Summary
When the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45° C, a synchronous round of DNA replication immediately ensues. Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication. Such treatments are necessary to achieve even modest synchrony in germinating spores. Our results showed that the first fragment to be replicated was a 4kb BamHI-SalI restriction fragment, BS6. In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS 6. Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB. The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores. We also discuss the possibility that B. subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baker TA, Sekimisiu K, Funnel BE, Kornberg A (1986) Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome. Cell 45:53–64
Clayton DA (1982) Replication of animal mitochondrial DNA. Cell 28:693–705
de Massy B, Fayet O, Kogoma T (1984) Multiple origin usage for DNA replication in sdrA(rnh) mutants of Escherichia coli K12: initiation in the absence of oriC. J Mol Biol 178:227–236
Donachie WD (1968) Relationship between cell size and time of initiation of DNA replication. Nature 219:1077–1079
Filutowicz M, Jonczyk P (1981) Essential role of the gyrB gene product in the transcriptional event coupled to dnaA-dependent initiation of Escherichia coli chromosome replication. Mol Gen Genet 183:134–138
Harford N, Lepesant-Kejzlarova J, Lepesant JA, Hamers R, Dedonder R (1976) Genetic circularity and mapping of the replication origin of the Bacillus subtilis chromosome. In: Schlessinger D (ed) Microbiology-1976 American Society for Microbiology, Washington DC, pp 28–34
Henckes G, Vannier F, Buu A, Séror-Laurent SJ (1982a) Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome. J Bacteriol 149:79–91
Henckes G, Vannier F, Seiki M, Ogasawara N, Yoshikawa H, Séror-Laurent SJ (1982b) Ribosomal RNA genes in the replication origin region of Bacillus subtilis chromosome. Nature 299:268–271
Kubo M, Kano Y, Nakamura H, Nagata A, Imamoto F (1979) In vivo enhancement of general and specific transcription in Escherichia coli by DNA gyrase activity. Gene 7:153–171
Lampe MF, Bott KF (1984) Cloning the gyrA gene of Bacillus subtilis. Nucleic Acids Res 12:6307–6323
Lampe MF, Bott KF (1985a) Genetic and physical organization of the cloned gyrA and gyrB genes of Bacillus subtilis. J Bacteriol 162:78–84
Lampe MF, Bott KF (1985b) Cloning of the Bacillus subtilis recF gene. Gene 38:139–144
Laskey RA, Mills AD (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56:335–341
Laurent SJ (1973) Initiation of deoxyribonucleic acid replication in a temperature-sensitve mutant of B. subtilis evidence for a transcriptional step. J Bacteriol 116:141–145
Laurent SJ, Vannier F (1973) Temperature-sensitive initiation of chromosome replication in a mutant of Bacillus subtilis. J Bacteriol 114:474–484
Marsh RC, Worcel A (1977) A DNA fragment containing the origin of replication of the Escherichia coli chromosome. Proc Natl Acad Sci USA 74:2720–2724
Menzel R, Gellert M (1983) Regulation of the genes for E. coli DNA gyrase: homeostatic control of DNA supercoiling. Cell 34:105–113
Moriya S, Ogasawara N, Yoshikawa H (1985) Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region. Nucleic Acids Res 13:2251–2265
Ogasawara N, Seiki M, Yoshikawa H (1979) Effect of novobiocin on initiation of DNA replication in Bacillus subtilis. Nature 281:702–704
Ogasawara N, Seiki M, Yoshikawa H (1983) Replication origin region of Bacillus subtilis chromosome contains two rRNA operons. J Bacteriol 154:50–57
Ogasawara N, Mizumuto S, Yoshikawa H (1983) Replication origin of the Bacillus subtilis chromosome determined by hybridization of the first-replicating DNA with cloned fragments from the replication origin region of the chromosome. Gene 30:173–182
Ogasawara N, Moriya S, von Meyenburg K, Hansen FG, Yoshikawa H (1985) Conservation of genes and their organization in the chromosomal replication origin region of Bacillus subtilis and Escherichia coli. EMBO J 4:3345–3350
Orr E, Fairweather NF, Holland BJ, Pritchard RH (1979) Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K 12. Mol Gen Genet 177:103–112
Pulleyblank DE, Shure M, Vinograd J (1977) The quantitation of fluorescence by photography. Nucleic Acids Res 4:1408–1418
Seiki M, Ogasawara N, Yoshikawa H (1981) Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. I. Isolation and characterization of plasmids containing the origin region. Mol Gen Genet 183:220–226
Séror JS, Vannier F, Levine A, Henckes G (1986) Stringent control of initiation of chromosomal replication in Bacillus subtilis. Nature 321:709–710
Séror-Laurent SJ, Henckes G (1985) An RNA-DNA copolymer whose synthesis is correlated with the transcriptional requirement for chromosomal initiation in Bacillus subtilis contains ribosomal RNA sequences. Proc Natl Acad Sci USA 82:3586–3590
Séror-Laurent SJ, Henckes G, Levine A, Vannier F (1986) Initiation of chromosomal replication in the Bacillus subtilis mutant dna37. In: Ganesan AT, Hoch JA (eds) Bacillus molecular genetics and biotechnology applications. Academic Press, New York, pp 55–71
Sicard N, Devoret R (1962) Effet de la carence en thymine sur des souches d'E. coli lysogènes K 12T− et colicinogènes 15T−. CR Séances Acad Sci [III] 255:1417–1419
Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci USA 44:1072–1078
Stewart GC, Wilson FE, Bott K (1982) Detailed physical mapping of the ribosomal RNA genes of Bacillus subtilis. Gene 19:153–162
Wake RG (1975) Bidirectional replication in Bacillus subtilis. In: Goulian MM, Hanawalt PC, Fox CF (eds) DNA synthesis and its regulation. Benjamin WA, Inc., Menlo Park, California pp 650–676
Walker GC (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 48:60–93
Author information
Authors and Affiliations
Additional information
Communicated by R. Devoret
Rights and permissions
About this article
Cite this article
Levine, A., Henckes, G., Vannier, F. et al. Chromosomal initiation in Bacillus subtilis may involve two closely linked origins. Mole Gen Genet 208, 37–44 (1987). https://doi.org/10.1007/BF00330419
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00330419