Characterization of a cryptic plasmid pSM429 and its application for heterologous expression in psychrophilic Pseudoalteromonas

Abstract

BackgroundPseudoalteromonas is an important genus widespread in marine environment, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. By now, there are only a few genetic systems for Pseudoalteromonas reported and no commercial Pseudoalteromonas genetic system is available, which impedes the study of Pseudoalteromonas, especially for psychrophilic strains. The aim of this study is to develop a heterologous expression system for psychrophilic Pseudoalteromonas.ResultsA cryptic plasmid pSM429 isolated from psychrophilic Pseudoalteromonas 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 (Escherichia coli, Pseudoalteromonas), 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 Pseudoalteromonas sp. SM20429, a plasmid-cured strain of Pseudoalteromonas sp. BSi20429, suggesting that the orf4 and its flanking intergenic regions are essential for plasmid replication. Although not essential, the sequence including some repeats between orf1 and orf2 plays important roles in segregational stability of the plasmid. With the aid of pWD-derived plasmid pWD2, the erythromycin resistance gene and the cd gene encoding the catalytic domain of a cold-adapted cellulase were successfully expressed in Pseudoalteromonas sp. SM20429.ConclusionsPlasmid 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 Pseudoalteromonas, offering new perspectives in the genetic manipulation of Pseudoalteromonas strains. With the aid of pWD-derived vector and its host, the erythromycin resistance gene and the cd 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.

Physiological and genetic analyses reveal a mechanistic insight into the multifaceted lifestyles of Pseudoalteromonas sp. SM9913 adapted to the deep‐sea sediment

“…pEV is a shuttle vector constructed based on the shuttle vector pWD (Zhao et al ., ), which was developed in our lab to express cold‐adapted proteins in psychrophilic Pseudoalteromonas strains. pEV contains a cold‐adapted promoter, multiple cloning sites (MCS) and the His tag, which can be transferred into a Pseudoalteromonas strain by conjugation.…”

Antarctic DNA moving forward: genomic plasticity and biotechnological potential

“…() and Zhao et al . () designed cold‐adapted expression vectors by cloning into mesophilic conjugative plasmids cold‐adapted replication sequences from cryptic plasmids isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 and the Arctic Pseudoalteromonas sp. BSi20429.…”

“…This plasmid duplicates in vivo by a rolling-circle mechanism, and several functional and structural features of the Rep initiator protein suggest the existence of a novel subfamily of RC replicons (Tutino et al, 2000). Later, Tutino et al (2001) and Zhao et al (2011) designed cold-adapted expression vectors by cloning into mesophilic conjugative plasmids cold-adapted replication sequences from cryptic plasmids isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 and the Arctic Pseudoalteromonas sp. BSi20429.…”

Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas

“…Genus Pseudoalteromonas belongs to the Gammaproteobacteria class with thirty-eight recognized species reported so far [ 1 , 2 ]. Pseudoalteromonas is ubiquitous in the marine environment [ 2 - 10 ], and many strains have been isolated from deep sea [ 8 ], polar sea [ 2 , 9 , 10 ], or other extreme marine habitats, highlighting their important and diverse role in marine ecosystems. Pseudoalteromonas strains also produce a range of bioactive compounds with antimicrobial, antifouling, or algicidal activities that have attracted global attentions from microbiologist, ecologists and chemists [ 11 ].…”