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.

Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection

“…Various electroporation parameters had been used in earlier S. oneidensis transformation attempts (Rachkevych et al 2014;Myers & Myers 1997;Zhou & Ng 2016;Saffarini et al 1994;Bendall et al 2013). We found that washing the cells with dH 2 O decreased both the transformation efficiency and frequency, ~80-and ~200-fold, respectively, compared to results using 10% glycerol.…”

“…Shewanella oneidensis strain MR-1 is a widely used Shewanella strain but displays a low efficiency of electrotransformation for heterologous plasmids derived from different bacterial species, due to its restriction-modification native system (Rachkevych et al 2014). Since the first attempt of electrotransformation in S. oneidensis in 1994 (Saffarini et al 1994), only a few studies have used electroporation as a method to transfer DNA in Shewanella (Rachkevych et al 2014;Myers & Myers 1997;Zhou & Ng 2016;Bendall et al 2013;Shi et al 2005), and no robust transformation protocol has been established to date.…”

Turn on the Mtr pathway genes under pLacI promoter in Shewanella oneidensis MR-1

“…1). As aforementioned, we have constructed two shuttle vectors for E. coli and Shewanella (i.e., pETSXM1 and pETSXM2) on the basis of the ori repB from pSXM33, and ori pMB1 from pET28a(+) (Zhou and Ng 2016). We observed that the plasmids pETSXM1 and pETSXM2 could be transformed into MR-1, but pET28a(+) could not.…”

“…The promoter proA and proB from the plasmid pSXM33 were identified as hypothetical promoters (Zhou and Ng 2016), while the promoter pLacI have been widely used in E. coli. Herein, we found that only promoter pLacI could drive the expression of GFP in S. oneidensis MR-1 among the above mentioned promoters.…”

“…In the previous study, repB from pSXM33 has been explored in S. xiamenensis BC01 and considering its propagation in S. oneidensis MR-1 (Zhou and Ng 2016). It has been ensured that pSXM33 is retained for cell division in Shewanella.…”