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

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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 Mg2+. The incorporation from uridine 5′-diphosphate-N-acetyl-[14C]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.