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.
Pseudoalteromonas is widespread in various marine environments, and most strains can affect invertebrate larval settlement and metamorphosis by forming biofilms. However, the impact and the molecular basis of population diversification occurring in Pseudoalteromonas biofilms are poorly understood. Here, we show that morphological diversification is prevalent in Pseudoalteromonas species during biofilm formation. Two types of genetic variants, wrinkled (frequency of 12 ± 5 %) and translucent (frequency of 5 ± 3 %), were found in Pseudoalteromonas lipolytica biofilms. The inducing activities of biofilms formed by the two variants on larval settlement and metamorphosis of the mussel Mytilus coruscus were significantly decreased, suggesting strong antifouling activities. Using whole-genome re-sequencing combined with genetic manipulation, two genes were identified to be responsible for the morphology alternations. A nonsense mutation in AT00_08765 led to a wrinkled morphology due to the overproduction of cellulose, whereas a point mutation in AT00_17125 led to a translucent morphology via a reduction in capsular polysaccharide production. Taken together, the results suggest that the microbial behavior on larval settlement and metamorphosis in marine environment could be affected by the self-generated variants generated during the formation of marine biofilms, thereby rendering potential application in biocontrol of marine biofouling.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6865-x) contains supplementary material, which is available to authorized users.
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