SmcR and cyclic AMP receptor protein coactivate Vibrio vulnificus vvpE encoding elastase through the RpoS-dependent promoter in a synergistic manner.
ABSTRACT The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE. We previously demonstrated that vvpE expression is differentially directed by two different promoters in a growth phase-dependent manner. The activity of the stationaryphase promoter (promoter S (PS)) is dependent on RpoS and is also under the positive control of cyclic AMP receptor protein (CRP). In this study, primer extension analyses revealed that SmcR, the Vibrio harveyi LuxR homolog, is also involved in the regulation of vvpE transcription by activating PS. Although the influence of CRP on PS is mediated by SmcR, the level of PS activity observed when CRP and SmcR function together was found to be greater than the sum of the PS activities achieved by each activator alone. Western blot analyses demonstrated that the cellular levels of RpoS, CRP, and SmcR were not significantly affected by one other, indicating that CRP and SmcR function cooperatively to activate PS rather than sequentially in a regulatory cascade. The binding sites for CRP and SmcR were mapped based on a deletion analysis of the vvpE promoter region and confirmed by in vitro DNase I protection assays. The binding sites for CRP and SmcR were juxtapositioned and centered 220 and 198 bp upstream of the transcription start site of PS, respectively. Accordingly, these results reveal that CRP and SmcR function synergistically to coactivate the expression of vvpE by the RpoS-dependent promoter (PS) and that the activators exert their effect by directly binding to the promoter in the stationary phase.
Article: Activation of the Vibrio vulnificus cadBA Operon by Leucine-responsive regulatory protein is mediated by CadC.[show abstract] [hide abstract]
ABSTRACT: The present study revealed that Lrp, a leucine-responsive regulatory protein, is involved in the regulation of cadBA transcription through activation of PcadBA. The influence of Lrp on PcadBA was mediated by CadC, and thereby, CadC was able to compensate for the lack of Lrp in the activation of PcadBA. Western blot analyses and EMSA demonstrated that the cellular level of CadC was not significantly affected by Lrp, and that Lrp exerted its effect by directly binding to PcadBA. These combined results suggested that CadC and Lrp function cooperatively to activate the PcadBA rather than sequentially in a regulatory cascade.Journal of Microbiology and Biotechnology 12/2008; 18(11):1755-61. · 1.38 Impact Factor
Article: Transcriptional regulation of opaR, qrr2-4 and aphA by the master quorum-sensing regulator OpaR in Vibrio parahaemolyticus.[show abstract] [hide abstract]
ABSTRACT: Vibrio parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis via the fecal-oral route. V. harveyi is a pathogen of fishes and invertebrates, and has been used as a model for quorum sensing (QS) studies. LuxR is the master QS regulator (MQSR) of V. harveyi, and LuxR-dependent expression of its own gene, qrr2-4 and aphA have been established in V. harveyi. Molecular regulation of target genes by the V. parahaemolyticus MQSR OpaR is still poorly understood. The bioinformatics analysis indicated that V. parahaemolyticus OpaR, V. harveyi LuxR, V. vulnificu SmcR, and V. alginolyticus ValR were extremely conserved, and that these four MQSRs appeared to recognize the same conserved cis-acting signals, which was represented by the consensus constructs manifesting as a position frequency matrix and as a 20 bp box, within their target promoters. The MQSR box-like sequences were found within the upstream DNA regions of opaR, qrr2-4 and aphA in V. parahaemolyticus, and the direct transcriptional regulation of these target genes by OpaR were further confirmed by multiple biochemical experiments including primer extension assay, gel mobility shift assay, and DNase I footprinting analysis. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and OpaR-binding sites were determined for the five target genes of OpaR, which gave a structural map of the OpaR-dependent promoters. Further computational promoter analysis indicated the above regulatory circuits were shared by several other closely related Vibrios but with slight exceptions. This study gave a comprehensive computational and characterization of the direct transcriptional regulation of five target genes, opaR, qrr2-4 and ahpA, by OpaR in V. parahaemolyticus. These characterized regulatory circuits were conserved in V. harveyi and V. parahaemolyticus.PLoS ONE 01/2012; 7(4):e34622. · 4.09 Impact Factor
Article: AphB influences acid tolerance of Vibrio vulnificus by activating expression of the positive regulator CadC.[show abstract] [hide abstract]
ABSTRACT: A mutant of Vibrio vulnificus that was more sensitive to low pH was screened from a library of mutants constructed by random transposon mutagenesis. By use of a transposon-tagging method, an open reading frame encoding a LysR homologue, AphB, was identified and cloned from V. vulnificus. The deduced amino acid sequence of AphB from V. vulnificus was 80% identical to that reported from V. cholerae. A mutational analysis demonstrated that the gene product of aphB contributes to acid tolerance of V. vulnificus. The lysine decarboxylase activity and cellular level of the cadA transcript were decreased in the aphB mutant, indicating that AphB exerts its effect on the acid tolerance of V. vulnificus by enhancing the expression of cadBA. Western blot analyses demonstrated that the cellular level of CadC, a transcription activator of the cadBA operon, was significantly reduced by aphB mutation, and a primer extension analysis revealed that the cadC promoter (P(cadC)) activity was under the positive control of AphB. A direct interaction between AphB and the P(cadC) DNA was demonstrated by gel mobility shift assays. The AphB binding site mapped by deletion analyses of the P(cadC) regulatory region and confirmed by a DNase I protection assay was centered at the 61.5 bp upstream of the transcription start site. Accordingly, these results demonstrate that AphB and CadC function sequentially in a regulatory cascade to activate cadBA expression and that AphB activates the expression of cadC by directly binding to an upstream region of P(cadC).Journal of Bacteriology 10/2006; 188(18):6490-7. · 3.83 Impact Factor