Vibrio vulnificus rpoS Expression Is Repressed by Direct Binding of cAMP-cAMP Receptor Protein Complex to Its Two Promoter Regions

Abstract

Vibrio vulnificus, a septicemia-causing pathogenic bacterium, acquires resistance against various stresses and expresses virulence factors via an rpoS gene product. In this study, we investigated the transcriptional characteristics of this global regulator. Two distinct transcriptional initiation sites for the rpoS gene, the proximal promoter (P(p)) and the distal promoter (P(d)), were defined by primer extension experiments. Various rpoS::luxAB transcriptional fusions indicated that P(d) is a major promoter of rpoS expression. Western blot analysis showed that RpoS levels were inversely correlated with intracellular levels of 3',5'-cyclic AMP (cAMP). The expressions of both P(d) and P(p) were increased in cya and crp mutants. The exogenous addition of cAMP to the cya mutant resulted in repressed expression of rpoS. In addition, rpoS expression was significantly lowered in the cpdA mutant, in which the level of cAMP was elevated because of the absence of 3',5'-cAMP phosphodiesterase. In vitro transcription assays using the V. vulnificus RNA polymerase showed that the transcripts from both promoters were reduced by addition of the cAMP-cAMP receptor protein (CRP). The cAMP-CRP was shown to bind to two rpoS promoters by electrophoretic mobility shift assays. The alteration of the putative CRP-binding site on each rpoS promoter, via site-directed mutagenesis, abolished the binding of cAMP-CRP as well as regulation by cAMP-CRP. Therefore, this study shows a relationship between the level of intracellular cAMP and the degree of rpoS expression and further demonstrates, for the first time, the direct binding of the cAMP-CRP complex to rpoS upstream regions, which results in repression of rpoS gene expression.