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 E. coli 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, orf35 and orf36 , were disabled. The constructed strains broaden the repertoire of tools for the introduction of DNA into the Gram-negative Alpha -, Beta -, and Gammaproteobacteria , as well as into Gram-positive bacteria such as Bacillus subtilis and Lactococcus lactis . The antibacterial procedure based on conjugation with the use of the orf35 - and orf36 -deficient strain lowered the recipient cell number by over 90% owing to the mobilizable plasmid-encoded toxin.