Abstract
Pseudoalteromonas is commonly found throughout the world’s oceans, and has gained increased attention due to the ecological and biological significance. Although over fifty Pseudoalteromonas genomes have been sequenced with an aim to explore the adaptive strategies in different habitats, in vivo studies are hampered by the lack of effective genetic manipulation systems for most strains in this genus. Here, nine Pseudoalteromonas strains isolated from different habitats were selected and used as representative strains to develop a universal genetic manipulation system. Erythromycin and chloramphenicol resistance were chosen as selection markers based on antibiotics resistance test of the nine strains. A conjugation protocol based on the RP4 conjugative machinery in E. coli WM3064 was developed to overcome current limitations of genetic manipulation in Pseudoalteromonas. Two mobilizable gene expression shuttle vectors (pWD2-oriT and pWD2Ery-oriT) were constructed, and conjugation efficiency of pWD2-oriT from E. coli to the nine Pseudoalteromonas strains ranged from 10−6 to 10−3 transconjugants per recipient cells. Two suicide vectors, pK18mobsacB-Cm and pK18mobsacB-Ery (with sacB for counter-selection), were constructed for gene knockout. To verify the feasibility of this system, we selected gene or operon that may lead to phenotypic change once disrupted as targets to facilitate in vivo functional confirmation. Successful deletions of two genes related to prodigiosin biosynthesis (pigMK) in P. rubra DSM 6842, one biofilm related gene (bsmA) in P. sp. SM9913, one gene related to melanin hyperproduction (hmgA) in P. lipolytica SCSIO 04301 and two flagella-related genes (fliF and fliG) in P. sp. SCSIO 11900 were verified, respectively. In addition, complementation of hmgA using shuttle vector pWD2-oriT was rescued the phenotype caused by deletion of chromosomal copy of hmgA in P. lipolytica SCSIO 04301. Taken together, we demonstrate that the vectors and the conjugative protocol developed here have potential to use in various Pseudoalteromonas strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0194-8) contains supplementary material, which is available to authorized users.
Highlights
Genus Pseudoalteromonas belongs to the Gammaproteobacteria class with thirty-eight recognized species reported so far [1,2]
Antibiotic resistance in different Pseudoalteromonas strains To develop a universal genetic manipulation system for a variety of Pseudoalteromonas strains, nine Pseudoalteromonas strains were selected as representative strains (Table 1)
Erythromycin and chloramphenicol resistance genes serve as good candidates of constructing vectors for universal gene expression and gene knockout in Pseudoalteromonas
Summary
Genus Pseudoalteromonas belongs to the Gammaproteobacteria class with thirty-eight recognized species reported so far [1,2]. Pseudoalteromonas strains produce a range of bioactive compounds with. Direct transfer of non-mobilizable pWD2 to other Pseudoalteromonas strains is constrained by the need for electroporation. Electroporation does not seem to work in majority of Pseudoalteromonas strains whose growth are usually saltdependent. Based on our current knowledge, to date, gene deletion systems have only been described for two Pseudoalteromonas strains, P. haloplanktis TAC125 and P. sp. SM9913 [14,15] Both protocols were designed for the construction of strain-specific isogenic knockouts, developing a widely applicable genetic manipulation system for Pseudoalteromonas becomes a priority
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