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

Abstract

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 oriTpCTX-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 1000-fold higher than that achieved with native pCTX-M3. We also constructed E. coli donor strains with chromosome-integrated conjugative transfer genes: S14 and S15, devoid of one putative regulator (orf35), and S25 and S26, devoid of two putative regulators (orf35 and orf36) of the pCTX-M3 tra genes. Strains S14 and S15, and S25 and S26 are, respectively, up to 100 and 1000 times more efficient in mobilization than pCTX-M3. Moreover, they also enable plasmid mobilization to 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. IMPORTANCE 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 1000-fold, two putative regulators of this system, orf35 and/or 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.

pCTX-M3—Structure, Function, and Evolution of a Multi-Resistance Conjugative Plasmid of a Broad Recipient Range

“…The oriT pCTX-M3 sequence lies upstream of nikA and comprises two pairs of inverted repeats and the nic site ACATCTTG↓T similar to that of the IncI1 plasmids [ 25 ]. Moreover, pCTX-M3 can mobilize plasmids bearing oriT of the IncI1 plasmid ColIb-P9, and ColIb-P9 can mobilize plasmids bearing oriT pCTX-M3 , albeit both with low efficiency [ 12 ].…”

“…pCTX-M3 acting as a helper plasmid can mobilize the oriT pCTX-M3 -bearing plasmids into recipients such as A. tumefaciens , Cupriavidus necator (previously Ralstonia eutropha ), and Pseudomonas putida , representatives of Alpha-, Beta-, and Gammaproteobacteria, respectively [ 21 ], and also into the Gram-positive bacteria Bacillus subtilis and Lactococcus lactis [ 25 ]. pCTX-M3 itself, when transferred, cannot be established in any these bacteria [ 21 , 25 ]. These indicate that the range of hosts of the conjugative transfer system of pCTX-M3 is much broader than the host range of its replicon, which is restricted to Enterobacteriaceae (Enterobacterales ord.…”

Molecular and Functional Characterization of MobK Protein—A Novel-Type Relaxase Involved in Mobilization for Conjugational Transfer of Klebsiella pneumoniae Plasmid pIGRK

“…A significant consequence of CT is the spreading of antibiotic resistance (AR) among pathogenic bacterial strains [ 4 , 5 , 6 , 7 ]. For these reasons, CT has been widely studied and extensive knowledge about the molecular bases of this process has been gained [ 3 , 8 , 9 , 10 , 11 ]. Thus, innovatory strategies for preventing CT-dependent AR spread have been proposed [ 12 ].…”