Award details

The role of protein complexes and protein localisation in regulation of bacterial nitrogen metabolism

Reference	BB/E022308/1
Principal Investigator / Supervisor	Professor Mike Merrick
Co-Investigators / Co-Supervisors
Institution	John Innes Centre
Department	Molecular Microbiology
Funding type	Research
Value (£)	350,933
Status	Completed
Type	Research Grant
Start date	15/10/2007
End date	14/10/2010
Duration	36 months

Abstract

This grant seeks to build on our recent work on the E. coli ammonia channel protein AmtB and on the importance of multi-protein complexes and protein localisation in the functioning and regulation of nitrogen metabolism. Whilst the role of protein localisation has been very clearly demonstrated in bacteria with regard to cell division processes there is still much to be learnt about the localisation, both static and dynamic, of proteins and protein complexes that carry out fundamental metabolic processes. We have demonstrated that the regulation of ammonia flux through AmtB is a very dynamic process involving sequestration of GlnK to the inner membrane (and we now know that this occurs in many bacterial species). We wish to determine exactly how changes in the intracellular concentrations of critical effectors, namely 2-oxoglutarate, ATP and ADP, regulate this process. Our recent studies, in Azospirillum, strongly suggest that this process may also be utilised to sequester GlnK (or GlnB) target proteins to the membrane and that relocalisation of these proteins may influence their activities or their access to their substrates. We now plan to investigate just how many proteins in E. coli might be PII targets and how these targets are localised in the cell under different growth conditions. We will utilise new techniques (sequential peptide affinity [SPA] tags coupled to mass spectrometry) for identifying protein complexes in E. coli. These approaches have already led to initial reports of novel GlnB targets. Finally we have some evidence that the primary ammonium assimilation enzyme, glutamine synthetase (GS), may also be partly membrane associated and we plan to explore the physiological significance of this by searching for the membrane proteins that might interact with GS in order to localise it to the membrane.

Summary

Until relatively recently most bacteria were considered to be single celled organisms within which there was little, if any, cellular organisation. They do not contain organelles such as mitochondria or nuclei and consequently it was not considered that proteins within the bacterial cell were specifically organised. However in the last ten years this picture has changed radically, largely as a consequence of studies on cell division where it is now apparent that proteins have very specific locations within the cell and that these can change dramatically and rapidly. A second major recent advance has been the ability to identify protein-protein interactions in bacteria on a whole cell scale and thereby to recognise that many, if not most, proteins are part of multi-protein complexes. The components of these complexes tend to reflect the activities of functionally related proteins and they may also be localised to specific positions within the cell. Furthermore these positions can also change according to the environment and metabolic state of the cell. Ammonium is a fundamental nitrogen source for most bacteria and in our recent studies we have described in some detail a novel membrane protein AmtB that acts as a channel to allow ammonium to enter the cell. This family of Amt proteins is ubiquitous in living organisms being found from bacteria to man. Furthermore we have shown that the flux of ammonia through this channel is controlled by the dynamic localisation of a regulatory protein, GlnK. In times of ammonium sufficiency GlnK binds to AmtB in such a way as to physically block the movement of ammonia into the cell, and this is reversed when the cellular demand for ammonium rises again. In this research we plan to investigate whether multi-protein complexes are a common feature of the way in which bacteria achieve and regulate the metabolism of ammonium and other nitrogen sources. Such work is fundamental to our understanding of the way in which bacteria, the most common life form on the earth, control their growth. It could also in the future identify new ways in which we might control that growth e.g. in pathogenic bacteria.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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