Mechanical and structural characterization of the relaxase and relaxosome of bacterial conjugation systems

传染病是对人类的持续威胁(Hutchings，Truman，and Wilkinson 2019；Podolsky 2018)。然而，病原体进化，细菌开发和传播赋予抗菌素耐药性(AMR)的基因(Ikuta等人。2022年)。近几十年来，新抗生素的开发已经放缓，需要探索新的替代战略来了解和减少耐药性的传播(Lewis 2020)。多重耐药性的传播和细菌适应通常通过细菌接合发生，这是水平基因转移的主要途径(Tatum和Lederberg 1947；Arnold，Huang和Hanage 2022)。在细菌接合过程中，通常情况下，接合的质粒被处理并通过IV型分泌系统(T4SS)中的狭窄通道从供体细胞转移到受体细胞(Waksman 2019)。接合的基本准备步骤之一包括在接合的质粒上建立蛋白质-DNA复合体，这被称为松弛小体。在松弛小体的形成过程中，蛋白松弛酶和辅助蛋白对结合的质粒进行加工，它们被认为是识别、重塑、切割和解开质粒的复合体，从而将单链DNA转移到受体细胞。松弛酶本身表现出DNA识别、DNA划痕和共价结合，以及调节的解旋酶活性(Waksman 2019；Gomis-Rüth和Coll 2006)。然而，对转移(ORIT)识别和重塑的起源以及松弛酶解旋酶活性的了解仍然有限。在将DNA转移到受体细胞的过程中，松弛酶保持与DNA的共价结合，并且可能通过去折叠酶机械地展开，因为它太大而不能通过狭窄的分泌通道转移(Cabezón等人)。2015年)。然而，到目前为止，人们还不清楚松弛酶有多稳定。转移后，松弛酶被假定为(部分)重新折叠到催化活性状态，以重新循环单链DNA质粒，以便在受体细胞中进一步处理。对于一个大的(&gt；900aa)，非重复的蛋白质，在机械去折叠后的再折叠还没有报道。目前对松弛小体和松弛酶本身的了解仍然有限，还需要进一步的研究。我们的目标是研究典型的T4SS松弛酶TrwC及其附属蛋白的基本机制。在这个项目中，我们将使用最先进的生化技术和单分子力谱、荧光相关光谱和相关显微镜来阐明(I)TrwC及其亚域的机械稳定性和去折叠/复性途径，(Ii)松弛小体复合体的逐步组装，(Iii)松弛酶解旋酶结构域受调控的DNA解旋活性。

Infectious diseases are a continuing threat to humans (Hutchings, Truman, and Wilkinson 2019; Podolsky 2018). However, pathogens evolve and bacteria develop and spread genes that grant antimicrobial resistance (AMR) (Ikuta et al. 2022). The development of new antibiotics has slowed in recent decades and new, alternative strategies to understand and reduce the spread of resistance need to be explored (Lewis 2020). Spread of multiple resistances and bacterial adaptation often happens through bacterial conjugation, the main pathway of horizontal gene transfer ( Tatum and Lederberg 1947; Arnold, Huang, and Hanage 2022). During bacterial conjugation, typically, a conjugative plasmid is processed and transferred from a donor cell to a recipient cell across a narrow channel in the type IV secretion system (T4SS) (Waksman 2019). One of the essential preparatory steps in conjugation includes establishment of a protein-DNA complex at the conjugative plasmid, which is called the relaxosome. During the relaxosome formation, the conjugative plasmid is processed by the protein relaxase and accessory proteins, which as a complex are thought to recognize, remodel, nick and unwind the plasmid for transferring a single-stranded DNA to the recipient cell. The relaxase itself showed DNA recognition, DNA nicking and covalent binding, as well as regulated helicase activity (Waksman 2019; Gomis-Rüth and Coll 2006). Yet, an understanding of the origin of transfer (oriT) recognition and remodeling and the relaxase helicase activity is still limited. During the transfer of the DNA to the recipient cell the relaxase remains covalently bound to the DNA and is likely mechanically unfolded by unfoldases, because it would be too large to be transferred through the narrow secretion channel (Cabezón et al. 2015). Yet, to date it remains unclear how stable relaxase is. After the transfer, the relaxase is assumed to (partially) refold into a catalytically active state to re-circularize the single-stranded DNA plasmid for further processing in the recipient cell. Refolding after mechanical unfolding for a large (>900 aa), non-repetitive protein has not been reported. The current state of knowledge of the relaxosome and relaxase itself remains still limited, and further research is necessary. We aim to study fundamental mechanisms of the relaxase TrwC, a prototypical T4SS relaxase, and its accessory proteins. In this project, we will use state-of-the art biochemical techniques and single-molecule force spectroscopy, fluorescence correlation spectroscopy and correlative microscopy to shed light on (i) the mechanical stability & unfolding/refolding pathways of TrwC and its subdomains, (ii) the stepwise assembly of the relaxosome complex, (iii) and the regulated DNA unwinding activity of the helicase domain of the relaxase.