Promoter Analysis and Regulatory Characteristics of vvhBAEncoding Cytolytic Hemolysin of Vibrio vulnificus

Abstract

Cytolytic hemolysin, a gene product of vvhA, is a putative virulence factor of the pathogenic bacterium Vibrio vulnificus. We have previously shown that hemolysin production is repressed by adding glucose to culture media and that production can be restored by adding cAMP. In this study, hemolysin activity and the level of vvh transcript were determined to reach a maximum in late exponential phase and were repressed when cells entered stationary phase. Northern blot and primer extension analyses revealed that vvhA is cotranscribed with a second gene, vvhB, located upstream of vvhA. Transcription of the vvhBA operon begins at a single site and is under the direction of a single promoter, P vvh . A crp null mutation decreased hemolysin production and the level of vvhBA transcript by reducing the activity of P vvh , indicating that the P vvh activity is under the positive control of cAMP receptor protein (CRP). A direct interaction between CRP and the regulatory region of the vvhBA operon was demonstrated by gel-mobility shift assays. The CRP binding site, centered at 59.5 bp upstream of the transcription start site, was mapped by deletion analysis of the vvhBA promoter region and confirmed by DNase I protection assays. These results demonstrate that the vvhBA expression is activated by CRP in a growth-dependent manner and that CRP exerts its effects by directly binding to DNA upstream of P vvh .

SmcR and Cyclic AMP Receptor Protein Coactivate Vibrio vulnificus vvpE Encoding Elastase through the RpoS-dependent Promoter in a Synergistic Manner

“…The 200-bp upstream region of vvpE, extending from residues Ϫ300 to Ϫ101 with respect to the ϩ1 transcription start site, was amplified by PCR using 32 P-labeled VVPE021 and unlabeled VVPE022 as the primers. The binding of CRP to the labeled DNA and electrophoretic analysis of the CRP⅐DNA complexes have already been described (16). The protein-DNA binding reactions with SmcR were the same as those with CRP, except that cAMP was omitted from the reaction buffer.…”

“…The His-tagged SmcR protein was then expressed in E. coli BL21(DE3), and the protein was purified by affinity chromatography according to the manufacturer's procedure (QIAGEN Inc., Valencia, CA). In a similar way, the expression and purification of His-tagged CRP and His-tagged RpoS were carried out using pHK0201 and pKP001, 3 carrying the V. vulnificus crp and rpoS genes, respectively, as described (16).…”

“…For the primer extension experiments, an end-labeled 24-base primer (VVPE9905) complementary to a coding region of vvpE was added to the RNA and then extended with SuperScript II RNase H Ϫ reverse transcriptase (Invitrogen) as described previously (9,16). The cDNA products were then purified and resolved on a sequencing gel alongside sequencing ladders generated with the same primer used for the primer extension.…”

“…The binding of CRP to the labeled DNA and DNase I digestion of the CRP⅐DNA complexes was carried out following the procedures described previously by Choi et al (16). After precipitation with ethanol, the digested DNA products were resolved on a sequencing gel alongside sequencing ladders of pKC980 generated using either VVPE023 (for the coding strand) or VVPE024 (for the noncoding strand) as the primer.…”

“…The vvpE-luxAB reporters were then transferred into ATCC29307 and the isogenic mutants by conjugation. The cellular luminescence of the cultures was measured with a luminometer (Lumat Model 9501, Berthold, Wildbad, Germany) and expressed in arbitrary relative light units as described previously (9,16).…”