We have developed a novel, high-throughput scintillation proximity assay to measure the membraneassociated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP-N-acetyl muramylpentapeptide and UDP-[ 3 H]-N-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinincoated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.

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The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was ϳ90 kDa and bound 3 H-benzylpenicillin and 125 I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli ⌬ponA ponB::spc r cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3 H-benzylpenicillin and 125 I-cephradine, the MIC of ␤-lactams for E. coli ⌬ponA ponB::spc r expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.The rod shape of Escherichia coli is maintained by the covalently bonded murein layer of the cell envelope (24). The final stages of murein synthesis are carried out by a group of specialized enzymes called penicillin-binding proteins (PBPs), and as the name implies, these proteins are characterized by the ability to form enzymatically inactive covalent complexes with ␤-lactam antibiotics (18). Eight PBPs have been identified in E. coli, of which PBP 1A and 1B are responsible for the synthesis of peptidoglycan at all stages of growth (17).PBP 1A and 1B are the major murein-synthesizing enzymes of E. coli, since inactivation of the genes encoding these PBPs, ponA and ponB, respectively, is lethal for E. coli (25). However, E. coli cells remain viable and grow if only one of these genes is inactivated, indicating functional complementarity of the two proteins (25). There is, however, growing evidence that the two PBP 1 proteins are not equivalent, although the precise physiological role for the E. coli PBP 1A and 1B proteins during peptidoglycan synthesis remains unclear (6).Structural analysis of the peptidoglycan has revealed that although the backbone of the peptidoglycan polymer is universally composed of alternating N-acetylglucosamine and N-acetylmuramic acid, the amino acids of the pentapeptide side chains, the degree of cross-linking, the presence of unique glycoproteins, and the length of the sugar residues can vary from one bacterial species to another. A number of genes encoding putative homologs of E. coli PBP 1A and 1B have been cloned and sequenced (13). However, the enzymatic properties of only the E. coli PBP 1A and 1B have been characterized, although, from overall homology, these proteins have been assigned to the family of high-molecular-weight PBPs. It is inte...

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