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Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA 总被引:1,自引:0,他引:1 下载免费PDF全文
Lee SE Shin SH Kim SY Kim YR Shin DH Chung SS Lee ZH Lee JY Jeong KC Choi SH Rhee JH 《Journal of bacteriology》2000,182(12):3405-3415
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 BglII-HindIII fragment and a 1.2-kb HindIII 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. 相似文献
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Southern blot analysis with a toxR-specific gene probe indicates that Vibrio cholerae 569B has a 1.2-kilobase deletion near the toxR gene. Heterologous conjugative crosses were carried out between the EI Tor strain RV79 and 569B tox mutants. Tox+ recombinants showed the same linkage properties to the his locus as to the previously mapped tox locus of 569B. Southern blot analysis with the toxR probe of the Tox+ recombinants obtained in these heterologous crosses showed that these recombinants had replaced the V. cholerae 569B (recipient) toxR DNA with the V. cholerae RV79 (donor) toxR DNA, indicating that tox and toxR are the same locus. However, the Tox+ recombinants synthesized an amount of toxin intermediate between the level observed for wild-type RV79 and 569B strains, suggesting there is a difference in the ability of toxR genes from different strains to activate ctx. About half of the mutations which suppress the phenotype of hypertoxinogenic locus htx are unlinked to htx and in addition have a hypotoxinogenic phenotype relative to that of the wild type. Most of these hypotoxinogenic, second-site suppressors show a linkage to his similar to the linkage of toxR to his and are therefore probably mutations in toxR. These results indicate that the toxR gene product is required for ctx expression and that a functional toxR gene is required for the effect of an htx mutation to be seen. 相似文献
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A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation 下载免费PDF全文
ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli. 相似文献
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DNA binding and ToxR responsiveness by the wing domain of TcpP,an activator of virulence gene expression in Vibrio cholerae 总被引:3,自引:0,他引:3
Virulence in Vibrio cholerae requires activation of toxT by two membrane-localized activators, TcpP and ToxR. We isolated 12 tcpP activation mutants that fell into two classes: class I mutants were inactive irrespective of the presence of ToxR, and class II mutants exhibited near wild-type activity when coexpressed with ToxR. Most class I mutants had lesions in the wing domain predicted by homology with the winged helix-turn-helix family of activators. Class I mutants bound promoter DNA poorly and were largely unable to interact with ToxR in a crosslinking assay, whereas class II mutants retained physical interaction with ToxR. One mutant constructed in vitro bound DNA poorly but nevertheless responded to ToxR by activating toxT and also maintained ToxR interaction. We propose that ToxR interaction, but not DNA binding, is essential for TcpP function and that the wing domain of TcpP enables contact with ToxR required for productive TcpP-RNA polymerase association. 相似文献