Summary
We have isolated a new mutant of Bacillus subtilis temperature sensitive in DNA replication; its properties are those of an initiation mutant. When liquid cultures are shifted to 48° DNA replication is the first macromolecular synthesis that stops, but only after synthesis of the amount of DNA predicted for the completion of one replication round. When spores of the mutant are germinated and shifted to 48° at subsequent times, one round of DNA replication is observed only when the shift occurs between 60 and 100 min; earlier shifts do not allow replication to start, later shifts allow more than one replication. The DNA replicated after a shift to high temperature is enriched in markers close to the terminus. The reinitiation of DNA replication stopped by the high temperature, takes place following a shift to a permissive temperature only if protein synthesis is allowed. Examination of DNA replication following toluene treatment shows that the elongation of DNA chains is not affected at the non-permissive temperature.
This mutant is shown by PBS-1 mapping to correspond to a new gene denominated dna P, which is located between the thy A and fur A genes and is distinct from all the mapped dna and rec genes of Bacillus subtilis. The mutation confers to the cells also a deficiency in the ability to be transformed, to be transfected with SPP1 phage DNA, and to survive treatment with methyl-methane sulfonate. These deficiencies, observed at the permissive temperature, are no more temperature dependent than in the parental strain. The ability to perform homologous and heterologous transduction with PBS-1 phage and the sensitivity to ultraviolet radiation or mitomycin C are normal.
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Abbreviations
- CAP:
-
chloramphenicol
- EDTA:
-
sodium ethylene-diamino-tetraacetate
- HPura:
-
6-(p-hydroxyphenylazo)-uracil
- KPB:
-
potassium phosphate buffer, 0.05 M, pH 7.4
- MMS:
-
methylmethane-sulfonate
- NTG:
-
N-methyl-N-nitro-N-nitrosoguanidine
- SSC:
-
standard saline citrate solution (0.15 M NaCl, 0.015 M sodium citrate)
- TCA:
-
trichloroacetic acid
- Tris:
-
tris-hydroxymethyl-aminomethane
- UV:
-
ultraviolet light
- mic :
-
minimum inhibitory concentration
- pfu :
-
plaque forming unit
- moi :
-
multiplicity of infection
References
Adelberg, E., Mandel, M., Chen, G. C.: Optimal conditions for mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine in Escherichia coli K12. Biochem. biophys. Res. Commun. 18, 788–795 (1965)
Bazill, G. W., Karamata, D.: Temperature sensitive mutants of Bacillus subtilis defective in Deoxyribonucleotide synthesis. Molec. gen. Genet. 117, 19–29 (1972)
Borenstein, S., Ephrati-Elizur, E.: Spontaneous release of DNA in sequential genetic order by B. subtilis. J. molec. Biol. 45, 137–152 (1969)
Brown, N. C.: Inhibition of bacterial DNA replication by 6-(p-hydroxyphenilazo)-uracil: differential effect on repair and semiconservative synthesis in Bacillus subtilis. J. molec. Biol. 59, 1–16 (1971)
Chestukhin, A. V., Shemyakin, M. F., Kalina, N. A., Prozorov, A. A.: Some properties of ATP-dependent deoxyribonucleases from normal and rec-mutant strains of Bacillus subtilis. Fed. Europ. Biochem. Soc. Letters 24, 121–125 (1972)
Clowes, R. C., Hayes, W.: Experiments in microbial genetics, p. 187. Oxford and Edinburgh: Blackwell Scientific Publications 1968
Davis, B. D., Mingioli, E. S.: Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bact. 60, 17–28 (1950)
Doly, J., Sasarman, E., Anagnostopoulos, C.: ATP-dependent deoxyribonuclease in Bacillus subtilis and a mutant deficient in this activity. Mutation Res. 22, 15–23 (1974)
Dubnau, D., Davidoff-Abelson, R., Smith, I.: Transformation and transduction in Bacillus subtilis: evidence for separate modes of recombinant formation. J. molec. Biol. 45, 155–179 (1969)
Ganesan, A. T., Laipis, P. J., Yehle, C. O.: In vitro DNA synthesis and function of the polymerases in Bacillus subtilis. DNA synthesis in vitro. R. D. Wells and R. B. Inman (eds.) Lancaster: Medical and Technical Publishing Co. Ltd. 1973
Gass, K. B., Cozzarelli, N. R.: Bacillus subtilis DNA polymerases. Methods in enzymology, vol. XXIX, p. 27–38. New York, London: Academic Press 1974
Gross, J.: DNA replication in bacteria. Current topics in microbiology and immunology, vol. 57, p. 39–74. Berlin-Heidelberg-New York: Springer 1972
Gross, J., Karamata, D., Hampstead, P. G.: Temperature-sensitive mutants of B. subtilis defective in DNA replication. Cold Spr. Harb. Symp. quant. Biol. 33, 307–312 (1968)
Hirota, Y., Mordoh, J., Scheffler, I., Jacob, F.: Genetic approach to DNA replication and its control in Escherichia coli. Fed. Proc. 31, 1422–1427 (1972)
Hoch, J. A., Anagnostopoulos, C.: Chromosomal location and properties of radiation sensitivity mutations in Bacillus subtilis. J. Bact. 103, 295–301 (1970)
Hoch, J. A., Barat, M., Anagnostopoulos, C.: Transformation and transduction in recombination defective mutants of Bacillus subtilis. J. Bact. 93, 1925–1937 (1967)
Karamata, D., Gross, J.: Isolation and genetic analysis of temperature sensitive mutants of B. subtilis defective in DNA synthesis. Molec. gen. Genet. 108, 277–287 (1970)
Laipis, P., Ganesan, A. T.: A deoxyribonucleic acid polymerase I deficient mutant of Bacillus subtilis. J. biol. Chem. 247, 5867–5871 (1972)
Laurent, S. J.: Initiation of deoxyribonucleic acid in a temperature-sensitive mutant of B. subtilis: evidence for a transcription step. J. Bact. 116, 141–145 (1973)
Laurent, S. J., Vannier, F. S.: Temperature-sensitive initiation of chromosome replication in a mutant of B. subtilis. J. Bact. 114, 474–484 (1973)
Marmur, J.: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. molec. Biol. 3, 208–218 (1961)
Matsushita, T., White, K. P., Sueoka, N.: Chromosome-replication in toluenized Bacillus subtilis cells. Nature (Lond.) New Biol. 232, 111–114 (1971)
Mazza, G., Fortunato, A., Ferrari, E., Canosi, U., Falaschi, A., Polsinelli, M.: Genetic and enzymic studies on the recombination process in Bacillus subtilis. Molec. gen. Genet. (1975) (in press)
Mendelson, N. H., Gross, J. D.: Characterization of a temperature sensitive mutant of B. subtilis defective in the deoxyribonucleic acid replication. J. Bact. 94, 1603–1608 (1967)
Moses, R. E.: Replicative deoxyribonucleic acid synthesis in a system diffusible for macromolecules. J. biol. Chem. 247, 6031–6038 (1972)
Ricard, M., Hirota, Y.: Effet des sels sur le processus de division cellulaire d'E. coli. C. R. Acad. Sci. (Paris) 268, 1335–1338 (1968)
Riva, S., Polsinelli, M., Falaschi, A.: A new phage of Bacillus subtilis with infectious DNA having separable strands. J. molec. Biol. 35, 347–356 (1968)
Spatz, H. Ch., Trautner, T. A.: The role of recombination in transfection of Bacillus subtilis. Molec. gen. Genet. 113, 174–190 (1971)
Spizizen, J.: Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. nat. Acad. Sci. (Wash.) 44, 1072–1078 (1958)
Stewart, C. R.: Physical heterogeneity among Bacillus subtilis deoxyribonucleic acid molecules carrying particular genetic markers. J. Bact. 98, 1239–1247 (1969)
Villani, G., Canosi, U., Fortunato, A., Mazza, G., Polsinelli, M., Falaschi, A.: Properties of Bacillus subtilis lacking DNA polymerase. I. Nucleic Acids Res. 1, 461–477 (1974)
White, K., Sueoka, N.: Temperature-sensitive DNA synthesis mutants of Bacillus subtilis. Genetics 73, 185–214 (1973)
Yamagishi, H., Takahashi, I.: Transducing particles of PBS-1. Virology 36, 639–645 (1968)
Yoshikawa, H.: DNA synthesis during germination of Bacillus subtilis spores. Proc. nat. Acad. Sci. (Wash.) 53, 1476–1482 (1965)
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Communicated by F. Gros
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Riva, S., van Sluis, C., Mastromei, G. et al. A new mutant of Bacillus subtilis altered in the initiation of chromosome replication. Molec. gen. Genet. 137, 185–202 (1975). https://doi.org/10.1007/BF00333015
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DOI: https://doi.org/10.1007/BF00333015