Novel Scintillation Proximity Assay for Measuring Membrane-Associated Steps of Peptidoglycan Biosynthesis in Escherichia coli

Abstract

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.

Bacterial Hash Function Using DNA-Based XOR Logic Reveals Unexpected Behavior of the LuxR Promoter

“…Following a bacterial cell transformation, satellite colonies can form around resistant colonies containing cells that successfully integrated an ampicillin-resistance plasmid. Ampicillin is often mistakenly thought to kill E. coli outright but ampicillin and other ß -lactams prevent cell wall peptidoglycan synthesis by the competitive inhibition of a transpeptidase 21 , thereby preventing the bacteria from forming new cell walls during cell division. Ampicillin-resistant cells can secrete up to 90% of the enzyme ß -lactamase beyond the periplasmic space when the enzyme is highly expressed 22 .…”

Microplate Assay for Inhibitors of the Transpeptidase Activity of PBP1b of Escherichia coli

“…The supernatant of a low-speed centrifugation was centrifuged at 150,000g, and the pellet was washed once and used as the enzyme source. The quality of each membrane batch was monitored by measuring the peptidoglycan synthesized by different quantities of protein 24 as well as by the counts per minute (cpm) obtained in the blank reaction (see below).…”

“…Membranes were prepared from E. coli AMA1004 as described earlier. 10,24 Briefly, the cells were suspended in 50 mM Tris-HCl pH 7.5, 0.1 mM MgCl 2 , and lysed in a French pressure cell. The supernatant of a low-speed centrifugation was centrifuged at 150,000g, and the pellet was washed once and used as the enzyme source.…”

“…10,24 Briefly, UDP-MurNAc(pp) was purified from a hot water extract of the cells by gel filtration and ion exchange chromatography. The purification was monitored by sugar estimation, and its concentration was estimated from its absorbance at 262 nM using a molar extinction coefficient of 10,000.…”

A High-Throughput Screening Assay for Simultaneous Selection of Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-Xylulose-5-Phosphate Synthase (Dxs) or 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase (Dxr)

“…24 in those cases, care is taken not to pick up inhibitors of the coupling enzyme. in a few exceptions (e.g., pathway assays 25 ), these are sensitive to inhibitors of each of the coupling enzymes.…”