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<p>Abstract</p> <p>Background</p> <p><it>Pseudoalteromonas </it>is an important genus widespread in marine environment, and a lot of psychrophilic <it>Pseudoalteromonas </it>strains thrive in deep sea and polar sea. By now, there are only a few genetic systems for <it>Pseudoalteromonas </it>reported and no commercial <it>Pseudoalteromonas </it>genetic system is available, which impedes the study of <it>Pseudoalteromonas</it>, especially for psychrophilic strains. The aim of this study is to develop a heterologous expression system for psychrophilic <it>Pseudoalteromonas</it>.</p> <p>Results</p> <p>A cryptic plasmid pSM429 isolated from psychrophilic <it>Pseudoalteromonas </it>sp. BSi20429 from the Arctic sea ice, was sequenced and characterized. The plasmid pSM429 is 3874 bp in length, with a G+C content of 28%. Four putative open reading frames (ORFs) were identified on pSM429. Based on homology, the ORF4 was predicted to encode a replication initiation (Rep) protein. A shuttle vector (<it>Escherichia coli, Pseudoalteromonas</it>), pWD, was constructed by ligating pSM429 and pUC19 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. To determine the minimal replicon of pSM429 and to check the functionality of identified ORFs, various pWD derivatives were constructed. All derivatives except the two smallest ones were shown to allow replication in <it>Pseudoalteromonas </it>sp. SM20429, a plasmid-cured strain of <it>Pseudoalteromonas </it>sp. BSi20429, suggesting that the <it>orf4 </it>and its flanking intergenic regions are essential for plasmid replication. Although not essential, the sequence including some repeats between <it>orf1 </it>and <it>orf2 </it>plays important roles in segregational stability of the plasmid. With the aid of pWD-derived plasmid pWD2, the erythromycin resistance gene and the <it>cd </it>gene encoding the catalytic domain of a cold-adapted cellulase were successfully expressed in <it>Pseudoalteromonas </it>sp. SM20429.</p> <p>Conclusions</p> <p>Plasmid pSM429 was isolated and characterized, and the regions essential for plasmid replication and stability were determined, helping the development of pSM429-based shuttle vectors. The shuttle vectors pWD and its derivatives could be used as cloning vectors for <it>Pseudoalteromonas</it>, offering new perspectives in the genetic manipulation of <it>Pseudoalteromonas </it>strains. With the aid of pWD-derived vector and its host, the erythromycin resistance gene and the <it>cd </it>gene of a cold-adapted protein were successfully expressed, indicating that the potential use of this system for recombinant protein production, especially for cold-adapted proteins.</p>