Skip to main content
SpringerLink
Log in

Isolation and characterization of comL, a transcription unit involved in competence development of Bacillus subtilis

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

Using the transformation-deficient mutant M465, which was previously isolated by means of insertional mutagenesis with plasmid pHV60, a transcription unit comL required for genetic competence of Bacillus subtilis was identified. A chromosomal DNA fragment flanking the inserted pHV60 was isolated and used to screen two different libraries of B. subtilis DNA in phage lambda EMBL4 and lambda EMBL12, respectively. With the aid of six recombinant phages that hybridize with this chromosomal fragment a restriction map of about 23 kb of B. subtilis chromosomal DNA was constructed. Using small adjoining pieces of this chromosomal DNA in Campbell integrations, the size of the transcription unit involved in competence development could be delimited to about 15 kb. By insertion of a promoterless lacZ gene into comL, the transcriptional regulation of comL was analysed and epistatic interactions among various other com genes were determined. The results of these experiments indicated that comL is optimally expressed in glucose-based minimal medium when the culture enters the stationary phase of growth and that the expression of late competence genes is dependent on previous transcription of comL, which in turn is dependent on the gene products of comA and comB.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Albano M, Hahn J, Dubnau D (1987) Expression of competence genes in Bacillus subtilis J Bacteriol 169:3110–3117

    Google Scholar 

  • Albano M, Dubnau D (1989) Cloning and characterization of a cluster of linked Bacillus subtilis late competence mutants. J Bacteriol 171:5376–5385

    Google Scholar 

  • Biswal N, Kleinschmidt AK, Spatz HC, Trautner TA (1967) Physical properties of the DNA of the bacteriophage SP50. Mol Gen Genet 100:39–55

    Google Scholar 

  • Bron S, Venema G (1972) Ultraviolet inactivation and excision repair in Bacillus subtilis. I. Construction and characterization of a transformable eightfold auxotrophic strain and two ultraviolet-sensitive derivatives. Mutat Res 15:1–10

    Google Scholar 

  • Casadaban MJ, Conen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207

    Google Scholar 

  • Chambers SP, Prior S, Barstow DB, Minton NP (1988) The pMTL series of cloning vectors: I. Improved polylinker regions to facilitate the generation of sonicated DNA for nucleotide sequencing. Gene 68:139–149

    Article  CAS  PubMed  Google Scholar 

  • Chomczynski P, Qasba PK (1984) Alkaline transfer of DNA to plastic membrane. Biochem Biophys Res Commun 122:340–344

    Google Scholar 

  • Dubnau D (1989) The competence regulon of Bacillus subtilis. In: Smith I, Slepecky R, Setlow P (eds) Regulation of prokaryotic development. American Society for Microbiology, Washington DC, pp 147–166

    Google Scholar 

  • Dubnau D, Roggiani M (1990) Growth medium-independent genetic competence mutants of Bacillus subtilis. J Bacteriol 172:4048–4055

    Google Scholar 

  • Dubnau E, Weir J, Nair G, Carter III, HL, Moran Jr, CP, Smith I (1988) Bacillus sporulation gene spoOH codes for σ30 (σH). J Bacteriol 170:1054–1062

    Google Scholar 

  • Frischauf AM, Lehrach H, Poustka A, Murray N (1983) Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170:827–842

    Google Scholar 

  • Gaur NK, Dubnau E, Smith I (1986) Characterization of a cloned Bacillus subtilis gene which inhibits sporulation in multiple copies. J Bacteriol 168:860–869

    Google Scholar 

  • Guillen N, Weinranch Y, Dubnau D (1989) Cloning and characterization of the regulatory Bacillus subtilis competence genes, comA and comB. J Bacteriol 171:5354–5361

    Google Scholar 

  • Hadden C, Nester EW (1968) Purification of competent cells in the Bacillus subtilis transformation system. J Bacteriol 95:876–885

    Google Scholar 

  • Haseltine Cahn F, Fox MS (1968) Fractionation of transformable bacteria from competent cultures of Bacillus subtilis on renografin gradients. J Bacteriol 95:867–875

    Google Scholar 

  • Henner DJ, Yang M, Ferrari E (1988) Localization of Bacilus subtilis sacU (HY) mutations to two linked genes with similarities to the conserved procaryotic family of two-component signaling systems. J Bacteriol 170:5102–5109

    Google Scholar 

  • Hoch JA, Trach K, Kawamura F, Saito H (1985) Identification of the transcriptional suppressor sof-1 as an alteration in the spoOA protein. J Bacteriol 161:552–555

    Google Scholar 

  • Horinouchi S, Weisblum B (1982) Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol 150:8I5–825

    Google Scholar 

  • Ish-Horowicz D, Burke FJ (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2999

    Google Scholar 

  • Jaacks KJ, Healy J, Losick R, Grossman AD (1989) Identification and characterization of genes controlled by the sporulationregulation gene spoOH in Bacillus subtilis. J Bacteriol 171:4121–4129

    Google Scholar 

  • Kiel JAKW, Vossen JPMJ, Venema G (1987) A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid seqregation. Mol Gen Genet 207:294–301

    Google Scholar 

  • Kunst F, Debarbouille M, Msadek T, Young M, Mauel C, Karamata D, Klier A, Rapoport G, Dedonder R (1988) Deduced polypeptides encoded by the Bacillus subtilis sacU locus share homology with two-component sensor-regulator systems. J Bacteriol 170:5093–5101

    Google Scholar 

  • Mandel M, Higa A (1970) Calcium-dependent bacteriophage DNA infection. J Mol Biol 53:159–162

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook F (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Marahiel MA, Krause M, Sharpeid H (1985) Cloning of the tyrocidine synthetase I gene from Bacillus brevis and its expression in Escherichia coli. Mol Gen Genet 201:231–236

    Google Scholar 

  • Messing J (1979) A multipurpose cloning system based on the single stranded DNA bacteriophage M13. Recombinant DNA Technical Bulletin, NIH Publication, No. 79–99, 2, No. 2, pp 43–48

  • Miller JH (1982) Experiments in molecular genetics Cold Spring Harbor Laboratory, Cold Spring Harbor New York

    Google Scholar 

  • Mittenhuber G, Weckermann R, Marahiel MA (1989) gene cluster containing the genes for tyrocidine synthetases 1 and 2 from Bacillus brevis: evidence for an operon. J Bacteriol 171:4881–4887

    Google Scholar 

  • Msadek T, Kunst F, Henner D, Klier A, Rapoport G, Dedonder R (1990) The signal transduction pathway controlling the synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analyses of mutations in degS and degU. J Bacteriol 170:824–834

    Google Scholar 

  • Nakano MM, Zuber P (1989) Cloning and characterization of srfB, a regulatory gene involved in surfactin production and competence in Bacillus subtilis. J Bacteriol 171:5347–5353

    Google Scholar 

  • Natt E, Scherer G (1986) EMBL12, a new lambda replacement vector with sites for Sall, XbaI, BamH1, Sst1 and EcoRI. Nucleic Acids Res 14:7128

    Google Scholar 

  • Perego M, Spiegelman GB, Hoch JA (1988) Structure of the gene for the transition state regulator abrB: regulator synthesis is controlled by the spoOA sporulation gene in Bacillus subtilis. Mol Microbiol 2:689–699

    Google Scholar 

  • Perkins JB, Youngman PJ (1986) Construction and properties of T017-lac, a transposon derivative that mediates transcriptional gene fusions in Bacillus subtilis. Proc Natl Acad Sci USA 83:140–144

    Google Scholar 

  • Rigby PWJ, Dieckman M, Rhodes C, Berg P (1977) Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251

    Google Scholar 

  • Roggiani M, Hahn J, Dubnau D (1990) Suppression of early competence mutations in Bacillus subtilis by mec mutations. J Bacteriol 172:4056–4063

    Google Scholar 

  • Rosenthal AL, Lacks SA (1977) Nuclease detection in SDS-polyacrylamide gel electrophoresis. Anal Biochem 80:76–90

    Google Scholar 

  • Sadaie Y, Kada T (1983) Formation of competent Bacillus subtilis cells. J Bacteriol 153:813–821

    Google Scholar 

  • Schaeffer P, Millet I, Aubert J (1965) Catabolite repression of bacterial sporulation. Proc Natl Acad Sci USA 54:704–761

    Google Scholar 

  • Spizizen J (1958) Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc Natl Acad Sci USA 44:1072–1078

    Google Scholar 

  • Stock JB, Ninfa AJ, Stock AM (1989) Protein phosphorylation and the regulation of adaptive responses in bacteria. Microbiol Rev 53:450–490

    Google Scholar 

  • Tanaka T, Kawata M (1988) Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases. J Bacteriol 170:3593–3600

    Google Scholar 

  • Venema G, Pritchard RH, Venema-Schröder T (1965) Fate of transforming deoxyribonucleic acid in Bacillus subtilis. J Bacteriol 89:1250–1255

    Google Scholar 

  • Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

    Google Scholar 

  • Vosman B, Kooistra J, Olijve J, Venema G (1986) Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism. Mol Gen Genet 204:524–531

    Google Scholar 

  • Vosman B, Kooistra J, Olijve J, Venema G (1987) Cloning in Escherichia coli of the gene specifying the DNA-entry nuclease of Bacillus subtilis. Gene 52:175–183

    Google Scholar 

  • Vosman B, Kuiken G, Kooistra J, Venema G (1988) Transformation in Bacillus subtilis: Involvement of the 17-kilodalton DNAentry nuclease and the competence specific 18-kilodalton protein. J Bacteriol 170:3703–3710

    Google Scholar 

  • Weckermann R, Fûrbass R, Marahiel MA (1988) Complete nucleotide sequence of the tycA gene coding the tyrocidine synthetase I from Bacillus brevis. nucleic Acids Res 16:11841

    Google Scholar 

  • Weinrauch Y, Penchev R, Dubnau E, Smith I, Dubnau D (1990) 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 4:860–872

    Google Scholar 

  • Weinstock GM, Berman ML, Silhavy TJ (1983) Chimeric genes with β-galactosidase. In: Papas TS, Rosenberg M, Chirikjian JG (eds) Gene amplification and analysis III. Elsevier, North Holland, New York, pp 27–64

    Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequence of the M13mpl8 and pUC19 vectors. Gene 33:103–119

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Genetics, University of Groningen, Kerklaan 30, NL-9751 NN, Haren, The Netherlands

    Douwe van Sinderen, Sebo Withoff, Hans Boels & Gerard Venema

Authors
  1. Douwe van Sinderen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sebo Withoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hans Boels
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gerard Venema
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by H. Böhme

Rights and permissions

Reprints and permissions

About this article

Cite this article

van Sinderen, D., Withoff, S., Boels, H. et al. Isolation and characterization of comL, a transcription unit involved in competence development of Bacillus subtilis . Molec. Gen. Genet. 224, 396–404 (1990). https://doi.org/10.1007/BF00262434

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00262434

Key words

Search

Navigation