Explored a cryptic plasmid pSXM33 from Shewanella xiamenensis BC01 and construction as the shuttle vector

Abstract

Plasmids are essential tools for gene transfer and manipulation in many kinds of microorganisms, but they remain mysterious in the Shewanella species. Herein, a novel cryptic plasmid pSXM33 was isolated from marine bacterium Shewanella xiamenensis BC01 (SXM) and followed by sequencing and characterization through bioinformatics approaches. At first, the plasmid DNA was digested to relatively short fragments, sub-cloned into vector pMD19T-Simple and pBluescript SK(II), then transformed into Escherichia coli (E. coli) DH5α for sequencing. A full-length pSXM33 nucleotide sequence revealed 8,068 bp with GC content of 44%, containing 12 putative open reading frames (ORFs) and several unique restriction sites. Based on the annotation of sequences, ORF1 and ORF4 showed the highest similarity to the integrase, while ORF3, ORF7 and ORF8 encoded the replication protein RepB, plasmid stabilization protein and CopG family transcriptional regulator, respectively. The promoter prediction and tandem repeats analyses suggested 15 promoters and multiple tandem repeats. Moreover, pETSXM1 and pETSXM2 were successfully constructed as shuttle vectors for E. coli and Shewanella species, based on the repB from pSXM33 and a kanamycin resistance gene from vector pET28a(+) as a selective marker. These results provide a useful genetic tool for new insight into molecular level study of the Shewanella species.