The capacity of newly transformed cells of Bacillus subtilis to synthesize deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein has been determined by folowing the kinetics of suicide after their exposure to tritiated precursors of each of these macromolecules. Competent cells, whether transformed or not, are heterogeneous with respect to DNA synthesis. About 40 to 50% are latent in DNA synthesis. This latency may persist for 2.5 to 3 hr since transformants are resistant to thymineless death for this period after DNA addition. The remainder of the transformants synthesize DNA at one-half the rate of the cells of the total population. Synthesis of stable RNA does not occur at an appreciable rate in newly transformed cells. Newly transformed cells, however, do synthesize protein extensively, as demonstrated by the lethality of incorporated tritiated amino acids. Either chloramphenicol or actinomycin D treatment during the time of exposure to the tritiated amino acid prevented the suicide of transformants.
Mycobacterium avium exhibits two colonial forms, transparent and opaque, on 7H10 agar. The transparent variant of M. avium B2900 undergoes a spontaneous transition to the opaque form at the rate of 4.7 X 10-5 to 3.5 X 10-4 per cell per generation. The two variants differ in cel morphology and mass; the opaque cells are two to three times as large as the transparent in terms of dry weight, total protein, deoxyribonucleic acid, or carbohydrate. A search for auxotrophic mutants resulted in the induction, by nitrosoguanidine or ultraviolet irradiation, of conditional, salt-sensitive mutants. One such mutant requires methionine and tryptophan supplementation for growth in a medium containing a buffer at high molarity. The clinical and physiological significance of these findings is discussed.
Mycobacterium avium, a facultative pathogen for humans, undergoes a life cycle in which selected small cells elongate and then fragment to form coccobacilli. M. avium cells of uniform size were selected by membrane filtration and tested for growth and division in the presence or absence of palmitic acid. Growth was measured by increased cellular protein, and cell division was determined by increased colony-forming units on agar or, electronically, by increased numbers of particles. Both growth and division rates of M. avium were found to be dependent upon the initial concentration of palmitic acid presented to the cells. The division constant varied from 0.05 to 0.13 when the concentration of palmitic acid ranged from 0 to 175 nmol/ml of medium. With ["4C ]palmitic acid as a tracer, it was found that rapid cell division began upon cessation of fatty acid uptake. During division, new lipid materials were released which contained "4C derived from ["4C]palmitic acid. Limited cell division and no fragmentation occurred in fatty acid-starved cultures. During fatty acid starvation, the transparent colony form, considered a pathogen, underwent a transition to the colony form considered a nonpathogen. The possible relationships between the organism's dependence on fatty acid and its ability to infect humans are discussed. 363 on August 5, 2020 by guest http://iai.asm.org/ Downloaded from 364 MCCARTHY on August 5, 2020 by guest http://iai.asm.org/ Downloaded from
Mycobacterium avium accumulates '4C-palmitic acid with saturation kinetics; the process is both temperature dependent and pH sensitive. The fatty acid is incorporated into triglyceride in vivo and the conversion is detectable within 5 min after exposure of the cells to '4C-palmitic acid. The triglyceride is rapidly utilized because 14CO2 evolution from it begins within 30 min after 14C-palmitic acid accumulation. Data from silicic acid column chromatography of extracts of cultures that have divided many times in medium containing '4C-palmitic acid indicate that a large proportion of the cell lipid is triglyceride, but the radioactivity is widely dispersed among the other lipids. It is estimated that about 5%, of the cell dry weight is triglyceride in a postexponential culture.The growth rate of most mycobacteria is stimulated if oleic acid is included in the medium. This feature was particularly emphasized by Schaefer and Lewis (15) and Hedgecock (7), who showed that growth of Mycobacterium kansasii was delayed 5 to 9 days if the organism was incubated in a defined medium containing only glycerol as the available carbon source. Oleic acid, alone, stimulated rapid growth which stopped when the fatty acid was depleted, but the inclusion of glycerol permitted a continuation of growth. Schaefer and Lewis (15) also demonstrated that M. kansasii accumulated globules when grown in a medium containing oleic acid.M. avium B2900 produces a transparent colony variant that requires either oleic or palmitic acid for growth and that is pathogenic for chickens and mice (14). The opaque colony variant derived from it is nonpathogenic (14) and does not require a fatty acid. Pathogenicity and requirement for a fatty acid are therefore associated in this strain. The kinetics with which a transparent colony variant of M. avium B2900 takes up and utilizes '4C-palmitic acid were determined as a first step in the elucidation of this organism's fatty acid requirement. MATERIALS AND METHODSOrganism and medium. Stock cultures of M. avium B2900, serotype II, were prepared and maintained at -40 C as described previously (12). Minimal S medium consisted of S salts (12), 0.5%0 dialyzed albumin, 0.005% palmitic acid, 0.5% glucose, 0.5% glycerol, and 0.1% Tween 80 (Polysorbate 80, Atlas Chem. Ind., Inc.). M. avium is unable to hydrolyze Tween 80 or to use it as a source of fatty acid (17). This organism did not increase in viability when incubated in medium containing only Tween 80 as a carbon source (unipublished observation).Chemicals. 14C-Palmitic acid, uniformly labeled (640 mCi/mmole); 2,5 diphenyloxazole (PPO); and 1 ,4-bis-2-(5-phenyloxazolyl)-benzene (POPOP) were purchased from New England Nuclear Corp. (Boston, Mass.). The palmitic acid remained chromatographically pure if it was used within 4 weeks after purchase.Accumulation experiments. The stock culture was diluted into minimal S medium and aerated at 37 C for 40 hr (1.5 doublings). The bacteria were harvested by centrifugation and washed with tris(hydroxymethyl)aminomethane (Tris)...
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