A Novel Mobilizing Tool Based on the Conjugative Transfer System of the IncM Plasmid pCTX-M3.

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 has been shown to enable efficient mobilization of -bearing plasmids into a broad range of hosts comprising -, -, and 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 donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator () of the pCTX-M3 genes, and S25 and S26, devoid of two putative regulators ( and ) of the pCTX-M3 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 and Additionally, the constructed 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 cells. The conjugation of donor and recipient bacterial cells resulting in conjugative transfer of mobilizable plasmids is the preferred method enabling the introduction of DNA into strains for which other transfer methods are difficult to establish (e.g., clinical strains). We have constructed strains carrying the conjugation system of the IncM plasmid pCTX-M3 integrated into the chromosome. To increase the mobilization efficiency up to 1,000-fold, two putative regulators of this system, and , were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative -, -, and , as well as into Gram-positive bacteria such as and The antibacterial procedure based on conjugation with the use of the - and -deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin.