title: Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas.
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.
authors.affiliation: 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, Guangzhou, 510301, China. wangpengxia@scsio.ac.cn.
authors.forename: Pengxia
authors.initials: P
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan, 250100, China. 1986yzc@163.com.
authors.forename: Zichao
authors.initials: Z
authors.lastname: Yu
authors.affiliation: 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, Guangzhou, 510301, China. lby245239@126.com.|University of Chinese Academy of Sciences, Beijing, 100049, China. lby245239@126.com.
authors.forename: Baiyuan
authors.initials: B
authors.lastname: Li
authors.affiliation: 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, Guangzhou, 510301, China. caixingsheng@scsio.ac.cn.
authors.forename: Xingsheng
authors.initials: X
authors.lastname: Cai
authors.affiliation: 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, Guangzhou, 510301, China. zengdongdi2005@126.com.|University of Chinese Academy of Sciences, Beijing, 100049, China. zengdongdi2005@126.com.
authors.forename: Zhenshun
authors.initials: Z
authors.lastname: Zeng
authors.affiliation: State Key Laboratory of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan, 250100, China. cxl0423@sdu.edu.cn.
authors.forename: Xiulan
authors.initials: X
authors.lastname: Chen
authors.affiliation: 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, Guangzhou, 510301, China. xxwang@scsio.ac.cn.
authors.forename: Xiaoxue
authors.initials: X
authors.lastname: Wang
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002701:Chloramphenicol / Q000494:pharmacology; D003227:Conjugation, Genetic; D024881:Drug Resistance, Bacterial / Q000235:genetics; D017753:Ecosystem; D004917:Erythromycin / Q000494:pharmacology; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D005822:Genetic Vectors / Q000235:genetics*; D019015:Geologic Sediments / Q000382:microbiology*; D009792:Oceans and Seas; D031161:Pseudoalteromonas / Q000187:drug effects / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D004917:Erythromycin; D002701:Chloramphenicol