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

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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.