細(xì)菌是存在于自然環(huán)境中的一個重要生物類群,，參與自然環(huán)境碳、氮和硫等元素的循環(huán),，另外,，細(xì)菌在人類的健康與疾病、工業(yè)微生物發(fā)酵及農(nóng)業(yè)生物病蟲害防治等領(lǐng)域也占有重要地位,。遺傳操作是研究細(xì)菌生理功能,、致病機(jī)理及構(gòu)建基因工程菌株的先決條件。迄今為止，僅有少數(shù)實(shí)驗(yàn)室的模式菌株實(shí)現(xiàn)了遺傳轉(zhuǎn)化,，而對直接從自然環(huán)境中分離的野生型細(xì)菌,、經(jīng)人工馴化的工業(yè)生產(chǎn)菌及大量的非模式菌株實(shí)現(xiàn)遺傳操作始終是困擾微生物學(xué)家的一個世界性難題。限制修飾（Restriction modification, RM）系統(tǒng)是外源DNA進(jìn)入細(xì)菌并實(shí)現(xiàn)穩(wěn)定遺傳的主要屏障,。在完成基因組測序的所有細(xì)菌中,，95%的菌株含有RM系統(tǒng)，而33%的菌株更是含有四套以上RM系統(tǒng),，含有多套RM系統(tǒng)使細(xì)菌的遺傳操作更加困難,。
典型的RM系統(tǒng)由DNA甲基轉(zhuǎn)移酶（DNA methyltransferases, MTase）和限制性內(nèi)切酶（Restriction endonucleases, REase）構(gòu)成。REase可特異性識別進(jìn)入細(xì)菌內(nèi)部的外源DNA并對其切割,、降解,，而MTase可通過甲基化修飾細(xì)菌自身的DNA而使其與外源DNA區(qū)別開來，不被REase降解,。
中國科學(xué)院微生物研究所溫廷益研究組的張國強(qiáng)博士建立了在大腸桿菌中模擬靶細(xì)菌DNA甲基化模式（Mimicking of DNA methylation patterns, MoDMP）,、穿越靶細(xì)菌限制屏障、實(shí)現(xiàn)含有多套RM系統(tǒng)的細(xì)菌遺傳操作的新方法,。在一株內(nèi)源限制修飾系統(tǒng)全部缺失的大腸桿菌EC135中,，研究人員克隆表達(dá)了來自漢氏硝化細(xì)菌（Nitrobacter hamburgensis）X14（研究細(xì)菌硝化作用的模式菌株，盡管已有100多年的純培養(yǎng)歷史,，因含有11套RM系統(tǒng)而難以進(jìn)行遺傳轉(zhuǎn)化）,、蠟樣芽胞桿菌（Bacillus cereus）ATCC 10987（含有與炭疽芽胞桿菌中編碼炭疽毒素合成蛋白類似的大質(zhì)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系統(tǒng)而導(dǎo)致來自于大腸桿菌的質(zhì)粒轉(zhuǎn)化效率極低,，難以實(shí)現(xiàn)遺傳操作）和解淀粉芽胞桿菌（Bacillus amyloliquefaciens）TA208（鳥嘌呤核苷工業(yè)生產(chǎn)菌，因含有5個推定的MTase而難以利用傳統(tǒng)方法實(shí)現(xiàn)基因敲除）的24個推定MTase,，經(jīng)活性驗(yàn)證后,，將13個具有活性的MTase通過釀酒酵母一步組裝法分別組裝至3個質(zhì)粒共表達(dá)。驗(yàn)證結(jié)果表明共表達(dá)了MTase的大腸桿菌具有與難轉(zhuǎn)化細(xì)菌相似的DNA甲基化模式,。
利用MoMDP系統(tǒng),，研究人員首次成功實(shí)現(xiàn)了硝化細(xì)菌X14的遺傳轉(zhuǎn)化和綠色熒光蛋白表達(dá)，首次實(shí)現(xiàn)了在鳥苷工業(yè)生產(chǎn)菌中利用整合質(zhì)粒進(jìn)行基因敲除,，另外,，穿梭質(zhì)粒對兩株芽胞桿菌的轉(zhuǎn)化效率也有了大幅提高，最高達(dá)到104倍,。最終,，通過實(shí)驗(yàn)證據(jù)，研究人員提出并驗(yàn)證了通過模擬頑固細(xì)菌DNA甲基化模式實(shí)現(xiàn)穿越其限制修飾屏障的理論模型,。（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
