An assay for exogenous sources of purified MurG, enabled by the complementation of Escherichia coli murG(Ts) by the Mycobacterium tuberculosis homologue.

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.

journal_name

FEMS Microbiol Lett

authors

Jha RK,Katagihallimath N,Hota SK,Das KS,de Sousa SM

doi

10.1111/j.1574-6968.2011.02446.x

subject

Has Abstract

pub_date

2012-01-01 00:00:00

pages

161-7

issue

2

eissn

0378-1097

issn

1574-6968

journal_volume

326

pub_type

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