JOURNAL OF BACTERIOLOGY, Nov. 1967, p. 1805-1806 ... and A. D. Hitchens, J. Gen. Microbiol. 33:412,. 1963; I. Takahashi, J. Bacteriol. 87:1799, 1964).
JOURNAL OF BACTERIOLOGY, Nov. 1967, p. 1805-1806 Copyright © 1967 American Society for Microbiology
Vol. 94, No. 5 Printed in U.S.A.
Extraction and Characterization of Deoxyribonucleic Acid from Spores and Vegetative Cells of Bacillus stearothermophilus' L. TABATABAI AND H. W. WALKER Department of Dairy and Food Industry, Iowa State University, Ames, Iowa 50010 Received for publication 5 September 1967
Exposure of bacterial spores to thioglycolate and 2-mercaptoethanol in 8 M urea has been used to facilitate rupture of the spore coat and extraction of cellular contents (G. W. Gould and A. D. Hitchens, J. Gen. Microbiol. 33:412, 1963; I. Takahashi, J. Bacteriol. 87:1799, 1964). The present report describes adaptation of the methods of Gould and Hitchens and of 0. Smithies (Science 150:1595, 1965) to the extraction of deoxyribonucleic acid (DNA) from spores of Bacillus stearothermophilus ATCC 7953 and presents a comparison of melting-temperature (Tm) values of DNA extracted from vegetative cells of B. stearothermophilus ATCC 7953 and 1503-4R and from spores of ATCC 7953. Vegetative cells were produced on nutrient agar incubated at 55 C for 24 hr; spores were produced on nutrient agar supplemented with MnCl2 and NaNO3 (2 mg/liter of medium). Spores and vegetative cells were washed several times in 0.15 M NaCl-0.1 M ethylenediaminetetraacetic acid (EDTA, pH 8.0). After the final wash, vegetative cells were maintained in the NaCl-EDTA buffer, and spores, in 0.1 M phosphate-buffered saline (PBS, pH 7.0). DNA was extracted from spores by suspending 10 g of wet, packed spores in 90 ml of 10% 2-mercaptoethanol (Sigma Chemical Co., St. Louis, Mo.) in 8 M urea (pH 8.0). The mixture was stirred and heated at 60 to 65 C for 15 min, after which N-ethylmaleimide (NEM) was added to a level of 0.15 M. The suspension was heated and stirred for an additional 1 hr. After cooling to room temperature, the spores were washed five times with PBS and suspended in 0.15 M NaCl-0.1 M EDTA (pH 8.0). Lysozyme (2 mg/ml) was added to the suspension, and the mixture was incubated overnight at 37 C. At this time, the spores were nonrefractile and the I Journal paper no. J-5712 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project no. 1393, Center for Economic and
Agricultural Development cooperating.
solution was highly viscous. Sodium lauryl sulfate was added to a final concentration of 2%; this mixture was heated at 60 C for 10 min. An equal volume of 0.15 M NaCI-0.015 M trisodium citrate (55 C, pH 7.0) was added with gentle stirring; the final solution was homogeneous but still viscous. DNA was extracted from vegetative cells by the method of J. Marmur (J. Mol. Biol. 3:208, 1961); his procedure was followed for purification of all DNA preparations. Tm values and guanosine-cytosine content (% G + C) of the DNA samples were determined by the method of J. Marmur and P. Doty (J. Mol. Biol. 5:109, 1962). Cuvettes containing the sample and blank were equilibrated for 10 min at each temperature, starting at 83 C and increasing by 2-C intervals. Changes in absorbance were followed with a Beckman DK-2A recording spectrophotometer fitted with a temperatureregulated cell holder. DNA extracted from spores of B. stearothermophilus ATCC 7953 had a Tm of 90.9 C and a content of 52.7% G + C. DNA extracted from vegetative cells of strain ATCC 7953 and 1503-4R had Tm values of 91.2 and 90.7 C and 53.4 and 52.2 % G + C, respectively. N. E. Welker and L. L. Campbell (J. Bacteriol. 89:175, 1965) reported values of 89 C and 50% for DNA from vegetative cells of strain 1503-4R. G. F. Saunders and L. L. Campbell (J. Bacteriol. 91:340, 1966) and B. Pace and L. L. Campbell (Proc. Natl. Acad. Sci., U.S. 57:1110, 1967) reported G + C values of 50 to 52.4% for the DNA of several other strains of B. stearothermophilus. No difference was evident between DNA extracted from vegetative cells and spores; this might be expected since DNA of the spore is derived from DNA of the vegetative cell (P. C. Fitz-James and I. E. Young, J. Bacteriol. 78:743, 1959; H. Hodson and J. V. Beck, J. Bacteriol. 79:661, 1960; M. Mandel and D. B. Rowley,
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NOTES
J. Bacteriol. 85:1445, 1964; I. Takahashi, J. Bacteriol. 87:1799, 1964). Addition of NEM to the spore suspension during treatment with 2-mercaptoethanol resulted in spores susceptible to attack by lysozyme. This method of rupturing the spore coat released DNA, which, when purified, appeared
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J. BACTrERIOL.
to be highly polymerized as deduced from the Tm profile. This investigation was supported by Public Health Service research grant EF-00354-08 from the Division of Environmental Engineering and Food Protection.
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