Conjugative plasmids are the main players in horizontal gene transfer in Gram-negative bacteria. DNA transfer tools constructed on the basis of such plasmids enable gene manipulation even in strains of clinical or environmental origin, which are often difficult to work with. The conjugation system of the IncM plasmid pCTX-M3 isolated from a clinical strain of Citrobacter freundii has been shown to enable efficient mobilization of oriT_pCTX-M3-bearing plasmids into a broad range of hosts comprising Alpha-, Beta-, and Gammaproteobacteria. We constructed a helper plasmid, pMOBS, mediating such mobilization with an efficiency up to 1,000-fold higher than that achieved with native pCTX-M3. We also constructed Escherichia coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35) of the pCTX-M3 tra genes, and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15 and strains S25 and S26 are, respectively, up to 100 and 1,000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization into the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis. Additionally, the constructed E. coli strains carried no antibiotic resistance genes that are present in pCTX-M3 to facilitate manipulations with antibiotic-resistant recipient strains, such as those of clinical origin. To demonstrate possible application of the constructed tool, an antibacterial conjugation-based system was designed. Strain S26 was used for introduction of a mobilizable plasmid coding for a toxin, resulting in the elimination of over 90% of recipient E. coli cells.

中文翻译：

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一种基于IncM质粒pCTX-M3的共轭转移系统的新型动员工具。

接合质粒是革兰氏阴性细菌水平基因转移的主要参与者。基于此类质粒构建的DNA转移工具即使在临床或环境起源的菌株中也能进行基因操作，而这些菌株通常很难使用。从弗氏柠檬酸杆菌的临床菌株中分离的IncM质粒pCTX-M3的偶联系统已显示能够将oriT _pCTX-M3的质粒有效地动员到包括Alpha，Beta和Gammaproteobacteria在内的多种宿主中。我们构建了一个辅助质粒pMOBS，以比天然pCTX-M3最高1000倍的效率介导了这种动员。我们还构建了带有染色体整合的共轭转移基因的大肠杆菌供体菌株：S14和S15，缺少一个pCTX-M3 tra基因的推定调控子（orf35），S25和S26，没有两个推定调控子（orf35和orf36） pCTX-M3 tra基因的表达 菌株S14和S15以及菌株S25和S26的动员效率分别比pCTX-M3高100到1000倍。而且，它们还使质粒能够动员到革兰氏阳性细菌枯草芽孢杆菌中和乳酸乳球菌。另外，构建的大肠杆菌菌株不携带存在于pCTX-M3中的抗生素抗性基因，以利于对具有抗生素抗性的受体菌株（例如临床来源的菌株）进行操作。为了证明所构建工具的可能应用，设计了一种基于抗菌结合的系统。S26菌株用于引入编码毒素的可动员质粒，从而消除了90％以上的受体大肠杆菌细胞。