Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Dec;172(12):6937–6941. doi: 10.1128/jb.172.12.6937-6941.1990

Use of integrational plasmid excision to identify cellular localization of gene expression during sporulation in Bacillus subtilis.

N Illing 1, M Young 1, J Errington 1
PMCID: PMC210813  PMID: 2123859

Abstract

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two sister cells, the prespore and the mother cell. Many of the genes that regulate sporulation (spo genes) are thought to be expressed differentially. However, direct demonstration of differential gene expression, by fractionation of prespore and mother cell proteins, is possible only at a relatively late stage of development. H. De Lencastre and P. J. Piggot (J. Gen. Microbiol. 114:377-389, 1979) have described a genetic method for determining the cellular location of the requirement for spo gene expression. Here we describe a similar method based on the use of integrational plasmids that can insertionally inactivate any given spo gene. Loss of the integrated plasmid by homologous recombination leads to the restoration of spo gene function. If this occurs just before sporulation begins, the phenotypes of the progeny of heat-resistant spores should depend on whether the gene is required in the prespore or the mother cell. Thus, we show that for known prespore-specific genes, such as spoIIIG and spoVA, only phenotypically Spo+ progeny that have lost the integrated plasmid are produced. In contrast, for mother-cell-specific genes, such as spoIIIC and spoVJ, a substantial proportion of the progeny are asporogenous, having retained the integrated plasmid. On the basis of our results, the spoIID and spoIIIA genes, which are expressed soon after division, appear to be required only in the mother cell compartment.

Full text

PDF
6941

Selected References

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

  1. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carlson H. C., Haldenwang W. G. The sigma E subunit of Bacillus subtilis RNA polymerase is present in both forespore and mother cell compartments. J Bacteriol. 1989 Apr;171(4):2216–2218. doi: 10.1128/jb.171.4.2216-2218.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clarke S., Lopez-Diaz I., Mandelstam J. Use of lacZ gene fusions to determine the dependence pattern of the sporulation gene spoIID in spo mutants of Bacillus subtilis. J Gen Microbiol. 1986 Nov;132(11):2987–2994. doi: 10.1099/00221287-132-11-2987. [DOI] [PubMed] [Google Scholar]
  4. Cutting S. M., Mandelstam J. Cloning and dependence pattern of the sporulation operon spoVH. J Bacteriol. 1988 Feb;170(2):802–809. doi: 10.1128/jb.170.2.802-809.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Errington J., Jones D. Cloning in Bacillus subtilis by transfection with bacteriophage vector phi 105J27: isolation and preliminary characterization of transducing phages for 23 sporulation loci. J Gen Microbiol. 1987 Mar;133(3):493–502. doi: 10.1099/00221287-133-3-493. [DOI] [PubMed] [Google Scholar]
  6. Errington J., Mandelstam J. Use of a lacZ gene fusion to determine the dependence pattern and the spore compartment expression of sporulation operon spoVA in spo mutants of Bacillus subtilis. J Gen Microbiol. 1986 Nov;132(11):2977–2985. doi: 10.1099/00221287-132-11-2977. [DOI] [PubMed] [Google Scholar]
  7. Errington J., Mandelstam J. Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIA in spo mutants of Bacillus subtilis. J Gen Microbiol. 1986 Nov;132(11):2967–2976. doi: 10.1099/00221287-132-11-2967. [DOI] [PubMed] [Google Scholar]
  8. Errington J., Wootten L., Dunkerley J. C., Foulger D. Differential gene expression during sporulation in Bacillus subtilis: regulation of the spoVJ gene. Mol Microbiol. 1989 Aug;3(8):1053–1060. doi: 10.1111/j.1365-2958.1989.tb00255.x. [DOI] [PubMed] [Google Scholar]
  9. Fort P., Errington J. Nucleotide sequence and complementation analysis of a polycistronic sporulation operon, spoVA, in Bacillus subtilis. J Gen Microbiol. 1985 May;131(5):1091–1105. doi: 10.1099/00221287-131-5-1091. [DOI] [PubMed] [Google Scholar]
  10. Foulger D., Errington J. The role of the sporulation gene spoIIIE in the regulation of prespore-specific gene expression in Bacillus subtilis. Mol Microbiol. 1989 Sep;3(9):1247–1255. doi: 10.1111/j.1365-2958.1989.tb00275.x. [DOI] [PubMed] [Google Scholar]
  11. Francesconi S. C., MacAlister T. J., Setlow B., Setlow P. Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis. J Bacteriol. 1988 Dec;170(12):5963–5967. doi: 10.1128/jb.170.12.5963-5967.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gholamhoseinian A., Piggot P. J. Timing of spoII gene expression relative to septum formation during sporulation of Bacillus subtilis. J Bacteriol. 1989 Oct;171(10):5747–5749. doi: 10.1128/jb.171.10.5747-5749.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Illing N., Errington J. The spoIIIA locus is not a major determinant of prespore-specific gene expression during sporulation in Bacillus subtilis. J Bacteriol. 1990 Dec;172(12):6930–6936. doi: 10.1128/jb.172.12.6930-6936.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jenkinson H. F. Altered arrangement of proteins in the spore coat of a germination mutant of Bacillus subtilis. J Gen Microbiol. 1983 Jun;129(6):1945–1958. doi: 10.1099/00221287-129-6-1945. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Kunkel B., Kroos L., Poth H., Youngman P., Losick R. Temporal and spatial control of the mother-cell regulatory gene spoIIID of Bacillus subtilis. Genes Dev. 1989 Nov;3(11):1735–1744. doi: 10.1101/gad.3.11.1735. [DOI] [PubMed] [Google Scholar]
  17. Kunkel B., Sandman K., Panzer S., Youngman P., Losick R. The promoter for a sporulation gene in the spoIVC locus of Bacillus subtilis and its use in studies of temporal and spatial control of gene expression. J Bacteriol. 1988 Aug;170(8):3513–3522. doi: 10.1128/jb.170.8.3513-3522.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lopez-Diaz I., Clarke S., Mandelstam J. spoIID operon of Bacillus subtilis: cloning and sequence. J Gen Microbiol. 1986 Feb;132(2):341–354. doi: 10.1099/00221287-132-2-341. [DOI] [PubMed] [Google Scholar]
  19. Mandelstam J., Errington J. Dependent sequences of gene expression controlling spore formation in Bacillus subtilis. Microbiol Sci. 1987 Aug;4(8):238–244. [PubMed] [Google Scholar]
  20. 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]
  21. Panzer S., Losick R., Sun D., Setlow P. Evidence for an additional temporal class of gene expression in the forespore compartment of sporulating Bacillus subtilis. J Bacteriol. 1989 Jan;171(1):561–564. doi: 10.1128/jb.171.1.561-564.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Piggot P. J., Curtis C. A., de Lencastre H. Use of integrational plasmid vectors to demonstrate the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis. J Gen Microbiol. 1984 Aug;130(8):2123–2136. doi: 10.1099/00221287-130-8-2123. [DOI] [PubMed] [Google Scholar]
  23. Rong S., Rosenkrantz M. S., Sonenshein A. L. Transcriptional control of the Bacillus subtilis spoIID gene. J Bacteriol. 1986 Mar;165(3):771–779. doi: 10.1128/jb.165.3.771-779.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sterlini J. M., Mandelstam J. Commitment to sporulation in Bacillus subtilis and its relationship to development of actinomycin resistance. Biochem J. 1969 Jun;113(1):29–37. doi: 10.1042/bj1130029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Stragier P., Bonamy C., Karmazyn-Campelli C. Processing of a sporulation sigma factor in Bacillus subtilis: how morphological structure could control gene expression. Cell. 1988 Mar 11;52(5):697–704. doi: 10.1016/0092-8674(88)90407-2. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Turner S. M., Errington J., Mandelstam J. Use of a lacZ gene fusion to determine the dependence pattern of sporulation operon spoIIIC in spo mutants of Bacillus subtilis: a branched pathway of expression of sporulation operons. J Gen Microbiol. 1986 Nov;132(11):2995–3003. doi: 10.1099/00221287-132-11-2995. [DOI] [PubMed] [Google Scholar]
  28. Vagner V., Ehrlich S. D. Efficiency of homologous DNA recombination varies along the Bacillus subtilis chromosome. J Bacteriol. 1988 Sep;170(9):3978–3982. doi: 10.1128/jb.170.9.3978-3982.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Young M., Ehrlich S. D. Stability of reiterated sequences in the Bacillus subtilis chromosome. J Bacteriol. 1989 May;171(5):2653–2656. doi: 10.1128/jb.171.5.2653-2656.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. de Lencastre H., Piggot P. J. Identification of different sites of expression for spo loci by transformation of Bacillus subtilis. J Gen Microbiol. 1979 Oct;114(2):377–389. doi: 10.1099/00221287-114-2-377. [DOI] [PubMed] [Google Scholar]

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

RESOURCES