Complete sequence of virulence plasmid pEIB1 from the marine fish pathogen Vibrio anguillarum strain MVM425 and location of its replication region.

AIMS: The aim of this study was to determine the whole DNA sequence of pEIB1, one pJM1-like virulence plasmid from Vibrio anguillarum MVM425 and locate the replication region.

METHODS AND RESULTS: DNA sequence of virulence plasmid pEIB1 from V. anguillarum MVM425 was determined using the methods of restriction endonuclease digestion, subcloning, and primer walking. The whole nucleotide sequence of pEIB1 comprises 66,164 bp, encoding 44 open reading frames (>400 bp) containing the genes of DNA replication, biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. With no demonstrated replication origin, the Sau3AI partial digested plasmid DNA fragments of pEIB1 were ligated into the BamHI-fragment containing the kanamycin-resistance gene (Kmr). For there is no effective transformation in V. anguillarum, the ligated DNA was first introduced into E. coli JM83, and the transfomants were selected for resistance to kanamycin. It was demonstrated with southern blotting and DNA sequencing that plasmid pEIB7 containing the Sau3AI DNA fragment of pEIB1 (from 12516 to 13957) has the ability to replicate in E. coli JM83 and V. anguillarum MVM425sh. The segregational stability of plasmid pEIB7 kept in 100 and 4% in E. coli JM83 and V. anguillarum MVM425sh respectively when the cells were cultured in 200th generation. In following experiments, we also found that plasmid pEIB7 replicated at a middle-copy number of 10-40 in JM83, while at a high-copy number of 100-300 in MVM425sh. Moreover, pEIB7 can survive in V. alginolyticus, another fish pathogenic.

CONCLUSIONS: With the whole DNA sequence of pEIB1 determining, it was found that pEIB1 showed microheterogeneity in its restriction endonuclease patterns with pJM1 though their DNA sequences had slight difference. According to the complete DNA sequence of pEIB1, its replication region was located from 12516 to 13957. And this replication region is compatible to pUC18 (pMB1), pKA3 (pSC101) and p15A: caiE (p15A).

SIGNIFICANCE AND IMPACT OF THE STUDY: The worldwide vibriosis marine pathogen V. anguillarum strains contain common virulence, pJM1-like plasmids, independent on the geographical source. The pEIB1 was the second common virulence plasmid, which sequence was determined. Its sequence is highly homologous to pJM1 as they both encode biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. Some interesting features as in pJM1 were also identified, such as transposon-like structures. So it can be deferred that the whole DNA sequences of virulent plasmid pEIB1 will be great helpful to future revealing these V. anguillarum virulence-related genes derived during evolution from transposition events or horizontal transfer of genes potentially originating in other organisms. Another result, replication region of pEIB1 locating is the first report about replication of pJM1-like plasmid. This work will be useful for researching pJM1-like plasmid replication mechanism in V. anguillarum.