Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1995 Dec;177(23):6928–6936. doi: 10.1128/jb.177.23.6928-6936.1995

The HPr protein of the phosphotransferase system links induction and catabolite repression of the Bacillus subtilis levanase operon.

J Stülke 1, I Martin-Verstraete 1, V Charrier 1, A Klier 1, J Deutscher 1, G Rapoport 1
PMCID: PMC177562  PMID: 7592487

Abstract

The LevR protein is the activator of expression of the levanase operon of Bacillus subtilis. The promoter of this operon is recognized by RNA polymerase containing the sigma 54-like factor sigma L. One domain of the LevR protein is homologous to activators of the NtrC family, and another resembles antiterminator proteins of the BglG family. It has been proposed that the domain which is similar to antiterminators is a target of phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent regulation of LevR activity. We show that the LevR protein is not only negatively regulated by the fructose-specific enzyme IIA/B of the phosphotransferase system encoded by the levanase operon (lev-PTS) but also positively controlled by the histidine-containing phosphocarrier protein (HPr) of the PTS. This second type of control of LevR activity depends on phosphoenolpyruvate-dependent phosphorylation of HPr histidine 15, as demonstrated with point mutations in the ptsH gene encoding HPr. In vitro phosphorylation of partially purified LevR was obtained in the presence of phosphoenolpyruvate, enzyme I, and HPr. The dependence of truncated LevR polypeptides on stimulation by HPr indicated that the domain homologous to antiterminators is the target of HPr-dependent regulation of LevR activity. This domain appears to be duplicated in the LevR protein. The first antiterminator-like domain seems to be the target of enzyme I and HPr-dependent phosphorylation and the site of LevR activation, whereas the carboxy-terminal antiterminator-like domain could be the target for negative regulation by the lev-PTS.

Full Text

The Full Text of this article is available as a PDF (332.7 KB).

Selected References

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

  1. Amster-Choder O., Houman F., Wright A. Protein phosphorylation regulates transcription of the beta-glucoside utilization operon in E. coli. Cell. 1989 Sep 8;58(5):847–855. doi: 10.1016/0092-8674(89)90937-9. [DOI] [PubMed] [Google Scholar]
  2. Arantes O., Lereclus D. Construction of cloning vectors for Bacillus thuringiensis. Gene. 1991 Dec 1;108(1):115–119. doi: 10.1016/0378-1119(91)90495-w. [DOI] [PubMed] [Google Scholar]
  3. Arnaud M., Vary P., Zagorec M., Klier A., Debarbouille M., Postma P., Rapoport G. Regulation of the sacPA operon of Bacillus subtilis: identification of phosphotransferase system components involved in SacT activity. J Bacteriol. 1992 May;174(10):3161–3170. doi: 10.1128/jb.174.10.3161-3170.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beijer L., Rutberg L. Utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in Bacillus subtilis. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):217–220. doi: 10.1111/j.1574-6968.1992.tb14043.x. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Cvitkovitch D. G., Boyd D. A., Thevenot T., Hamilton I. R. Glucose transport by a mutant of Streptococcus mutans unable to accumulate sugars via the phosphoenolpyruvate phosphotransferase system. J Bacteriol. 1995 May;177(9):2251–2258. doi: 10.1128/jb.177.9.2251-2258.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Deutscher J., Pevec B., Beyreuther K., Kiltz H. H., Hengstenberg W. Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr. Biochemistry. 1986 Oct 21;25(21):6543–6551. doi: 10.1021/bi00369a031. [DOI] [PubMed] [Google Scholar]
  10. Deutscher J., Reizer J., Fischer C., Galinier A., Saier M. H., Jr, Steinmetz M. Loss of protein kinase-catalyzed phosphorylation of HPr, a phosphocarrier protein of the phosphotransferase system, by mutation of the ptsH gene confers catabolite repression resistance to several catabolic genes of Bacillus subtilis. J Bacteriol. 1994 Jun;176(11):3336–3344. doi: 10.1128/jb.176.11.3336-3344.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deutscher J., Saier M. H., Jr ATP-dependent protein kinase-catalyzed phosphorylation of a seryl residue in HPr, a phosphate carrier protein of the phosphotransferase system in Streptococcus pyogenes. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6790–6794. doi: 10.1073/pnas.80.22.6790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Deutscher J., Sauerwald H. Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase system. J Bacteriol. 1986 Jun;166(3):829–836. doi: 10.1128/jb.166.3.829-836.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Débarbouillé M., Martin-Verstraete I., Kunst F., Rapoport G. The Bacillus subtilis sigL gene encodes an equivalent of sigma 54 from gram-negative bacteria. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9092–9096. doi: 10.1073/pnas.88.20.9092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Eisermann R., Deutscher J., Gonzy-Treboul G., Hengstenberg W. Site-directed mutagenesis with the ptsH gene of Bacillus subtilis. Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site. J Biol Chem. 1988 Nov 15;263(32):17050–17054. [PubMed] [Google Scholar]
  16. Fujita Y., Miwa Y. Catabolite repression of the Bacillus subtilis gnt operon mediated by the CcpA protein. J Bacteriol. 1994 Jan;176(2):511–513. doi: 10.1128/jb.176.2.511-513.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gay P., Cordier P., Marquet M., Delobbe A. Carbohydrate metabolism and transport in Bacillus subtilis. A study of ctr mutations. Mol Gen Genet. 1973 Mar 19;121(4):355–368. doi: 10.1007/BF00433234. [DOI] [PubMed] [Google Scholar]
  18. Gonzy-Tréboul G., Steinmetz M. Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene. J Bacteriol. 1987 May;169(5):2287–2290. doi: 10.1128/jb.169.5.2287-2290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gonzy-Tréboul G., de Waard J. H., Zagorec M., Postma P. W. The glucose permease of the phosphotransferase system of Bacillus subtilis: evidence for IIGlc and IIIGlc domains. Mol Microbiol. 1991 May;5(5):1241–1249. doi: 10.1111/j.1365-2958.1991.tb01898.x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Kohlbrecher D., Eisermann R., Hengstenberg W. Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: molecular cloning and nucleotide sequence of the Staphylococcus carnosus ptsI gene and expression and complementation studies of the gene product. J Bacteriol. 1992 Apr;174(7):2208–2214. doi: 10.1128/jb.174.7.2208-2214.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Kunst F., Rapoport G. Salt stress is an environmental signal affecting degradative enzyme synthesis in Bacillus subtilis. J Bacteriol. 1995 May;177(9):2403–2407. doi: 10.1128/jb.177.9.2403-2407.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kunst F., Steinmetz M., Lepesant J. A., Dedonder R. Presence of a third sucrose hydrolyzing enzyme in Bacillus subtilis: constitutive levanase synthesis by mutants of Bacillus subtilis Marburg 168. Biochimie. 1977;59(3):289–292. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Lindner C., Stülke J., Hecker M. Regulation of xylanolytic enzymes in Bacillus subtilis. Microbiology. 1994 Apr;140(Pt 4):753–757. doi: 10.1099/00221287-140-4-753. [DOI] [PubMed] [Google Scholar]
  29. Lopez J. M., Thoms B. Role of sugar uptake and metabolic intermediates on catabolite repression in Bacillus subtilis. J Bacteriol. 1977 Jan;129(1):217–224. doi: 10.1128/jb.129.1.217-224.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lévy S., Zeng G. Q., Danchin A. Cyclic AMP synthesis in Escherichia coli strains bearing known deletions in the pts phosphotransferase operon. Gene. 1990 Jan 31;86(1):27–33. doi: 10.1016/0378-1119(90)90110-d. [DOI] [PubMed] [Google Scholar]
  31. Martin-Verstraete I., Débarbouillé M., Klier A., Rapoport G. Interactions of wild-type and truncated LevR of Bacillus subtilis with the upstream activating sequence of the levanase operon. J Mol Biol. 1994 Aug 12;241(2):178–192. doi: 10.1006/jmbi.1994.1487. [DOI] [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., Débarbouillé M., Klier A., Rapoport G. Mutagenesis of the Bacillus subtilis "-12, -24" promoter of the levanase operon and evidence for the existence of an upstream activating sequence. J Mol Biol. 1992 Jul 5;226(1):85–99. doi: 10.1016/0022-2836(92)90126-5. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Martin I., Débarbouillé M., Ferrari E., Klier A., Rapoport G. Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase. Mol Gen Genet. 1987 Jun;208(1-2):177–184. doi: 10.1007/BF00330439. [DOI] [PubMed] [Google Scholar]
  37. Mattoo R. L., Waygood E. B. An enzymatic method for [32P]phosphoenolpyruvate synthesis. Anal Biochem. 1983 Jan;128(1):245–249. doi: 10.1016/0003-2697(83)90372-x. [DOI] [PubMed] [Google Scholar]
  38. Postma P. W., Lengeler J. W., Jacobson G. R. Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. Microbiol Rev. 1993 Sep;57(3):543–594. doi: 10.1128/mr.57.3.543-594.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reizer J., Sutrina S. L., Saier M. H., Stewart G. C., Peterkofsky A., Reddy P. Mechanistic and physiological consequences of HPr(ser) phosphorylation on the activities of the phosphoenolpyruvate:sugar phosphotransferase system in gram-positive bacteria: studies with site-specific mutants of HPr. EMBO J. 1989 Jul;8(7):2111–2120. doi: 10.1002/j.1460-2075.1989.tb03620.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Roossien F. F., Brink J., Robillard G. T. A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium. Biochim Biophys Acta. 1983 Oct 4;760(1):185–187. doi: 10.1016/0304-4165(83)90141-1. [DOI] [PubMed] [Google Scholar]
  41. Roy A., Haziza C., Danchin A. Regulation of adenylate cyclase synthesis in Escherichia coli: nucleotide sequence of the control region. EMBO J. 1983;2(5):791–797. doi: 10.1002/j.1460-2075.1983.tb01502.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Saier M. H., Jr Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system. Microbiol Rev. 1989 Mar;53(1):109–120. doi: 10.1128/mr.53.1.109-120.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Schnetz K., Rak B. Beta-glucoside permease represses the bgl operon of Escherichia coli by phosphorylation of the antiterminator protein and also interacts with glucose-specific enzyme III, the key element in catabolite control. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5074–5078. doi: 10.1073/pnas.87.13.5074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Tangney M., Buchanan C. J., Priest F. G., Mitchell W. J. Maltose uptake and its regulation in Bacillus subtilis. FEMS Microbiol Lett. 1992 Oct 1;76(1-2):191–196. doi: 10.1016/0378-1097(92)90385-2. [DOI] [PubMed] [Google Scholar]
  48. Trieu-Cuot P., Courvalin P. Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III. Gene. 1983 Sep;23(3):331–341. doi: 10.1016/0378-1119(83)90022-7. [DOI] [PubMed] [Google Scholar]
  49. Weigel N., Powers D. A., Roseman S. Sugar transport by the bacterial phosphotransferase system. Primary structure and active site of a general phosphocarrier protein (HPr) from Salmonella typhimurium. J Biol Chem. 1982 Dec 10;257(23):14499–14509. [PubMed] [Google Scholar]
  50. Weinrauch Y., Msadek T., Kunst F., Dubnau D. Sequence and properties of comQ, a new competence regulatory gene of Bacillus subtilis. J Bacteriol. 1991 Sep;173(18):5685–5693. doi: 10.1128/jb.173.18.5685-5693.1991. [DOI] [PMC free article] [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]

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

RESOURCES