Dalia Triana N, Machouri Mérick, Lacrouts Céline, Fauconnet Yoann, Guerois Raphaël, Andreani Jessica, Radicella J Pablo, Dalia Ankur B
bioRxiv. 2025 Feb 9:2024.10.21.619469. doi: 10.1101/2024.10.21.619469.
Natural transformation (NT) represents one of the major modes of horizontal gene transfer in bacterial species. During NT, cells can take up free DNA from the environment and integrate it into their genome by homologous recombination. While NT has been studied for >90 years, the molecular details underlying this recombination remain poorly understood. Recent work has demonstrated that ComM is an NT-specific hexameric helicase that promotes recombinational branch migration in Gram-negative bacteria. How ComM is loaded onto the post-synaptic recombination intermediate during NT, however, remains unclear. Another NT-specific recombination mediator protein that is ubiquitously conserved in both Gram-positive and Gram-negative bacteria is DprA. Here, we uncover that DprA homologs in Gram-negative species contain a C-terminal winged helix domain that is predicted to interact with ComM by AlphaFold. Using and as model systems, we demonstrate that ComM directly interacts with the DprA winged-helix domain, and that this interaction is critical for DprA to recruit ComM to the recombination site to promote branch migration during NT. These results advance our molecular understanding of recombination during this conserved mode of horizontal gene transfer. Furthermore, they demonstrate how structural modeling can help uncover unexpected interactions between well-studied proteins to provide deep mechanistic insight into the molecular coordination required for their activity.
Bacteria can acquire novel traits like antibiotic resistance and virulence through horizontal gene transfer by natural transformation. During this process, cells take up free DNA from the environment and integrate it into their genome by homologous recombination. Many of the molecular details underlying this process, however, remain incompletely understood. In this study, we identify a new protein-protein interaction between ComM and DprA, two factors that promote homologous recombination during natural transformation in Gram-negative species. Through a combination of bioinformatics, structural modeling, cell biological assays, and complementary genetic approaches, we demonstrate that this interaction is required for DprA to recruit ComM to the site of homologous recombination.
自然转化（NT）是细菌物种水平基因转移的主要方式之一。在自然转化过程中，细胞可以从环境中摄取游离DNA，并通过同源重组将其整合到基因组中。虽然自然转化已经研究了90多年，但这种重组的分子细节仍知之甚少。最近的研究表明，ComM是一种自然转化特异性的六聚体解旋酶，可促进革兰氏阴性菌中的重组分支迁移。然而，在自然转化过程中ComM如何加载到突触后重组中间体上仍不清楚。另一种在革兰氏阳性菌和革兰氏阴性菌中普遍保守的自然转化特异性重组介导蛋白是DprA。在这里，我们发现革兰氏阴性菌中的DprA同源物含有一个C端带翼螺旋结构域，预测该结构域可通过AlphaFold与ComM相互作用。以[具体菌名1]和[具体菌名2]为模型系统，我们证明ComM直接与DprA带翼螺旋结构域相互作用，并且这种相互作用对于DprA在自然转化过程中将ComM招募到重组位点以促进分支迁移至关重要。这些结果推进了我们对这种保守的水平基因转移模式下重组的分子理解。此外，它们展示了结构建模如何有助于揭示研究充分的蛋白质之间意想不到的相互作用，从而深入了解其活性所需的分子协调机制。
细菌可以通过自然转化的水平基因转移获得诸如抗生素抗性和毒力等新特性。在此过程中，细胞从环境中摄取游离DNA，并通过同源重组将其整合到基因组中。然而，这一过程背后的许多分子细节仍未完全了解。在本研究中，我们鉴定了ComM和DprA之间一种新的蛋白质-蛋白质相互作用，这两个因子在革兰氏阴性菌的自然转化过程中促进同源重组。通过生物信息学、结构建模、细胞生物学实验和互补遗传方法的结合，我们证明这种相互作用是DprA将ComM招募到同源重组位点所必需的。
