Showing papers by "Matthew K. Waldor published in 2007"

Distribution of Centromere-Like parS Sites in Bacteria: Insights from Comparative Genomics

Abstract: Partitioning of low-copy-number plasmids to daughter cells often depends on ParA and ParB proteins acting on centromere-like parS sites. Similar chromosome-encoded par loci likely also contribute to chromosome segregation. Here, we used bioinformatic approaches to search for chromosomal parS sites in 400 prokaryotic genomes. Although the consensus sequence matrix used to search for parS sites was derived from two gram-positive species, putative parS sites were identified on the chromosomes of 69% of strains from all branches of bacteria. Strains that were not found to contain parS sites clustered among relatively few branches of the prokaryotic evolutionary tree. In the vast majority of cases, parS sites were identified in origin-proximal regions of chromosomes. The widespread conservation of parS sites across diverse bacteria suggests that par loci evolved very early in the evolution of bacterial chromosomes and that the absence of parS, parA, and/or parB in certain strains likely reflects the loss of one of more of these loci much later in evolution. Moreover, the highly conserved origin-proximal position of parS suggests par loci are primarily devoted to regulating processes that involve the origin region of bacterial chromosomes. In species containing multiple chromosomes, the parS sites found on secondary chromosomes diverge significantly from those found on their primary chromosomes, suggesting that chromosome segregation of multipartite genomes requires distinct replicon-specific par loci. Furthermore, parS sites on secondary chromosomes are not well conserved among different species, suggesting that the evolutionary histories of secondary chromosomes are more diverse than those of primary chromosomes.

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

Abstract: 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.

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

Abstract: Summary 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 s E also plays a critical role in determining V. cholerae resistance to AP and that OmpU and s E lie in the same pathway. In fact, we found that OmpU is a key determinant of basal s E

par genes and the pathology of chromosome loss in Vibrio cholerae

Abstract: 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.

RNase E‐dependent processing stabilizes MicX, a Vibrio cholerae sRNA

Abstract: In Vibrio cholerae, bioinformatic approaches have been used to predict the locations of numerous small RNA (sRNA)-encoding genes, but biological roles have been determined for very few. Here, we describe the expression, processing and biological role of an sRNA (previously known as A10) that was identified through such analyses. We have renamed this sRNA MicX as, like the Escherichia coli sRNAs MicA, MicC and MicF, it regulates expression of an outer membrane protein (OMP). MicX appears to be a direct negative regulator of vc0972, which encodes an uncharacterized OMP, and vc0620, which encodes the periplasmic component of a peptide ABC transporter. Hfq is apparently not required for MicX's interactions with and regulation of these targets. The sequence encoding MicX overlaps with vca0943; however, primary transcripts of MicX are processed in an RNase E- and Hfq-dependent fashion to a shorter, still active and much more stable form consisting largely of the vca0943 3' untranslated region. Our data suggest that processing of MicX enhances its effectiveness, and that sRNA cleavage is not simply a means to sRNA inactivation and clearance.

Enterohemorrhagic Escherichia coli O157:H7 gal Mutants Are Sensitive to Bacteriophage P1 and Defective in Intestinal Colonization

Abstract: 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.

Distinct centromere-like parS sites on the two chromosomes of Vibrio spp.

Abstract: Vibrio cholerae, the cause of cholera, has two circular chromosomes. The parAB genes on each V. cholerae chromosome act to control chromosome segregation in a replicon-specific fashion. The chromosome I (ChrI) parAB genes (parAB1) govern the localization of the origin region of ChrI, while the chromosome II (ChrII) parAB genes (parAB2) control the segregation of ChrII. In addition to ParA and ParB proteins, Par systems require ParB binding sites (parS). Here we identified the parS sites on both V. cholerae chromosomes. We found three clustered origin-proximal ParB1 binding parS1 sites on ChrI. Deletion of these three parS1 sites abrogated yellow fluorescent protein (YFP)-ParB1 focus formation in vivo and resulted in mislocalization of the ChrI origin region. However, as observed in a parA1 mutant, mislocalization of the ChrI origin region in the parS1 mutant did not compromise V. cholerae growth, suggesting that additional (non-Par-related) mechanisms may mediate the partitioning of ChrI. We also identified 10 ParB2 binding parS2 sites, which differed in sequence from parS1. Fluorescent derivatives of ParB1 and ParB2 formed foci only with the cognate parS sequence. parABS2 appears to form a functional partitioning system, as we found that parABS2 was sufficient to stabilize an ordinarily unstable plasmid in Escherichia coli. Most parS2 sites were located within 70 kb of the ChrII origin of replication, but one parS2 site was found in the terminus region of ChrI. In contrast, in other sequenced vibrio species, the distribution of parS1 and parS2 sites was entirely chromosome specific.

Determinants of Entry Exclusion within Eex and TraG Are Cytoplasmic

Abstract: We report here functional and topological analyses of TraG and Eex, the donor and recipient cell inner membrane proteins that mediate entry exclusion in the SXT/R391 family of integrative conjugative elements. We found that the exclusion-determining regions of the Eex variants EexS (SXT) and EexR (R391) are located in distinct yet overlapping regions of the proteins. Unexpectedly, the carboxyl-terminal regions of TraG and Eex, which contain the residues essential for exclusion activity and specificity, were found to localize in the cell cytoplasm. These observations suggest that complex topological rearrangements of conjugative proteins must occur during mating to enable these domains to interact.

The SXT/R391 Family of Integrative Conjugative Elements Is Composed of Two Exclusion Groups

Abstract: Conjugative elements often encode entry exclusion systems that convert host cells into poor recipients for identical or similar elements. The diversity of exclusion systems within families of conjugative elements has received little attention. We report here the most comprehensive study to date of the diversity of exclusion determinants within a single family of conjugative elements. Unexpectedly, our analyses indicate that there are only two exclusion groups among the diverse members of the SXT/R391 family of integrative conjugative elements.

Global Gene Expression and Phenotypic Analysis of a Vibrio cholerae rpoH Deletion Mutant

Abstract: 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. σ32, 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 σ32 in V. cholerae physiology. In aggregate, our findings suggest that σ32 promotes V. cholerae growth at temperatures ranging at least from 15°C to 42°C. Growth of the rpoH mutant was severely attenuated within the suckling mouse intestine, suggesting that σ32-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 σ32 consensus binding sequence and subsequently to identify genes that are likely to be directly regulated by RpoH in the whole V. cholerae genome.

Adrenergic regulation of bacterial virulence.

Abstract: Complex interactions between pathogen and host occur during the course of most infectious diseases. Microbes sense the host environment and produce factors, such as toxins, that usually promote their growth and often lead to symptoms in the host. Hosts sense the microbe and respond in various ways, such as activation of the innate immune response, to eliminate the pathogen. The host molecules that microbes sense to trigger production of virulence factors are poorly understood. Accumulating observations made in the past few years suggest that host adrenergic agonists like norepinephrine (NE) can promote the virulence of enterohemorrhagic Escherichia coli (EHEC) [1-4]. In this issue of the Journal, there is an intriguing paper by Nakano et al. [5] that shows that the pathogenicity of another enteric pathogen, Vibrio parahaemolyticus, is also augmented by NE. V. parahaemolyticus is an important cause of foodborne illness. In Japan, this gram-negative rod is the most common foodborne bacterial cause of gastroenteritis. In the United States, V. parahaemolyticus is the most frequent cause of Vibrio-associated gastroenteritis, and most cases are associated with consumption of undercooked shellfish [6]. This summer the Centers for Disease Control and Prevention issued a dispatch detailing elevations in the number of V. parahaemolyticus–associated cases of gastroenteritis in New York, Oregon, and Washington [7]. The mechanisms of V. parahaemolyticus pathogenicity are not well understood, in part because of the lack of a good animal model of infection. The V. parahaemolyticus thermostable hemolysin, Tdh, has long been considered one of its key virulence factors. The V. parahaemolyticus genome sequence revealed the unexpected finding that each of the 2 V. parahaemolyticus chromosomes codes for a type III secretion system (TTSS1 and TTSS2) [8]. TTSSs are found in many enteric pathogens such as Salmonella enterica, EHEC, and enteropathogenic E. coli, Shigella species, and Yersinia species. These specialized multiprotein component secretion systems mediate the transfer of protein effectors directly from the bacterial to the host cell cytoplasm. In many cases, the targets and activities of the translocated proteins are unknown. Functional studies have suggested that the 2 V. parahaemolyticus TTSSs may mediate distinct aspects of this organism's pathogenicity. TTSS1 appears to mediate the organism's cytotoxicity, whereas TTSS2 appears to account for its enterotoxicity [9]. In this issue of the Journal, Nakano et al. show that NE promotes the pathogenic effects of both V. parahaemolyticus TTSSs. They found that addition of NE to tissue-cultured cells shortly before the addition of V. parahaemolyticus augmented the pathogen's cytotoxic effects. Because either α- or β-adrenergic antagonists inhibited NE stimulation of V. parahaemolyticus cytotoxicity, they argue that NE is acting via adrenergic receptors on the cell line. However, recent work has demonstrated that NE can act directly on EHEC [10], and it is plausible that V. parahaemolyticus might also directly respond to this adrenergic agonist. The authors also show that V. parahaemolyticus TTSS1 and not TTSS2 was required for NE to promote the pathogen's cytotoxicity. Interestingly, this adrenergic agonist was found to increase the transcript levels of several TTSS1 loci but not of TTSS2 loci or tdh. The mechanism by which NE stimulates transcription of TTSS1 genes was not explored. Taken together, these observations suggest that NE either acts directly or indirectly to increase transcription of V. parahaemolyticus TTSS1 genes thereby augmenting its cytotoxicity. The authors used a rat ileal loop model to investigate the influence of NE on V. parahaemolyticus enterotoxicity. They found that NE increased V. parahaemolyticus induced fluid accumulation in this model. Unlike the case for NE stimulation of V. parahaemolyticus cytotoxicity, NE stimulation of V. parahaemolyticus’ enterotoxicity only required the pathogen's TTSS2 and not its TTSS1. α-adrenergic antagonists blocked this effect, but β-adrenergic antagonists did not, suggesting that NE-stimulation of V. parahaemolyticus enterotoxicity requires only host cell α-adrenergic receptors. NE does not appear to elevate transcripts of TTSS2 genes. Deciphering the mechanisms by which NE stimulates V. parahaemolyticus cytotoxicity and enterotoxicity is a key challenge for the future. As in V. parahaemolyticus, NE induces expression of TTSS genes in EHEC [3]. In EHEC, NE has been shown to act directly on the pathogen. This adrenergic agonist directly binds to QseC, an EHEC sensor kinase, increasing its autophosphorylation and thereby initiating a complex signaling cascade that activates transcription of the genes encoding the TTSS, flagella, and Shiga toxin [3, 10]. The binding of NE to QseC can be inhibited by the α-adrenergic antagonist phentolamine, suggesting that QseC functions as a bacterial adrenergic receptor analog [10]. Thus, even though V. parahaemolyticus, like EHEC, lacks a homologue of a mammalian adrenergic receptor, it may, like EHEC, encode a protein that can recognize and respond to host adrenergic agonists. In both of these enteric pathogens, type III secretion is also regulated by quorum sensing signaling [3, 11, 12]. In EHEC, the QseC sensor responds to both the quorum sensing signal AI-3 and the host signal NE by increasing its autophosphorylation, leading to increased expression of the TTSS genes [10, 13]. Exploration of the cross-talk between quorum sensing signaling and host adrenergic signaling has only just begun. Regardless of the mechanisms by which adrenergic agonists augment virulence, the observations of Nakano et al. provide a remarkable example of interkingdom signaling between host and pathogen. Furthermore, these observations suggest that andrenergic antagonists may someday become part of the therapeutic armamentarium to treat enteric infections.

Eschericia coli mutants and methods of use thereof

Abstract: The present invention provides methods and compositions for production of gram- negative bacterial mutants that are defective in intestinal colonization capacity and sensitive to infection by bacteriophage Pl. Thus the present invention provides immunogenic compositions for the prevention or attenuation of food- and water-borne illnesses associated with ingestion of bacteria such as enterohemorrhagic Escherichia coli.