Sfx and TraN homologs.

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.

排斥现象可使接合性质粒（conjugative plasmids）选择性阻碍相同或相关遗传元件进入宿主细胞，从而避免由此引发的基因组不稳定。进入排斥（entry exclusion）会阻断DNA转位进入受体细胞，而表面排斥（surface exclusion）则会破坏接合配对的结构稳定性。IncC型接合性质粒在γ-变形菌纲（Gammaproteobacteria）的抗生素抗性基因传播过程中发挥了核心作用。已知IncC质粒可对其亲缘质粒产生排斥效应，涵盖IncC与IncA型质粒，但仅排斥因子eexC无法完全阐释该排斥表型。本研究采用转座子定向插入测序技术，鉴定出sfx是介导剩余排斥表型的必要且充分条件。Sfx是一种迄今尚未被报道的新型排斥因子。细胞分级分离实验证实，Sfx定位于细菌外膜。逆转录PCR（reverse transcription PCR）与β-半乳糖苷酶实验结果显示，sfx的组成型表达水平高于eexC。对γ-变形菌纲基因组的同源检索发现，Sfx的同源蛋白编码于IncC、IncA及相关未分型接合性质粒，以及一类未被表征的整合型可移动元件——这类元件可能依赖IncC质粒实现自身移动。接合实验证实，供体菌中的sfx并非排斥反应所必需，这排除了Sfx作为排斥靶标的可能性。与之相反，互补实验表明，供体菌中的推定黏附素TraN介导了表面排斥的特异性。利用来自气单胞菌属（Aeromonas spp.）和海鱼发光杆菌（Photobacterium damselae）的相关未分型质粒的TraN同源蛋白开展接合实验，可将其划分为两类表面排斥组，且每类Sfx仅对同组TraN同源蛋白具有识别特异性。综上，本研究结果深化了我们对IncA与IncC质粒间表观不相容性的认知，并提出了IncC质粒及相关元件中Sfx介导的表面排斥作用机制模型，该研究对抗生素抗性的猖獗传播具有重要的理论与应用启示。