The AraC homolog ToxT coordinately regulates virulence gene expression in Vibrio cholerae. ToxT is required for transcriptional activation of the genes encoding cholera toxin and the toxin coregulated pilus, among others. In this work we focused on the interaction of ToxT with the tcpA promoter and investigated the mechanism of ToxT-dependent transcriptional activation at tcpA. Deletion analysis showed that a region from ؊95 to ؉2 was sufficient for ToxT binding and activation, both of which were simultaneously lost when the deletion was extended to ؊63. A collection of point mutations generated by error-prone PCR revealed two small regions required for ToxT-dependent transactivation. Binding studies performed with representative mutations showed that the two regions define sites at which ToxT binds to the tcpA promoter region, most likely as a dimer. Results obtained by using a rpoA truncation mutation showed that ToxT-dependent activation at tcpA involves the C-terminal domain of the RNA polymerase alpha subunit. A model of ToxT-dependent transcriptional activation at tcpA is proposed, in which ToxT interacts with two A-rich regions of tcpA centered at ؊72 and ؊51 and requires the alpha C-terminal domain of RNA polymerase.

Expression of the ctx and tcp genes, which encode cholera toxin and the toxin coregulated pilus, the Vibrio cholerae O1 virulence determinants having the largest contribution to cholera disease, is repressed by the nucleoid-associated protein H-NS and activated by the AraC-like transcriptional regulator ToxT. To elucidate the molecular mechanism by which H-NS controls transcription of the ctxAB operon, H-NS repression and binding were characterized by using a promoter truncation series, gel mobility shift assays, and DNase I footprinting. Promoter regions found to be important for H-NS repression correlated with in vitro binding. Four main H-NS binding regions are present at ctx. One region overlaps the high-affinity ToxT binding site and extends upstream, another overlaps the ToxT low-affinity binding site around the ؊35 element, and the remaining two are located adjacent to one another downstream of the transcriptional start site. Competition for binding to the overlapping H-NS/ToxT binding sites was observed in gel mobility shift assays, where ToxT was found to displace H-NS from the ctx promoter region. In addition, regulatory differences between the ctx and tcpA promoters were examined. H-NS was found to have a higher relative binding affinity for the ctx promoter than for the tcpA promoter in vitro. In contrast to ToxT-dependent activation of the tcpA promoter, ToxT activation of ctx did not require the C-terminal domain of the ␣-subunit of RNA polymerase. These findings demonstrate that transcriptional regulation of ctx and tcpA by H-NS and ToxT is mechanistically distinct, and this may lead to important differences in the expression of these coregulated genes.Vibrio cholerae is the etiological agent of the human diarrheal disease cholera. Two virulence factors that are produced by V. cholerae and are essential for disease are toxin-coregulated pilus (TCP) (62) and cholera toxin (CT) (28). TCP is a type IV pilus that is assembled by polymerization of the pilin subunit, TcpA, and forms long filaments that laterally associate into bundles. Expression of TCP in vitro results in autoagglutination of the bacterium, and TCP-mediated bacterium-bacterium interactions in vivo facilitate microcolony formation on the intestinal epithelium. Pilus biogenesis requires at least 9 other proteins in addition to TcpA, which are encoded in an operon located on the Vibrio pathogenicity island (VPI). The pilus biogenesis apparatus plays a second role in colonization by secreting the cotranscribed, soluble colonization factor TcpF, which is also essential for colonization but for which a mechanism remains unknown (29, 30). The second main virulence factor, CT, is a potent A 1 B 5 subunit, ADP-ribosylating toxin that is responsible for the severe watery diarrhea that is associated with cholera. CT is encoded by the ctxAB operon, which is located on the lysogenic bacteriophage CTX. The VPI and CTX were both acquired by horizontal gene transfer. TCP serves as the high-affinity receptor for CTX that links TCP production to ctx acq...

Intracellular polysaccharides (IPS) are glycogen-like storage polymers which contribute significantly to Streptococcus mutans-induced cariogenesis. We previously identified and cloned a locus from the S. mutans chromosome which is required for the accumulation of IPS. Sequencing of this locus revealed at least four contiguous open reading frames, all of which are preceded by a common promoter region and are transcribed in the same direction. Analysis of the amino acid sequence deduced from the first of these open reading frames (ORF1) revealed domains which are highly conserved among d-alanine-activating enzymes (DltA) inLactobacillus rhamnosus (formerlyLactobacillus casei) and Bacillus subtilis. The deduced amino acid sequences derived from ORF2, -3, and -4 also exhibit extensive similarity to DltB, -C, and -D, respectively, in these microorganisms. However, Southern hybridization experiments indicate that this operon maps to a locus on the S. mutanschromosome which is separate from the glgP,glgA, and glgD genes, whose products are known mediators of bacterial IPS accumulation. We therefore assigned a newdlt designation to the locus which we had formerly calledglg. We maintain that the dlt genes are involved in S. mutans IPS accumulation, however, since they complement a mutation in trans which otherwise rendersS. mutans IPS deficient. In this study, we found that expression of the S. mutans dlt genes is growth phase dependent and is modulated by carbohydrates internalized via the phosphoenolpyruvate phosphotransferase system (PTS). We demonstrated that the S. mutans dlt genes are expressed constitutively when non-PTS sugars are provided as the sole source of carbohydrate. Consistent with a role for the PTS in dltexpression is a similar constitutive expression of the dltgenes in an S. mutans PTS mutant grown in a chemically defined medium supplemented with glucose. In summary, these findings support a novel role for the dlt gene products inS. mutans IPS accumulation and suggest thatdlt expression in this oral pathogen is subject to complex mechanisms of control imposed by growth phase, dietary carbohydrate, and other factors present in the plaque environment.

The causative agent of the respiratory disease whooping cough, Bordetella pertussis, is a nutritionally fastidious microorganism but can be grown with relative ease in research laboratories. Stainer‐Scholte synthetic broth medium and Bordet‐Gengou blood agar both support growth of B. pertussis and are commonly used. B. pertussis prefers aerobic conditions and a temperature range of 35° to 37°C. Appropriate laboratory safety protocols are required to prevent the generation of aerosols, which could potentially spread this highly infectious agent. Curr. Protoc. Microbiol. 15:4B.1.1‐4B.1.9. © 2009 by John Wiley & Sons, Inc.

The BvgAS two-component system controls virulence in the human respiratory pathogen Bordetella pertussis, the etiological agent of whooping cough. BvgAS is unlike orthodox two-component signal transduction systems in that it employs a four step phosphorelay from the sensor protein BvgS to the response regulator BvgA, instead of the more common two step phosphotransfer. Further, B. pertussis displays at least three distinct phenotypic phases, each characterized by maximal expression of some genes and minimal expression of others. We are engaged in the development of a computational model of this signal transduction and gene expression pathway, which we intend to employ in exploring the following questions. First, what are the levels of BvgA~P (phosphorylated response regulator) in each phenotypic phase? Does the concentration of BvgA~P vary in a stepwise or continuous fashion in response to changes in signal compound concentration? Second, what is the advantage of employing a 4-step phosphorelay instead of a 2step phosphotransfer system? Can a 2-step phosphorelay mediate three phenotypic phases? Third, BvgAS regulates its own transcription; what is the effect of this autoregulation? The computational model is being developed in a methodical fashion. In vitro experiments are modeled by ordinary differential equations of chemical kinetics in order to obtain kinetic parameter estimates. This suggests gaps in the experimental literature and experiments best suited to fill those gaps, which are then performed. Completed versions of the model are then simulated using deterministic (ODE), stochastic (Gillespie's algorithm) or multiscale (tau leaping, slow-scale SSA or hybrid) chemical kinetics algorithms, depending on what is appropriate.Preliminary results indicate that the full complexity of the three phenotypic phases of B. pertussis cannot be achieved without incorporating the phosphorelay (i.e. by simple two-step phosphotransfer) or BvgAS autoregulation in the model.