Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Mar;175(6):1735–1744. doi: 10.1128/jb.175.6.1735-1744.1993

Molecular cloning and characterization of the Bacillus subtilis spore photoproduct lyase (spl) gene, which is involved in repair of UV radiation-induced DNA damage during spore germination.

P Fajardo-Cavazos 1, C Salazar 1, W L Nicholson 1
PMCID: PMC203968  PMID: 8449881

Abstract

Upon UV irradiation, Bacillus subtilis spore DNA accumulates the novel thymine dimer 5-thyminyl-5,6-dihydrothymine. Spores can repair this "spore photoproduct" (SP) upon germination either by the uvr-mediated general excision repair pathway or by the SP-specific spl pathway, which involves in situ monomerization of SP to two thymines by an enzyme named SP lyase. Mutants lacking both repair pathways produce spores that are extremely sensitive to UV. For cloning DNA that can repair a mutation in the spl pathway called spl-1, a library of EcoRI fragments of chromosomal DNA from B. subtilis 168 was constructed in integrative plasmid pJH101 and introduced by transformation into a mutant B. subtilis strain that carries both the uvrA42 and spl-1 mutations, and transformants whose spores exhibited UV resistance were selected by UV irradiation. With a combination of genetic and physical mapping techniques, the DNA responsible for the restoration of UV resistance was shown to be present on a 2.3-kb EcoRI-HindIII fragment that was mapped to a new locus in the metC-pyrD region of the B. subtilis chromosome immediately downstream from the pstI gene. The spl coding sequence was localized on the cloned fragment by analysis of in vitro-generated deletions and by nucleotide sequencing. The spl nucleotide sequence contains an open reading frame capable of encoding a 40-kDa polypeptide that shows regional amino acid sequence homology to DNA photolyases from a number of bacteria and fungi.

Full text

PDF
1741

Images in this article

1742Image
on p.1742

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amjad M., Castro J. M., Sandoval H., Wu J. J., Yang M., Henner D. J., Piggot P. J. An SfiI restriction map of the Bacillus subtilis 168 genome. Gene. 1991 May 15;101(1):15–21. doi: 10.1016/0378-1119(91)90219-2. [DOI] [PubMed] [Google Scholar]
  2. Antoniewski C., Savelli B., Stragier P. The spoIIJ gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes. J Bacteriol. 1990 Jan;172(1):86–93. doi: 10.1128/jb.172.1.86-93.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arraj J. A., Marinus M. G. Phenotypic reversal in dam mutants of Escherichia coli K-12 by a recombinant plasmid containing the dam+ gene. J Bacteriol. 1983 Jan;153(1):562–565. doi: 10.1128/jb.153.1.562-565.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Boylan R. J., Mendelson N. H., Brooks D., Young F. E. Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid. J Bacteriol. 1972 Apr;110(1):281–290. doi: 10.1128/jb.110.1.281-290.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Byrne C. R., Monroe R. S., Ward K. A., Kredich N. M. DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK regions to ptsH. J Bacteriol. 1988 Jul;170(7):3150–3157. doi: 10.1128/jb.170.7.3150-3157.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donnellan J. E., Jr, Setlow R. B. Thymine Photoproducts but not Thymine Dimers Found in Ultraviolet-Irradiated Bacterial Spores. Science. 1965 Jul 16;149(3681):308–310. doi: 10.1126/science.149.3681.308. [DOI] [PubMed] [Google Scholar]
  9. Donnellan J. E., Jr, Stafford R. S. The ultraviolet photochemistry and photobiology of vegetative cells and spores of Bacillus megaterium. Biophys J. 1968 Jan;8(1):17–28. doi: 10.1016/S0006-3495(68)86471-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feavers I. M., Foulkes J., Setlow B., Sun D., Nicholson W., Setlow P., Moir A. The regulation of transcription of the gerA spore germination operon of Bacillus subtilis. Mol Microbiol. 1990 Feb;4(2):275–282. doi: 10.1111/j.1365-2958.1990.tb00594.x. [DOI] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Ferrari F. A., Nguyen A., Lang D., Hoch J. A. Construction and properties of an integrable plasmid for Bacillus subtilis. J Bacteriol. 1983 Jun;154(3):1513–1515. doi: 10.1128/jb.154.3.1513-1515.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. George D. G., Barker W. C., Hunt L. T. Mutation data matrix and its uses. Methods Enzymol. 1990;183:333–351. doi: 10.1016/0076-6879(90)83022-2. [DOI] [PubMed] [Google Scholar]
  14. Gonzy-Tréboul G., Steinmetz M. Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene. J Bacteriol. 1987 May;169(5):2287–2290. doi: 10.1128/jb.169.5.2287-2290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gonzy-Tréboul G., Zagorec M., Rain-Guion M. C., Steinmetz M. Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: nucleotide sequence of ptsX, ptsH and the 5'-end of ptsI and evidence for a ptsHI operon. Mol Microbiol. 1989 Jan;3(1):103–112. doi: 10.1111/j.1365-2958.1989.tb00109.x. [DOI] [PubMed] [Google Scholar]
  16. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  17. Karmazyn-Campelli C., Bonamy C., Savelli B., Stragier P. Tandem genes encoding sigma-factors for consecutive steps of development in Bacillus subtilis. Genes Dev. 1989 Feb;3(2):150–157. doi: 10.1101/gad.3.2.150. [DOI] [PubMed] [Google Scholar]
  18. Kobayashi T., Takao M., Oikawa A., Yasui A. Molecular characterization of a gene encoding a photolyase from Streptomyces griseus. Nucleic Acids Res. 1989 Jun 26;17(12):4731–4744. doi: 10.1093/nar/17.12.4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kohlbrecher D., Eisermann R., Hengstenberg W. Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: molecular cloning and nucleotide sequence of the Staphylococcus carnosus ptsI gene and expression and complementation studies of the gene product. J Bacteriol. 1992 Apr;174(7):2208–2214. doi: 10.1128/jb.174.7.2208-2214.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LiCalsi C., Crocenzi T. S., Freire E., Roseman S. Sugar transport by the bacterial phosphotransferase system. Structural and thermodynamic domains of enzyme I of Salmonella typhimurium. J Biol Chem. 1991 Oct 15;266(29):19519–19527. [PubMed] [Google Scholar]
  21. Li Y. F., Heelis P. F., Sancar A. Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro. Biochemistry. 1991 Jun 25;30(25):6322–6329. doi: 10.1021/bi00239a034. [DOI] [PubMed] [Google Scholar]
  22. Mason J. M., Hackett R. H., Setlow P. Regulation of expression of genes coding for small, acid-soluble proteins of Bacillus subtilis spores: studies using lacZ gene fusions. J Bacteriol. 1988 Jan;170(1):239–244. doi: 10.1128/jb.170.1.239-244.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  24. Mueller J. P., Sonenshein A. L. Role of the Bacillus subtilis gsiA gene in regulation of early sporulation gene expression. J Bacteriol. 1992 Jul;174(13):4374–4383. doi: 10.1128/jb.174.13.4374-4383.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Munakata N. Genetic analysis of a mutant of Bacillus subtilis producingltraviolet-sensitive spores. Mol Gen Genet. 1969 Jul 3;104(3):258–263. doi: 10.1007/BF02539290. [DOI] [PubMed] [Google Scholar]
  26. Munakata N., Ikeda Y. A mutant of Bacillus subtilis producing ultraviolet-sensitive spores. Biochem Biophys Res Commun. 1968 Nov 8;33(3):469–475. doi: 10.1016/0006-291x(68)90597-4. [DOI] [PubMed] [Google Scholar]
  27. Munakata N., Rupert C. S. Dark repair of DNA containing "spore photoproduct" in Bacillus subtilis. Mol Gen Genet. 1974 May 31;130(3):239–250. doi: 10.1007/BF00268802. [DOI] [PubMed] [Google Scholar]
  28. Munakata N., Rupert C. S. Genetically controlled removal of "spore photoproduct" from deoxyribonucleic acid of ultraviolet-irradiated Bacillus subtilis spores. J Bacteriol. 1972 Jul;111(1):192–198. doi: 10.1128/jb.111.1.192-198.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nelson S. O., Schuitema A. R., Benne R., van der Ploeg L. H., Plijter J. S., Aan F., Postma P. W. Molecular cloning, sequencing, and expression of the crr gene: the structural gene for IIIGlc of the bacterial PEP:glucose phosphotransferase system. EMBO J. 1984 Jul;3(7):1587–1593. doi: 10.1002/j.1460-2075.1984.tb02015.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nicholson W. L., Setlow B., Setlow P. Ultraviolet irradiation of DNA complexed with alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species makes spore photoproduct but not thymine dimers. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8288–8292. doi: 10.1073/pnas.88.19.8288. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nicholson W. L., Sun D. X., Setlow B., Setlow P. Promoter specificity of sigma G-containing RNA polymerase from sporulating cells of Bacillus subtilis: identification of a group of forespore-specific promoters. J Bacteriol. 1989 May;171(5):2708–2718. doi: 10.1128/jb.171.5.2708-2718.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Perego M., Cole S. P., Burbulys D., Trach K., Hoch J. A. Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis. J Bacteriol. 1989 Nov;171(11):6187–6196. doi: 10.1128/jb.171.11.6187-6196.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. ROMIG W. R., WYSS O. Some effects of ultraviolet radiation of sporulating cultures of Bacillus cereus. J Bacteriol. 1957 Sep;74(3):386–391. doi: 10.1128/jb.74.3.386-391.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  36. STUY J. H. Photoreactivation of ultraviolet-inactivated bacilli. Biochim Biophys Acta. 1955 Jun;17(2):206–211. doi: 10.1016/0006-3002(55)90351-6. [DOI] [PubMed] [Google Scholar]
  37. Saffen D. W., Presper K. A., Doering T. L., Roseman S. Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia coli ptsH, ptsI, and crr genes. J Biol Chem. 1987 Nov 25;262(33):16241–16253. [PubMed] [Google Scholar]
  38. Sancar G. B. Sequence of the Saccharomyces cerevisiae PHR1 gene and homology of the PHR1 photolyase to E. coli photolyase. Nucleic Acids Res. 1985 Nov 25;13(22):8231–8246. doi: 10.1093/nar/13.22.8231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sancar G. B., Smith F. W., Lorence M. C., Rupert C. S., Sancar A. Sequences of the Escherichia coli photolyase gene and protein. J Biol Chem. 1984 May 10;259(9):6033–6038. [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schaeffer P., Millet J., Aubert J. P. Catabolic repression of bacterial sporulation. Proc Natl Acad Sci U S A. 1965 Sep;54(3):704–711. doi: 10.1073/pnas.54.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schwartz M. Quantum yield determinations of photosynthetic reactions. Methods Enzymol. 1972;24:139–146. doi: 10.1016/0076-6879(72)24064-2. [DOI] [PubMed] [Google Scholar]
  43. Setlow P. DNA in dormant spores of Bacillus species is in an A-like conformation. Mol Microbiol. 1992 Mar;6(5):563–567. doi: 10.1111/j.1365-2958.1992.tb01501.x. [DOI] [PubMed] [Google Scholar]
  44. Setlow P. I will survive: protecting and repairing spore DNA. J Bacteriol. 1992 May;174(9):2737–2741. doi: 10.1128/jb.174.9.2737-2741.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Setlow P. Small, acid-soluble spore proteins of Bacillus species: structure, synthesis, genetics, function, and degradation. Annu Rev Microbiol. 1988;42:319–338. doi: 10.1146/annurev.mi.42.100188.001535. [DOI] [PubMed] [Google Scholar]
  46. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  47. Spizizen J. TRANSFORMATION OF BIOCHEMICALLY DEFICIENT STRAINS OF BACILLUS SUBTILIS BY DEOXYRIBONUCLEATE. Proc Natl Acad Sci U S A. 1958 Oct 15;44(10):1072–1078. doi: 10.1073/pnas.44.10.1072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sun D. X., Cabrera-Martinez R. M., Setlow P. Control of transcription of the Bacillus subtilis spoIIIG gene, which codes for the forespore-specific transcription factor sigma G. J Bacteriol. 1991 May;173(9):2977–2984. doi: 10.1128/jb.173.9.2977-2984.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sun D. X., Stragier P., Setlow P. Identification of a new sigma-factor involved in compartmentalized gene expression during sporulation of Bacillus subtilis. Genes Dev. 1989 Feb;3(2):141–149. doi: 10.1101/gad.3.2.141. [DOI] [PubMed] [Google Scholar]
  50. Sun D., Fajardo-Cavazos P., Sussman M. D., Tovar-Rojo F., Cabrera-Martinez R. M., Setlow P. Effect of chromosome location of Bacillus subtilis forespore genes on their spo gene dependence and transcription by E sigma F: identification of features of good E sigma F-dependent promoters. J Bacteriol. 1991 Dec;173(24):7867–7874. doi: 10.1128/jb.173.24.7867-7874.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sussman M. D., Setlow P. Cloning, nucleotide sequence, and regulation of the Bacillus subtilis gpr gene, which codes for the protease that initiates degradation of small, acid-soluble proteins during spore germination. J Bacteriol. 1991 Jan;173(1):291–300. doi: 10.1128/jb.173.1.291-300.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sutrina S. L., Reddy P., Saier M. H., Jr, Reizer J. The glucose permease of Bacillus subtilis is a single polypeptide chain that functions to energize the sucrose permease. J Biol Chem. 1990 Oct 25;265(30):18581–18589. [PubMed] [Google Scholar]
  53. Takao M., Kobayashi T., Oikawa A., Yasui A. Tandem arrangement of photolyase and superoxide dismutase genes in Halobacterium halobium. J Bacteriol. 1989 Nov;171(11):6323–6329. doi: 10.1128/jb.171.11.6323-6329.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Van Wang T. C., Rupert C. S. Evidence for the monomerization of spore photoproduct to two thymines by the light-independent "spore repair" process in Bacillus subtilis. Photochem Photobiol. 1977 Jan;25(1):123–127. doi: 10.1111/j.1751-1097.1977.tb07432.x. [DOI] [PubMed] [Google Scholar]
  55. Varghese A. J. 5-Thyminyl-5,6-dihydrothymine from DNA irradiated with ultraviolet light. Biochem Biophys Res Commun. 1970 Feb 6;38(3):484–490. doi: 10.1016/0006-291x(70)90739-4. [DOI] [PubMed] [Google Scholar]
  56. Yajima H., Inoue H., Oikawa A., Yasui A. Cloning and functional characterization of a eucaryotic DNA photolyase gene from Neurospora crassa. Nucleic Acids Res. 1991 Oct 11;19(19):5359–5362. doi: 10.1093/nar/19.19.5359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  58. Yasui A., Takao M., Oikawa A., Kiener A., Walsh C. T., Eker A. P. Cloning and characterization of a photolyase gene from the cyanobacterium Anacystis nidulans. Nucleic Acids Res. 1988 May 25;16(10):4447–4463. doi: 10.1093/nar/16.10.4447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Youngman P., Perkins J. B., Losick R. A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions. Mol Gen Genet. 1984;195(3):424–433. doi: 10.1007/BF00341443. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES