Preparation of sphaeroplasts from Lactobacillus casei by growth with low concentration of benzylpenicillin.
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Six marine bacteria which synthesize macromolecular antibiotics were isolated from neritic waters on the French Mediterranean coast, and their frequency recorded over two successive years. They appeared in relatively large numbers during the period August to December, and can be identified as marine pseudomonads; however, the low guaninecytosine ratio of their DNA, lack of catalase and specific self-inhibition are not compatible with the characteristics of the genus Pseudomonas. Two produced violacein, usually synthesized by bacteria belonging to the genus Chromobacterium. Their taxonomic position is discussed.
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Mycoplasma mycoides subsp. capri was grown in different media. The amounts of the lipids in these media were varied, resulting in altered lipid compositions of the cells. Lowering the amounts of cholesterol in the media resulted in less cholesterol incorporation into the cell lipid, with a concomitant decrease in the amount of phospholipid. The changes in the phospholipid/cholesterol ratio in the mycoplasmas were very small compared with the large changes in the amounts of cholesterol which occurred when the cholesterol content in the medium was altered. Changes in the amounts of glycolipid and glyceride in the cell lipid also resulted from alterations of the cholesterol concentration in the media. Under these conditions cell with reduced cholesterol contents were more sensitive to lysis by digitonin. No changes were observed in the polyacrylamide gel electrophoretic pattern of cellular or membrane proteins when the cholesterol was replaced by other sterols.
The activities of polymyxin B sulphate, colistin (polymyxin E) sulphate and their sulphomethyl derivatives were compared by continuous turbidimetric monitoring of dense cultures of an Escherichia coli strain exposed to these agents. Judged by the concentration of antibiotic which caused a rapid fall in opacity of the culture, polymyxin B sulphate and colistin sulphate had similar activities, but the sulphomethyl compounds differed considerably: sulphomyxin sodium induced lysis of the culture at a concentration four times that of the parent compound, whereas colistin sulphomethate sodium induced a delayed fall in opacity consistent with recruitment of activity as the inactive sulphomethyl derivative was broken down to the parent compound. Durign overnight incubation, regrowth of cultures which had initially succumbed to polymyxin action occurred, apparently due to the selection of phenotypically resistant variants from within the population. In this way cultures could easily be adapted to growth in concentrations of antibiotic well above the conventionally-determined minimum inhibitory concentration. The comparative ease of adaptation was in the order: colistin sulphomethate greater than sulphomyxin greater than colistin sulphate greater than polymyxin B sulphate.
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Several type C and D strains of Clostridium botulinum, which had been converted to the toxgenic state by phages, were serially transferred through cooked meat medium with and without specific anti-phage serum. Most of the converted strains lost their toxigenicity even during transfer without antiserum, and the non-toxigenic variants that appeared were resistant to lysis and conversion by the original phage. However, in some combinations of phage and host bacteria toxigenicity was stable after ten transfers, though it showed a transient decrease, and the non-toxigenic variants that arose remained sensitive to lysis and conversion. When converted strains were transferred in medium containing anti-phage serum, toxigenicity was lost more rapidly than in the absence of serum and the non-toxigenic variants that appeared remained sensitive to lysis and conversion by the parent phage. Filtrates of the supernatants of culture fluids of strains transferred without anti-phase serum converted non-toxigenic strains to toxigenicity at varying rates.
A toxic substance, which destroyed leucocytes from man but was inactive against erythrocytes, was demonstrated in cultures of four out of 110 strains of Pseudomonas aeruginosa tested. The toxin, designated 'leucocidin', was cell-bound as a precursor toxin, exhibiting little or no toxicity. It was converted into toxin with maximum activity by various proteases including an endogenous elastase. The production of leucocidin was directly proportional to the number of bacteria and was not influenced by variations in media, iron concentration, pH or temperature. The best method for large-scale production of leucocidin was autolysis of washed bacteria.
Leucocidin from Pseudomonas aeruginosa strain 158 was released from bacteria by autolysis and purified 19-fold by ammonium sulphate precipitation (20% saturation) and combined 'tandem' gel filtration on Sephadex G-100 superfine and Bio Gel P-100. The product gave a single band (mol. wt. 27000) after poly-acrylamide gel electrophoresis with sodium dodecyl sulphate (SDS). However, it was separated into two active peaks (pI 5-0 and 5-2) by isoelectric focusing, and into five bands by disc electrophoresis without SDS. All bands contained leucocidic activity of about the same specific activity and retained their homogeneity. The purified toxin was thermolabile and was inactivated by pronase, but not by several other proteases. The ultraviolet light absorbancy was typical of proteins. Antibodies directed against leucocidin were detected by passive haemagglutination and by toxin-neutralization. These antibodies inhibited the cytotoxic action of leucocidin bound to granulocytes. The toxin damaged all tested leucocytes (granulocytes of various animal species and lymphocytes of humans) and a number of tissue cultures, but was ineffective against erythrocytes, thrombocytes and isolated granules from polymorphonuclear leucocytes. The intravenous lethal dose for mice was about 1 mug.
Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na2HPO4/NaH2PO4 buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCC15909) was maximal in 0-05 TO 0-2 M buffer, while the two S. mutans strains and S. mitis ATCC15912 showed maximal autolysis in 0-5 and 1-0 M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 degrees C Than at 45 degrees C and 10 degrees C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, ATCC15909 and ATCC15912) was maximal at pH 7-0 AND 7-5 and in 1-0 M buffers. Streptococcus mitis ATCC15909 also showed maximal lysis in 0-01 M and 0-5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCC15912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quanitity.