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Research Letter
An assay for exogenous sources of purified MurG, enabled by the complementation of Escherichia coli murG(Ts) by the Mycobacterium tuberculosis homologue
Article first published online: 30 NOV 2011
DOI: 10.1111/j.1574-6968.2011.02446.x
© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved
Additional Information
How to Cite
Jha, R. K., Katagihallimath, N., Hota, S. K., Das, K. S. and de Sousa, S. M. (2012), An assay for exogenous sources of purified MurG, enabled by the complementation of Escherichia coli murG(Ts) by the Mycobacterium tuberculosis homologue. FEMS Microbiology Letters, 326: 161–167. doi: 10.1111/j.1574-6968.2011.02446.x
Publication History
- Issue published online: 15 DEC 2011
- Article first published online: 30 NOV 2011
- Accepted manuscript online: 31 OCT 2011 12:59PM EST
- Manuscript Accepted: 18 OCT 2011
- Manuscript Revised: 30 SEP 2011
- Manuscript Received: 20 JUL 2011
- Abstract
- Article
- References
- Cited By
Keywords:
- enzyme assay;
- peptidoglycan;
- purification;
- cell wall;
- scintillation proximity assay;
- drug discovery
Abstract
The Mycobacterium tuberculosis murG gene, Rv2153, was expressed in Escherichia coli murG(Ts) strain OV58 on a plasmid under the control of the arabinose-inducible araBAD promoter. Mycobacterium tuberculosis murG rescued the growth of E. coli murG(Ts) at the nonpermissive temperature: transformants were only obtained in the presence of 0.2% arabinose at 42 °C, and their growth rate was dependent on arabinose concentrations. However, no MurG activity was detected in membranes from the transformant grown in arabinose at 42 °C, while MraY activity was normal. This observation led to the development of a membrane-based scintillation proximity assay for exogenous sources of MurG. Addition of purified E. coli MurG resulted in the reconstitution of MurG and peptidoglycan synthesis in these membranes. MurG is an attractive target for drug discovery, but assays to measure the activity of purified MurG are challenging. This presents an easy method to measure the activity of exogenous sources of MurG for structure–activity studies of mutant MurG proteins. It can also be used to compare the activity of, or effect of inhibitors on, MurG from other bacterial species.