细菌是存在于自然环境中的一个重要生物类群,参与自然环境碳、氮和硫等元素的循环,另外,细菌在人类的健康与疾病、工业微生物发酵及农业生物病虫害防治等领域也占有重要地位。遗传操作是研究细菌生理功能、致病机理及构建基因工程菌株的先决条件。迄今为止,仅有少数实验室的模式菌株实现了遗传转化,而对直接从自然环境中分离的野生型细菌、经人工驯化的工业生产菌及大量的非模式菌株实现遗传操作始终是困扰微生物学家的一个世界性难题。限制修饰(Restriction modification, RM)系统是外源DNA进入细菌并实现稳定遗传的主要屏障。在完成基因组测序的所有细菌中,95%的菌株含有RM系统,而33%的菌株更是含有四套以上RM系统,含有多套RM系统使细菌的遗传操作更加困难。
典型的RM系统由DNA甲基转移酶(DNA methyltransferases, MTase)和限制性内切酶(Restriction endonucleases, REase)构成。REase可特异性识别进入细菌内部的外源DNA并对其切割、降解,而MTase可通过甲基化修饰细菌自身的DNA而使其与外源DNA区别开来,不被REase降解。
中国科学院微生物研究所温廷益研究组的张国强博士建立了在大肠杆菌中模拟靶细菌DNA甲基化模式(Mimicking of DNA methylation patterns, MoDMP)、穿越靶细菌限制屏障、实现含有多套RM系统的细菌遗传操作的新方法。在一株内源限制修饰系统全部缺失的大肠杆菌EC135中,研究人员克隆表达了来自汉氏硝化细菌(Nitrobacter hamburgensis)X14(研究细菌硝化作用的模式菌株,尽管已有100多年的纯培养历史,因含有11套RM系统而难以进行遗传转化)、蜡样芽胞杆菌(Bacillus cereus)ATCC 10987(含有与炭疽芽胞杆菌中编码炭疽毒素合成蛋白类似的大质粒,是研究炭疽病的理想的非致死菌株,由于含有9套推定的RM系统而导致来自于大肠杆菌的质粒转化效率极低,难以实现遗传操作)和解淀粉芽胞杆菌(Bacillus amyloliquefaciens)TA208(鸟嘌呤核苷工业生产菌,因含有5个推定的MTase而难以利用传统方法实现基因敲除)的24个推定MTase,经活性验证后,将13个具有活性的MTase通过酿酒酵母一步组装法分别组装至3个质粒共表达。验证结果表明共表达了MTase的大肠杆菌具有与难转化细菌相似的DNA甲基化模式。
利用MoMDP系统,研究人员首次成功实现了硝化细菌X14的遗传转化和绿色荧光蛋白表达,首次实现了在鸟苷工业生产菌中利用整合质粒进行基因敲除,另外,穿梭质粒对两株芽胞杆菌的转化效率也有了大幅提高,最高达到104倍。最终,通过实验证据,研究人员提出并验证了通过模拟顽固细菌DNA甲基化模式实现穿越其限制修饰屏障的理论模型。(生物谷Bioon.com)
doi:10.1371/journal.pgen.1002987
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A Mimicking-of-DNA-methylation-patterns Pipeline for Overcoming the Restriction Barrier of Bacteria
ZHANG Guoqiang, WANG Wenzhao, DENG Aihua, SUN Zhaopeng, ZHANG Yun , LIANG Yong, CHE Yongsheng, WEN Tingyi
Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in theE. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase ( upp ) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform