Radiolabelling of penicillin-binding proteins (PBPs) in intact Pseudomonas aeruginosa cells: consequences of beta-lactamase activity by PBP-5.
The time-course of labelling of penicillin-binding proteins (PBPs) was compared for intact and sonicated cell preparations of nine Pseudomonas aeruginosa strains, all of which gave identical PBP profiles. Saturation of all the PBPs in cell-sonicates occurred within 2 min of exposure to 35 mg/l 14[C] benzylpenicillin. PBP-5 formed an unstable penicilloyl-complex: the other PBPs formed highly stable complexes. Saturation of PBP-4 in intact cells occurred within 2 min of exposure to the antibiotic, correlating with the high affinity of this protein for penicillin. Labelling of PBPs 1a, 1b and 3 was slow but progressive, suggesting that these proteins were shielded by the permeability barrier(s) of the cell. Labelling of PBP-5 in intact cells achieved 10-20% saturation within 2-10 min of exposure to 35 mg/l 14[C] benzylpenicillin, but did not increase subsequently. This behaviour may indicate the establishment of a steady state between the formation and breakdown of the PBP-5-penicillin complex, suggesting that PBP-5, potentiated by the permeability barrier, functions as a feeble beta-lactamase. Such activity may distort the labelling of other PBPs by reducing the concentration of penicillin in the periplasm, thus invalidating the PBP accessibility experiments that have been recommended as probes of bacterial permeability. The beta-lactamase activity also may be significant in resistance, since Noguchi et al. (Journal of Antibiotics, 1980, 33, 1521-6) have previously associated the loss of PBP-5-beta-lactamase activity with hypersusceptibility to beta-lactams in P. aeruginosa.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000032:analysis*; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis*; D010400:Penicillin G / Q000378:metabolism; D010403:Penicillin Resistance; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D011550:Pseudomonas aeruginosa / Q000032:analysis* / Q000187:drug effects; D001618:beta-Lactamases / Q000032:analysis*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases; D010400:Penicillin G