Sep 5, 1972 - The aminopterin technique was adapted for the isolation of thymidine auxotrophs of Pseudomonas acidovorans. All the mutants isolated ...
Vol. 113, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOG.Y, January 1973, p. 510-511 Copyright 0 1973 American Society for Microbiology
Obligate Thymidine Auxotrophs of Pseudomonas acidovorans' ROD A. KELLN AND R. A. J. WARREN
Department of Microbiology, University of British Columbia, Vancouver 8, British Columbia, Canada Received for publication 5 September 1972
The aminopterin technique was adapted for the isolation of thymidine auxotrophs of Pseudomonas acidovorans. All the mutants isolated responded to thymidine but not to thymine.
A thymine auxotroph was first isolated from an Escherichia coli culture that had been exposed to X rays (11). Subsequently, the folic acid antagonists aminopterin (APN) (8-10, 14) or trimethoprim (TMBP) (12) were used to select thymine auxotrophs in several genera of bacteria. A prerequisite for the isolation of thymine auxotrophs is the presence of enzymes capable of converting exogenous thymine to thymidylic acid. Work in this laboratory (R. A. Kelln, unpublished data) indicated that Pseudomonas acidovorans is unable to convert thymine to thymidine. It can use thymine as sole nitrogen source. Furthermore, wild-type cells incorporate thymidine very poorly even though the cells contain thymidine kinase activity. Since we needed to label specifically its deoxyribonucleic acid (DNA), we attempted to isolate thymidine auxotrophs of this organism. The minimal salts medium (2; without NaCl) was supplemented with 2 g of succinic acid per liter for liquid culture and with 6.8 g of disodium succinate .6H2O and 15 g of agar per liter for plates. A culture of P. acidovorans strain 29 (6) was grown to 5 x 108 cells per ml in minimal medium, and 0.1-ml samples were spread on plates of minimal medium containing (g/liter): APN, 0.6; TMBP, 0.015; thymidine, 2.0. Further 0.1-ml samples were spread on plates of the same medium without aminopterin. The plates were incubated at 30 C. Only those colonies appearing more than 48 hr later were screened for thymidine auxotrophy. All colonies that developed on plates containing both TMBP and APN as selective agents required thymidine for growth. Only 10% of the colonies appearing after 48 hr on TMBP plates IThis work was presented in part at the 3rd Annual Meeting of the Gulf Coast Molecular Biology Conference, Corpus Christi, Tex., 28-30 January 1972.
510
were thymidine requiring. All of the mutants which were screened required very high concentrations of thymidine for growth (Table 1). Attempts to isolate secondary mutants of several of these strains which could grow with low levels of thymidine were only partially successful. Strains could be isolated which grew very poorly with 50 jg of thymidine per ml. The high requirement for thymidine was not a consequence of extensive catabolism of the added thymidine (R. A. Kelln, unpublished data). Thymine at concentrations up to 2 mg/ml did not support growth of any of the mutants, not even in the presence of an added deoxyribonucleoside. This confirmed that P. acidovorans was unable to convert thymine to thymidine. Therefore, all the mutants were obligate thymidine auxotrophs. Thymidine starvation (7) of strain 3L resulted in exponential cell death. After 30 min of starvation, viability was reduced by 50%. The DNA of strain 3L could be labeled with radioactive thymidine. Radioactive uracil labeled both the cytosine and thymidine residues of wild-type DNA but only the cytosine residues of mutant 3L DNA. This suggested that the mutants were defective in thymidylate synthetase. Enzyme assays confirmed this conclusion. The isolation of a thymidine-requiring mutant in a Pseudomonas species has been reported previously (4). The mutant required a very low level of thymidine (1 ,ug/ml) for growth, and it was not determined whether thymine could substitute for thymidine. However, this bacterium, a marine halophile, cannot be assigned to any of the subgeneric catagories (3) described by Stanier et al. (13). Obligate thymidine auxotrophs have been isolated in Diplococcus pneumoniae (1, 5). These mutants required at least 20 to 25 ,ug of
NOTES
VOL 113, 1973
TABLE 1. Concentration of thymidine required for growtha Thymidine concnc (jug/ml) Mutant" 2,000
1,000
6A 1B 1C 1D 1E 5A 5B 6A 6B
+ + + +
+ + + +
+ +
+ +
+
+
+
+
+
is
+
+ +
lil1L 12 2S 13 2L
+ + + + +
+ + + + +
8 2 3 4 5 6 7 8 9 10
14 38 15 3L
500
250
0
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+ -
-
-
_
_
+
-
+ +
-
+
-, n + + +
-, no growth in 24 aSymbols: +,growth hr. b Mutants 1 to 9 selected on medium containing both aminopterin and trimethoprim; mutants 10 to 15 on medium containing only trimethoprim. c Final concentration of thymidine in mannitol broth (6).
in 24 hr;
thymidine per ml for growth. We are indebted to G. A. O'Donovan for his suggestions and encouragement and for making available to us the facilities and materials of his laboratory where the assay of thymidylate synthetase was performed. This investigation was supported by grant A-3686 from the National Research Council of Canada. One of us (R.A.K.) wishes to thank the H. R. MacMillan Family Fund for a Frank F. Wesbrook Fellowship and to also thank J. J. R. Campbell of this Department for a travel grant to Texas A&M University.
511
LITERATURE CITED 1. Brunel, F., A. M. Sicard, and N. Sicard. 1971. Transforming ability of bacterial deoxyribonucleic acid in relation to the marker efficiencies in Diplococcus pneumoniae during thymidine starvation. J. Bacteriol. 106:904-907. 2. Clark, D. J. 1968. Regulation of deoxyribonucleic acid replication and cell division in Escherichia coli B/r. J. Bacteriol. 96:1214-1224. 3. Espejo, R. T., and E. S. Canelo. 1968. Properties and characterization of the host bacterium of bacteriophage PM2. J. Bacteriol. 95:1887-1891. 4. Espejo, R. T., E. S. Canelo, and R. L. Sinsheimer. 1971. Replication of bacteriophage PM2 deoxyribonucleic acid: a closed circular duplex molecule. J. Mol. Biol. 56:597-621. 5. Friedman, L. R., and A. W. Ravin. 1972. Genetic and biochemical properties of thymidine-dependent mutants of pneumococcus. J. Bacteriol. 109:459-461. 6. Kropinski, A. M. B., and R. A. J. Warren. 1970. Isolation and properties of a Pseudomonas acidovorans bacteriophage. J. Gen. Virol. 6:85-93. 7. O'Donovan, G. A., and J. Neuhard. 1970. Pyrimidine metabolism of microorganisms. Bacteriol. Rev. 34:278-343. 8. Okada, T., J. Homma, and H. Sonohara. 1962. Improved method for obtaining thymineless mutants of Escherichia coli and Salmonella thyphimurium. J. Bacteriol. 84:602-603. 9. Okada, T., K. Yanagisawa, and F. J. Ryan. 1960. Elective production of thymine-less mutants. Nature (London) 188:340-341. 10. Okada, T., K. Yanagisawa, and F. J. Ryan. 1961. A method of securing thymineless mutants from strains of E. coli. Z. Vererbungslehre 92:403-412. 11. Roepke, R. R., and F. E. Mercer. 1947. Lethal and sublethal effects of X-rays on Escherichia coli as related to the yield of bicohemical mutants. J. Bacteriol. 54:731-743. 12. Stacey, K. A., and E. Simson. 1965. Improved method for the isolation of thymine requiring mutants of Escherichia coli. J. Bacteriol. 90:554-555. 13. Stanier, R. Y., M. J. Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159-271. 14. Wilson, M. C., J. L. Farmer, and F. Rothman. 1966. Thymidylate synthesis and aminopterin resistance in Bacillus subtilis. J. Bacteriol. 92:186-196.
