Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Sep;178(17):5144–5152. doi: 10.1128/jb.178.17.5144-5152.1996

Plasmid-amplified comS enhances genetic competence and suppresses sinR in Bacillus subtilis.

L Liu 1, M M Nakano 1, O H Lee 1, P Zuber 1
PMCID: PMC178310  PMID: 8752331

Abstract

The establishment of genetic competence in Bacillus subtilis is controlled by a vast signal transduction network involving the products of genes that function in several postexponential-phase processes. Two of these proteins, SinR and DegU, serve as molecular switches that influence a cell's decision to undergo either sporulation or genetic competence development. In order to determine the roles of SinR and DegU in competence control, multicopy suppression experiments with plasmid-amplified comS, SinR, and degU genes were undertaken. Multicopy comS was found to elevate competence gene transcription and transformation efficiency in both wild-type and sinR mutant cells but not in degU mutant cells. Multicopy degU failed to suppress comS or sinR mutations. No suppression of comS or degU by multicopy sinR was observed. The expression of a comS'::'lacZ translational fusion and srf-lacZ operon fusion was examined in sinR cells and cells bearing plasmid-amplified sinR. The expression of comS'::'lacZ gene fusion was reduced by the sinR mutation, but both comS'::'lacZ and srf-lacZ were repressed by multicopy sinR. Cells bearing plasmid-amplified sinR were poorly competent. These results suggest that sinR is required for optimal comS expression but not transcription from the srf promoter and that SinR at high concentrations represses srf transcription initiation.

Full Text

The Full Text of this article is available as a PDF (294.9 KB).

Selected References

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

  1. Albano M., Hahn J., Dubnau D. Expression of competence genes in Bacillus subtilis. J Bacteriol. 1987 Jul;169(7):3110–3117. doi: 10.1128/jb.169.7.3110-3117.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arima K., Kakinuma A., Tamura G. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun. 1968 May 10;31(3):488–494. doi: 10.1016/0006-291x(68)90503-2. [DOI] [PubMed] [Google Scholar]
  3. Bai U., Mandic-Mulec I., Smith I. SinI modulates the activity of SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction. Genes Dev. 1993 Jan;7(1):139–148. doi: 10.1101/gad.7.1.139. [DOI] [PubMed] [Google Scholar]
  4. Cosmina P., Rodriguez F., de Ferra F., Grandi G., Perego M., Venema G., van Sinderen D. Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis. Mol Microbiol. 1993 May;8(5):821–831. doi: 10.1111/j.1365-2958.1993.tb01629.x. [DOI] [PubMed] [Google Scholar]
  5. D'Souza C., Nakano M. M., Corbell N., Zuber P. Amino-acylation site mutations in amino acid-activating domains of surfactin synthetase: effects on surfactin production and competence development in Bacillus subtilis. J Bacteriol. 1993 Jun;175(11):3502–3510. doi: 10.1128/jb.175.11.3502-3510.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. D'Souza C., Nakano M. M., Frisby D. L., Zuber P. Translation of the open reading frame encoded by comS, a gene of the srf operon, is necessary for the development of genetic competence, but not surfactin biosynthesis, in Bacillus subtilis. J Bacteriol. 1995 Jul;177(14):4144–4148. doi: 10.1128/jb.177.14.4144-4148.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. D'Souza C., Nakano M. M., Zuber P. Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9397–9401. doi: 10.1073/pnas.91.20.9397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dahl M. K., Msadek T., Kunst F., Rapoport G. Mutational analysis of the Bacillus subtilis DegU regulator and its phosphorylation by the DegS protein kinase. J Bacteriol. 1991 Apr;173(8):2539–2547. doi: 10.1128/jb.173.8.2539-2547.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dahl M. K., Msadek T., Kunst F., Rapoport G. The phosphorylation state of the DegU response regulator acts as a molecular switch allowing either degradative enzyme synthesis or expression of genetic competence in Bacillus subtilis. J Biol Chem. 1992 Jul 15;267(20):14509–14514. [PubMed] [Google Scholar]
  10. Dubnau D., Davidoff-Abelson R. Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J Mol Biol. 1971 Mar 14;56(2):209–221. doi: 10.1016/0022-2836(71)90460-8. [DOI] [PubMed] [Google Scholar]
  11. Dubnau D., Hahn J., Roggiani M., Piazza F., Weinrauch Y. Two-component regulators and genetic competence in Bacillus subtilis. Res Microbiol. 1994 Jun-Aug;145(5-6):403–411. doi: 10.1016/0923-2508(94)90088-4. [DOI] [PubMed] [Google Scholar]
  12. Dubnau D., Roggiani M. Growth medium-independent genetic competence mutants of Bacillus subtilis. J Bacteriol. 1990 Jul;172(7):4048–4055. doi: 10.1128/jb.172.7.4048-4055.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dubnau J., Struhl G. RNA recognition and translational regulation by a homeodomain protein. Nature. 1996 Feb 22;379(6567):694–699. doi: 10.1038/379694a0. [DOI] [PubMed] [Google Scholar]
  14. Fouet A., Sonenshein A. L. A target for carbon source-dependent negative regulation of the citB promoter of Bacillus subtilis. J Bacteriol. 1990 Feb;172(2):835–844. doi: 10.1128/jb.172.2.835-844.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fuma S., Fujishima Y., Corbell N., D'Souza C., Nakano M. M., Zuber P., Yamane K. Nucleotide sequence of 5' portion of srfA that contains the region required for competence establishment in Bacillus subtilus. Nucleic Acids Res. 1993 Jan 11;21(1):93–97. doi: 10.1093/nar/21.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Galli G., Rodriguez F., Cosmina P., Pratesi C., Nogarotto R., de Ferra F., Grandi G. Characterization of the surfactin synthetase multi-enzyme complex. Biochim Biophys Acta. 1994 Mar 16;1205(1):19–28. doi: 10.1016/0167-4838(94)90087-6. [DOI] [PubMed] [Google Scholar]
  17. Gaur N. K., Cabane K., Smith I. Structure and expression of the Bacillus subtilis sin operon. J Bacteriol. 1988 Mar;170(3):1046–1053. doi: 10.1128/jb.170.3.1046-1053.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gaur N. K., Dubnau E., Smith I. Characterization of a cloned Bacillus subtilis gene that inhibits sporulation in multiple copies. J Bacteriol. 1986 Nov;168(2):860–869. doi: 10.1128/jb.168.2.860-869.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gaur N. K., Oppenheim J., Smith I. The Bacillus subtilis sin gene, a regulator of alternate developmental processes, codes for a DNA-binding protein. J Bacteriol. 1991 Jan;173(2):678–686. doi: 10.1128/jb.173.2.678-686.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gryczan T., Shivakumar A. G., Dubnau D. Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis. J Bacteriol. 1980 Jan;141(1):246–253. doi: 10.1128/jb.141.1.246-253.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hahn J., Dubnau D. Growth stage signal transduction and the requirements for srfA induction in development of competence. J Bacteriol. 1991 Nov;173(22):7275–7282. doi: 10.1128/jb.173.22.7275-7282.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hahn J., Kong L., Dubnau D. The regulation of competence transcription factor synthesis constitutes a critical control point in the regulation of competence in Bacillus subtilis. J Bacteriol. 1994 Sep;176(18):5753–5761. doi: 10.1128/jb.176.18.5753-5761.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hahn J., Roggiani M., Dubnau D. The major role of Spo0A in genetic competence is to downregulate abrB, an essential competence gene. J Bacteriol. 1995 Jun;177(12):3601–3605. doi: 10.1128/jb.177.12.3601-3605.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hamoen L. W., Eshuis H., Jongbloed J., Venema G., van Sinderen D. A small gene, designated comS, located within the coding region of the fourth amino acid-activation domain of srfA, is required for competence development in Bacillus subtilis. Mol Microbiol. 1995 Jan;15(1):55–63. doi: 10.1111/j.1365-2958.1995.tb02220.x. [DOI] [PubMed] [Google Scholar]
  25. Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815–825. doi: 10.1128/jb.150.2.815-825.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kong L., Dubnau D. Regulation of competence-specific gene expression by Mec-mediated protein-protein interaction in Bacillus subtilis. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5793–5797. doi: 10.1073/pnas.91.13.5793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kong L., Siranosian K. J., Grossman A. D., Dubnau D. Sequence and properties of mecA, a negative regulator of genetic competence in Bacillus subtilis. Mol Microbiol. 1993 Jul;9(2):365–373. doi: 10.1111/j.1365-2958.1993.tb01697.x. [DOI] [PubMed] [Google Scholar]
  28. Kramer W., Fritz H. J. Oligonucleotide-directed construction of mutations via gapped duplex DNA. Methods Enzymol. 1987;154:350–367. doi: 10.1016/0076-6879(87)54084-8. [DOI] [PubMed] [Google Scholar]
  29. Kunst F., Pascal M., Lepesant-Kejzlarova J., Lepesant J. A., Billault A., Dedonder R. Pleiotropic mutations affecting sporulation conditions and the syntheses of extracellular enzymes in Bacillus subtilis 168. Biochimie. 1974;56(11-12):1481–1489. doi: 10.1016/s0300-9084(75)80270-7. [DOI] [PubMed] [Google Scholar]
  30. Lorenz M. G., Wackernagel W. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev. 1994 Sep;58(3):563–602. doi: 10.1128/mr.58.3.563-602.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Luttinger A., Hahn J., Dubnau D. Polynucleotide phosphorylase is necessary for competence development in Bacillus subtilis. Mol Microbiol. 1996 Jan;19(2):343–356. doi: 10.1046/j.1365-2958.1996.380907.x. [DOI] [PubMed] [Google Scholar]
  32. Magnuson R., Solomon J., Grossman A. D. Biochemical and genetic characterization of a competence pheromone from B. subtilis. Cell. 1994 Apr 22;77(2):207–216. doi: 10.1016/0092-8674(94)90313-1. [DOI] [PubMed] [Google Scholar]
  33. Mandic-Mulec I., Gaur N., Bai U., Smith I. Sin, a stage-specific repressor of cellular differentiation. J Bacteriol. 1992 Jun;174(11):3561–3569. doi: 10.1128/jb.174.11.3561-3569.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mohan S., Dubnau D. Transcriptional regulation of comC: evidence for a competence-specific transcription factor in Bacillus subtilis. J Bacteriol. 1990 Jul;172(7):4064–4071. doi: 10.1128/jb.172.7.4064-4071.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Msadek T., Kunst F., Rapoport G. MecB of Bacillus subtilis, a member of the ClpC ATPase family, is a pleiotropic regulator controlling competence gene expression and growth at high temperature. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5788–5792. doi: 10.1073/pnas.91.13.5788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mukai K., Kawata M., Tanaka T. Isolation and phosphorylation of the Bacillus subtilis degS and degU gene products. J Biol Chem. 1990 Nov 15;265(32):20000–20006. [PubMed] [Google Scholar]
  37. Nakano M. M., Corbell N., Besson J., Zuber P. Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Mol Gen Genet. 1992 Mar;232(2):313–321. doi: 10.1007/BF00280011. [DOI] [PubMed] [Google Scholar]
  38. Nakano M. M., Magnuson R., Myers A., Curry J., Grossman A. D., Zuber P. srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis. J Bacteriol. 1991 Mar;173(5):1770–1778. doi: 10.1128/jb.173.5.1770-1778.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nakano M. M., Marahiel M. A., Zuber P. Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. J Bacteriol. 1988 Dec;170(12):5662–5668. doi: 10.1128/jb.170.12.5662-5668.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nakano M. M., Xia L. A., Zuber P. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in Bacillus subtilis. J Bacteriol. 1991 Sep;173(17):5487–5493. doi: 10.1128/jb.173.17.5487-5493.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Nakano M. M., Zuber P. Mutational analysis of the regulatory region of the srfA operon in Bacillus subtilis. J Bacteriol. 1993 May;175(10):3188–3191. doi: 10.1128/jb.175.10.3188-3191.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nakano M. M., Zuber P. The primary role of comA in establishment of the competent state in Bacillus subtilis is to activate expression of srfA. J Bacteriol. 1991 Nov;173(22):7269–7274. doi: 10.1128/jb.173.22.7269-7274.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Niaudet B., Ehrlich S. D. In vitro genetic labeling of Bacillus subtilis cryptic plasmid pHV400. Plasmid. 1979 Jan;2(1):48–58. doi: 10.1016/0147-619x(79)90005-2. [DOI] [PubMed] [Google Scholar]
  44. Perego M., Higgins C. F., Pearce S. R., Gallagher M. P., Hoch J. A. The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol Microbiol. 1991 Jan;5(1):173–185. doi: 10.1111/j.1365-2958.1991.tb01838.x. [DOI] [PubMed] [Google Scholar]
  45. Perego M., Spiegelman G. B., Hoch J. A. Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis. Mol Microbiol. 1988 Nov;2(6):689–699. doi: 10.1111/j.1365-2958.1988.tb00079.x. [DOI] [PubMed] [Google Scholar]
  46. Retallack D. M., Friedman D. I. A role for a small stable RNA in modulating the activity of DNA-binding proteins. Cell. 1995 Oct 20;83(2):227–235. doi: 10.1016/0092-8674(95)90164-7. [DOI] [PubMed] [Google Scholar]
  47. Roggiani M., Dubnau D. ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA. J Bacteriol. 1993 May;175(10):3182–3187. doi: 10.1128/jb.175.10.3182-3187.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Roggiani M., Hahn J., Dubnau D. Suppression of early competence mutations in Bacillus subtilis by mec mutations. J Bacteriol. 1990 Jul;172(7):4056–4063. doi: 10.1128/jb.172.7.4056-4063.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rudner D. Z., LeDeaux J. R., Ireton K., Grossman A. D. The spo0K locus of Bacillus subtilis is homologous to the oligopeptide permease locus and is required for sporulation and competence. J Bacteriol. 1991 Feb;173(4):1388–1398. doi: 10.1128/jb.173.4.1388-1398.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Solomon J. M., Magnuson R., Srivastava A., Grossman A. D. Convergent sensing pathways mediate response to two extracellular competence factors in Bacillus subtilis. Genes Dev. 1995 Mar 1;9(5):547–558. doi: 10.1101/gad.9.5.547. [DOI] [PubMed] [Google Scholar]
  51. Stewart G. J., Carlson C. A. The biology of natural transformation. Annu Rev Microbiol. 1986;40:211–235. doi: 10.1146/annurev.mi.40.100186.001235. [DOI] [PubMed] [Google Scholar]
  52. Strauch M. A., Spiegelman G. B., Perego M., Johnson W. C., Burbulys D., Hoch J. A. The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein. EMBO J. 1989 May;8(5):1615–1621. doi: 10.1002/j.1460-2075.1989.tb03546.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Strauch M., Webb V., Spiegelman G., Hoch J. A. The SpoOA protein of Bacillus subtilis is a repressor of the abrB gene. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1801–1805. doi: 10.1073/pnas.87.5.1801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vollenbroich D., Kluge B., D'Souza C., Zuber P., Vater J. Analysis of a mutant amino acid-activating domain of surfactin synthetase bearing a serine-to-alanine substitution at the site of carboxylthioester formation. FEBS Lett. 1993 Jul 5;325(3):220–224. doi: 10.1016/0014-5793(93)81077-d. [DOI] [PubMed] [Google Scholar]
  55. Weinrauch Y., Guillen N., Dubnau D. A. Sequence and transcription mapping of Bacillus subtilis competence genes comB and comA, one of which is related to a family of bacterial regulatory determinants. J Bacteriol. 1989 Oct;171(10):5362–5375. doi: 10.1128/jb.171.10.5362-5375.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weinrauch Y., Penchev R., Dubnau E., Smith I., Dubnau D. A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems. Genes Dev. 1990 May;4(5):860–872. doi: 10.1101/gad.4.5.860. [DOI] [PubMed] [Google Scholar]
  57. Weir J., Predich M., Dubnau E., Nair G., Smith I. Regulation of spo0H, a gene coding for the Bacillus subtilis sigma H factor. J Bacteriol. 1991 Jan;173(2):521–529. doi: 10.1128/jb.173.2.521-529.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yansura D. G., Henner D. J. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc Natl Acad Sci U S A. 1984 Jan;81(2):439–443. doi: 10.1073/pnas.81.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. van Sinderen D., Galli G., Cosmina P., de Ferra F., Withoff S., Venema G., Grandi G. Characterization of the srfA locus of Bacillus subtilis: only the valine-activating domain of srfA is involved in the establishment of genetic competence. Mol Microbiol. 1993 May;8(5):833–841. doi: 10.1111/j.1365-2958.1993.tb01630.x. [DOI] [PubMed] [Google Scholar]
  60. van Sinderen D., Luttinger A., Kong L., Dubnau D., Venema G., Hamoen L. comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis. Mol Microbiol. 1995 Feb;15(3):455–462. doi: 10.1111/j.1365-2958.1995.tb02259.x. [DOI] [PubMed] [Google Scholar]
  61. van Sinderen D., Venema G. comK acts as an autoregulatory control switch in the signal transduction route to competence in Bacillus subtilis. J Bacteriol. 1994 Sep;176(18):5762–5770. doi: 10.1128/jb.176.18.5762-5770.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. van Sinderen D., Withoff S., Boels H., Venema G. Isolation and characterization of comL, a transcription unit involved in competence development of Bacillus subtilis. Mol Gen Genet. 1990 Dec;224(3):396–404. doi: 10.1007/BF00262434. [DOI] [PubMed] [Google Scholar]

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

RESOURCES