Award details

A highly conserved essential pathway in bacteria

Reference	BB/D000386/1
Principal Investigator / Supervisor	Professor Tracy Palmer
Co-Investigators / Co-Supervisors	Dr Berengere Ize
Institution	University of East Anglia
Department	Biological Sciences
Funding type	Research
Value (£)	133,860
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	31/12/2007
Duration	27 months

Abstract

The yjeE gene is highly conserved across all eubacterial species. In E. coli, and other bacteria examined so far, it is essential for viability. It encodes a small nucleotide-binding protein that binds ATP with a kD in the region of 80 micromolar, and we propose that it acts as an ATP dependent switch. We have shown that YjeE interacts with another highly conserved essential protein YeaZ and we aim to define the essential cellular process that we propose is regulated by YjeE. The project aims to: [a] Define the network of proteins that interact with YjeE, YeaZ and YgjD (a YeaZ homologue). This will involve a number of approaches including bacterial 2-hybrid studies, immunoprecipitation and affinity purification. Proteins will be purified and interactions studied in vitro using surface plasmon resonance. The effect of nucleotides on complex formation will be assessed. [b] Define the essential pathway in which YjeE, YeaZ and YgjD are involved. This will involve a number of approaches including (i) sub-cellular localisation of YjeE, YeaZ and YgjD using cell fractionation and fluorescence tagging; (ii) transcriptome analysis of cognate depletion strains and (iii) identification of the substrates of YeaZ and YgjD by proteomics and other approaches.

Summary

Bacteria kill 5 million people on average every year, accounting for 10 per cent of human deaths. Bacterial infection is usually treated with antibiotics which kill the organisms by blocking essential metabolic processes. Unfortunately over time many antibiotics have lost their effectiveness against certain types of bacteria because resistant strains have developed. In order to develop new types of antibiotics, it is essential that we understand the basic cellular processes in bacteria. We have been studying an essential protein, called YjeE, in the model bacterium E. coli. Although YjeE is found in all bacteria, currently almost nothing is known about its function and why it is essential for survival of E. coli. We have shown that this protein can interact with a compound called ATP. We have also shown that it binds to another protein in the cell called YeaZ. YeaZ is also essential for survival of E. coli, and we know that YeaZ is normally found in a complex with another partner, YgjD. Our hypothesis is that YjeE acts as a regulator or switch and that when it binds compounds such as ATP it changes shape allowing it to interact with other proteins (for example YeaZ) and to affect their activity. In this work we want to identify the essential process that is regulated by YjeE. Firstly we would like to identify all of the different proteins in E. coli with which YjeE can interact. Once we have identified these we wish to prevent them from being produced by knocking out the DNA that codes for each one. This will allow us to find out which of the proteins is essential for the survival of E. coli. We also want to find out which proteins interact with YeaZ and YgjD. This information will allow us to build up a picture or network of all of the different interactions that occur in this metabolic pathway. Secondly we would like to purify the YjeE, YeaZ and YgjD proteins away from all of the other proteins in the cell. We can then use a technique called Surface Plasmon Resonance to examine the different complexes that form between these purified proteins. If we work with purified proteins we can also then add compounds such as ATP to our mixtures and look at the effect this has on the types of complexes that form. Finally we would like to understand the essential cellular process that is regulated by YjeE. In order to do this we need to understand what it is that the YeaZ and YgjD proteins do in the cell. They appear to be very similar to a protein called gcp, that has already been studied by scientists. Gcp is a protein that destroys other proteins in the cell, but only if those proteins have been modified by addition of sugar residues. We will use techniques that allow us to separate and see all of the different proteins from E. coli cells. We can stain these proteins with a dye that will only react with them if they are modified by attachment of sugars. We can then add some of our purified YeaZ and YgjD proteins to this and look to see if they can destroy sugar-modified proteins.

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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