Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Jan;179(2):496–506. doi: 10.1128/jb.179.2.496-506.1997

Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis.

S Tobisch 1, P Glaser 1, S Krüger 1, M Hecker 1
PMCID: PMC178721  PMID: 8990303

Abstract

A new catabolic system in Bacillus subtilis involved in utilization of beta-glucosidic compounds has been investigated. It consists of five genes encoding phosphotransferase system (PTS) enzyme II (licB and licC) and enzyme IIA (licA), a presumed 6-phospho-beta-glucosidase (licH), as well as a putative regulator protein (licR). The genes map around 334 degrees of the B. subtilis chromosome, and their products are involved in the uptake and utilization of lichenan degradation products. These five genes are organized in two transcriptional units. A weak promoter precedes gene licR, and transcription is obviously terminated at a secondary structure immediately downstream of the reading frame, as shown by Northern RNA blot analysis. Genes licB, licC, licA, and licH constitute an operon. Initiation of transcription at the promoter in front of this operon presumably requires activation by the gene product of licR. The LicR protein shows an unusual domain structure, i.e., similarities to (i) the conserved transcriptional antiterminator BgIG family signature and (ii) PTS enzyme II. Using RNA techniques and transcriptional lacZ fusions, we have shown that the expression of the licBCAH operon is inducible by products of lichenan hydrolysis, lichenan and cellobiose. The presence of excess glucose prevents the induction of this operon, indicating the control by carbon catabolite repression. Moreover, the expression of the operon requires the general PTS components and seems to be negatively controlled by the specific lic PTS enzymes.

Full Text

The Full Text of this article is available as a PDF (377.2 KB).

Selected References

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

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Arnaud M., Débarbouillé M., Rapoport G., Saier M. H., Jr, Reizer J. In vitro reconstitution of transcriptional antitermination by the SacT and SacY proteins of Bacillus subtilis. J Biol Chem. 1996 Aug 2;271(31):18966–18972. doi: 10.1074/jbc.271.31.18966. [DOI] [PubMed] [Google Scholar]
  3. Aymerich S., Gonzy-Tréboul G., Steinmetz M. 5'-noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis. J Bacteriol. 1986 Jun;166(3):993–998. doi: 10.1128/jb.166.3.993-998.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aymerich S., Steinmetz M. Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10410–10414. doi: 10.1073/pnas.89.21.10410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beck von Bodman S., Hayman G. T., Farrand S. K. Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):643–647. doi: 10.1073/pnas.89.2.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  7. Breidt F., Jr, Hengstenberg W., Finkeldei U., Stewart G. C. Identification of the genes for the lactose-specific components of the phosphotransferase system in the lac operon of Staphylococcus aureus. J Biol Chem. 1987 Dec 5;262(34):16444–16449. [PubMed] [Google Scholar]
  8. Crutz A. M., Steinmetz M., Aymerich S., Richter R., Le Coq D. Induction of levansucrase in Bacillus subtilis: an antitermination mechanism negatively controlled by the phosphotransferase system. J Bacteriol. 1990 Feb;172(2):1043–1050. doi: 10.1128/jb.172.2.1043-1050.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Debarbouille M., Arnaud M., Fouet A., Klier A., Rapoport G. The sacT gene regulating the sacPA operon in Bacillus subtilis shares strong homology with transcriptional antiterminators. J Bacteriol. 1990 Jul;172(7):3966–3973. doi: 10.1128/jb.172.7.3966-3973.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deutscher J., Küster E., Bergstedt U., Charrier V., Hillen W. Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria. Mol Microbiol. 1995 Mar;15(6):1049–1053. doi: 10.1111/j.1365-2958.1995.tb02280.x. [DOI] [PubMed] [Google Scholar]
  11. Débarbouillé M., Martin-Verstraete I., Klier A., Rapoport G. The transcriptional regulator LevR of Bacillus subtilis has domains homologous to both sigma 54- and phosphotransferase system-dependent regulators. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2212–2216. doi: 10.1073/pnas.88.6.2212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fischer R., von Strandmann R. P., Hengstenberg W. Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the enzyme IIIMtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes. J Bacteriol. 1991 Jun;173(12):3709–3715. doi: 10.1128/jb.173.12.3709-3715.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fisher S. H., Sonenshein A. L. Control of carbon and nitrogen metabolism in Bacillus subtilis. Annu Rev Microbiol. 1991;45:107–135. doi: 10.1146/annurev.mi.45.100191.000543. [DOI] [PubMed] [Google Scholar]
  14. Glaser P., Kunst F., Arnaud M., Coudart M. P., Gonzales W., Hullo M. F., Ionescu M., Lubochinsky B., Marcelino L., Moszer I. Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325 degrees to 333 degrees. Mol Microbiol. 1993 Oct;10(2):371–384. [PubMed] [Google Scholar]
  15. Hall B. G., Xu L. Nucleotide sequence, function, activation, and evolution of the cryptic asc operon of Escherichia coli K12. Mol Biol Evol. 1992 Jul;9(4):688–706. doi: 10.1093/oxfordjournals.molbev.a040753. [DOI] [PubMed] [Google Scholar]
  16. Henikoff S., Haughn G. W., Calvo J. M., Wallace J. C. A large family of bacterial activator proteins. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6602–6606. doi: 10.1073/pnas.85.18.6602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Henkin T. M., Grundy F. J., Nicholson W. L., Chambliss G. H. Catabolite repression of alpha-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lacl and galR repressors. Mol Microbiol. 1991 Mar;5(3):575–584. doi: 10.1111/j.1365-2958.1991.tb00728.x. [DOI] [PubMed] [Google Scholar]
  18. Hoch J. A. Genetic analysis in Bacillus subtilis. Methods Enzymol. 1991;204:305–320. doi: 10.1016/0076-6879(91)04015-g. [DOI] [PubMed] [Google Scholar]
  19. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  20. Hueck C. J., Hillen W. Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria? Mol Microbiol. 1995 Feb;15(3):395–401. doi: 10.1111/j.1365-2958.1995.tb02252.x. [DOI] [PubMed] [Google Scholar]
  21. Hueck C. J., Hillen W., Saier M. H., Jr Analysis of a cis-active sequence mediating catabolite repression in gram-positive bacteria. Res Microbiol. 1994 Sep;145(7):503–518. doi: 10.1016/0923-2508(94)90028-0. [DOI] [PubMed] [Google Scholar]
  22. Itaya M. Construction of a novel tetracycline resistance gene cassette useful as a marker on the Bacillus subtilis chromosome. Biosci Biotechnol Biochem. 1992 Apr;56(4):685–686. doi: 10.1271/bbb.56.685. [DOI] [PubMed] [Google Scholar]
  23. Kraus A., Hueck C., Gärtner D., Hillen W. Catabolite repression of the Bacillus subtilis xyl operon involves a cis element functional in the context of an unrelated sequence, and glucose exerts additional xylR-dependent repression. J Bacteriol. 1994 Mar;176(6):1738–1745. doi: 10.1128/jb.176.6.1738-1745.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krüger S., Gertz S., Hecker M. Transcriptional analysis of bglPH expression in Bacillus subtilis: evidence for two distinct pathways mediating carbon catabolite repression. J Bacteriol. 1996 May;178(9):2637–2644. doi: 10.1128/jb.178.9.2637-2644.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krüger S., Hecker M. Regulation of the putative bglPH operon for aryl-beta-glucoside utilization in Bacillus subtilis. J Bacteriol. 1995 Oct;177(19):5590–5597. doi: 10.1128/jb.177.19.5590-5597.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Krüger S., Stülke J., Hecker M. Catabolite repression of beta-glucanase synthesis in Bacillus subtilis. J Gen Microbiol. 1993 Sep;139(9):2047–2054. doi: 10.1099/00221287-139-9-2047. [DOI] [PubMed] [Google Scholar]
  27. Kunst F., Vassarotti A., Danchin A. Organization of the European Bacillus subtilis genome sequencing project. Microbiology. 1995 Feb;141(Pt 2):249–255. doi: 10.1099/13500872-141-2-249. [DOI] [PubMed] [Google Scholar]
  28. Lai X., Ingram L. O. Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli. J Bacteriol. 1993 Oct;175(20):6441–6450. doi: 10.1128/jb.175.20.6441-6450.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Le Coq D., Lindner C., Krüger S., Steinmetz M., Stülke J. New beta-glucoside (bgl) genes in Bacillus subtilis: the bglP gene product has both transport and regulatory functions similar to those of BglF, its Escherichia coli homolog. J Bacteriol. 1995 Mar;177(6):1527–1535. doi: 10.1128/jb.177.6.1527-1535.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lee C. A., Saier M. H., Jr Mannitol-specific enzyme II of the bacterial phosphotransferase system. III. The nucleotide sequence of the permease gene. J Biol Chem. 1983 Sep 10;258(17):10761–10767. [PubMed] [Google Scholar]
  31. Liljeström P. L., Liljeström P. Nucleotide sequence of the melA gene, coding for alpha-galactosidase in Escherichia coli K-12. Nucleic Acids Res. 1987 Mar 11;15(5):2213–2220. doi: 10.1093/nar/15.5.2213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Martin-Verstraete I., Débarbouillé M., Klier A., Rapoport G. Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon. J Mol Biol. 1990 Aug 5;214(3):657–671. doi: 10.1016/0022-2836(90)90284-S. [DOI] [PubMed] [Google Scholar]
  33. Martin-Verstraete I., Stülke J., Klier A., Rapoport G. Two different mechanisms mediate catabolite repression of the Bacillus subtilis levanase operon. J Bacteriol. 1995 Dec;177(23):6919–6927. doi: 10.1128/jb.177.23.6919-6927.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Martin I., Debarbouille M., Klier A., Rapoport G. Induction and metabolite regulation of levanase synthesis in Bacillus subtilis. J Bacteriol. 1989 Apr;171(4):1885–1892. doi: 10.1128/jb.171.4.1885-1892.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Murphy N., McConnell D. J., Cantwell B. A. The DNA sequence of the gene and genetic control sites for the excreted B. subtilis enzyme beta-glucanase. Nucleic Acids Res. 1984 Jul 11;12(13):5355–5367. doi: 10.1093/nar/12.13.5355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Parker L. L., Hall B. G. Characterization and nucleotide sequence of the cryptic cel operon of Escherichia coli K12. Genetics. 1990 Mar;124(3):455–471. doi: 10.1093/genetics/124.3.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Parker L. L., Hall B. G. Mechanisms of activation of the cryptic cel operon of Escherichia coli K12. Genetics. 1990 Mar;124(3):473–482. doi: 10.1093/genetics/124.3.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Priest F. G. Extracellular enzyme synthesis in the genus Bacillus. Bacteriol Rev. 1977 Sep;41(3):711–753. doi: 10.1128/br.41.3.711-753.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rosey E. L., Stewart G. C. Nucleotide and deduced amino acid sequences of the lacR, lacABCD, and lacFE genes encoding the repressor, tagatose 6-phosphate gene cluster, and sugar-specific phosphotransferase system components of the lactose operon of Streptococcus mutans. J Bacteriol. 1992 Oct;174(19):6159–6170. doi: 10.1128/jb.174.19.6159-6170.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Saier M. H., Jr, Reizer J. Proposed uniform nomenclature for the proteins and protein domains of the bacterial phosphoenolpyruvate: sugar phosphotransferase system. J Bacteriol. 1992 Mar;174(5):1433–1438. doi: 10.1128/jb.174.5.1433-1438.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schnetz K., Stülke J., Gertz S., Krüger S., Krieg M., Hecker M., Rak B. LicT, a Bacillus subtilis transcriptional antiterminator protein of the BglG family. J Bacteriol. 1996 Apr;178(7):1971–1979. doi: 10.1128/jb.178.7.1971-1979.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schnetz K., Toloczyki C., Rak B. Beta-glucoside (bgl) operon of Escherichia coli K-12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus subtilis genes. J Bacteriol. 1987 Jun;169(6):2579–2590. doi: 10.1128/jb.169.6.2579-2590.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stülke J., Hanschke R., Hecker M. Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool. J Gen Microbiol. 1993 Sep;139(9):2041–2045. doi: 10.1099/00221287-139-9-2041. [DOI] [PubMed] [Google Scholar]
  46. Stülke J., Martin-Verstraete I., Charrier V., Klier A., Deutscher J., Rapoport G. The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon. J Bacteriol. 1995 Dec;177(23):6928–6936. doi: 10.1128/jb.177.23.6928-6936.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Völker U., Engelmann S., Maul B., Riethdorf S., Völker A., Schmid R., Mach H., Hecker M. Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology. 1994 Apr;140(Pt 4):741–752. doi: 10.1099/00221287-140-4-741. [DOI] [PubMed] [Google Scholar]
  48. Wetzstein M., Völker U., Dedio J., Löbau S., Zuber U., Schiesswohl M., Herget C., Hecker M., Schumann W. Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. J Bacteriol. 1992 May;174(10):3300–3310. doi: 10.1128/jb.174.10.3300-3310.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Witt E., Frank R., Hengstenberg W. 6-Phospho-beta-galactosidases of gram-positive and 6-phospho-beta-glucosidase B of gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of Staphylococcus aureus. Protein Eng. 1993 Nov;6(8):913–920. doi: 10.1093/protein/6.8.913. [DOI] [PubMed] [Google Scholar]
  50. Zhang J., Aronson A. A Bacillus subtilis bglA gene encoding phospho-beta-glucosidase is inducible and closely linked to a NADH dehydrogenase-encoding gene. Gene. 1994 Mar 11;140(1):85–90. doi: 10.1016/0378-1119(94)90735-8. [DOI] [PubMed] [Google Scholar]
  51. Zukowski M. M., Miller L., Cosgwell P., Chen K., Aymerich S., Steinmetz M. Nucleotide sequence of the sacS locus of Bacillus subtilis reveals the presence of two regulatory genes. Gene. 1990 May 31;90(1):153–155. doi: 10.1016/0378-1119(90)90453-x. [DOI] [PubMed] [Google Scholar]
  52. de Vos W. M., Boerrigter I., van Rooyen R. J., Reiche B., Hengstenberg W. Characterization of the lactose-specific enzymes of the phosphotransferase system in Lactococcus lactis. J Biol Chem. 1990 Dec 25;265(36):22554–22560. [PubMed] [Google Scholar]

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

RESOURCES