Construction of Pseudoalteromonas - Escherichia coli shuttle vector based on a small plasmid from the marine organism Pseudoalteromonas

Abstract

A small plasmid (pDK4) from the Antarctic marine organism<em> Pseudoalteromonas</em> sp. PAMC 21150, was purified, sequenced and analyzed. pDK4 was determined to be 3,480 bp in length with a G+C content of 41.64% and contains three open reading frames encoding a replication initiation protein (RepA), a conjugative mobilization protein (Mob) and a hypothetical protein. PCR-amplified pDK4 was cloned in high-copy pUC19 to yield the fusion vector pDOC153. The chloramphenicol resistance gene was inserted into pDOC153 to give an ampicillin and chloramphenicol-resistant, <em>Pseudoalteromonas ？ Escherichia coli</em> shuttle vector (7,216 bp; pDOC155). The TonB-dependent receptor<em> (chi22718_IV )</em> and exochitinase<em> (chi22718_III ) </em>genes from Arctic marine<em> P. issachenkonii </em>PAMC 22718 were cloned into pDOC155 to produce pDOC158 and pDOC165, respectively. Both vector derivatives were transferred into plasmid-free <em>Pseudoalteromonas</em> sp. PAMC 22137 by the triparental mating method. PCR experiments showed that the genes were stably maintained both in Pseudoalteromonas sp. PAMC 22137 and<em> E. coli </em>DH5α cells, indicating the potential use of pDOC155 as a new gene transfer system into marine <em> Pseudoalteromonas</em> spp.