Mom Jérémy, Valette Odile, Pieulle Laetitia, Pelicic Vladimir
Laboratoire de Chimie Bactérienne, Aix-Marseille Université/CNRS (UMR7283), Institut de Microbiologie de la Méditerranée, Marseille, France.
mBio. 2025 Jun 11;16(6):e0085125. doi: 10.1128/mbio.00851-25. Epub 2025 May 23.
Transformation is a mechanism of horizontal gene transfer widespread in bacteria. The first step in transformation-capture of exogenous DNA-is mediated by surface-exposed filaments belonging to the type 4 filament (T4F) superfamily. How these protein polymers, composed of major and minor pilin subunits, interact with DNA remains poorly understood. Here, we address this question for the Com pilus, a widespread T4F mediating DNA capture in competent monoderm species. Our functional analysis, performed in , was guided by a complete structural model of the Com pilus. We show that the major pilin ComGC does not bind DNA. In contrast, a systematic mutational analysis of electropositive residues exposed at the filament surface in the four minor pilins (ComGD, ComGE, ComGF, and ComGG) reveals that the interface between ComGD and ComGF is important for DNA capture. Sequential mutations in these two interacting subunits lead to complete abolition of transformation, without affecting piliation. We further demonstrate the physical interaction between ComGD and ComGF using disulfide crosslinking, upon mutagenesis of two strategically positioned residues into cysteines. A structural model of the Com pilus tip interacting with DNA recapitulates all these findings and highlights a novel mode of DNA-binding, conserved in hundreds of monoderm species.
Bacteria are capable of evolving and diversifying very rapidly by acquiring new genetic material via horizontal gene transfer (HGT). Transformation is a widespread mechanism of HGT, which results from the capture of extracellular DNA by surface-exposed pili belonging to the type 4 filament (T4F) superfamily. How T4F-composed of major and minor pilins-interact with DNA remains poorly understood, especially in monoderm species that use a unique T4F for DNA capture, known as Com pilus or T4dP. The significance of this work is in characterizing a novel mode of DNA-binding by showing that the interface between two minor pilins, part of a tip-located complex of four pilins-found in different T4F-has been functionalized in monoderms to capture DNA. This is an evolutionary mechanism promoting the exceptional functional versatility of T4F.
转化是细菌中广泛存在的一种水平基因转移机制。转化的第一步——捕获外源DNA——由属于4型菌毛（T4F）超家族的表面暴露细丝介导。这些由主要和次要菌毛蛋白亚基组成的蛋白质聚合物如何与DNA相互作用仍知之甚少。在这里，我们针对Com菌毛解决了这个问题，Com菌毛是一种广泛存在的T4F，介导感受态单细胞膜菌物种中的DNA捕获。我们在[具体实验环境]中进行的功能分析以Com菌毛的完整结构模型为指导。我们发现主要菌毛蛋白ComGC不结合DNA。相比之下，对四种次要菌毛蛋白（ComGD、ComGE、ComGF和ComGG）细丝表面暴露的正电残基进行的系统突变分析表明，ComGD和ComGF之间的界面对于DNA捕获很重要。这两个相互作用亚基中的连续突变导致转化完全丧失，而不影响菌毛形成。在将两个战略定位的残基突变为半胱氨酸后，我们使用二硫键交联进一步证明了ComGD和ComGF之间的物理相互作用。Com菌毛尖端与DNA相互作用的结构模型概括了所有这些发现，并突出了一种在数百种单细胞膜菌物种中保守的新型DNA结合模式。
细菌能够通过水平基因转移（HGT）获取新的遗传物质，从而非常迅速地进化和多样化。转化是一种广泛存在的HGT机制，它源于属于4型菌毛（T4F）超家族的表面暴露菌毛捕获细胞外DNA。由主要和次要菌毛蛋白组成的T4F如何与DNA相互作用仍知之甚少，特别是在使用独特的T4F进行DNA捕获的单细胞膜菌物种中，这种T4F被称为Com菌毛或T4dP。这项工作的意义在于通过表明在不同T4F中发现的由四个菌毛蛋白组成的尖端定位复合物的一部分的两个次要菌毛蛋白之间的界面在单细胞膜菌中已被功能化以捕获DNA，从而表征一种新型的DNA结合模式。这是一种进化机制，促进了T4F卓越的功能多样性。
