Characteristics of the Conjugative Transfer System of the IncM Plasmid pCTX-M3 and Identification of Its Putative Regulators.
Characteristics of the Conjugative Transfer System of the IncM Plasmid pCTX-M3 and Identification of Its Putative Regulators.
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DOI：
10.1128/jb.00234-18
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发表时间：
2018-09-15
影响因子：
3.2
通讯作者：
Kern-Zdanowicz I
中科院分区：
文献类型：
--
作者：
Dmowski M;Gołębiewski M;Kern-Zdanowicz I
基因水平转移导致细菌基因组的快速变化，而质粒的接合转移对细菌的可塑性有很大影响。在这里，我们提出了一个缺失分析的接合转移系统基因的pCTX-M3质粒的IncM组，这是负责在肠杆菌科抗生素耐药基因的传播。我们发现，orf 35和orf 36基因的缺失，这是接合转移，增加了质粒动员效率。实时荧光定量PCR（RT-qPCR）分析表明orf 35和orf 36参与调节转移基因的表达。我们还修改了pCTX-M3的宿主范围，表明其接合转移系统具有比其复制子更广泛的宿主范围。质粒接合转移系统包括与DNA加工和复制偶联的IV型分泌系统（T4 SS）。T4 SS可分为两个系统发育亚家族，即IVA和IVB，或根据VirB 4 ATP酶的系统发育分为8个组。来自弗氏柠檬酸杆菌的IncM组质粒pCTX-M3的接合系统被分类为IVB亚家族和MPFI组，IncI 1组质粒的接合系统也是如此。虽然IncM和IncI 1质粒的接合基因大多数显示保守的同线性，但存在几个差异。在这里，我们提出了27个基因的pCTX-M3接合转移区的缺失分析。值得注意的是，缺失接合转移的两个基因中的任一个，即orf 35和orf 36，导致质粒动员效率增加。orf 35和orf 36缺失突变体的转录分析表明，这些基因参与调节接合转移基因的表达。我们还修改了pCTX-M3复制子的宿主范围，发现该复制子不能支持在根癌土壤杆菌、真养罗尔斯通氏菌和恶臭假单胞菌中的复制，尽管其接合系统能够将携带oriTpCTX-M3的质粒引入这些细菌中，这些细菌分别是α-、β-和γ-变形菌的代表。因此，pCTX-M3的接合转移系统具有比其复制子更宽的宿主范围。重要性水平基因转移是细菌基因组快速变化的原因，质粒的接合转移对细菌的可塑性有很大影响。在这里，我们提出了一个缺失分析的接合转移系统基因的pCTX-M3质粒的IncM组，这是负责在肠杆菌科抗生素耐药基因的传播。我们发现，orf 35和orf 36基因的缺失，这是接合转移，增加了质粒动员效率。实时荧光定量PCR（RT-qPCR）分析表明orf 35和orf 36参与调节转移基因的表达。我们还修改了pCTX-M3的宿主范围，表明其接合转移系统具有比其复制子更广泛的宿主范围。
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 Enterobacteriaceae. We found that the deletion of either of the orf35 and orf36 genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. Real-time quantitative PCR (RT-qPCR) analysis suggested the involvement of orf35 and orf36 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. 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 Citrobacter freundii, is classified in the IVB subfamily and in the MPFI 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, orf35 and orf36, resulted in an increased plasmid mobilization efficiency. Transcriptional analysis of the orf35 and orf36 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 Agrobacterium tumefaciens, Ralstonia eutropha, and Pseudomonas putida, though its conjugation system is capable of introducing plasmids bearing oriTpCTX-M3 into these bacteria, which are representatives of Alpha-, Beta-, and Gammaproteobacteria, respectively. Thus, the conjugative transfer system of pCTX-M3 has a much broader host range than its replicon. IMPORTANCE 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 Enterobacteriaceae. We found that the deletion of either of the orf35 and orf36 genes, which are dispensable for conjugative transfer, increased the plasmid mobilization efficiency. Real-time quantitative PCR (RT-qPCR) analysis suggested the involvement of orf35 and orf36 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.