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Mohan, RaamR. (Warner-Lambert Research Institute, Morris Plains, N.J.), Donald P. Kronish, Roland S. Pianotti, Ray L. Epstein, and Benjamin S. Schwartz. Autolytic mechanism for spheroplast formation in Bacillus cereusand Escherichia coli. J. Bacteriol. 90:1355–1364. 1965.—Spheroplasts of Bacillus cereusstrain T and Escherichia coliB were prepared by incubating early log-phase cells in appropriate buffers and stabilizers for 3 hr at 30 and 37 C, respectively. Upon incubation in 0.05 mtris(hydroxymethyl)aminomethane buffer osmotically stabilized with 16% polyethylene glycol at pH 7.5, 99% of the B. cereuscells formed spheroplasts; 90% of the E. colicells were converted to spheroplasts in 0.4 msodium acetate buffer osmotically stabilized with 1.6 msucrose at pH 6.0. The extent of spheroplast formation was determined by phase-contrast microscopic examination, by measuring the rate of fall of optical density in the reaction mixture when subjected to osmotic shock, and by viable intact cell counts. The effect of a selected group of metabolic inhibitors on the autolytic system of B. cereusand E. colihas been examined. B. cereusand E. coliwall components comprising 26% of the dry weight of the original cellular material were recovered from dialyzed fractions by precipitation in 70% ethyl alcohol. Chemical and chromatographic analysis of cell-wall hydrolysates from B. cereusand E. coliindicated the presence of glucosamine, alanine, lysine, glycine, aspartic acid, diaminopimelic acid, glutamic acid, and muramic acid.

Kronish, DonaldP. (Warner-Lambert Research Institute, Morris Plains, N.J.), Raam R. Mohan, and Benjamin S. Schwartz. Distribution of radioactivity in autolyzed cell wall of Bacillus cereusduring spheroplast formation. J. Bacteriol. 87:581–587. 1964.—Spheroplasts of Bacillus cereusstrain T were produced from cells grown in the presence of uniformly labeled C14-glucose. At regular intervals during spheroplast formation, enzymatically degraded cell wall was isolated by a new procedure. Radioactivity of solubilized cell wall in cell-free material increased from 2.5 to 42% of the total incorporated label during spheroplast formation. The rate of cell-wall degradation as measured by increase in radioactivity was biphasic with relative slopes of 2.0 and 5.0. During autolytic depolymerization of B. cereuscell wall, two major components were solubilized at different rates. Chemical fractionation revealed these to be a peptide and a mucopeptide. The possibility of two enzymes being involved in spheroplast formation and cell-wall degradation is discussed.

Oxolinic acid (1-ethyl-1,4-dihydro-6,7-methylenedioxy-4-oxo-3-quinolinecarboxylic acid) is an antimicrobial agent effective against a variety of gram-negative pathogens, including Proteus. With the exception of Staphylococcus aureus, oxolinic acid is inactive against gram-positive bacteria and against fungi. Our results suggest that oxolinic acid exerted its primary action on synthesis of deoxyribonucleic acid (DNA). The rate of thymidine-2-14Cincorporation into DNA was significantly depressed in the presence of 0.1 μg of oxolinic acid per ml and was markedly inhibited at 1 μg/ml. No evidence of complexing with DNA was observed. Pulse labeling with radioactive precursors revealed that at levels approximating the minimal inhibitory concentration, oxolinic acid had no effect on rate of incorporation of 14C-valine into protein, uracil-2-14Cinto ribonucleic acid, or sodium acetate-1-14Cinto lipid. Filamentous forms of P. vulgarisATCC 881 were observed after in vitro exposure to subinhibitory levels of oxolinic acid. Concentrations of oxolinic acid in excess of the minimal inhibitory concentration (0.39 μg/ml) did not cause lysis of cells of P. vulgarisor leakage of cytoplasmic materials. Mg++ions diminished the in vitro activity of oxolinic acid, possibly through formation of Mg++chelates

Fox, AlfredE. (Warner-Lambert Research Institute, Morris Plains, N.J.), Joachim Anschel, George L. Evans, Raam R. Mohan, and Benjamin S. Schwartz. Isolation of a soluble resistance-enhancing factor from Mycobacterium phlei. J. Bacteriol. 92:285–290. 1966.—Extraction of a crude cell wall preparation from Mycobacterium phleiwith 20% urea yielded a fraction which induced a state of enhanced resistance to microbial challenge. The resulting soluble extract, after removal of the urea, represented a 15% yield of solids with the separation of the biologically active component(s) and elimination of toxicity. Single oral or subcutaneous submicrogram doses of this material induced a prolonged state of increased resistance to subsequent challenge with Salmonella enteritidisin mice. This effect appeared as early as 2 hr after oral administration and persisted for at least 30 days. Protection against experimental infection with Staphylococcus aureuswas also demonstrated. Resistance to viral challenge with influenza type A was observed after intranasal administration of the M. phleiextract to mice. The isolated material was found to contain carbohydrate, protein, nucleic acids, and lipids. The lipids represented 60% of the total solids, and were all short-chain fatty acids. No toxic effects, including pyrogenicity, could be demonstrated after oral or parenteral administration of this preparation.