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Molecular and General Genetics MGG

, Volume 200, Issue 2, pp 220–228 | Cite as

The DNA sequence of the gene for the secreted Bacillus subtilis enzyme levansucrase and its genetic control sites

  • Michel Steinmetz
  • Dominique Le Coq
  • Stéphane Aymerich
  • Geneviève Gonzy-Tréboul
  • Philippe Gay
Article

Summary

We present the sequence of a 2 kb fragment of the Bacillus subtilis Marburg genome containing sacB, the structural gene of levansucrase, a secreted enzyme inducible by sucrose. The peptide sequence deduced for the secreted enzyme is very similar to that directly determined by Delfour (1981) for levansucrase of the non-Marburg strain BS5. The peptide sequence is preceded by a 29 amino acid signal peptide. Codon usage in sacB is rather different from that in the sequenced genes of other secreted enzymes in B. subtilis, especially α-amylase.

Genetic evidence has shown that the sacB promotor is rather far from the beginning of sacB (200 bp or more). The 200 bp region preceding sacB shows some of the features of an attenuator. A preliminary discussion of the putative workings and roles of this attenuator-like structure is proposed. sacRc mutations, which allow constitutive expression of levansucrase, have been located within the 450 bp upstream of sacB. It is shown that sacRc and sacR+ alleles control in cis the expression of the adjacent sacB gene.

Keywords

Enzyme Codon Bacillus Subtilis Peptide Sequence Codon Usage 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

bp

base pairs

Cm, R/S

chloramphenicol resistant(ce)/sensitive(ity)

His +/

prototroph/auxotroph for histidine

kb

kilobase

LS, +/

levansucrase, plus/minus

LSi/LSc

inducible/constitutive synthesis of levansucrase

TU

transcriptional unit

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References

  1. Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in B. subtilis. J Bacteriol 81:741–746Google Scholar
  2. Bauer C, Carey J, Kasper L, Lynn S, Waechter D, Gardner J (1983) Attenuation in bacterial operons. In: Beckwith J, Davies J, Gallant J (eds) Gene function in prokaryotes. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 65Google Scholar
  3. Brosius JA, Cate R, Perlmutter A (1982) Precise location of two promoters for the β-lactamase gene of pBR322. J Biol Chem 257:9205–9210Google Scholar
  4. Chambert R, Gonzy-Tréboul G (1976) Levansucrase of B. subtilis: kinetic and thermodynamic aspects of transfructosylation process. Eur J Biochem. 62:55–64Google Scholar
  5. Dedonder R (1966) Levansucrase from B. subtilis. Methods Enzymol 8:500–505Google Scholar
  6. Delfour A (1981) Approche tactique pour une analyse par spectroscopie de masse de la structure primaire des proteines: application à la levanesaccharase de B. subtilis. Thèse de doctorat d'Etat. Université Paris VII. ParisGoogle Scholar
  7. Ferrari, F, Nguyen A, Lang D, Hoch J (1983) Construction and properties of an integrable plasmid for B. subtilis. J Bacteriol 154:1513–1515Google Scholar
  8. Fouet A, Aubert E, Arnaud M, Le Coq D, Klier A, Rapoport G (1984) The sucrose system as a model of genetic regulation in B. subtilis. In: Ganesan AT, Hoch JA (eds) Genetics and biotechnology of Bacilli. Academic Press, New York, p 113Google Scholar
  9. Gay P, Le Coq D, Steinmetz M, Ferrari E, Hoch JA (1983) Cloning structural gene sacB, which codes for exoenzyme levansucrase of B. subtilis: expression of the gene in E. coli. J Bacteriol 153:1424–1431Google Scholar
  10. Gouy M, Gautier C (1982) Codons usage in bacteria: correlation with gene expressivity. Nucleic Acids Res 10:7055–7074Google Scholar
  11. Gryczan TJ, Israeli-Reches M Dubnau D (1984) Induction of M.L.S. resistance requires ribosomes able to bind inducer. Mol Gen Genet 194:357–361Google Scholar
  12. Haldenwang WG, Banner CDBN, Ollington JF, Losick R, Hoch JA, O'Connor MB, Sonenscheim AL (1981) Mapping a gene under sporulation control by insertion of a drug resistance marker into B. subtilis chromosome. J Bacteriol 142:90–98Google Scholar
  13. Hironouchi S, Weisblum B (1982) Nucleotide sequence and map of pC194, a plasmid that specifies inducible resistance to chloramphenicol. J Bacteriol 150: 815–825Google Scholar
  14. Iglesias A, Trautner TA (1983) Plasmid transformation in B. Subtilis: symmetry of gene conversion in transformation with a hybrid plasmid containing chromosomal DNA. Mol Gen Genet 189:73–76Google Scholar
  15. Ikemura T, Ozeki H (1982) Codon usage and transfer-RNA content: organism specific codon-choice patterns in reference to the isoacceptor contents. Cold Spring Harbor Symp Quant Biol 47:1087–1097Google Scholar
  16. Kersten H (1984) On the biological significance of modified nucleosides in tRNA. In: Progress in nucleic acid research and molecular biology, vol 31, Academic Press, New YorkGoogle Scholar
  17. Kunst F, Pascal M, Lepesant-Kejzlarovà J, Lepesant JA Billault A, Dedonder R (1974) Pleiotropic mutations affecting sporulation conditions and the syntheses of extracellular enzymes in B. subtilis 168. Biochimie 56:1481–1489Google Scholar
  18. Le Brun E, Van Rapenbusch R (1980) The structure of B. subtilis levansucrase at 3.8 A resolution. J Biol Chem 255:12034–12036Google Scholar
  19. Le Coq D, Ratet P, Steinmetz M, Gay P (1984) A genetic approach to levansucrase secretion in B. subtilis. In: Ganesan AT, Hoch JA (eds) Genetics and biotechnology of Bacilli. Academic Press, New York, p 141Google Scholar
  20. Lepesant JA, Kunst F, Lepesant-Kejzlarovà J, Dedonder R (1972) Chromosomal location of mutations affecting sucrose metabolism in B. subtilis Marburg. Mol Gen Genet 118:135–160Google Scholar
  21. Lepesant JA, Kunst F, Pascal M, Lepesant-Kejzlarova J, Steinmetz M, Dedonder R (1976) Specific and pleiotropic regulatory mechanisms in the sucrose system of B. subtilis 168. In: Schlessinger D (ed) Microbiology-1976. Academican Society for Microbiology, Washington, DC p 58Google Scholar
  22. Mitra SK, Ninio J (1978) Recognition between codon and anticodon. The limit of our knowledge. In: Rapoport S (ed) Gene function: Proceedings of the 12th FEBS Meeting. Pergamon Press, Oxford, New York, p 437Google Scholar
  23. Moran CP Jr, Lang N, LeGrice SFJ, lee J, Stephens M, Sonenshein AL, Pero J, Losick R (1982) Nucleotide sequences that signal the inhitiation of transcription and translation in B. subtilis. Mol Gen Genet 186:339–346Google Scholar
  24. Murphy N, McConnell DJ, Cantwell BA (1984) The DNA sequence and genetic control sites for the excreted B. subtilis β-glucanase. Nucleic Acids Res 12:5355–5367Google Scholar
  25. Normak S, Bergstrom S, Edlund T, Grundstrom T, Jaurin, B Lindberg FP, Olsson O (1983) Overlapping genes. Annu Rev Genet 17:499–525Google Scholar
  26. Ohtsubo H, Ohtsubo E (1978) Nucleotid sequence of an insertion element IS1. Proc Natl Acad Sci USA 75:615–619Google Scholar
  27. Pratt T, Bear DG (1983) Role of RNA-polymerase, p factor, and ribosomes in transcription termination. In: Beckwith J, Davies J, Gallant J (eds) Gene function in prokariotes. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 123Google Scholar
  28. Priest FG (1977) Extracellular enzymes in the genus Bacillus. Bacteriol Rev 41:711–753Google Scholar
  29. Sanger FS, Nicklen S, Coulson AR (1977) DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  30. Stahl ML, Ferrati E (1984) Replacement of the B. subtilis subtilisine structural gene with an in-vitro-derived deletion mutation. J Bacteriol 158:411–418Google Scholar
  31. Starzyk R (1984) tRNA base modifications and gene regulation. Trends Biochem Sci 9:333–334Google Scholar
  32. Steinmetz M, Kunst F, Dedonder R (1976) Mapping of mutations affecting synthesis of exocellular enzymes in B. subtilis. Mol Gen Genet 148:281–285Google Scholar
  33. Steinmetz M, Le Coq D, Ben Djemia H, Gay P (1983) Analyse genetique de sacB, gène de structure d'une enzyme secrétée, la levane-saccharase de B. subtilis. Mol Gen Genet 191:138–144Google Scholar
  34. Vold B (1973) Analysis of isoaccepting tRNA species of B. subtilis: chromatographic differences between tRNA from spores and cells in exponential growth. J Bacteriol 113:825–833Google Scholar
  35. Weisblum B (1983) Inducible resistance to macrolides, lincosamides, and streptogramin type-B antibiotics: the resistance phenotype, its biological diversity, and structural elements that regulate expression. In: Beckwith J, Davies J, Gallant J (eds) Gene function in prokariotes. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p 91Google Scholar
  36. Wrinkler ME, Mullis K, Barnett J, Stoynowski I, Yanofsky C (1982) Transcription termination at the tryptophan operon attenuator is decreased in-vitro by an oligomer complementary to a segment of the leader transcript. Proc Natl Acad Sci USA 79:2181–2185Google Scholar
  37. Yang M, Gallizzi A, Henner D (1983) Nucleotide sequence of the amylase gene from B. subtilis. Nucleic Acids Res 11:237–249Google Scholar
  38. Yoneda Y (1982) Regulation of α-amylase production in B. subtilis. In: Ganesan AT, Chang S, Dubnau D (eds) Molecular cloning and gene regulation in Bacilli. Academic Press, New York, p 111Google Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • Michel Steinmetz
    • 1
  • Dominique Le Coq
    • 1
  • Stéphane Aymerich
    • 1
  • Geneviève Gonzy-Tréboul
    • 1
  • Philippe Gay
    • 1
  1. 1.Laboratoire Génétique et Membranes, Institut Jacques MonodCNRSParis Cedex 05France

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