Vibrio vulnificus Has the Transmembrane Transcription Activator ToxRS Stimulating the Expression of the Hemolysin Gene vvhA

Abstract

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus , we tried to identify the V. cholerae transmembrane virulence regulator toxRS ( toxRS _Vc ) homologs in V. vulnificus . By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus ( toxRS _Vp ), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb Bgl II- Hin dIII fragment and a 1.2-kb Hin dIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxR _Vv , toxS _Vv , and htpG _Vv were cloned. ToxR _Vv shared 55.0 and 63.0% sequence homology with ToxR _Vc and ToxR _Vp , respectively. ToxS _Vv was 71.5 and 65.7% homologous to ToxS _Vc and ToxS _Vp , respectively. The amino acid sequences of ToxRS _Vv showed transmembrane and activity domains similar to those observed in ToxRS _Vc and ToxRS _Vp . Western blot analysis proved the expression of ToxR _Vv in V. vulnificus . ToxRS _Vv enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene ( vvhA ) fivefold. ToxRS _Vv also activated the ToxR _Vc -regulated ctx promoter incorporated into an E. coli chromosome. A toxR _Vv null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxR _Vv mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR _Vv may regulate the virulence expression of V. vulnificus .