Oligonucleotides used in this study.

Contact-dependent growth inhibition (CDI) is a widespread form of inter-bacterial competition mediated by CdiA effector proteins. CdiA is presented on the inhibitor cell surface and delivers its toxic C-terminal region (CdiA-CT) into neighboring bacteria upon contact. Inhibitor cells also produce CdiI immunity proteins, which neutralize CdiA-CT toxins to prevent auto-inhibition. Here, we describe a diverse group of CDI ionophore toxins that dissipate the transmembrane potential in target bacteria. These CdiA-CT toxins are composed of two distinct domains based on AlphaFold2 modeling. The C-terminal ionophore domains are all predicted to form five-helix bundles capable of spanning the cell membrane. The N-terminal "entry" domains are variable in structure and appear to hijack different integral membrane proteins to promote toxin assembly into the lipid bilayer. The CDI ionophores deployed by E. coli isolates partition into six major groups based on their entry domain structures. Comparative sequence analyses led to the identification of receptor proteins for ionophore toxins from groups 1 & 3 (AcrB), group 2 (SecY) and groups 4 (YciB). Using forward genetic approaches, we identify novel receptors for the group 5 and 6 ionophores. Group 5 exploits homologous putrescine import proteins encoded by puuP and plaP, and group 6 toxins recognize di/tripeptide transporters encoded by paralogous dtpA and dtpB genes. Finally, we find that the ionophore domains exhibit significant intra-group sequence variation, particularly at positions that are predicted to interact with CdiI. Accordingly, the corresponding immunity proteins are also highly polymorphic, typically sharing only ~30% sequence identity with members of the same group. Competition experiments confirm that the immunity proteins are specific for their cognate ionophores and provide no protection against other toxins from the same group. The specificity of this protein interaction network provides a mechanism for self/nonself discrimination between E. coli isolates.接触依赖性生长抑制（Contact-dependent growth inhibition, CDI）是一类广泛分布的细菌间竞争机制，由CdiA效应蛋白介导。CdiA展示于杀伤性细菌的细胞表面，可在细胞接触时将其毒性C端区域（CdiA-CT）递送至邻近细菌体内。杀伤性细菌同时会合成CdiI免疫蛋白，通过中和CdiA-CT毒素以避免自身发生自抑制。本文报道了一类多样化的CDI离子载体毒素，这类毒素可破坏靶细菌的跨膜电位。基于AlphaFold2建模结果，这些CdiA-CT毒素由两个截然不同的结构域构成：其C端离子载体结构域经预测均能形成可跨细胞膜的五螺旋束；而N端"进入"结构域的结构具有高度变异性，似乎通过劫持不同的整合膜蛋白来促进毒素组装进入脂质双层。大肠杆菌（E. coli）菌株所部署的CDI离子载体毒素，可根据其进入结构域的结构划分为六大类群。通过比较序列分析，我们鉴定出了类群1与3、类群2以及类群4的离子载体毒素对应的受体蛋白，分别为AcrB、SecY与YciB。借助正向遗传学方法，我们还鉴定出了类群5与6离子载体毒素的新型受体：类群5利用由puuP与plaP基因编码的同源腐胺转运蛋白，而类群6毒素则识别由旁系同源基因dtpA与dtpB编码的二/三肽转运蛋白。进一步研究发现，离子载体结构域在类群内存在显著的序列变异，尤其是在预测与CdiI相互作用的位点上。相应地，对应的免疫蛋白也呈现高度多态性，同一类群的免疫蛋白间通常仅共享约30%的序列同一性。竞争实验证实，免疫蛋白仅对其同源对应的离子载体毒素具有特异性，无法为同一类群中的其他毒素提供保护。这一蛋白质相互作用网络的特异性，为大肠杆菌菌株间的自我/非自我识别提供了分子机制。