Construction Of Genomic Fosmid Library Of Vibrio Anguillarum And Its Application In Screnning Of Putative Virulence-associated Genes

Vibrio anguillarum causes a terminal haemorrhagic septicaemia in marine fish resulting in severe economic losses in the fish farming industry. The whole genomic sequence of this bacterium is not available so far. In this study, we constructed a genomic fosmid library of V.anguillarum strain M3, and predicted 24 putative virulence-assocaited genes according to the bioinformatic analyses of this library. Additionally, we cloned the aroA gene from this library, and identified a protease (prtV ) gene. The major results are presented as follows:1. Construction and characterization of genomic fosmid library of V. anguillarum M3A V. anguillarum genomic fosmid library was constructed. It consists of 960 clones with average insertion size of about 40 kb, covering 9.1 times of genome (estimation of 5Mb). A total of 11 target genes were screened in the library, yielding 2 to 11 positive clones for each gene. Thus, the fosmid library can serve as an effective library for screening the taget gene.2. Bioinformatic analysis of partial genomic sequence of V. anguillarumAbout 600 individual fosmid clones were carried out end-sequencing, resulting in 927 DNA fragment sequences. Together with 442 DNA fragment sequences we obtained from a random genomic library of V. anguillarum, we obtained a total of 1369 DNA fragment sequence with 646, 472bp, representing approximately 19.35% of the V. anguillarum genome. A total of 718 sequences were found to significantly hit to sequences in GenBank database using BLASTN searches (E < e-5). There were 1,106 sequence hitting to nr database, and 826 sequence hitting to EST database by using BLASTX searches. According to the bioinformatics information and published references, 24 genes are predicted as putative virulence-related gene, including 3 haemolysin genes, 4 RTX (Repeat in toxin) genes, 9 flagellum assemble genes, 2 pilu genes and 6 protease genes.3. Clonning the aroA gene of V. anguillarum from M3 fosmid LibraryThe aroA gene of V. anguillarum was cloned from the M3 fosmid library by using the genomic walking. The full-length sequence of aroA consists of 1281 bp encoding for 427 amino acids with a calculated molecular mass of 46,177 Dalton. The amino acids of aroA contains a putative signal peptide of 11 amino acid residues and a 416 amino acid extracellular polypeptide, showing high homology with those of other bacteria, with the identities of 78%-82%. Phylogenetic analyse showed that aroA of V. anguillarum has a close relationship with the other bacteria of Vibiro sp.4. Functional identification of PrtV gene of V. anguillarumA new protease gene prtV was cloned from the fosmid library. The full-length sequence of PrtV consists of 2778 bp encoding for 926 amino with a calculated molecular mass of 102.52 kDa. A prtV insertion mutant and its complementation strain were made. When the prtV gene was inserted, the ability of growth, the glutin enzyme and N-acetylglucosamine enzyme activities were declined. The prtV gene also can inhibit the hemolytic activities of V. anguillarum. The LD50 of the mutant was 100 times higher than the wild-type in the rainbow trout (Scoophthamus maximusinfection) experiments, which indicated that prtV contributes to the virulence of V. anguillarum.