The resulting V. anguillarum colonies were transferred to TSA-sheep blood agar (Northeast Laboratories Service, Waterville, ME) and screened for none-hemolytic colonies (vah1 rtxA). The resulting colonies were checked for the desired allelic exchange this website using PCR amplification. Complementation of mutants The various mutants were complemented by cloning the appropriate target gene fragment into the shuttle vector pSUP202 (GenBank accession no. AY428809) as described previously by [8]. Briefly, primers (Table 3) were designed with EcoRI and AgeI sites

and then used to amplify the entire target gene plus ~500 bp of the 5′ and ~200 bp 3′flanking regions from genomic PKC412 DNA of V. anguillarum M93Sm. The DNA fragment was then ligated into pSUP202 after digestion with EcoRI and AgeI, and the ligation mixture was introduced into E. coli Sm10 by electroporation using a BioRad Gene Pulser II. Transformants were selected on LB10 Tc15 Ap100 agar plates. The complementing plasmid was transferred from E. coli Sm10 into the V. anguillarum mutant by conjugation. Transconjugants were selected by tetracycline resistance (Tc2). The transconjugants were then confirmed by PCR amplification and restriction digestion. Bacterial

conjugation Bacterial conjugation were carried out using the procedure modified from Varina et al.[39]. Briefly, 100 μl V. anguillarum grown overnight was added into 2.5 ml nine salts solution (NSS) [40]; 100 μl E. coli culture overnight was added into 2.5 ml 10 mM MgSO4. The resulting V. anguillarum and E. coli suspension was mixed, vacuum filtered onto an autoclaved 0.22-μm-pore-diameter nylon membrane (Millipore, USA), placed on an LB15 agar plate (LB-plus-1.5% NaCl), and allowed to incubate overnight at 27°C. Following incubation, the cells were removed from the filter by vigorous vortex mixing in 1 ml NSS. Cell suspensions (70 μl) were spread on LB20 plated with appropriate antibiotics and the plates were incubated at 27°C until V. anguillarum colonies were observed

aminophylline (usually 24 to 48 h). Cloning, over-expression, purification, and refolding of the Plp protein The whole length of the plp gene (stop codon not included) was amplified by PCR with a sense primer introducing a BamHI site and an antisense primer introducing BglII site, respectively. Genomic DNA extracted from V. anguillarum M93Sm was used as template. The amplified PCR product was digested with BamHI and BglII, and ligated into a pQE60 (QIAGEN, USA) vector, which was also cut with BamHI and BglII. The ligation mix was transformed into E. coli M15 (pREP4) and clones with pQE60-plp were selected on LB10 agar containing kanamycin and ampicillin. A clone harboring plasmid pQE60-plp was selected and the plasmid DNA sequence isolated from the clone confirmed by sequencing.