Bacillus Subtilis Chaperone/protease Mechanisms In Metabolic Shutdown

In many types of bacteria there is a rotary-shredder machine called ClpCP that acts as waste disposal for proteins that are no longer needed. It is built of two parts: ClpC, which is a 6-membered ring that uses energy from ATP hydrolysis to unwind the three dimensional shapes of proteins into a single chain that looks like beads on a wire. This is then fed into ClpP, which takes the form of a stacked pair of 7-membered rings, to be chopped up into small reusable parts. The ClpCP machine is recruited to particular jobs within cells by adaptor proteins. One of these jobs is to destroy many proteins that are important for metabolism, when bacteria form long-lived hardy spores to survive harsh conditions as the spores need to become metabolically inactive.

In this study my group will examine a newly discovered protein, MicA, which acts an adaptor to ClpCP during bacterial spore formation. Sporulation is partially responsible for the persistence of 'hospital superbugs' as spores are a long-lived bacterial form, resistant to cleaning agents and thriving in patients depleted of natural gut microflora.

We intend to uncover the detailed molecular shapes of MicA when it is bound to ClpCP and substrates using indirect techniques as they are too small to see even using powerful microscopes. We specialise in measuring protein shapes and the way they fit together by producing them artificially in large quantities, with the help of bacteria which act as our 'protein factories'. We then deduce the proteins' molecular structures by processing their behaviour when we bounce X-rays or electrons off them or put them in strong magnetic fields. Each of these techniques has its strengths and weaknesses but our combined approach can yield complementary information filling in the gaps left by using just one of the methods. Collaborating with microbiologists, electron microscopists and chaperone scientists, we will feed information into each others' experiments to build up a mechanistic picture of this important step in bacterial spore formation. For example, if we identify a mutation in one of our proteins that makes it bind more tightly to its partner our collaborator can make the same mutation within bacteria to test whether it has the predicted effect in living systems.

By solving this jigsaw puzzle we hope to be in a stronger position to design novel antibiotics to attack the increasing problem of bacterial drug resistance and the project also has longer term implications for understanding metabolism and protein quality control in many aspects of health and disease.

In health and disease cells sometimes change their identities by closing down one set of genes and activating another, removing obsolete proteins using AAA+ chaperone protease systems. Studying these tightly-controlled transitions at a molecular level is vital for defining their precise mechanisms and ultimately designing drugs that can interfere with them. A protein, MicA, discovered by our collaborator, acts as a novel adaptor for the chaperone/protease, ClpCP, and plays a vital role in effecting metabolic shutdown in a canonical identity change in the tractable model organism Bacillus subtilis, whereby the bacteria become dormant long-lived spores to survive stress conditions. From a genetics perspective this transition has been well-characterised in B. subtilis but the mechanistic gaps that remain can only be filled using the molecular approach proposed here. Forespore metabolism must shut down for the spore to become dormant but the mechanisms for this process are still elusive. MicA is involved in shutting down metabolism in the forespore while components still required to complete the process are supplied by the mother cell through a 'feeding tube'. Here we will investigate this system by solving structures of MicA and its complex with ClpCP using cryoEM and other biophysics methods. We will probe substrate interactions using biophysical techniques to learn precisely how MicA effects its function. In concert with my microbiology collaborators we will shed light on this important part of the sporulation process and investigate the clear likelihood of MicA/ClpCP being a target for antimicrobial development. This work will increase basic understanding of proteostasis with specific implications for combating the problem of 'hospital superbugs' which can persist in hardy spore form evading heat and disinfectant.