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.

Highlights

  • Cold-adapted bacteria are excellent candidates for investigations of protein evolution and molecular adaptations to extreme conditions

  • Pseudoalteromonas is a genus of gamma-proteobacteria that is widespread in the world’s oceans, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea

  • There are only a few genetic systems for Pseudoalteromonas reported [9,10,11,12] and no commercial Pseudoalteromonas genetic system is available, which impedes the study of Pseudoalteromonas, especially for psychrophilic strains

Read more

Summary

Introduction

Cold-adapted bacteria are excellent candidates for investigations of protein evolution and molecular adaptations to extreme conditions. Native plasmids from cold-adapted bacteria are of particular interest, because plasmids contribute directly in the adaptation of bacteria to their natural environments and provide easy model systems for investigation of basic molecular processes [3]. Pseudoalteromonas is a genus of gamma-proteobacteria that is widespread in the world’s oceans, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. The genus Pseudoalteromonas contains over 30 marine species [4], which play an important role in marine ecosystem. Pseudoalteromonas species are important for investigation of microbe-host interactions in the sea. Pseudoalteromonas is an important genus widespread in marine environment, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. The aim of this study is to develop a heterologous expression system for psychrophilic Pseudoalteromonas

Objectives
Methods
Results
Discussion
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call