Molecular Mechanism Of DNA Processing For Antibiotic Resistance Gene Transfer In Bacteria

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The discovery of antibiotics in the earlier part of the 20th century has had a significant impact on humans and has saved millions of lives over the years. These antibiotics however are increasingly becoming ineffective and multi drug resistant so called "superbugs" are on the rise. Recent studies have suggested, in the year 2019 alone, drug resistance infections have claimed 51,000 lives in western Europe and an estimated 1.27 million lives globally, and these numbers are expected to rise. In the UK, drug resistant infections are expected to double the treatment cost of patients and will become a big burden on the NHS. The extensive use of antibiotics in the healthcare, veterinary and agricultural settings has contributed to the development of resistance against such drugs and this situation requires urgent attention. The reason for this increase in resistance is because bacteria can develop resistance through evolution and/or by obtaining these resistance causing genes horizontally from other bacteria. Conjugation is a process that mediate horizontal gene transfer and is ubiquitous in all bacteria. This highly sophisticated process of conjugation in bacteria is facilitated by a set of molecular machines. These includes a large macromolecular complex called the 'Type 4 secretion system' (T4SS), an extra cellular conduit called the 'pilus' and a DNA processing machinery called the 'relaxosome'. Reasonable progress has been made towards understanding the T4SS machinery, however little is known about how the DNA, harbouring antibiotic resistance genes, is processed and transported.For conjugation to occur, the double stranded state of the DNA has to be disrupted and opened to enable its transport. This DNA opening is mediated by the relaxosome, a multi-protein complex that facilitates access to a central enzyme called relaxase. This relaxase performs the initiation and DNA unwinding steps and possibly pilots the DNA transfer. It is unclear how these steps occur to enable transfer of DNA by conjugation. We will be studying the relaxosome complex and relaxase protein in detail to derive a fundamental understanding into the conjugative DNA processing mechanism. To address the molecular mechanism behind conjugative DNA processing, we will exploit the recent advances in high-resolution cryo electron microscopy technique to obtain structural details of these complexes. This technique allows us to see the three dimensional arrangement of this complex to a resolution where individual components such as the amino acids and the nucleobases could be located. Further to this we will carry out biochemical and cellular studies to obtain a holistic understanding into this process. Through our work we will be able to provide insights into the mechanism of DNA processing that enables bacterial conjugation. The outcome from this work could be exploited for drug discovery that will ultimately help tackle the spread of antibiotic resistance in bacteria.