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

Microbial Cell Factories

Research

Xiaoxue Wang¹  Xingsheng Cai¹  Pengxia Wang¹  Zhenshun Zeng²  Baiyuan Li²  Zichao Yu³  Xiulan Chen³ 

[1] Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, the South China Sea Institute of Oceanology, Chinese Academy of Sciences, 510301, Guangzhou, China;Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, the South China Sea Institute of Oceanology, Chinese Academy of Sciences, 510301, Guangzhou, China;University of Chinese Academy of Sciences, 100049, Beijing, China;State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, 250100, Jinan, China;

关键词: Pseudoalteromonas;    Conjugation;    Genetic manipulation;    Marine bacteria;   

DOI  :  10.1186/s12934-015-0194-8

 received in 2014-10-28, accepted in 2015-01-10,  发布年份 2015

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Wang et al.; licensee BioMed Central. 2015