This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ramachandran, V. Articles by de Sousa, S. M. Search for Related Content PubMed PubMed Citation Articles by Ramachandran, V. Articles by de Sousa, S. M.

Screen for Inhibitors of the Coupled Transglycosylase-Transpeptidase of Peptidoglycan Biosynthesis in Escherichia coli

Vasanthi Ramachandran, B. Chandrakala, Vidya P. Kumar, Veeraraghavan Usha, Suresh M. Solapure, and Sunita M. de Sousa^*

AstraZeneca India Pvt. Ltd., Bangalore 560 024, India

Received 29 October 2005/ Returned for modification 11 January 2006/ Accepted 3 February 2006

Class A high-molecular-weight penicillin-binding protein 1a (PBP1a) and PBP1b of Escherichia coli have both transglycosylase (TG) and transpeptidase (TP) activity. These enzymes are difficult to assay, since their substrates are difficult to prepare. We show the activity of PBP1a or PBP1b can be measured in membranes by cloning the PBP into an E. coli ponB::Spc^r strain. Using this assay, we show that PBP1a is 10-fold more sensitive to penicillin than PBP1b and that the 50% inhibitory concentration (IC₅₀) of moenomycin, a TG inhibitor, is 10-fold higher in the PBP transformants than in wild-type membranes; this increase in IC₅₀ in transformants can be used to test the specificity of test compounds for inhibition of the TG. Alternatively, the coupled TG-TP activity of PBP1b can be directly measured in a two-step microplate assay. In the first step, radiolabeled lipid II, the TG substrate, was made in membranes of the E. coli ponB::Spc^r strain by incubation with the peptidoglycan sugar precursors. In the second step, the TG-TP activity was assayed by adding a source of PBP1b to the membranes. The coupled TG-TP activity converts lipid II to cross-linked peptidoglycan, which was specifically captured by wheat germ agglutinin-coated scintillation proximity beads in the presence of 0.2% Sarkosyl (B. Chandrakala et al., Antimicrob. Agents Chemother. 48:30-40, 2004). The TG-TP assay was inhibited by penicillin and moenomycin as expected. Surprisingly, tunicamycin and nisin also inhibited the assay, and paper chromatography analysis revealed that both inhibited the transglycosylase. The assay can be used to screen for novel antibacterial agents.

Present address: School of Biosciences, University of Birmingham, Birmingham, United Kingdom.

This article has been cited by other articles:

• Jha, R. K., de Sousa, S. M. (2006). Microplate Assay for Inhibitors of the Transpeptidase Activity of PBP1b of Escherichia coli. J Biomol Screen 11: 1005-1014 [Abstract]