1887

Abstract

, a bacterium of biotechnological interest due to its ability to produce mosquitocidal toxins, is unable to use sugars as carbon source. However, genes encoding HPr and EI proteins belonging to a PTS were cloned, sequenced and characterized. Both HPr and EI proteins were fully functional for phosphoenolpyruvate-dependent transphosphorylation in complementation assays using extracts from mutants for one of these proteins. HPr(His) was purified from wild-type and a Ser46/Gln mutant of , and used for phosphorylation experiments using extracts from either or as kinase source. The results showed that both phosphorylated forms, P-Ser46-HPr and P-His15-HPr, could be obtained. The findings also proved indirectly the existence of an HPr kinase activity in . The genetic structure of these genes has some unusual features, as they are co-transcribed with genes encoding metabolic enzymes related to -acetylglucosamine (GlcNAc) catabolism (, and an undetermined ). In fact, this bacterium was able to utilize this amino sugar as carbon and energy source, but a null mutant had lost this characteristic. Investigation of GlcNAc uptake and streptozotocin inhibition in both a wild-type and a null mutant strain led to the proposal that GlcNAc is transported and phosphorylated by an EII element of the PTS, as yet uncharacterized. In addition, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase activities were determined; both were induced in the presence of GlcNAc. These results, together with the authors' recent findings of the presence of a phosphofructokinase activity, are strongly indicative of a glycolytic pathway in . They also open new possibilities for genetic improvements in industrial applications.

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2003-07-01
2019-10-18
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References

  1. Alexander, B. & Priest, F. G. ( 1990; ). Numerical classification and identification of Bacillus sphaericus including some strains pathogenic for mosquito larvae. J Gen Microbiol 136, 367–376.[CrossRef]
    [Google Scholar]
  2. Alice, A. F. ( 2001; ). Sistema de fosfotransferasa dependiente del fosfoenolpiruvato (PTS) y represión catabólica en Bacillus subtilis. PhD Thesis, University of Buenos Aires.
  3. Alice, A. F., Perez-Martinez, G. & Sanchez-Rivas, C. ( 2002; ). Existence of a true phosphofructokinase in Bacillus sphaericus: cloning and sequencing of the pfk gene. Appl Environ Microbiol 68, 6410–6415.[CrossRef]
    [Google Scholar]
  4. Altschul, S. F., Madden, T. L., Schaeffer, A. A., Zhang, A., Miller, W. & Lipman, D. J. ( 1997; ). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3398–3402.
    [Google Scholar]
  5. Ammer, J., Brennenstuhl, M., Schindler, P., Höltje, J. V. & Zähner, H. ( 1979; ). Phosphorylation of streptozotocin during uptake via the phosphoenolpyruvate : sugar phosphotransferase system in Escherichia coli. Antimicrob Agents Chemother 16, 801–807.[CrossRef]
    [Google Scholar]
  6. Bates, C. J. & Pasternak, C. A. ( 1965; ). Further studies on the regulation of amino sugar metabolism in Bacillus subtilis. Biochem J 96, 147–154.
    [Google Scholar]
  7. Baumann, P., Clark, M. A., Baumann, L. & Broadwell, A. H. ( 1991; ). Bacillus sphaericus as a mosquito pathogen: properties of the organism and its toxins. Microbiol Rev 55, 425–436.
    [Google Scholar]
  8. Charles, J. F., Nielsen-Le Roux, C. & Delécluse, A. ( 1996; ). Bacillus sphaericus toxins: molecular biology and mode of action. Annu Rev Entomol 41, 451–472.[CrossRef]
    [Google Scholar]
  9. Charrier, V., Buckley, E., Parsonage, D., Galinier, A., Darbon, E., Jaquinod, M., Forest, E., Deutscher, J. & Clairbone, A. ( 1997; ). Cloning and sequencing of two enterococcal glpK genes and regulation of the encoded glycerol kinases by phosphoenolpyruvate-dependent, phosphotransferase system-catalyzed phosphorylation of a single histidyl residue. J Biol Chem 272, 14166–14174.[CrossRef]
    [Google Scholar]
  10. Clarke, J. S. & Pasternak, C. A. ( 1962; ). The regulation of amino sugar metabolism in Bacillus subtilis. Biochem J 84, 185–191.
    [Google Scholar]
  11. Couch, T. L. ( 2000; ). Industrial fermentation and formulation of entomopathogenic bacteria. In Entomopathogenic Bacteria: from Laboratory to Field Application, pp. 297–316. Edited by J. F. Charles and others. Dordrecht:: Kluwer.
  12. deBarjac, H., Veron, M. & Cosmao Dumanoir, V. ( 1980; ). Caractérisation biochimique et sérologique de souches de Bacillus sphaericus pathogènes ou non pour les moustiques. Ann Microbiol 131B, 191–201.
    [Google Scholar]
  13. Deutscher, J., Küster, E., Bergstedt, U., Charrier, V. & Hillen, W. ( 1995; ). Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to catabolite repression in Gram-positive bacteria. Mol Microbiol 15, 1049–1053.[CrossRef]
    [Google Scholar]
  14. Dossonnet, V., Monedero, V., Zagorec, M., Galinier, A., Perez-Martinez, G. & Deutscher, J. ( 2000; ). Phosphorylation of HPr by the bifunctional HPr kinase/P-Ser-HPr phosphatase from Lactobacillus casei controls catabolite repression and inducer exclusion but not inducer expulsion. J Bacteriol 182, 2582–2590.[CrossRef]
    [Google Scholar]
  15. Fujita, Y., Miwa, Y. Galinier A. & Deutscher, J. ( 1995; ). Specific recognition of the Bacillus subtilis gnt cis-acting catabolite-responsive element by a protein complex formed between CcpA and seryl-phosphorylated HPr. Mol Microbiol 17, 953–960.[CrossRef]
    [Google Scholar]
  16. Galinier, A., Kravanja, M., Engelmann, R., Hengstenberg, W., Kilhoffer, M. C., Deutscher, J. & Hiech, J. ( 1998; ). New protein kinase and protein phosphatase families mediate signal transduction in bacterial catabolite repression. Proc Natl Acad Sci U S A 95, 1823–1828.[CrossRef]
    [Google Scholar]
  17. Gonzy-Tréboul, G., Zagorec, M., Rain-Guian, M. C. & Steinmetz, M. ( 1989; ). Phosphoenolpyruvate : sugar phosphotransferase system of Bacillus subtilis: nucleotide sequence of ptsX, ptsH and the 5′ end of the ptsI and evidence for a ptsHI operon. Mol Microbiol 3, 103–112.[CrossRef]
    [Google Scholar]
  18. Guérout-Fleury, A. M., Shazand, K., Frandsen, N. & Stragier, P. ( 1995; ). Antibiotic-resistance cassettes for Bacillus subtilis. Gene 167, 335–336.[CrossRef]
    [Google Scholar]
  19. Haldenwang, W. G. ( 1995; ). The sigma factors of Bacillus subtilis. Microbiol Rev 59, 1–30.
    [Google Scholar]
  20. Hengstenberg, W., Penberthy, W., Hill, K. & Morse, M. ( 1969; ). Phosphotransferase system of Staphylococcus aureus: its requirement for the accumulation and metabolism of galactosides. J Bacteriol 99, 383–388.
    [Google Scholar]
  21. Henkin, T. M., Grundy, F. J., Nicholson, W. L. & Chamblis, G. H. (1991; ). Catabolite repression of α-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli LacI and GalR repressors. Mol Microbiol 5, 575–584.[CrossRef]
    [Google Scholar]
  22. Himmelreich, R., Hilbert, H., Plagens, H., Pirkl, E., Li, B. C. & Herrmann, R. ( 1996; ). Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res 24, 4420–4449.[CrossRef]
    [Google Scholar]
  23. Hoischen, C., Dijkstra, A., Rottem, S., Reizer, J. & Saier, M. H., Jr ( 1993; ). Presence of protein constituents of the Gram-positive bacterial phosphotransferase regulatory system in Acholeplasma laidlawii. J Bacteriol 175, 6599–6604.
    [Google Scholar]
  24. Iwamoto, R. & Imanaga, Y. ( 1991; ). Direct evidence of the Entner–Doudoroff pathway operating in the metabolism of d-glucosamine in bacteria. J Biochem 109, 66–69.
    [Google Scholar]
  25. Jacobson, G. R., Poy, F. & Lengeler, J. W. ( 1990; ). Inhibition of Streptococcus mutans by the antibiotic streptozotocin: mechanisms of uptake and the selection of carbohydrate-negative mutants. Infect Immun 58, 543–549.
    [Google Scholar]
  26. Jault, J. M., Fieulaine, S., Nessler, S., Gonzalo, P., Di Pietro, A., Deutscher, J. & Galinier, A. ( 2000; ). The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding. J Biol Chem 275, 1773–1780.[CrossRef]
    [Google Scholar]
  27. Jones-Mortimer, M. C. & Kornberg, H. L. ( 1980; ). Amino-sugar transport systems in Escherichia coli K12. J Gen Microbiol 117, 369–376.
    [Google Scholar]
  28. Kravanja, M., Engelmann, R., Dossonnet, V. & 7 other authors (1999; ). The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase. Mol Microbiol 31, 59–66.[CrossRef]
    [Google Scholar]
  29. Krych, V. K., Johnson, J. L. & Yousten, A. A. ( 1980; ). Deoxyribonucleic acid homologies among strains of Bacillus sphaericus. Int J Syst Bacteriol 30, 476–482.[CrossRef]
    [Google Scholar]
  30. Lai, X. & Ingram, L. O. ( 1995; ). Discovery of a ptsHI operon, which includes a third gene (ptsT), in the thermophile Bacillus stearothermophilus. Microbiology 141, 1443–1449.[CrossRef]
    [Google Scholar]
  31. Landt, O., Grunert, H. P. & Hanh, U. ( 1990; ). A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96, 125–128.[CrossRef]
    [Google Scholar]
  32. Lengeler, J. ( 1980; ). Analysis of the physiological effects of the antibiotic streptozotocin on Escherichia coli K12 and other sensitive bacteria. Arch Microbiol 128, 196–203.[CrossRef]
    [Google Scholar]
  33. Ludwig, H., Homuth, G., Schmalisch, M., Dyka, F. M., Hecker, M. & Stülke, J. ( 2001; ). Transcription of glycolytic genes and operons in Bacillus subtilis: evidence for the presence of multiple levels of control of the gapA operon. Mol Microbiol 41, 409–422.[CrossRef]
    [Google Scholar]
  34. Martin-Verstraete, I., Débarbouillé, M., Klier, A. & Rapoport, G. ( 1990; ). Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon. J Mol Biol 214, 657–671.[CrossRef]
    [Google Scholar]
  35. Mijakovic, I., Poncet, S., Galinier, A. & 9 other authors ( 2002; ). Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: a relic of early life? Proc Natl Acad Sci U S A 99, 13442–13447.[CrossRef]
    [Google Scholar]
  36. Mitchell, W., Reizer, J., Herring, C., Hoischen, C. & Saier, M. H., Jr ( 1993; ). Identification of a phosphoenolpyruvate : fructose phosphotransferase system (fructose-1-phosphate forming) in Listeria monocytogenes. J Bacteriol 175, 2758–2761.
    [Google Scholar]
  37. Mobley, H. T., Doyle, R. J., Streips, U. N. & Langemeier, S. O. ( 1982; ). Transport and incorporation of N-acetylglucosamine in Bacillus subtilis. J Bacteriol 150, 8–15.
    [Google Scholar]
  38. Peri, K. G. & Waygood, E. B. ( 1988; ). Sequence of cloned Enzyme IINag of the phosphoenolpyruvate : N-acetylglucosamine phosphotransferase system of Escherichia coli. Biochemistry 27, 6054–6061.[CrossRef]
    [Google Scholar]
  39. Plumbridge, J. ( 1989; ). Sequence of the nagBACD operon in Escherichia coli K12 and pattern of transcription within the nag regulon. Mol Microbiol 3, 506–515.
    [Google Scholar]
  40. Plumbridge, J. ( 1995; ). Co-ordinated regulation of amino sugar biosynthesis and degradation: the NagC repressor acts as both an activator and a repressor for the transcription of the glmUS operon and requires two separated NagC binding sites. EMBO J 15, 3958–3965.
    [Google Scholar]
  41. Plumbridge, J. ( 2001; ). Regulation of PTS gene expression by the homologous transcriptional regulators, Mlc and NagC, in Escherichia coli (or how two similar repressors can behave differently). J Mol Microbiol Biotechnol 3, 371–380.
    [Google Scholar]
  42. Porter, A. G., Davidson, E. W. & Liu, J. W. ( 1993; ). Mosquitocidal toxins of Bacilli and their genetic manipulation for effective biological control of mosquitoes. Microbiol Rev 57, 838–861.
    [Google Scholar]
  43. Postma, P. W., Lengeler, J. W. & Jacobson, G. R. ( 1993; ). Phosphoenolpyruvate : carbohydrate phosphotransferase systems of bacteria. Microbiol Rev 57, 543–594.
    [Google Scholar]
  44. Priest, F. G. ( 2000; ). Biodiversity of the entomopathogenic, endospore-forming bacteria. In Entomopathogenic Bacteria: from Laboratory to Field Application, pp. 1–22. Edited by J. F. Charles and others. Dordrecht: Kluwer.
  45. Reizer, J., Peterkofsky, A. & Romano, A. H. ( 1988; ). Evidence for the presence of heat-stable protein (HPr) and ATP-dependent HPr kinase in heterofermentative lactobacilli lacking phosphoenolpyruvate : glycose phosphotransferase activity. Proc Natl Acad Sci U S A 85, 2041–2045.[CrossRef]
    [Google Scholar]
  46. Reizer, J., Hoischen, C., Titgemeyer, F., Rivolta, C., Rabus, R., Stülke, J., Karamata, D., Saier, M. H., Jr & Hillen, W. ( 1998; ). A novel protein kinase that controls carbon catabolite repression in bacteria. Mol Microbiol 27, 1157–1169.[CrossRef]
    [Google Scholar]
  47. Reizer, J., Bachem, S., Reizer, A., Arnaud, M., Saier, M. H., Jr & Stülke, J. ( 1999a; ). Novel phosphotransferase system genes revealed by genome analysis – the complete complement of PTS proteins encoded within the genome of Bacillus subtilis. Microbiology 145, 3419–3429.
    [Google Scholar]
  48. Reizer, J., Reizer, A., Lagrou, M. J., Folger, K. R., Kendall Stover, C. & Saier, M. H., Jr ( 1999b; ). Novel phosphotransferase systems revealed by bacterial genome analysis: the complete repertoire of pts genes in Pseudomonas aeruginosa. J Mol Microbiol Biotechnol 1, 289–293.
    [Google Scholar]
  49. Rippere, K. E., Johnson, J. L. & Yousten, A. A. ( 1997; ). DNA similarities among mosquito-pathogens and nonpathogenic strains of Bacillus sphaericus. Int J Syst Bacteriol 47, 214–216.[CrossRef]
    [Google Scholar]
  50. Romano, A. & Saier, M. H., Jr ( 1992; ). Evolution of bacterial phosphoenolpyruvate : sugar phosphotransferase system. I. Physiological and organismic considerations. In The Evolution of Metabolic Function, pp. 143–170. Edited by R. P. Mortlock. Boca Raton, FL: CRC Press.
  51. Roossien, F. F., Brink, J. & Robillard, G. T. ( 1983; ). A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium. Biochim Biophys Acta 760, 185–187.[CrossRef]
    [Google Scholar]
  52. Russell, B. L., Jelley, S. A. & Yousten, A. A. ( 1989; ). Carbohydrate metabolism in the mosquito pathogen Bacillus sphaericus 2362. Appl Environ Microbiol 55, 294–297.
    [Google Scholar]
  53. Saier, M. H., Jr, Chavaux, S., Cook, G. M., Deutscher, J., Paulsen, I. T., Reizer, J. & Ye, J. J. ( 1996; ). Catabolite repression and inducer control in Gram-positive bacteria. Microbiology 142, 217–230.[CrossRef]
    [Google Scholar]
  54. Sambrook, J., Fritsh, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor NY: Cold Spring Harbor Laboratory.
  55. Steinhauer, K., Jepp, T., Hillen, W. & Stülke, J. ( 2002; ). A novel mode of control of Mycoplasma pneumoniae HPr kinase/phosphatase activity reflects its parasitic lifestyle. Microbiology 148, 3277–3284.
    [Google Scholar]
  56. Stülke, J. & Hillen, W. ( 1999; ). Carbon catabolite repression in bacteria. Curr Opin Microbiol 2, 195–201.[CrossRef]
    [Google Scholar]
  57. Stülke, J., Martin-Verstraete, I., Zagorec, M., Rose, M., Klier, A. & Rapoport, G. ( 1997; ). Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT. Mol Microbiol 25, 65–78.[CrossRef]
    [Google Scholar]
  58. Stülke, J., Arnaud, M., Rapoport, G. & Martin-Verstraete, I. ( 1998; ). PRD – a protein domain involved in PTS-dependent induction and carbon catabolite repression of catabolic operons in bacteria. Mol Microbiol 28, 865–874.[CrossRef]
    [Google Scholar]
  59. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. ( 1997; ). The CLUSTAL X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.[CrossRef]
    [Google Scholar]
  60. Titgemeyer, F. & Hillen, W. ( 2002; ). Global control of sugar metabolism: a gram-positive solution. Antonie Van Leeuwenhoek 82, 59–71.[CrossRef]
    [Google Scholar]
  61. Titgemeyer, F., Walkenhorst, J., Reizer, J., Stuiver, M., Cui, X. & Saier, M. H., Jr ( 1995; ). Identification and characterization of phosphoenolpyruvate : fructose phosphotransferase in three Streptomyces species. Microbiology 141, 51–58.[CrossRef]
    [Google Scholar]
  62. Vogler, A. P. & Lengeler, J. W. ( 1989; ). Analysis of the nag regulon from Escherichia coli K12 and Klebsiella pneumoniae and of its regulation. Mol Gen Genet 219, 97–105.
    [Google Scholar]
  63. Wagner, A., Küster-Schöck, E. & Hillen, W. ( 2000; ). Sugar uptake and carbon catabolite repression in Bacillus megaterium strains with inactivated ptsHI. J Mol Microbiol Biotechnol 2, 587–592.
    [Google Scholar]
  64. Wang, F., Xiao, X., Saito, A. & Schrempf, H. ( 2002; ). Streptomyces olivaceoviridis possesses a phosphotransferase system that mediates specific, phosphoenolpyruvate-dependent uptake of N-acetylglucosamine. Mol Genet Genomics 268, 344–351.[CrossRef]
    [Google Scholar]
  65. Zhu, P. P., Herzberg, O. & Peterkofsky, A. ( 1998; ). Topography of the interaction of HPr(Ser) kinase with HPr. Biochemistry 37, 11762–11770.[CrossRef]
    [Google Scholar]
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