cholerae and V. vulnificus, our study found that this locus in V. parahaemolyticus was not involved in O-antigen biosynthesis. We also showed that gene cluster referred to as “”capsule”" genes by Guvener et al (VPA1403-VPA1412) was not related to either K-antigen capsule polysaccharide or O-antigen but was instead related to exopolysaccharide production, which causes rugose phase variation. We suggest reserving the term “”capsule”" for K-antigen polysaccharides and referring to the rugose related polysaccharide exopolysaccharide. Our understanding of the major surface polysaccharides in V. parahaemolyticus had been limited, in part, due to our limited ability to perform genetic manipulations in this species. Genetic

manipulation check details of genes in V. parahaemolyticus was

previously Doramapimod supplier achieved by first cloning the DNA of interest into a suicide plasmid that cannot replicate in V. parahaemolyticus, propagating the plasmid in an E. coli host, then transferring the plasmid from E. coli to V. parahaemolyticus by conjugation, followed by counter selection against the E. coli host and screening for mutants of V. parahaemolyticus [23]. The procedure is tedious and time consuming. There are few reports using electroporation in V. parahaemolyticus and no report of successful chemical transformation [24, 25]. We tested electroporation on V. parahaemolyticus and had limited success with plasmid DNA but no success with linear DNA (data not shown). Chemical transformation was also not successful. Obatoclax Mesylate (GX15-070) Therefore we sought alternative methods for targeted gene deletion in V. parahaemolyticus. Meibom et al. reported that V. cholerae became competent and took up foreign DNA when cultured with chitin [26]. The chitin based transformation

method was later successfully adapted for V. vulnificus [27]. We modified the chitin based transformation technique and developed a rapid method to mutate genes in V. parahaemolyticus. On average, 150 mutants were obtained from each transformation. Since only one mutant is needed in most cases, this transformation efficiency will satisfy most deletion applications in V. parahaemolyticus. Capsule biogenesis in E. coli is classified into 4 groups. Exportation of group 1 and 4 capsules rely on Wza proteins, while group 2 and 3 may rely on CPSM and CPST proteins [28]. Previous research has shown that capsules in V. cholerae O31 and V. vulnificus have similarities to E. coli group 1- or group 4 capsules; with a wza gene inside the capsule gene cluster [6, 7, 19]. Genomic analysis also revealed that a wza gene was present in the putative capsule regions in the other published genomes of V. vulnificus and non-O1, non-O139 V. cholerae [29]. In contrast, the wza gene was present in V. parahaemolyticus, but was not within the capsular polysaccharide region. Furthermore, mutagenesis of this gene showed it was not required for K antigen biosynthesis.