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Matthew K Waldor

Publications and source records attributed to Matthew K Waldor.

At least 19 recordsLinked to original sources

par genes and the pathology of chromosome loss in Vibrio cholerae.

The causes and consequences of chromosome loss in bacteria with multiple chromosomes are unknown. Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, has two circular chromosomes. Like many other bacterial chromosomes, both V. cholerae chromosomes contain homologues of plasmid partitioning (par) genes. In plasmids, par genes act to segregate plasmid molecules to daughter cells and thereby ensure plasmid maintenance; however, the contribution of par genes to chromosome segregation is not clear. Here, we show that the chromosome II parAB2 genes are essential for the segregation of chromosome II but not chromosome I. In a parAB2 deletion mutant, chromosome II is mislocalized and frequently fails to segregate, yielding cells with only chromosome I. These cells divide once; their progeny are not viable. Instead, chromosome II-deficient cells undergo dramatic cell enlargement, nucleoid condensation and degradation, and loss of membrane integrity. The highly consistent nature of these cytologic changes suggests that prokaryotes, like eukaryotes, may possess characteristic death pathways.

Bacterial Proteins↗

Antimicrobial peptides activate the Vibrio cholerae sigmaE regulon through an OmpU-dependent signalling pathway.

Vibrio cholerae, an enteric pathogen, is subject to assault by several membrane-acting, host gut-derived antimicrobial peptides (AP). We previously found that a major V. cholerae outer membrane protein, OmpU, confers resistance to polymyxin B and to a bioactive peptide (P2) derived from the human bactericidal/permeability-increasing protein. Here, we report that the alternative sigma factor sigma(E) also plays a critical role in determining V. cholerae resistance to AP and that OmpU and sigma(E) lie in the same pathway. In fact, we found that OmpU is a key determinant of basal sigma(E) expression. We also found that sublethal AP exposure activates sigma(E) and the sigma(E)-mediated periplasmic stress response. sigma(E) is not activated by P2 in V. cholerae cells lacking OmpU or DegS, a periplasmic protease that controls sigma(E) activity. The lack of AP-elicited sigma(E) activation in a strain harbouring a point mutation in OmpU's putative DegS-binding residues provides support for a link between OmpU and DegS-mediated activation of sigma(E). We propose that AP-induced membrane perturbations change the conformation of OmpU to trigger a DegS-dependent sigma(E)-activating cascade. Thus, OmpU appears to act as a sensor component in a signal transduction pathway.

Adhesins, Bacterial↗

Enterohemorrhagic Escherichia coli O157:H7 gal mutants are sensitive to bacteriophage P1 and defective in intestinal colonization.

Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal(+) strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal(+) parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal(+) revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

Animals↗

A dynamic, mitotic-like mechanism for bacterial chromosome segregation.

The mechanisms that mediate chromosome segregation in bacteria are poorly understood. Despite evidence of dynamic movement of chromosome regions, to date, mitotic-like mechanisms that act on the bacterial chromosome have not been demonstrated. Here we provide evidence that the Vibrio cholerae ParAI and ParBI proteins are components of an apparatus that pulls the origin region of the large V. cholerae chromosome to the cell pole and anchors it there. ParBI interacts with a conserved origin-proximal, centromere-like site (parSI) that, following chromosome replication, segregates asymmetrically from one pole to the other. While segregating, parSI stretches far away from neighboring chromosomal loci. ParAI forms a dynamic band that extends from the pole to the segregating ParBI/parSI complex. Movement of ParBI/parSI across the cell occurs in concert with ParAI retraction. Deletion of parAI disrupts proper origin localization and segregation dynamics, and parSI no longer separates from nearby regions. These data suggest that ParAI forms a dynamic structure that pulls the ParBI-bound chromosome to the pole in a process analogous to anaphase of eukaryotic mitosis.

Bacterial Proteins↗

Characterization of a higBA toxin-antitoxin locus in Vibrio cholerae.

Toxin-antitoxin (TA) loci, which were initially characterized as plasmid stabilization agents, have in recent years been detected on the chromosomes of numerous free-living bacteria. Vibrio cholerae, the causative agent of cholera, contains 13 putative TA loci, all of which are clustered within the superintegron on chromosome II. Here we report the characterization of the V. cholerae higBA locus, also known as VCA0391/2. Deletion of higA alone was not possible, consistent with predictions that it encodes an antitoxin, and biochemical analyses confirmed that HigA interacts with HigB. Transient exogenous expression of the toxin HigB dramatically slowed growth of V. cholerae and Escherichia coli and reduced the numbers of CFU by several orders of magnitude. HigB toxicity could be counteracted by simultaneous or delayed production of HigA, although HigA's effect diminished as the delay lengthened. Transcripts from endogenous higBA increased following treatment of V. cholerae with translational inhibitors, presumably due to reduced levels of HigA, which represses the higBA locus. However, no higBA-dependent cell death was observed in response to such stimuli. Thus, at least under the conditions tested, activation of endogenous HigB does not appear to be bactericidal.

Amino Acid Sequence↗

Global gene expression and phenotypic analysis of a Vibrio cholerae rpoH deletion mutant.

Vibrio cholerae, the cause of cholera, can grow in a variety of environments outside of human hosts. During infection, this pathogen must adapt to significant environmental alterations, including the elevated temperature of the human gastrointestinal tract. Sigma(32), an alternative sigma factor encoded by rpoH, activates transcription of genes involved in the heat shock response in several bacterial species. Here, we assessed the role of sigma(32) in V. cholerae physiology. In aggregate, our findings suggest that sigma(32) promotes V. cholerae growth at temperatures ranging at least from 15 degrees C to 42 degrees C. Growth of the rpoH mutant was severely attenuated within the suckling mouse intestine, suggesting that sigma(32)-regulated genes are critical for V. cholerae adaptation to conditions within the gastrointestinal tract. We defined the V. cholerae RpoH regulon by comparing the whole-genome transcription profiles of the wild-type and rpoH mutant strains after a temperature up-shift. Most of the V. cholerae genes expressed in an RpoH-dependent manner after heat shock encode proteins that influence protein fate, such as proteases and chaperones, or are of unknown function. Bioinformatic analyses of the microarray data were used to define a putative sigma(32) consensus binding sequence and subsequently to identify genes that are likely to be directly regulated by RpoH in the whole V. cholerae genome.

Animals↗

LexA represses CTXphi transcription by blocking access of the alpha C-terminal domain of RNA polymerase to promoter DNA.

CTXPhi is a Vibrio cholerae-specific temperate filamentous phage that encodes cholera toxin. CTXPhi lysogens can be induced with DNA damage-inducing agents such as UV light, leading to the release of CTXPhi virions and the rapid dissemination of cholera toxin genes to new V. cholerae hosts. This environmental regulation is directly mediated by LexA, the host-encoded global SOS transcription factor. LexA and a phage-encoded repressor, RstR, both repress transcription from P(rstA), the primary CTXPhi promoter. Because the LexA binding site is located upstream of the core P(rstA) promoter and overlaps with A-tract sequences, we speculated that LexA represses P(rstA) by occluding a promoter UP element, a binding site for the C-terminal domain of the alpha subunit of RNA polymerase (RNAP) (alphaCTD). Using in vitro transcription assays, we have shown that the LexA binding site stimulates maximal rstA transcription in the absence of any added factors. The alphaCTD of RNAP is required for this stimulation, demonstrating that the LexA site contains, or overlaps with, a promoter UP element. LexA represses rstA transcription by normal RNAP but fails to repress rstA transcription catalyzed by RNAP lacking the alphaCTD. DNase I footprint analysis mapped the alphaCTD binding site to the upstream promoter region that includes the LexA binding site. The addition of free alpha subunits blocked the binding of LexA to rstA promoter DNA, indicating that LexA and the alphaCTD directly compete for binding to their respective sites. To our knowledge, this is the first report of a repressor blocking transcription initiation by occluding a promoter UP element.

Bacterial Proteins↗

The current ICE age: biology and evolution of SXT-related integrating conjugative elements.

SXT is an integrating conjugative element (ICE) that was initially isolated from a 1992 Vibrio cholerae O139 clinical isolate from India. This approximately 100-kb ICE encodes resistance to multiple antibiotics. SXT or closely related ICEs are now present in most clinical and some environmental V. cholerae isolates from Asia and Africa. SXT-related ICEs are not limited to V. cholerae. It is now clear that so-called IncJ elements such as R391 are closely related to SXT. More than 25 members of the SXT/R391 family of ICEs have now been identified in environmental and clinical isolates of diverse species of gamma-proteobacteria worldwide. In this review, we discuss the diversity, evolution and biology of this family of ICEs.

Bacterial Proteins↗

Identification of 17 Pseudomonas aeruginosa sRNAs and prediction of sRNA-encoding genes in 10 diverse pathogens using the bioinformatic tool sRNAPredict2.

sRNAs are small, non-coding RNA species that control numerous cellular processes. Although it is widely accepted that sRNAs are encoded by most if not all bacteria, genome-wide annotations for sRNA-encoding genes have been conducted in only a few of the nearly 300 bacterial species sequenced to date. To facilitate the efficient annotation of bacterial genomes for sRNA-encoding genes, we developed a program, sRNAPredict2, that identifies putative sRNAs by searching for co-localization of genetic features commonly associated with sRNA-encoding genes. Using sRNAPredict2, we conducted genome-wide annotations for putative sRNA-encoding genes in the intergenic regions of 11 diverse pathogens. In total, 2759 previously unannotated candidate sRNA loci were predicted. There was considerable range in the number of sRNAs predicted in the different pathogens analyzed, raising the possibility that there are species-specific differences in the reliance on sRNA-mediated regulation. Of 34 previously unannotated sRNAs predicted in the opportunistic pathogen Pseudomonas aeruginosa, 31 were experimentally tested and 17 were found to encode sRNA transcripts. Our findings suggest that numerous genes have been missed in the current annotations of bacterial genomes and that, by using improved bioinformatic approaches and tools, much remains to be discovered in 'intergenic' sequences.

Computational Biology↗

SXT-related integrating conjugative element in New World Vibrio cholerae.

SXT-related integrating conjugative elements (ICEs) became prevalent in Asian Vibrio cholerae populations after V. cholerae O139 emerged. Here, we describe an SXT-related ICE, ICEVchMex1, in a Mexican environmental V. cholerae isolate. Identification of ICEVchMex1 represents the first description of an SXT-related ICE in the Western Hemisphere. The significant differences between the SXT and ICEVchMex1 genomes suggest that these ICEs have evolved independently.

Conjugation, Genetic↗

Activation of the Vibrio cholerae SOS response is not required for intestinal cholera toxin production or colonization.

Cholera toxin, one of the main virulence factors of Vibrio cholerae, is encoded in the genome of CTXphi, a V. cholerae-specific lysogenic filamentous bacteriophage. Although the genes encoding cholera toxin, ctxAB, are known to have their own promoter, the toxin genes can also be transcribed from an upstream CTXphi promoter, PrstA. The V. cholerae SOS response to DNA damage induces the CTX prophage by stimulating gene expression initiating from PrstA. Here, we investigated whether ctxA mRNA levels increase along with the levels of the transcripts for the other CTXphi genes following stimulation of the V. cholerae SOS response. Treatment of V. cholerae with the SOS-inducing agent mitomycin C increased the level of ctxA mRNA approximately sevenfold, apparently by augmenting the activity of PrstA. However, using suckling mice as a model host, we found that intraintestinal ctxA transcription does not depend on PrstA. In fact, the suckling mouse intestine does not appear to be a potent inducer of the V. cholerae SOS response. Furthermore, alleviation of LexA-mediated repression of the V. cholerae SOS regulon was not required for V. cholerae growth in the suckling mouse intestine. Our observations suggest that pathogenicity of V. cholerae does not depend on its SOS response.

Animals↗

Requirement for Vibrio cholerae integration host factor in conjugative DNA transfer.

The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. In the absence of IHF, V. cholerae displayed a modest defect for serving as an SXT recipient. Surprisingly, SXT integration into or excision from the V. cholerae chromosome, which requires an SXT-encoded integrase related to lambda integrase, did not require IHF. Therefore, the defect in SXT transmission in the V. cholerae IHF mutant is probably not related to IHF's ability to promote DNA recombination. The V. cholerae IHF mutant was also highly impaired as a donor of RP4, a broad-host-range conjugative plasmid. Thus, the V. cholerae IHF mutant appears to have a general defect in conjugation. Escherichia coli IHF mutants were not impaired as donors or recipients of SXT or RP4, indicating that IHF is a V. cholerae-specific conjugation factor.

Bacterial Proteins↗

Independent control of replication initiation of the two Vibrio cholerae chromosomes by DnaA and RctB.

Although the two Vibrio cholerae chromosomes initiate replication in a coordinated fashion, we show here that each chromosome appears to have a specific replication initiator. DnaA overproduction promoted overinitiation of chromosome I and not chromosome II. In contrast, overproduction of RctB, a protein that binds to the origin of replication of chromosome II, promoted overinitiation of chromosome II and not chromosome I.

Bacterial Proteins↗

Autorepression of RctB, an initiator of Vibrio cholerae chromosome II replication.

The RctB protein binds to the origin of replication of Vibrio cholerae chromosome II (chrII) and is required for oriCIIVc-based replication. Here, we found that RctB acts as an autorepressor, inhibiting rctB transcription. Integration host factor promotes rctB transcription, while Dam and DnaA, factors required for replication of both V. cholerae chromosomes, influence RctB autorepression. Thus, RctB appears to regulate chrII replication as both an initiator and a transcription repressor, and its synthesis is modulated by factors that govern replication of both chromosomes.

Chromosomes, Bacterial↗

sRNAPredict: an integrative computational approach to identify sRNAs in bacterial genomes.

Small non-coding bacterial RNAs (sRNAs) play important regulatory roles in a variety of cellular processes. Nearly all known sRNAs have been identified in Escherichia coli and most of these are not conserved in the majority of other bacterial species. Many of the E.coli sRNAs were initially predicted through bioinformatic approaches based on their common features, namely that they are encoded between annotated open reading frames and are flanked by predictable transcription signals. Because promoter consensus sequences are undetermined for most species, the successful use of bioinformatics to identify sRNAs in bacteria other than E.coli has been limited. We have created a program, sRNAPredict, which uses coordinate-based algorithms to integrate the respective positions of individual predictive features of sRNAs and rapidly identify putative intergenic sRNAs. Relying only on sequence conservation and predicted Rho-independent terminators, sRNAPredict was used to search for sRNAs in Vibrio cholerae. This search identified 9 of the 10 known or putative V.cholerae sRNAs and 32 candidates for novel sRNAs. Small transcripts for 6 out of 9 candidate sRNAs were observed by Northern analysis. Our findings suggest that sRNAPredict can be used to efficiently identify novel sRNAs even in bacteria for which promoter consensus sequences are not available.

Algorithms↗

LexA cleavage is required for CTX prophage induction.

The physiologic conditions and molecular interactions that control phage production have been studied in few temperate phages. We investigated the mechanisms that regulate production of CTXphi, a temperate filamentous phage that infects Vibrio cholerae and encodes cholera toxin. In CTXphi lysogens, the activity of P(rstA), the only CTXphi promoter required for CTX prophage development, is repressed by RstR, the CTXvphi repressor. We found that the V. cholerae SOS response regulates CTXvphi production. The molecular mechanism by which this cellular response to DNA damage controls CTXphi production differs from that by which the E. coli SOS response controls induction of many prophages. UV-stimulated CTXphi production required RecA-dependent autocleavage of LexA, a repressor that controls expression of numerous host DNA repair genes. LexA and RstR both bind to and repress P(rstA). Thus, CTXphi production is controlled by a cellular repressor whose activity is regulated by the cell's response to DNA damage.

Antibiotics, Antineoplastic↗

Interactions between inner membrane proteins in donor and recipient cells limit conjugal DNA transfer.

Conjugation enables horizontal transmission of DNA among bacteria, thereby facilitating the rapid spread of genes such as those conferring resistance to antibiotics. Cell-cell contact is required for conjugative DNA transfer but does not ensure its success. The presence of certain plasmids in potential recipient cells inhibits redundant transfer of these plasmids from competent donors despite contact between donor and recipient cells. Here, we used two closely related integrating conjugative elements (ICEs), SXT and R391, to identify genes that inhibit redundant conjugative transfer. Cells containing SXT exclude transfer of a second copy of SXT but not R391 and vice versa. The specific exclusion of SXT and R391 is dependent upon variants of TraG and Eex, ICE-encoded inner membrane proteins in donor and recipient cells, respectively. We identified short sequences within each variant that determine the exquisite specificity of self-recognition; these data suggest that direct interactions between TraG and Eex mediate exclusion.

Amino Acid Sequence↗