Cross-linked peptidoglycan synthesis has been monitored in Escherichia coli (Eco) membranes by incubation with the two sugar precursors UDP-N-acetyl-muramylpentapeptide [UDP-MurNAc(pp)] and UDP-GlcNAc, one of which is radiolabelled (Chandrakala et al., 2001). In the membranes, the disaccharide unit of peptidoglycan is synthesized on a lipid carrier by the MraY and MurG enzymes and subsequently polymerized by the transglycosylase and cross-linked to pre-existing peptidoglycan by the transpeptidase (Fig. 1). The radiolabelled,

newly synthesized cross-linked peptidoglycan formed can be monitored by paper this website chromatography or a microplate scintillation proximity assay (SPA) using wheat germ agglutinin (WGA)-coated SPA beads (Chandrakala et al., 2001, 2004). To monitor MurG activity,

the pathway of reactions must be stopped at lipid II (Mengin-Lecreulx et al., 1991) (Fig. 1a) using an inhibitor of the transglycosylase (Ravishankar et al., 2005). Typically, in a first step, the MurG substrate is synthesized in situ; in a second step, transfer of radiolabelled GlcNAc by MurG occurs (Fig. 1b). The product lipid II can be separated from UDP-GlcNAc by paper chromatography (Mengin-Lecreulx et al., 1991) or by an SPA (Ravishankar et al., 2005) (Fig. 1b). We intended setting up an assay GW-572016 mw for Mycobacterium tuberculosis (Mtu) MurG by introducing it into an E. coli background, so that an established SPA (Ravishankar et al., 2005) could be used. Strain OV58 has an amber mutation in murG and a temperature-sensitive amber suppressor, so that practically no E. coli protein is made at 42 °C (Salmond et al., 1980; Mengin-Lecreulx et al., 1991). A key question was

whether the Mtu murG would functionally replace the E. coli homologue. Wheat germ agglutinin-coated (WGA) beads for the SPAs were from Amersham International plc. U.K. UDP-[3H]-N-acetyl glucosamine was from NEN Dupont, USA. Moenomycin was gifted by Hoechst India. Ni-NTA resin was from Qiagen, USA. Other chemicals were from Sigma-Aldrich. Thiamine-diphosphate kinase UDP-N-acetyl muramyl pentapeptide [UDP-MurNAc(pp)] was purified from Bacillus cereus 6A1 (Chandrakala et al., 2001) and radiolabelled by incubation with [3H]-NHS-propionate (Solapure et al., 2005). Escherichia coli murG(Ts) (Salmond et al., 1980) was a gift from W.D. Donachie. pRSETA and E. coli BL21(DE3) were from Novagen; pBAD/Myc-HisA and PMOSBlue were from Stratagene. L-broth (LB) was used for bacterial growth medium, and ampicillin was added at 50 or 100 μg mL−1 when required (LB-amp). The murG gene was PCR-amplified from Mtu genomic DNA with forward (5′- AAG GAC ACG GTC AGC CAG CC -3′) and reverse primers (5′- TCT AAA GCT TCG TCG TTG TCC TGG CAC CGG -3′) and cloned into pBAD/Myc-His A (Guzman et al., 1995) between the NcoI and HindIII sites. The resulting plasmid pAZI8952 has Mtu murG gene under the control of BAD promoter.