VOLUME 4 NUMBER 1 (January to June 2011)

Isolation and sequence analysis of the full-length toxR gene of type strain Vibrio campbellii and use of the toxR gene sequence to evaluate variation and relatedness with other Vibrio species

Availability of complete sequences of the toxR gene (which are universally present yet exhibit significant variations) from type strains of different vibrios will be valuable in evaluating phylogenetic relatedness as well as developing a rapid species-specific identification and differential detection protocols for unknown vibrios. This study focused on the isolation and determination of the complete sequence of the toxR gene from type strain Vibrio campbellii NBRC 15631. Also, the effectiveness of toxR in the evaluation of variation and relatedness among Vibrio and Photobacterium species is compared with the 16S rRNA gene. Using a combination of toxR- and toxS-targeted PCR primers, the full-length toxR gene homologue from type strain V. campbellii NBRC 15631 was amplified and amplicons were subjected to sequencing. Nucleotide sequence analysis of the full-length 873-bp toxR revealed highest sequence similarity with the previously sequenced partial toxR of V. campbellii CAIM 519 (100%) and exhibited lower sequence similarity with toxR from V. harveyi (79%), V. parahaemolyticus (75%), V. anguillarum (64%), V. vulnificus (63%), V. cholerae (59%), V. fischeri (53%), V. hollisae (50%), and Photobacterium profundum (26%). The gene encodes for a 290-amino acid polypeptide, and multiple alignment of the V. campbellii ToxR with reported full-length sequences from other vibrios in the database revealed sequence similarity with V. harveyi (85%) and other vibrios (38-76%), but a fairly conserved transcription activation (62-98% between V. campbellii and other Vibrio and Photobacterium species) and transmembrane and periplasmic domains (30-85% between V. campbellii and other species) flanking a highly divergent membrane “tether” region (6-62% between V. campbellii and other species) is observed. The use of the toxR and 16S rRNA genes to evaluate the relatedness among Vibrio and Photobacterium species were compared. The phylogenetic tree based on toxR sequence variation showed that the different vibrios formed separate clusters, including the closely related V. campbellii and V. harveyi, illustrating that V. campbellii and V. harveyi are indeed distinct species. In contrast, the phylogenetic tree based on the 16S rRNA gene is less specific as indicated by lower bootstrap values. Because of greater sequence variation among species, the use of the toxR gene becomes more effective than the 16S rRNA gene in distinguishing different species of Vibrio. The divergent region in toxR, the membrane “tether” region, could be targeted in designing V. campbellii-specific and other species-specific toxR-targeted PCR primer pairs for the rapid differential detection of closely related vibrios.