Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas.
Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas.
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开发基于接合的高效假交替单胞菌遗传操作系统
DOI：
10.1186/s12934-015-0194-8
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发表时间：
2015-01-23
影响因子：
6.4
通讯作者：
Wang X
中科院分区：
文献类型：
--
作者：
Wang P;Yu Z;Li B;Cai X;Zeng Z;Chen X;Wang X
假交替单胞菌在世界海洋中普遍存在，由于其重要的生态学和生物学意义而受到越来越多的关注。虽然已经对50多个假交替单胞菌的基因组进行了测序，以探索其在不同生境中的适应策略，但由于缺乏有效的遗传操作系统，该属大多数菌株的体内研究受到阻碍。本研究选择了9株分离自不同生境的交替假单胞菌作为代表菌株，建立了一个通用的遗传操作系统。通过对9株菌株的耐药性测定，选择红霉素和氯霉素作为选择标记。基于E. coliWM 3064的基因工程改造，以克服目前在假交替单胞菌中遗传操作的局限性。构建了两个可移动的基因表达穿梭载体（pWD 2-oriT和pWD 2 Ery-oriT），并对pWD 2-oriT与E.大肠杆菌对9株交替假单胞菌的转化率范围为每个受体细胞10−6至10−3个转化接合子。构建了两个自杀载体pK 18 mobsacB-Cm和pK 18 mobsacB-Ery（具有sacB用于反选择）用于基因敲除。为了验证该系统的可行性，我们选择了一旦被破坏就可能导致表型改变的基因或操纵子作为靶标，以便于体内功能确认。成功地删除了红色假单胞菌DSM 6842中与灵菌红素生物合成相关的两个基因（pigMK）、P.sp.SM9913中与生物膜相关的一个基因（bsmA）、P. lipolytica SCSIO 04301中与黑色素过度产生相关的一个基因（hmgA）以及P. sp. SCSIO 11900中与鞭毛相关的两个基因（fliF和fliG）。此外，使用穿梭载体pWD 2-oriT的hmgA互补挽救了由解脂毕赤酵母SCSIO 04301中hmgA的染色体拷贝缺失引起的表型。两者合计，我们表明，在这里开发的载体和接合协议有可能在各种假交替单胞菌菌株中使用。本文的在线版本（doi：10.1186/s12934-015-0194-8）包含补充材料，可供授权用户使用。
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. The online version of this article (doi:10.1186/s12934-015-0194-8) contains supplementary material, which is available to authorized users.
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影响因子：
7
作者：
Médigue, C;Krin, E;Danchin, A
通讯作者：
Danchin, A
影响因子：
4.9
作者：
GAUTHIER, MJ
通讯作者：
GAUTHIER, MJ
影响因子：
3.6
作者：
Pratt, LA;Kolter, R
通讯作者：
Kolter, R
DOI：
10.1007/978-1-61779-433-9_11
发表时间：
2012-01-01
期刊：
RECOMBINANT GENE EXPRESSION: REVIEWS AND PROTOCOLS, THIRD EDITION
影响因子：
--
作者：
Giuliani, Maria;Parrilli, Ermenegilda;Tutino, Maria Luisa
通讯作者：
Tutino, Maria Luisa
从北极海冰中分离出的六种假交替单胞菌菌株的基因组序列
影响因子：
3.2
作者：
Bian, Fei;Xie, Bin-Bin;Zhang, Yu-Zhong
通讯作者：
Zhang, Yu-Zhong