Stringent response interacts with the ToxR regulon to regulate Vibrio cholerae virulence factor expression.

The epidemic diarrheal disease cholera is caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae virulence factors include the toxin-coregulated pilus (TCP) and cholera toxin, which are major factors responsible for host colonization and production of diarrhea. Expression of cholera toxin and TCP genes is controlled by the ToxR regulon. The ToxR regulon includes the transcriptional activators ToxR, TcpP, and ToxT. ToxT directly initiates transcription of the cholera toxin and TCP genes. TcpP and ToxR are necessary for expression of toxT. TcpP and ToxR activity requires TcpH and ToxS, respectively. Additionally, ToxR is able to directly initiate transcription of the cholera toxin genes independent of TcpP and ToxT. TCP is required early in infection to colonize the small intestine, then cholera toxin is expressed later in infection to produce diarrhea. We tested whether stringent response, the low nutrient stress response, was involved in regulation of virulence genes. Using an infant mouse model, we found that V. cholerae strains with deletions of the stringent response genes were unable to colonize the small intestine. We further tested these stringent response-null mutants and found that stringent response was necessary for TCP expression, although effects on cholera toxin expression were not significant. We then tested whether stringent response regulation of TCP occurred through the ToxR regulon. We found that stringent response induced toxT and tcpPH expression, while repressing toxRS. This differential regulation of ToxR and TcpP may explain the differential expression of TCP and cholera toxin in vivo.

(p)ppGpp, a Small Nucleotide Regulator, Directs the Metabolic Fate of Glucose in Vibrio cholerae.

When V. cholerae encounters nutritional stress, it activates (p)ppGpp-mediated stringent response. The genes relA and relV are involved in the production of (p)ppGpp, whereas the spoT gene encodes an enzyme that hydrolyzes it. Herein, we show that the bacterial capability to produce (p)ppGpp plays an essential role in glucose metabolism. The V. cholerae mutants defective in (p)ppGpp production (i.e. ΔrelAΔrelV and ΔrelAΔrelVΔspoT mutants) lost their viability because of uncontrolled production of organic acids, when grown with extra glucose. In contrast, the ΔrelAΔspoT mutant, a (p)ppGpp overproducer strain, exhibited better growth in the presence of the same glucose concentration. An RNA sequencing analysis demonstrated that transcriptions of genes consisting of an operon for acetoin biosynthesis were markedly elevated in N16961, a seventh pandemic O1 strain, but not in its (p)ppGpp(0) mutant during glucose-stimulated growth. Transposon insertion in acetoin biosynthesis gene cluster resulted in glucose-induced loss of viability of the ΔrelAΔspoT mutant, further suggesting the crucial role of acetoin production in balanced growth under glucose-rich environments. Additional deletion of the aphA gene, encoding a negative regulator for acetoin production, failed to rescue the (p)ppGpp(0) mutant from the defective glucose-mediated growth, suggesting that (p)ppGpp-mediated acetoin production occurs independent of the presence of AphA. Overall, our results reveal that (p)ppGpp, in addition to its well known role as a stringent response mediator, positively regulates acetoin production that contributes to the successful glucose metabolism and consequently the proliferation of V. cholerae cells under a glucose-rich environment, a condition that may mimic the human intestine.

Cholera toxin production during anaerobic trimethylamine N-oxide respiration is mediated by stringent response in Vibrio cholerae.

As a facultative anaerobe, Vibrio cholerae can grow by anaerobic respiration. Production of cholera toxin (CT), a major virulence factor of V. cholerae, is highly promoted during anaerobic growth using trimethylamine N-oxide (TMAO) as an alternative electron acceptor. Here, we investigated the molecular mechanisms of TMAO-stimulated CT production and uncovered the crucial involvement of stringent response in this process. V. cholerae 7th pandemic strain N16961 produced a significantly elevated level of ppGpp, the bacterial stringent response alarmone, during anaerobic TMAO respiration. Bacterial viability was impaired, and DNA replication was also affected under the same growth condition, further suggesting that stringent response is induced. A ΔrelA ΔspoT ppGpp overproducer strain produced an enhanced level of CT, whereas anaerobic growth via TMAO respiration was severely inhibited. In contrast, a ppGpp-null strain (ΔrelA ΔspoT ΔrelV) grew substantially better, but produced no CT, suggesting that CT production and bacterial growth are inversely regulated in response to ppGpp accumulation. Bacterial capability to produce CT was completely lost when the dksA gene, which encodes a protein that works cooperatively with ppGpp, was deleted. In the ΔdksA mutant, stringent response growth inhibition was alleviated, further supporting the inverse regulation of CT production and anaerobic growth. In vivo virulence of ΔrelA ΔspoT ΔrelV or ΔdksA mutants was significantly attenuated. The ΔrelA ΔspoT mutant maintained virulence when infected with exogenous TMAO despite its defective growth. Together, our results reveal that stringent response is activated under TMAO-stimulated anaerobic growth, and it regulates CT production in a growth-dependent manner in V. cholerae.

Stringent response regulation of biofilm formation in Vibrio cholerae.

Biofilm formation is a key factor in Vibrio cholerae environmental survival and host colonization. Production of biofilm enables V. cholerae to survive and persist in aquatic environments and aids in the passage through the gastric acid barrier to allow access to the small intestine. The genes involved in biofilm formation are regulated by the transcriptional activators vpsR and vpsT, which are in turn transcriptionally regulated by a number of environmental signals. In this study, the role of the stringent response in biofilm formation was examined. V. cholerae mutants deficient in stringent response had a reduced ability to form biofilms, although they were not completely deficient in biofilm formation. There are three (p)ppGpp synthases in V. cholerae: RelA, SpoT, and RelV. All three synthases were necessary for vpsR transcription, with RelV showing the strongest effect. RelA was the only synthase that was necessary for vpsT expression. Stringent response regulation of vpsR and vpsT was shown to partially occur through rpoS. Biofilm formation in V. cholerae is controlled by a complex regulatory apparatus, with negative regulators of biofilm gene expression, such as quorum sensing, and positive regulators of biofilm genes, including stringent response, interacting to ensure that biofilm formation is coordinated with the environment.

Regulation of the stringent response is the essential function of the conserved bacterial G protein CgtA in Vibrio cholerae.

The gene encoding the conserved bacterial G protein CgtA (Obg) is essential for viability in every organism in which it has been studied. CgtA has been reported to be involved in several diverse bacterial functions, including ribosome assembly, DNA repair, sporulation, and morphological development. However, none of these functions have been identified as essential. Here we show that depletion of CgtA in Vibrio cholerae causes global changes in gene expression that are consistent with induction of a classical low nutrient stress response or "stringent" response. We show that depletion of CgtA leads to increased ppGpp levels that correlate with induction of the global stress response and cessation of growth. The enzyme RelA is responsible for synthesis of the alarmone ppGpp during the stringent response. We show that CgtA is no longer essential in a relA deletion mutant and thus conclude that the essentiality of CgtA is directly linked to its ability to affect ppGpp levels. The enzyme SpoT degrades ppGpp, and here we show that SpoT is essential in a RelA+ CgtA+ background but not in a relA deletion mutant. We also confirmed that CgtA interacts with SpoT in a two-hybrid assay. We suggest that the essential function of CgtA is as a repressor of the stringent response that acts by regulating SpoT activity to maintain low ppGpp levels when bacteria are growing in a nutrient-rich environment.

Bacterial genomics and pathogen evolution.

The availability of hundreds of bacterial genome sequences has altered the study of bacterial pathogenesis, affecting both design of experiments and analysis of results. Comparative genomics and genomic tools have been used to identify virulence factors and genes involved in environmental persistence of pathogens. However, a major stumbling block in the genomics revolution has been the large number of genes with unknown function that have been identified in every organism sequenced to date.

ATP-dependent interactions between Escherichia coli Min proteins and the phospholipid membrane in vitro.

Proper placement of the division apparatus in Escherichia coli requires pole-to-pole oscillation of the MinC division inhibitor. MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles. To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions. We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg(2+), whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked. The results support and extend recent work by Z. Hu et al. (Z. Hu, E. P. Gogol, and J. Lutkenhaus, Proc. Natl. Acad. Sci. USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP). The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide.