Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913.
Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913.
复制标题
深海嗜冷细菌假交替单胞菌遗传系统的开发。
DOI：
10.1186/1475-2859-13-13
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发表时间：
2014-01-22
影响因子：
6.4
通讯作者：
Zhang YZ
中科院分区：
文献类型：
--
作者：
Yu ZC;Zhao DL;Ran LY;Mi ZH;Wu ZY;Pang X;Zhang XY;Su HN;Shi M;Song XY;Xie BB;Qin QL;Zhou BC;Chen XL;Zhang YZ
背景：假交替单胞菌是深海沉积物中常见的一类海洋γ-变形菌，在深海沉积物生态系统中具有重要作用。虽然假交替单胞菌的基因组序列分析揭示了一些与适应极端深海环境相关的特定特征，但由于缺乏遗传操作系统，研究假交替单胞菌如何适应深海环境仍然很困难。本研究的目的是建立深海沉积细菌Pseudoalteromonassp.SM9913的遗传系统，为利用同源重组进行基因突变提供可能。构建穿梭载体pOriT-4 Em，通过属间接合法将其转入交替假单胞菌SM 9913，转化效率为1.8 × 10-3，可用于基因敲除试验。以pOriT-4 Em为骨架载体，sacB基因为反选择标记，构建了自杀载体pMT。选择编码UDP-葡萄糖脂质载体转移酶的epsT基因作为通过框内缺失失活的靶基因。在将自杀载体pMT转入假交替单胞菌SM 9913后，使用两步整合-分离策略将epsT框内删除。结果表明，ΔepsT突变株的胞外多糖产量下降了73%，表明epsT基因是该菌株胞外多糖产量的重要调控基因。结论：构建的接合转移系统具有较宽的选择温度范围和较高的转移效率，为该菌株的遗传操作奠定了基础。成功地将SM 9913的epsT基因敲除，在宿主染色体上没有选择标记，从而为敲除同一宿主中的其他基因提供了可能。假交替单胞菌SM 9913基因敲除系统的构建将有助于了解假交替单胞菌适应深海环境的分子机制。
Background:Pseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem. Although genome sequence analysis of Pseudoalteromonas has revealed some specific features associated with adaptation to the extreme deep-sea environment, it is still difficult to study how Pseudoalteromonas adapt to the deep-sea environment due to the lack of a genetic manipulation system. The aim of this study is to develop a genetic system in the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913, making it possible to perform gene mutation by homologous recombination.Results:The sensitivity of Pseudoalteromonas sp. SM9913 to antibiotic was investigated and the erythromycin resistance gene was chosen as the selective marker. A shuttle vector pOriT-4Em was constructed and transferred into Pseudoalteromonas sp. SM9913 through intergeneric conjugation with an efficiency of 1.8 × 10-3, which is high enough to perform the gene knockout assay. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and sacB gene as the counterselective marker. The epsT gene encoding the UDP-glucose lipid carrier transferase was selected as the target gene for inactivation by in-frame deletion. The epsT was in-frame deleted using a two-step integration-segregation strategy after transferring the suicide vector pMT into Pseudoalteromonas sp. SM9913. The ΔepsT mutant showed approximately 73% decrease in the yield of exopolysaccharides, indicating that epsT is an important gene involved in the EPS production of SM9913.Conclusions:A conjugal transfer system was constructed in Pseudoalteromonas sp. SM9913 with a wide temperature range for selection and a high transfer efficiency, which will lay the foundation of genetic manipulation in this strain. The epsT gene of SM9913 was successfully deleted with no selective marker left in the chromosome of the host, which thus make it possible to knock out other genes in the same host. The construction of a gene knockout system for Pseudoalteromonas sp. SM9913 will contribute to the understanding of the molecular mechanism of how Pseudoalteromonas adapt to the deep-sea environment.
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影响因子：
3.2
作者：
Lauro, Federico M.;Tran, Khiem;Bartlett, Douglas H.
通讯作者：
Bartlett, Douglas H.
压电希瓦氏菌WP3冷激调控基因的筛选及冷调控基因的时程表达
影响因子：
2.8
作者：
Li, Shengkang;Xiao, Xiang;Wang, Fengping
通讯作者：
Wang, Fengping
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
DOI：
10.1023/a:1022425621742
发表时间：
2002-11-01
期刊：
JOURNAL OF PROTEIN CHEMISTRY
影响因子：
--
作者：
Chen, XL;Sun, CY;Gao, PJ
通讯作者：
Gao, PJ
影响因子：
5.1
作者：
Cui Z;Lai Q;Dong C;Shao Z
通讯作者：
Shao Z