It could also be used to compare the effect of inhibitors on MurG from different bacteria, especially as all other membrane components of the system Dasatinib mouse and nonspecific effects would be similar. An added advantage is that the assay described measures MurG activity in its natural lipid environment. The assay is easy to perform and reagents
can be bought or easily prepared, unlike the reported solution-based assays (Auger et al., 2003). A solution assay is not the natural environment for MurG: the natural lipid substrate is less preferred than a short-chain synthetic substrate and unusual assay conditions may be required, for example 35% DMSO (Auger et al., 2003) or 15% methanol (Chen et al., 2002). Hence, it is possible that compounds that inhibit MurG in solution may be ineffective in the natural environment (Silva et al., 2000), misleading the structure–activity relationship and running the risk that enzyme inhibition may be divergent from whole-cell antibacterial activity. Importantly, the reconstituted MurG assay can be used to monitor the specific activity of the protein during purification or that of mutant MurG proteins to elucidate structure–activity Seliciclib cell line relationships. In summary, the Mtu
murG gene can support the growth of an E. coli strain, which is devoid of the murG gene product. The surprising lack of MurG activity in the membranes of this strain enabled a novel microplate assay to measure the activity of external sources of MurG in an E. coli membrane background. We thank Dwarakanath Prahlad and R. Philomena for the cloning and overexpression of E. coli MurG. We thank Dr Noel D’Souza of Hoechst, India, for the gift of moenomycin and Dr W.D. Donachie Thymidylate synthase for the
gift of the E. coli murG(Ts) strain. K.D. designed research and wrote the gene complementation part of this manuscript. “
“Dibutyl phosphite, an organophosphorous compound, finds applications in different chemical industries and processes. Here, we report an efficient approach of biodegradation to be eventually used in bioremediation of dibutyl phosphite. Aerobic granules capable of dibutyl phosphite biodegradation were cultivated in a sequencing batch reactor (SBR). The SBR was operated with a 24-h cycle by feeding with dibutyl phosphite as a cosubstrate along with acetate. During the course of the SBR operation, aerobic granules of 0.9 ± 0.3 mm size were developed. Complete biodegradation of 1.4, 2 and 3 mM of dibutyl phosphite was achieved in 4, 5 and 8 h, respectively, accompanied by stoichiometric release of phosphite (H3PO3). Phosphatase activity in the dibutyl phosphite-degrading granular biomass was 3- and 1.5-fold higher as compared to the activated sludge (seed biomass) and acetate-fed aerobic granules, respectively, indicating involvement in the hydrolysis of dibutyl phosphite. Microbial community analysis by t-RFLP showed the presence of 12 different bacterial types.