Functional Characterization of the Stringent Response Regulatory GenedksAof Vibrio cholerae and Its Role in Modulation of Virulence Phenotypes

Abstract

In bacteria, nutrient deprivation evokes the stringent response, which is mediated by the small intracellular signaling molecule ppGpp. In Gram negatives, the RelA enzyme synthesizes and SpoT hydrolyzes ppGpp, although the latter protein also has weak synthetase activity. DksA, a recently identified RNA polymerase binding transcription factor, acts as a coregulator along with ppGpp for controlling the stringent response. Recently, we have shown that three genes, relA, spoT, and relV, govern cellular levels of ppGpp during various starvation stresses in the Gram-negative cholera pathogen Vibrio cholerae. Here we report functional characterization of the dksA gene of V. cholerae (dksA Vc ), coding for the protein DksA Vc . Extensive genetic analyses of the ⌬dksA Vc mutants suggest that DksA Vc is an important component involved in the stringent response in V. cholerae. Further analysis of mutants revealed that DksA Vc positively regulates various virulence-related processes, namely, motility, expression of the major secretory protease, called hemagglutinin protease (HAP), and production of cholera toxin (CT), under in vitro conditions. We found that DksA Vc upregulates expression of the sigma factor FliA ( 28 ), a critical regulator of motility in V. cholerae. Altogether, it appears that apart from stringent-response regulation, DksA Vc also has important roles in fine regulation of virulence-related phenotypes of V. cholerae.

Cholera Toxin Production during Anaerobic Trimethylamine N-Oxide Respiration Is Mediated by Stringent Response in Vibrio cholerae

“…Therefore, stringent response-mediated ctxAB gene expression is predicted to be controlled by the indirect mechanism, known as -factor competition. This hypothesis was supported by previous studies demonstrating that recruitment of alternative -factors ( 54 , 38 , 28 , and E ) affected survival and virulence under starvation and other stress conditions in V. cholerae (22,49,50).…”

“…DksA (VC0596, 17.2 kDa) binds to RNAP and facilitates the effects of ppGpp on the transcriptional control in bacterial cells (22, 31, 36 -38). Recently, DksA has also been suggested to play a critical role in regulating V. cholerae virulence (22). To test whether CT production elicited by stringent response during anaerobic TMAO respiration was also affected by the deficiency of DksA, we constructed a dksA in-frame deletion mutant.…”

“…It has been suggested that CgtA most likely modulates the SpoT function to maintain low ppGpp levels (20,21). In addition, DksA, an RNA polymerase binding transcription factor, also plays several important roles in modulating stringent response and regulation of other genes in V. cholerae (22,23).…”

Genetic and mutational characterization of the small alarmone synthetase gene relV of Vibrio cholerae

“…We have (Pal et al, 2012). In this study, we have further examined the toxic effect of relV gene expression through pRelV BAD in E. coli background.…”

“…For reverse transcriptase (RT)-PCR assay of the relV gene, total cellular RNA was prepared from bacterial cells grown in LB medium to an OD 600 value of~1.0 using TRI Reagent (Sigma) essentially as described by the supplier. RT-PCR was done with the OneStep RT-PCR kit (Qiagen) essentially as described previously (Pal et al, 2012) using purified RNA and the primers 1224int-F/1224int-R (Table S1). …”