A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation

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.     

Proteomic analysis of pathogenic bacterium Vibrio vulnificus

In this study we have constructed a proteome reference map of the pathogenic bacterium Vibrio vulnificus. From the reference map, we identified several virulence-related proteins, such as ToxR and ToxS, as well as numerous proteins involved in diverse cellular functions. To search for additional virulence-related proteins, we compared the whole proteomes from the wild-type and toxR mutant of V. vulnificus and found that several proteins were up- or down-regulated in the toxR mutant. We suggest that these differentially regulated proteins whose expression is coordinately controlled by a virulence regulator ToxR, some of which are already implicated in virulence, play roles in the pathogenesis of V. vulnificus.     

Amplification and sequence analysis of the full length toxR gene in Vibrio harveyi

This study was focused on obtaining the complete gene sequence of the toxR gene in V. harveyi by using toxR-targeted PCR to amplify 5' and 3' regions flanking the 576-bp Vibrio harveyi (NBRC 15634) toxR gene fragment previously amplified using degenerate PCR. To obtain the 5' flanking sequences, a forward PCR primer (VhtoxRpv) was designed based on known sequences upstream of toxR in V. parahaemolyticus and V. vulnificus. The reverse primer (VctoxR2R) was based on the sequence of the 576-bp Vibrio harveyi toxR fragment. The resulting 750-bp amplicon was sequenced, providing the 5' sequences of the V. harveyi (NBRC 15634) toxR gene. The 3' flanking region was amplified using a primer pair toxRS1 and toxRS2 based on V. parahaemolyticus and V. vulnificus toxR and toxS, resulting in a 900-bp amplicon that contained the remaining 3' sequences of the V. harveyi NBRC 15634 toxR. This paper reports, for the first time, a complete 882-bp nucleotide sequence for toxR in Vibrio harveyi. Sequence analysis and alignment revealed that the complete toxR gene in V. harveyi shares 87% sequence similarity with toxR of V. parahaemolyticus, 84% similarity with V. fluvialis, 83% with V. vulnificus and partial sequence of V. campbellii. The phylogenetic trees revealed wider divergence in toxR compared to 16S rRNA genes, so that V. harveyi could easily be distinguished from V. campbellii and V. parahaemolyticus.     

The ToxR protein of Vibrio cholerae forms homodimers and heterodimers.

The ToxR protein of Vibrio cholerae regulates the expression of several virulence factors that play important roles in the pathogenesis of cholera. Previous experiments with ToxR-alkaline phosphatase (ToxR-PhoA) fusion proteins suggested a model for gene regulation in which the inactive form of ToxR was a monomer and the active form of ToxR was a dimer (V. L. Miller, R. K. Taylor, and J. J. Mekalanos, Cell 48:271-279, 1987). In order to examine whether ToxR exists in a dimeric form in vivo, biochemical cross-linking analyses were carried out. Different dimeric cross-linked species were detected depending on the expression level of ToxR: when overexpressed, ToxR+ToxR homodimers and ToxR+ToxS heterodimers were detected, and when ToxR was expressed at normal levels, exclusively ToxR+ToxS heterodimers were detected. The amount of overexpression was quantitated by using ToxR-PhoA fusion proteins and was found to correspond to 2.7-fold the normal level of ToxR. The formation of both homodimeric ToxR species and heterodimeric ToxR+ToxS species is consistent with previously reported genetic data that suggested that both types of ToxR oligomeric interactions occur. However, variation in the amount of either the homodimeric or heterodimeric form detectable by this cross-linking analysis was not observed to correlate with laboratory culture conditions known to modulate ToxR activity. Thus, genetic and biochemical data indicate that ToxR is able to interact with both itself and ToxS but that these interactions may not explain mechanistically the observed changes in ToxR activity that occur in response to environmental conditions.     

Analysis of a gene (vch) encoding hemolysin isolated and sequenced from Vibrio campbellii

This study describes the amplification, localization, and sequence analysis of a hemolysin gene from type strain V. campbellii NBRC 15631--the first report of a full-length hemolysin gene for the species. An amplicon ( approximately 600 bp) of polymerase chain reaction performed using V. campbellii DNA template and primers previously designed to target a fragment of V. harveyi hemolysin gene (vhh) was shotgun-cloned and sequenced, generating 576 bp nucleotide sequences of the V. campbellii hemolysin gene. PCR primers designed based on these initial sequences were used to amplify a 551-bp V. campbellii hemolysin gene fragment that was used as probe in Southern hybridization, which localized the complete hemolysin gene within a 3.5-kb HindIII restriction fragment of the V. campbellii genomic DNA. To obtain the remaining DNA sequences upstream and downstream of the 576-bp hemolysin gene sequences, inverse PCR was performed using a self-ligated (circularized) V. campbellii HindIII restriction fragment as the template and PCR primers designed to amplify flanking regions of the 576-bp gene fragment. Nucleotide sequences from the terminal regions of the 3.1-kb product of inverse PCR provided the flanking sequences, resulting in the complete sequence for the V. campbellii hemolysin gene. A VCH PCR primer set was designed to amplify a 1.3-kb region containing the entire hemolysin gene even from other V. campbellii strains, which was sequenced to confirm the V. campbellii hemolysin gene sequence. An open reading frame (ORF) of 1,254 bp (designated as vch) was identified, sharing 79% sequence identity with V. harveyi hemolysin gene vhh, representing 262 base substitutions between V. campbellii and V. harveyi. The deduced amino acid sequence of V. campbellii hemolysin (VCH) shows homologies to the V. harveyi hemolysin (VHH), thermolabile hemolysin of V. parahaemolyticus, proteins such as phospholipase of V. vulnificus and lecithinases of V. mimicus and V. cholerae. The VCH primer set did not produce any amplicon in PCR using V. harveyi DNA, and may therefore be used to distinguish environmental strains of V. campbellii from V. harveyi.     

Analysis of membrane protein interaction: ToxR can dimerize the amino terminus of phage lambda repressor

The ToxR protein of Vibrio cholerae is an integral membrane protein that co-ordinately regulates virulence determinant expression. ToxR directiy activates the cholera toxin operon, but maximal activation is achieved in the presence of ToxS, an integral membrane protein thought to interact with ToxR periplasmic sequences. Studies that substitute alkaline phosphatase sequences for the periplasmic domain of ToxR have led to a model for ToxR activation based on dimerization and ToxS interaction. We constructed λ-ToxR chimeric proteins using the DNA-binding domain of the phage λ repressor, which cannot effectively dimerize by itself, to assess the ability of ToxR to form dimers in Escherichia coli The results suggest that ToxR sequences can propagate dimerization, and that ToxS can influence the ability to dimerize.