JAMES L. FARMER AND FRANK ROTHMAN. Biology Department, Brown University, Providence, Rhode Island. Received for publication 11 September 1964.
Transformable Thymine-Requiring Mutant of Bacillus subtilis JAMES L. FARMER
AND
FRANK ROTHMAN
Biology Department, Brown University, Providence, Rhode Island
Received for publication 11 September 1964
In recent years, an increasing number of investigations of molecular genetics and the regulation of macromolecular synthesis have been carried out with Bacillus subtilis. Some features of B. subtilis which often make it the organism of choice are the ability to transfer genetic markers by transformation (Spizizen, Proc. Nat. Acad. Sci. U.S. 44:1072, 1958), sensitivity to actinomycin D which selectively inhibits deoxyribonucleic acid (DNA)-dependent ribonucleic acid synthesis, and a size which permits the observation of considerable detail of cell structure by electron microscopy. This report describes the isolation and properties of a thymine-requiring mutant of B. subtilis strain 168, which can serve as either donor or recipient in genetic transformation. This strain is very useful in studies demanding experimental control of DNA synthesis and for the preparation of radioactive- or density-labeled transforming DNA. It is currently in use in several laboratories (e.g., Bodmer and Ganesan, Genetics 50:236, 1964). A spontaneous, thymine-requiring (thy-) mutant of B. subtilis wild-type strain 23 (Spizizen, Proc. Nat. Acad. Sci. U.S. 44:1072, 1958) was isolated by a modification of the technique of Okada, Homma, and Sonohara (J. Bacteriol. 84:602, 1962). Strain 23 was grown at 37 C in the selective GS-AM-thymine medium which contains 200 jig of thymine and 200 jig of aminopterin per ml. A sample of this culture was diluted into Penassay broth (Difco Antibiotic Medium % 3) and incubated overnight. Dilutions of the broth culture were plated on GS-AM-thymine plates. After 48 hr of incubation, the largest colonies were picked, tested, and found to require thymine or thymidine for growth. The thy- marker was transferred from strain 23 thy- to strain 168 ind- (which requires indole or L-tryptophan) by transformation; thy- transformants were selected on GS-AM-thymine plates supplemented with 20 Aug of L-tryptophan per ml. Studies by E. S. and F. Kahan (personal com-
munication) have shown that the thymine dependence of strain 168 thy- ind- results from the absence of thymidylate synthetase activity (as does the similar requirement of Escherichia coli 15 T-, demonstrated by Barner and Cohen, J. Biol. Chem. 234:2987, 1959). Assays of crude extracts of strain 168 thy- ind- by the spectrophotometric method of Wahba and Friedkin (J. Biol. Chem. 236:PC11, 1961) and by a radioisotopic procedure employing C14-labeled formaldehyde showed no detectable thymidylate synthetase activity. The more sensitive isotopic assay sets an upper limit of 0.2 m,mole of thymidylate synthesized per mg of protein per hr (at 37 C); identically prepared extracts of B. subtilis var. Marburg (ATCC 6051) were found by both assays to synthesize 40 mAmoles. The latter activity is comparable to that reported in E. coli B by Mathews and Cohen (J. Biol. Chem. 238:367, 1963). When deprived of thymine, cells of strain 168 thy- ind- undergo "thymineless death" similar to that of E. coli 15 T- (Cohen and Barner, Proc. Nat. Acad. Sci. U.S. 40:885, 1954). Growth-response studies show that 5 ,ug/ml of thymine or thymidine will support full growth in minimal medium, whereas 2 4g/ml is insufficient. Cultures are routinely supplemented with 20 ,g/ ml of thymine. Thymine-requiring mutants of strain 168 can serve as either donors or recipients for transformation. The transformation technique of Anagnostopoulos and Spizizen (J. Bacteriol. 81:741, 1961) was used with the modification that cells were not diluted between the two growth phases. The growth medium was supplemented with 50 ,ug of thymine per ml in the first growth phase and 5 ,g/ml in the second. When transformed with wild-type DNA, strain 168 thy- ind- gives the same frequency of transformants (0.05 to 0.4%) for the thy+ and ind+ markers. It was of some interest to examine the competence of strain 168 thy- ind- to undergo 262
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NOTES
transformation in the absence of thymine. Thymine starvation of a competent culture (5 X 108 cells per milliliter) in minimal medium containing 5 X 10-3 M MgSO4 and 20 Ag of L-tryptophan per ml for 3 hr did not lead to any loss of competence or viability.
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We are indebted to Bernard Strauss for introducing us to the art of transformation and for his generous hospitality during two long visits by F. R. This work was supported by grant G-18928 from the National Science Foundation and by Public Health Service training grant 8TI-HD00019 (to J. F).
