Summary
Two spore genes, spoOB and spoIIG have been cloned from the B. subtilis genome library, constructed by ligating Sau3A partially digested DNA to the dephosphorylated pHV33 plasmid vector at its BamH1 site.
An hybrid plasmid pGsOB2, carrying a 1.7 Kb insert of B. subtilis DNA amplifiable in E. coli was cloned. This recombinant plasmid was capable of transforming the appropriate B. subtilis Rec+ and Rec- recipients to Spo+ at very high efficiency. The pGsOB2 was further subcloned and four hybrid plasmids, pGsOB8, pGsOB9, pGsOB10 and pGsOB11 were selected and their restriction enzyme maps established. The four subcloned hybrid plasmids retained their entire transforming activity in both Rec+ and Rec- recipients although two of them carry the insert in an inverse orientation, indicating thus, that the spoOB gene in these plasmids is being transcribed by the B. subtilis RNA polymerase using an internal promotor of the cloned DNA fragment. The adjacent genes spoIVF and pheA, mapped respectively to the right and left of the spoOB locus, that normally show 90% cotransformation, are absent on the cloned DNA fragments. The cloned hybrid plasmids have been expressed in E. coli minicells and it was shown that the spoOB locus encoded a polypeptide of 24 K.
We have also cloned the spoIIG gene in two hybrid plasmids, pGsIIG24 and pGsIIG26, carrying respectively inserts of 2 and 3 Kb. From the transforming activity and the endonuclease cleavage maps it was shown that these two hybrid plasmids do not carry the entire spoIIG locus. The use of these plasmids for further cloning of this gene is discussed.
Similar content being viewed by others
References
Alton NK, Hautala JA, Giles NH, Kushner SR, Vapnek D (1978) Transcription and translation in E. coli of hybrid plasmids containning the catabolic dehydrogenase gene from Neurospora crassa. Gene 4:241–259
Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81:74–76
Birboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523
Bolivar F, Backman K (1979) Plasmids of Escherichia coli as cloning vectors. In: Wu R (ed) Methods in Enzymology — Recombinant DNA. vol 68, pp 245–267
Bonamy C, Keryer E, Szulmajster J (1978) Cloning of Bacillus subtilis DNA fragments carrying spore genes. In: Chambliss G, Vary JC (eds) Spores VII. Am Soc Microbiol, Washington DC, pp 139–143
Donnellan JE, Nags EN, Levinson NS (1964) Chemically defined synthetic media for sporulation and for germination and growth of Bacillus subtilis. J Bacteriol 87:332–336
Dubnau D, Davidoff-Abelson R (1971) Fate of transforming DNA following uptake by competent Bacillus subtilis. J Mol Biol 56:209–221
Dubnau E, Ramakrishna N, Cabane K, Smith I (1981) Cloning of a sporulation gene in Bacillus subtilis. In: Levinson HS, Sonsenshein AL, Tibber DJ (eds) Sporulation and germination. Am Soc Microbiol. Washington DC, pp 104–108
Ehrlich SD (1977) Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc Natl Acad Sci USA 74:1620–1628
Ehrlich SD (1978) DNA cloning in Bacillus subtilis. Proc Natl Acad Sci USA 75:1433–1436
Ferrari E, Henner DJ, Hoch JA (1981) Isolation of Bacillus subtilis genes from a Charon 4A library. J Bacteriol 146:430–432
Gryczan TJ, Dubnau D (1978) Construction and properties of chimeric plasmids in Bacillus subtilis. Proc Natl Acad Sci USA 75:1428–1432
Harris-Warrick RM, Elkana Y, Ehrlich SD, Lederberg J (1975) Electrophoretic separation of Bacillus subtilis genes. Proc Natl Acad Sci USA 72:2207–2211
Hirochika H, Kobayashi Y, Kawamura F, Saito H (1981) Cloning of sporulation gene spoOB of Bacillus subtilis and its genetic and biochemical analysis. J Bacteriol 146:494–505
Hoch JA (1971) Selection of cells transformed to prototrophy for sporulating markers. J Bacteriol 105:1200–1201
Hoch JA, Mathwes JL (1973) Chromosomal location of pleiotropic negative sporulation mutations in Bacillus subtilis. Genetics 73:215–228
Hoch JA, Shiflett MA, Trowsdale J, Chen SMH (1978) Stage O genes and their products. In: Chambliss G, Vary JC (eds) Spores VII. Am Soc Microbiol, Washington DC, pp 127–130
Jayaraman K, Keryer E, Szulmajster J (1981) Cloning of a fragment of B. subtilis DNA carrying the spoOB and pheA genes. FEMS Lett 10:273–276
Kawamura F, Shimotsu H, Saito H, Hirochika H, Kobayashi Y (1981) Cloning of spoO genes with bacteriophage and plasmid vectors in Bacillus subtilis. In: Levinson HS, Sonenshein AL, Tipper EJ (eds) Sporulation and Germination, Am Soc Microbiol, Washington DC, pp 109–113
Klein RD, Selsing E, Wells RD (1980) A rapid microscale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis. Plasmid 3:88–91
Klotz LC, Zimm BH (1972) Size of DNA determined by viscoelastic measurements: results on bacteriophages, Bacillus subtilis and Escherichia coli. J Mol Biol 72:779–800
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Losick R (1981) Sigma factor, stage O genes and sporulation. In: Levinson HS, Sonenshein AL, Tipper DJ (eds) Sporulation and Germination. Am Soc Microbiol, Washington DC, pp 48–56
Lovett PS (1981) Cloning strategies in Bacillus subtilis. In: Levinson HS, Sonenshein AL, Tipper DJ (eds) Sporulation and Germination. Am Soc Microbiol, Washington DC, pp 40–47
Mandel M, Higa A (1970) Clacium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162
Michel B, Palla E, Niaudet B, Ehrlich SD (1980) DNA cloning in Bacillus subtilis. III. Efficiency of random-segment cloning and insertional inactivation factors. Gene 12:147–154
Piggot PJ, Coote JG (1976) Genetic aspects of bacterial endospore formation. Bacteriol Rev 40:908–962
Primrose SB, Ehrlich SD (1981) Isolation of plasmid deletion mutants and study of their instability. Plasmid 6:193–201
Schaeffer P, Millet J, Aubert JP (1965) Catabolic repression of bacterial sporulation. Proc Natl Acad Sci USA 54:704–711
Uhlen M, Flock JI, Philipson L (1981) RecE independent deletions of recombinant plasmids in Bacillus subtilis. Plasmid 5:161–169
Ullrich A, Shine J, Chirgwin J, Pictet R, Tischer E, Rutter WJ, Goodman HM (1977) Rat insulin genes: construction of plasmids containing the coding sequences. Science 196:1313–1318
Young M (1975) Genetic mapping of sporulation operons in Bacillus subtilis using a thermosensitive sporulation mutant. J Bacteriol 122:1109–1116
Author information
Authors and Affiliations
Additional information
Communicated by W. Gajewski
Rights and permissions
About this article
Cite this article
Bonamy, C., Szulmajster, J. Cloning and expression of Bacillus subtilis spore genes. Mol Gen Genet 188, 202–210 (1982). https://doi.org/10.1007/BF00332676
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00332676