Slow Edward Coli Genome Fosmid Library Construction, Construction Of The Opp Gene Cluster Cloned Aroa Gene Deletion Mutant

The genomic fosmid library of Edwardsiella tarda LSE40 strain was constructed in this study. The obtained library contained approximate 2 500 clones in which the average size of insertion DNA was 33.6 kb. The total capacity of this library was about 84 Mb, which covered more than 16 times of the genome of E.tarda LSE40 (estimation of 5Mb). About 1 000 clones were selected randomly from this fosmid library to perform end-pairing sequencing. A total of 1 741 high-quality sequences were obtained, and each had the average length of 546 bp. The sequences are sum up to 949 997bp, which covered about 19% of the E.tarda genome. KO(KEGG Orthology) annotations were acquired by the KEGG automatic server KAAS for analysis of the metabolic pathway of the sequences. Results indicated that there were 932, 283, 220, 64 and 16 sequences related to pathway of Metabolism, Environmental Information Processing, Genetic Information Processing, Cellular Processing and Human Diseases, respectively. Meanwhile, a total of 61 sequences were found to be virulence-related by BlastX, which closely corresponds to results in the"Common Themes in Microbial Pathogenicity".The Oligopeptide Permease (opp) gene cluster was screened and cloned from the fosmid library of E.tarda. The obtained opp gene cluster spanned 6 741bp which contains five ORFs encoding five putative proteins, OppA, OppB, OppC, OppD and OppF, respectively. Two potential stem-loop structures located in the spacer region between oppA and oppB, and at downstream of oppF, are presumed as the terminators of oppA and opp, respectively. Phylogenetic tree was constructed based on the OppA conserved domain SBP_bac₅. Results showed that E.tarda LSE40 is most closely related to E. ictaluri, closely related to other Enterobacteriaceae bacteria, and less related to Gram positive bacteria, suggesting that the SBP_bac₅ domain of OppA can be used as a molecular marker in the bacterial identification.The full length sequence of E.tarda LSE40 aroA was screened and cloned from the fosmid library. It had a full-length of 1 287bp, encoding 428 amino acids, which showed 94% identity with that of Edwardsiella ictaluri, 73%-74% identity with that of other Enterobacteriaceae bacteria. The in-frame deletion mutant of aroA was constructed. This mutant showed 62 times increase of 50 percent lethal dose (LD50) in comparison to the wild-type strain. All the flounder fish died in 6 days post-injection of ¹06 cfu/ml of wild-type bacteria, and the moribund fish showed 7.97×108cfu/100mg bacteria in kidney tissues. While no mortalities were found in fish injected with ¹06 cfu/ml of aroA mutant bacteria, and almost no aroA mutant bacteria were re-isolated from the kidney tissue 28 days post-injection. Thus, the aroA mutant will be used in the future study for identification its potential efficacy as a live attenuated vaccine against E. tarda infection.