems, it remains underused in the mapping of bacterial transcriptional regulator-binding sites. Using the virulence-required iron-responsive ferric uptake regulator (Fur), we report a simple, broadly applicable ChIP-seq method in the pathogen Vibrio cholerae. Combining our ChIP-seq results with available microarray data, we clarify direct and indirect Fur regulation of known iron-responsive genes. We validate a subset of Fur-binding sites in vivo and show a common motif present in all Fur ChIP-seq peaks that has enhanced binding affinity for purified V. cholerae Fur. Further analysis shows that V. cholerae Fur directly regulates several additional genes associated with Fur-binding sites, expanding the role of this transcription factor into the regulation of ribosome formation, additional transport functions, and unique sRNAs.

PLoS Pathogens, 2011

Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. ... more Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.

Nature Reviews Microbiology, 2007

Molecular Microbiology, 2010

The UPF0054 protein family is highly conserved with homologs present in nearly every sequenced ba... more The UPF0054 protein family is highly conserved with homologs present in nearly every sequenced bacterium. In some bacteria, the respective gene is essential, while in others its loss results in a highly pleiotropic phenotype. Despite detailed structural studies, a cellular role for this protein family has remained unknown. We report here that deletion of the Escherichia coli homolog, YbeY, causes striking defects that affect ribosome activity, translational fidelity and ribosome assembly. Mapping of 16S, 23S and 5S rRNA termini reveals that YbeY influences the maturation of all three rRNAs, with a particularly strong effect on maturation at both the 5′-and 3′-ends of 16S rRNA as well as maturation of the 5′-termini of 23S and 5S rRNAs. Furthermore, we demonstrate strong genetic interactions between ybeY and rnc (encoding RNase III), ybeY and rnr (encoding RNase R), and ybeY and pnp (encoding PNPase), further suggesting a role for YbeY in rRNA maturation. Mutation of highly conserved amino acids in YbeY, allowed the identification of two residues (H114, R59) that were found to have a significant effect in vivo. We discuss the implications of these findings for rRNA maturation and ribosome assembly in bacteria.

Molecular Cell, 2008

MutS homologs function in several cellular pathways including mismatch repair (MMR), the process ... more MutS homologs function in several cellular pathways including mismatch repair (MMR), the process by which mismatches introduced during DNA replication are corrected. We demonstrate that the Cterminus of Bacillus subtilis MutS is necessary for an interaction with β-clamp. This interaction is required for MutS-GFP focus formation in response to mismatches. Reciprocally, we show a mutant of the β-clamp causes elevated mutation frequencies and is reduced for MutS-GFP focus formation. MutS mutants defective for interaction with β-clamp failed to support the next step of MMR, MutL-GFP focus formation. We conclude that the interaction between MutS and β is the major molecular interaction facilitating focus formation and that β-clamp aids in the stabilization of MutS at a mismatch in vivo. The striking ability of the MutS C-terminus to direct focus formation at replisomes by itself, suggests that it is mismatch recognition that licenses MutS's interaction with β-clamp.

Molecular Cell, 2013

Quality control of ribosomes is critical for cellular function since protein mistranslation leads... more Quality control of ribosomes is critical for cellular function since protein mistranslation leads to severe physiological consequences. We report evidence of a previously unrecognized ribosome quality control system in bacteria that operates at the level of 70S to remove defective ribosomes. YbeY, a previously unidentified endoribonuclease, and the exonuclease RNase R act together by a process mediated specifically by the 30S ribosomal subunit, to degrade defective 70S ribosomes but not properly matured 70S ribosomes or individual subunits. Furthermore, there is essentially no fully matured 16S rRNA in a DybeY mutant at 45 C, making YbeY the only endoribonuclease to be implicated in the critically important processing of the 16S rRNA 3 0 terminus. These key roles in ribosome quality control and maturation indicate why YbeY is a member of the minimal bacterial gene set and suggest that it could be a potential target for antibacterial drugs.

Molecular Cell, 2009

Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP poo... more Hydroxyurea (HU) specifically inhibits class I ribonucleotide reductase (RNR), depleting dNTP pools and leading to replication fork arrest. While HU inhibition of RNR has been recognized for decades, the mechanism by which it leads to cell death remains unknown. To investigate the mechanism of HU-induced cell death we used a systems-level approach to determine the genomic and physiological responses of E. coli to HU treatment. Our results suggest a model by which HU treatment rapidly induces a set of protective responses to manage genomic instability in the majority of the cell population. Continued HU stress activates iron uptake as well as the toxins MazF and RelE whose activity causes the synthesis of incompletely translated proteins and stimulation of the envelope stress response system. These effects alter the properties of one of the cell's two terminal cytochrome oxidases in the electron transport chain, causing an increase in the production of superoxide. The increased superoxide production from the respiratory chain together with the increased iron uptake fuels the formation of hydroxyl radicals that contribute to HUinduced cell death. This work significantly expands our understanding of HU-mediated cell death and more broadly suggests a pathway whereby replication fork arrest leads to cell death.

Journal of Bacteriology, 2007

During the initial stages of symbiosis with the host plant Medicago sativa, Sinorhizobium melilot... more During the initial stages of symbiosis with the host plant Medicago sativa, Sinorhizobium meliloti must overcome an oxidative burst produced by the plant in order for proper symbiotic development to continue. While identifying mutants defective in symbiosis and oxidative stress defense, we isolated a mutant with a transposon insertion mutation of sitA, which encodes the periplasmic binding protein of the putative iron/ manganese ABC transporter SitABCD. Disruption of sitA causes elevated sensitivity to the reactive oxygen species hydrogen peroxide and superoxide. Disruption of sitA leads to elevated catalase activity and a severe decrease in superoxide dismutase B (SodB) activity and protein level. The decrease in SodB level strongly correlates with the superoxide sensitivity of the sitA mutant. We demonstrate that all free-living phenotypes of the sitA mutant can be rescued by the addition of exogenous manganese but not iron, a result that strongly implies that SitABCD plays an important role in manganese uptake in S. meliloti.

Journal of Bacteriology, 2009

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instabilit... more DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell.

Journal of Bacteriology, 2008

We report here the first characterization of the Sinorhizobium meliloti open reading frame SMc011... more We report here the first characterization of the Sinorhizobium meliloti open reading frame SMc01113. The SMc01113 protein is a member of a highly conserved protein family, universal among bacteria. We demonstrate that the SMc01113 gene is absolutely required for S. meliloti symbiosis with alfalfa and also for the protection of the bacterium from a wide range of environmental stresses.

Journal of Bacteriology, 2007

Employing a novel two-part screen, we identified Sinorhizobium meliloti mutants that were both se... more Employing a novel two-part screen, we identified Sinorhizobium meliloti mutants that were both sensitive to hydrogen peroxide and symbiotically defective on the host plant Medicago sativa. The mutations affect a wide variety of cellular processes and represent both novel and previously identified genes important in symbiosis.