Identification of the binding protein which may be the target of penicillin action in Bacillus megaterium

Abstract

PENICILLINS and cephalosporins (β-lactam antibiotics) are believed to kill bacteria by inhibiting reactions involved in the final stages of cell wall assembly¹. It is widely assumed that inhibition of a transpeptidase involved in the insertion of nascent peptidoglycan strands into the cell wall directly leads to cell death. One approach to the study of the lethal action of penicillin has involved the investigation of penicillin-binding proteins (PBPs) which, with few exceptions, are located exclusively in the cytoplasmic membrane of bacteria^2–4. PBPs form covalent complexes with benzyl-(¹⁴C)-penicillin, and can be detected by fluorography after sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. All species of bacteria examined contain multiple PBPs. We have shown that the isolated membranes of Bacillus megaterium contain five proteins which bind benzylpenicillin covalently and we have measured their relative affinities for the antibiotic⁴. However, the accessibility of the PBPs and their affinities for penicillin may be different in whole cells, and to identify the PBP which represents the lethal target for benzylpenicillin, it is necessary to investigate the binding of the antibiotic to the PBPs of whole cells. We report here that only one of the five PBPs in B. megaterium reacts with benzylpenicillin to any significant extent at concentrations just sufficient to kill the cells, and that the characteristics of the inhibition and of the breakdown of the PBP–penicillin complex are consistent with its being the protein which catalyses transpeptidation.