Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas
Wang, Pengxia1; Yu, Zichao1; Li, Baiyuan1; Cai, Xingsheng1; Zeng, Zhenshun1; Chen, Xiulan1; Wang, Xiaoxue1;
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.
关键词Pseudoalteromonas Conjugation Genetic Manipulation Marine Bacteria
学科领域Biotechnology & Applied Microbiology
作者单位1.[Wang, Pengxia
2.Li, Baiyuan
3.Cai, Xingsheng
4.Zeng, Zhenshun
5.Wang, Xiaoxue] Chinese Acad Sci, Guangdong Key Lab Marine Mat Med, Key Lab Trop Marine Bioresources & Ecol, RNAM Ctr Marine Microbiol,South China Sea Inst Oc, Guangzhou 510301, Guangdong, Peoples R China
6.[Yu, Zichao
7.Chen, Xiulan] Shandong Univ, Marine Biotechnol Res Ctr, State Key Lab Microbial Technol, Jinan 250100, Peoples R China
8.[Li, Baiyuan
9.Zeng, Zhenshun] Univ Chinese Acad Sci, Beijing 100049, Peoples R China
GB/T 7714
Wang, Pengxia,Yu, Zichao,Li, Baiyuan,et al. Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas[J]. MICROBIAL CELL FACTORIES,2015,14:11-.
APA Wang, Pengxia.,Yu, Zichao.,Li, Baiyuan.,Cai, Xingsheng.,Zeng, Zhenshun.,...& of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas.MICROBIAL CELL FACTORIES,14,11-.
MLA Wang, Pengxia,et al."Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas".MICROBIAL CELL FACTORIES 14(2015):11-.
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