Département de biologie, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Instituto Tecnológico y de Estudios Superiores de Monterrey, Monterrey, Nuevo León, Mexico.
PLoS Genet. 2024 Oct 9;20(10):e1011442. doi: 10.1371/journal.pgen.1011442. eCollection 2024 Oct.
The phenomenon of exclusion allows conjugative plasmids to selectively impede the entry of identical or related elements into their host cell to prevent the resulting instability. Entry exclusion blocks DNA translocation into the recipient cell, whereas surface exclusion destabilizes the mating pair. IncC conjugative plasmids largely contribute to the dissemination of antibiotic-resistance genes in Gammaproteobacteria. IncC plasmids are known to exert exclusion against their relatives, including IncC and IncA plasmids, yet the entry exclusion factor eexC alone does not account for the totality of the exclusion phenotype. In this study, a transposon-directed insertion sequencing approach identified sfx as necessary and sufficient for the remaining exclusion phenotype. Sfx is an exclusion factor unrelated to the ones described to date. A cell fractionation assay localized Sfx in the outer membrane. Reverse transcription PCR and beta-galactosidase experiments showed that sfx is expressed constitutively at a higher level than eexC. A search in Gammaproteobacteria genomes identified Sfx homologs encoded by IncC, IncA and related, untyped conjugative plasmids and an uncharacterized family of integrative and mobilizable elements that likely rely on IncC plasmids for their mobility. Mating assays demonstrated that sfx is not required in the donor for exclusion, ruling out Sfx as the exclusion target. Instead, complementation assays revealed that the putative adhesin TraN in the donor mediates the specificity of surface exclusion. Mating assays with TraN homologs from related untyped plasmids from Aeromonas spp. and Photobacterium damselae identified two surface exclusion groups, with each Sfx being specific of TraN homologs from the same group. Together, these results allow us to better understand the apparent incompatibility between IncA and IncC plasmids and to propose a mechanistic model for surface exclusion mediated by Sfx in IncC plasmids and related elements, with implications for the rampant dissemination of antibiotic resistance.
排除现象使可接合质粒能够选择性地阻止相同或相关元件进入宿主细胞，以防止由此产生的不稳定性。进入排除阻止 DNA 易位进入受体细胞，而表面排除则使交配对不稳定。IncC 可接合质粒在很大程度上促进了γ变形菌中抗生素耐药基因的传播。已知 IncC 质粒对其亲缘质粒，包括 IncC 和 IncA 质粒，施加排除作用，但单独的外排因子 eexC 并不能解释全部的排除表型。在这项研究中，转座子定向插入测序方法确定 sfx 对于剩余的排除表型是必要和充分的。Sfx 是一种与迄今为止描述的无关的排除因子。细胞分馏测定将 Sfx 定位在外膜中。逆转录 PCR 和β-半乳糖苷酶实验表明，sfx 的表达水平比 eexC 更高，且组成型表达。在γ变形菌基因组中的搜索发现，Sfx 同源物由 IncC、IncA 和相关的、未分型的可接合质粒以及未表征的整合和可移动元件家族编码，这些质粒可能依赖 IncC 质粒来实现其移动性。交配实验表明，在供体中 sfx 不是排除所必需的，排除了 Sfx 作为排除靶标。相反，互补实验表明，供体中的假定粘附素 TraN 介导了表面排除的特异性。与气单胞菌属和发光杆菌属的相关未分型质粒的 TraN 同源物的交配实验确定了两个表面排除组，每个 Sfx 都与来自同一组的 TraN 同源物特异性相关。总之，这些结果使我们能够更好地理解 IncA 和 IncC 质粒之间明显的不兼容性，并提出一个由 Sfx 在 IncC 质粒和相关元件中介导的表面排除的机制模型，这对抗生素耐药性的猖獗传播具有重要意义。
