title: 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.
authors.affiliation: AstraZeneca India Pvt. Ltd, Bangalore, India.
authors.forename: Ramesh K
authors.initials: RK
authors.lastname: Jha
authors.forename: Nainesh
authors.initials: N
authors.lastname: Katagihallimath
authors.forename: Swetansu K
authors.initials: SK
authors.lastname: Hota
authors.forename: Kaveri S
authors.initials: KS
authors.lastname: Das
authors.forename: Sunita M
authors.initials: SM
authors.lastname: de Sousa
mesh_terms: D001089:Arabinose / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D005816:Genetic Complementation Test; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D017351:N-Acetylglucosaminyltransferases / Q000032:analysis* / Q000037:antagonists & inhibitors; D010457:Peptidoglycan / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014169:Transformation, Bacterial
chemical_list: D001425:Bacterial Outer Membrane Proteins; D010457:Peptidoglycan; D011994:Recombinant Proteins; D001089:Arabinose; D017351:N-Acetylglucosaminyltransferases; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase