Understanding the rules of life

Category: Standard Studentships

Characterisation of an atypical kinase regulating bacterial metabolism

Project No.2227

Primary Supervisor

Dr Stephen Hare – University of Sussex

Co-Supervisor(s)

Dr Roger Draheim – University of Portsmouth

 

Summary

When faced with metabolic, environmental or antibiotic stress, bacteria can enter a non-dividing, quiescent state, wherein a bacterial cell’s metabolic requirement is greatly reduced, enabling it to endure until conditions are more favourable.

Notwithstanding the importance of this stress response in bacterial pathogens, our understanding of the regulation of quiescence is lacking. In E. coli, an atypical kinase, YeaG, has been shown to control the response to nitrogen starvation and to increase the viability of cells following stress. YeaG comprises an AAA+ ATPase domain linked with a kinase domain. We have revealed that the structure of the kinase domain is unique and that this domain alone does not constitute a functional kinase (Hare, unpublished data). Hypothetically, the active site of the kinase is reconstituted

upon oligomerisation of the protein and to this end we have demonstrated by negative stain electron microscopy and SEC-MALLS that recombinant YeaG is hexameric. This project will characterise the structure-function relationship of YeaG by solving the full-length structure. The protein can be purified to homogeneity and is amenable to study by electron microscopy. Characterisation of the structure will reveal how the kinase active site is formed and suggest mechanisms of regulation. The student will also undertake microbiology experiments to validate conclusions drawn from structural information. These will include growth assays of E. coli under normal and stress conditions and critically upon recovery from stress, where YeaG is key. We will perform site-directed mutagenesis to modify and monitor the dynamics of the domains in vivo. In addition, we will look for other interaction partners of YeaG, determine its oligomeric state and probe for its activating stimulus. Results from this work will impact our understanding of stress response in pathogenic bacteria and potentially lead to ways to overcome persistent and recurrent infections caused by metabolically quiescent bacteria.