In Vitro Synthesis of Peptidoglycan by β-Lactam-Sensitive and -Resistant Strains of Neisseria gonorrhoeae : Effects of β-Lactam and Other Antibiotics

Abstract

The synthesis in vitro of peptidoglycan by Neisseria gonorrhoeae was studied in organisms made permeable to nucleotide precursors by treatment with ether. Optimum synthesis occurred at 30°C in tris(hydroxymethyl)aminomethane-maleate buffer (0.05 M; pH 6) in the presence of 20 mM Mg 2+ . The incorporation from uridine 5′-diphosphate- N -acetyl-[ 14 C]glucosamine into peptidoglycan, measured after precipitation of the cells with trichloroacetic acid, was sensitive to the β-lactam antibiotics, bacitracin, diumycin, and tunicamycin and relatively resistant to spectinomycin and tetracycline. Differences in sensitivity between preparations from a β-lactamase producer and a laboratory segregant derived from it were not great. Synthesized peptidoglycan was also fractionated into sodium dodecyl sulfate-soluble and -insoluble portions. β-Lactam antibiotics at concentrations equivalent to the minimal inhibitory concentrations for growth of the organisms did not inhibit peptidoglycan synthesis, but rather caused a small enhancement. At higher concentrations, above about 0.5 μg/ml, incorporation into sodium dodecyl sulfate-insoluble material was progressively inhibited, whereas the amount of sodium dodecyl sulfate-soluble product increased greatly, more than compensating for the loss of the precipitable fraction. Similar observations were made with three strains, and also with the β-lactam clavulanic acid, normally considered as a β-lactamase inhibitor rather than as itself an effective antibiotic.