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

Abstract

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.