Louvain Institute of Biomolecular Science and Technology, Biochemistry and Genetics of Microorganisms, Université catholique de Louvain, B-1348 Louvain-La-Neuve, Belgium.
Institute of Integrative Biology of the Cell (I2BC), University Paris-Sarclay, CEA, CNRS, 91198, Gif-sur-Yvette, France.
Proc Natl Acad Sci U S A. 2020 Apr 7;117(14):7745-7754. doi: 10.1073/pnas.1916085117. Epub 2020 Mar 20.
Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence traits. In most streptococci, competence is regulated by ComRS signaling, a system based on the mature ComS pheromone (XIP), which is internalized to activate the (R)RNPP-type ComR sensor by triggering dimerization and DNA binding. Cross-talk analyses demonstrated major differences of selectivity between ComRS systems and raised questions concerning the mechanism of pheromone-sensor recognition and coevolution. Here, we decipher the molecular determinants of selectivity of the closely related ComRS systems from and Despite high similarity, we show that the divergence in ComR-XIP interaction does not allow reciprocal activation. We perform the structural analysis of the ComRS system from Comparison with its ortholog from reveals an activation mechanism based on a toggle switch involving the recruitment of a key loop by the XIP C terminus. Together with a broad mutational analysis, we identify essential residues directly involved in peptide binding. Notably, we generate a ComR mutant that displays a fully reversed selectivity toward the heterologous pheromone with only five point mutations, as well as other ComR variants featuring XIP bispecificity and/or neofunctionalization for hybrid XIP peptides. We also reveal that a single XIP mutation relaxes the strictness of ComR activation, suggesting fast adaptability of molecular communication phenotypes. Overall, this study is paving the way toward the rational design or directed evolution of artificial ComRS systems for a range of biotechnological and biomedical applications.
细菌具有将外源 DNA 片段内化的能力，从而获得新的表型，如抗生素抗性或毒力特性。在大多数链球菌中，感应系统（ComRS）通过 ComS 成熟信号肽（XIP）来调节感受态，该信号肽被内化后通过触发二聚化和 DNA 结合来激活（R）RNPP 型 ComR 传感器。串扰分析表明 ComRS 系统之间存在选择性的显著差异，这引发了关于信号肽-传感器识别和共同进化机制的问题。在这里，我们解析了来自 和 的密切相关的 ComRS 系统的选择性的分子决定因素。尽管存在高度相似性，但我们表明 ComR-XIP 相互作用的差异不允许相互激活。我们对 中的 ComRS 系统进行结构分析。与来自 的同源物的比较揭示了一种基于Toggle Switch 的激活机制，涉及由 XIP C 末端募集关键环。与广泛的突变分析相结合，我们确定了直接参与肽结合的必需残基。值得注意的是，我们生成了一个 ComR 突变体，它只需要五个点突变就能对异源信号肽表现出完全相反的选择性，并且还生成了其他具有 XIP 双特异性和/或混合 XIP 肽的新功能化的 ComR 变体。我们还揭示了单个 XIP 突变可放宽 ComR 激活的严格性，表明分子通讯表型具有快速适应性。总体而言，这项研究为一系列生物技术和生物医学应用中的人工 ComRS 系统的合理设计或定向进化铺平了道路。
