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An integrated post-genomic characterisation of the Escherichia coli 'metalome' with special reference to zinc

Reference	BB/C509831/1
Principal Investigator / Supervisor	Professor Robert K Poole
Co-Investigators / Co-Supervisors	Dr Renli Ma, Professor Cameron McLeod
Institution	University of Sheffield
Department	Molecular Biology and Biotechnology
Funding type	Research
Value (£)	288,040
Status	Completed
Type	Research Grant
Start date	07/03/2005
End date	06/03/2008
Duration	36 months

Abstract

Integrative biology requires a complete, holistic understanding of the cell; this includes not only genes, nucleic acids, lipids, proteins and other organic biomolecules but also individual elements, particularly metals - the metalome. In recent years, zinc (Zn) has come to the forefront of biological research - the galvanization of biology. Zn is absolutely required for the structure and function of key cellular proteins and is believed to be important in cellular processes as diverse as bacterial pathogenicity and brain function. Zn availability is controlled in part by the inducible synthesis of specific uptake and efflux pumps, many examples of which have been identified in Escherichia coli, in addition, the intracellular availability of Zn is regulated by coordination of the metal by proteins. In fact, it has been calculated that, in a single bacterial cell, there is less than one atom of free Zn. This project exploits our leading position acquired through integrating molecular genetic and physical analytical tools for metal analysis. We propose a global, integrated approach to studying the responses to zinc stress in E. coli, with which we have obtained novel preliminary data using both microarray and ICP-MS methodologies. Preliminary experiments under carefully combined chemostat growth conditions show that supra-optimal but non-toxic concentrations of Zn increase expression of 64 genes. Although some of these are known or anticipated to be directly involved in Zn resistance mechanisms, others are involved in antibiotic resistance, strongly suggesting that environmental Zn levels modulate resistance to antimicrobial agents. Laser ablation-ICP-MS results also point to a remarkable complexity in bacterial responses to Zn stress. We will use genome-wide transcriptional profiling under carefully controlled chemostat growth conditions to identify those genes whose regulation is modulated by exposure to supra- or sub-optimal concentrations of Zn in the environment.We will define the roles of genes already identified as being up-regulated by excess zinc (mdt, basRS, for example) and identify and characterise additional genes involved in responding to Zn stresses. In parallel, we will characterise Zn-binding proteins by gel electrophoresis linked with LA-ICP-MS and MALDI-TOF-MS. For the former, an enriched stable isotope 68Zn will be used to differentiate Zn-protein complexes newly synthesised under stress from the proteome of unstressed cells. We will also exploit Surface-Enhanced Laser Desorption/Ionisation - Time of Flight - Mass Spectrometry (SELDI-TOF-MS), integrating protein extraction/fractionation and MALDI-TOF-MS in one platform, namely a protein chip, to provide rapid protein expression profiling. The further development of powerful analytical tools proposed should find wide utility at the biology/chemistry interface. Using these methods, we hope to identify proteins that may be involved in Zn transport, sequestration, and regulation.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
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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