Characteristics of the Conjugative Transfer System of the IncM Plasmid pCTX-M3 and Identification of Its Putative Regulators

Abstract

Plasmid conjugative transfer systems comprise type IV secretion systems (T4SS) coupled to DNA processing and replication. The T4SSs are divided into two phylogenetic subfamilies, namely, IVA and IVB, or on the basis of the phylogeny of the VirB4 ATPase, into eight groups. The conjugation system of the IncM group plasmid pCTX-M3, from <i>Citrobacter freundii</i>, is classified in the IVB subfamily and in the MPF_I group, as are the conjugation systems of IncI1 group plasmids. Although the majority of the conjugative genes of the IncM and IncI1 plasmids display conserved synteny, there are several differences. Here, we present a deletion analysis of 27 genes in the conjugative transfer regions of pCTX-M3. Notably, the deletion of either of two genes dispensable for conjugative transfer, namely, <i>orf35</i> and <i>orf36</i>, resulted in an increased plasmid mobilization efficiency. Transcriptional analysis of the <i>orf35</i> and <i>orf36</i> deletion mutants suggested an involvement of these genes in regulating the expression of conjugative transfer genes. We also revised the host range of the pCTX-M3 replicon by finding that this replicon is unable to support replication in <i>Agrobacterium tumefaciens</i>, <i>Ralstonia eutropha</i>, and <i>Pseudomonas putida</i>, though its conjugation system is capable of introducing plasmids bearing <i>oriT</i>_pCTX-M3 into these bacteria, which are representatives of <i>Alpha</i>-, <i>Beta</i>-, and <i>Gammaproteobacteria</i>, respectively. Thus, the conjugative transfer system of pCTX-M3 has a much broader host range than its replicon.<b>IMPORTANCE</b> Horizontal gene transfer is responsible for rapid changes in bacterial genomes, and the conjugative transfer of plasmids has a great impact on the plasticity of bacteria. Here, we present a deletion analysis of the conjugative transfer system genes of the pCTX-M3 plasmid of the IncM group, which is responsible for the dissemination of antibiotic resistance genes in <i>Enterobacteriaceae</i> We found that the deletion of either of the <i>orf35</i> and <i>orf36</i> genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. Real-time quantitative PCR (RT-qPCR) analysis suggested the involvement of <i>orf35</i> and <i>orf36</i> in regulating the expression of transfer genes. We also revised the host range of pCTX-M3 by showing that its conjugative transfer system has a much broader host range than its replicon.