1887

Abstract

A transcriptome comparison of a wild-type strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a reporter fusion to the promoter, it was shown that was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that is cotranscribed with the downstream gene. The gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP ( / 102 and 10 s mM, respectively). However, physiological tests with single and multiple mutant strains affected in and/or in paralogues showed that does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of expression. Genetic analysis and studies with purified YufM protein showed that YufM binds just upstream of promoter and activates transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. and / could thus be renamed for lic nzyme and / for ate inase sensor/ate response egulator, respectively.

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2003-09-01
2019-10-17
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References

  1. Anagnostopoulos, C. & Spizizen, J. ( 1961; ). Requirements for transformation in Bacillus subtilis. J Bacteriol 81, 741–746.
    [Google Scholar]
  2. Ansanay, V., Dequin, S., Blondin, B. & Barre, P. ( 1993; ). Cloning, sequence and expression of the gene encoding the malolactic enzyme from Lactococcus lactis. FEBS Lett 332, 74–80.[CrossRef]
    [Google Scholar]
  3. Arthurs, C. E. & Lloyd, D. ( 1999; ). Kinetics, stereospecificity, and expression of the malolactic enzyme. Appl Environ Microbiol 65, 3360–3363.
    [Google Scholar]
  4. Asai, K., Baik, S. H., Kasahara, Y., Moriya, S. & Ogasawara, N. ( 2000; ). Regulation of the transport system for C4-dicarboxylic acids in Bacillus subtilis. Microbiology 146, 263–271.
    [Google Scholar]
  5. Aymerich, S., Gonzy-Treboul, G. & Steinmetz, M. ( 1986; ). 5′-noncoding region sacR is the target of all identified regulation affecting the levansucrase gene in Bacillus subtilis. J Bacteriol 166, 993–998.
    [Google Scholar]
  6. Bartolucci, S., Rella, R., Guagliardi, A., Raia, C. A., Gambacorta, A., De Rosa, M. & Rossi, M. ( 1987; ). Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties. J Biol Chem 262, 7725–7731.
    [Google Scholar]
  7. Berka, R. M., Hahn, J., Albano, M., Draskovic, I., Persuh, M., Cui, X., Sloma, A., Widner, W. & Dubnau, D. ( 2002; ). Microarray analysis of the Bacillus subtilis K-state: genome-wide expression changes dependent on ComK. Mol Microbiol 43, 1331–1345.[CrossRef]
    [Google Scholar]
  8. Bott, M., Meyer, M. & Dimroth, P. ( 1995; ). Regulation of anaerobic citrate metabolism in Klebsiella pneumoniae. Mol Microbiol 18, 533–546.[CrossRef]
    [Google Scholar]
  9. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248–254.[CrossRef]
    [Google Scholar]
  10. Denayrolles, M., Aigle, M. & Lonvaud-Funel, A. ( 1994; ). Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: relationships with malic enzymes. FEMS Microbiol Lett 116, 79–86.[CrossRef]
    [Google Scholar]
  11. Dervyn, E., Suski, C., Daniel, R., Bruand, C., Chapuis, J., Errington, J., Janniere, L. & Ehrlich, S. D. ( 2001; ). Two essential DNA polymerases at the bacterial replication fork. Science 294, 1716–1719.[CrossRef]
    [Google Scholar]
  12. Diesterhaft, M. D. & Freese, E. ( 1973; ). Role of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and malic enzyme during growth and sporulation of Bacillus subtilis. J Biol Chem 248, 6062–6070.
    [Google Scholar]
  13. Doan, T. & Aymerich, S. ( 2003; ). Regulation of the central glycolytic genes in Bacillus subtilis: binding of the repressor CggR to its single DNA target sequence is modulated by fructose-1, 6-bisphosphate. Mol Microbiol 47, 1709–1721.[CrossRef]
    [Google Scholar]
  14. Driscoll, B. T. & Finan, T. M. ( 1993; ). NAD+-dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation. Mol Microbiol 7, 865–873.[CrossRef]
    [Google Scholar]
  15. Driscoll, B. T. & Finan, T. M. ( 1996; ). NADP+-dependent malic enzyme of Rhizobium meliloti. J Bacteriol 178, 2224–2231.
    [Google Scholar]
  16. Driscoll, B. T. & Finan, T. M. ( 1997; ). Properties of NAD+- and NADP+-dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti and differential expression of their genes in nitrogen-fixing bacteroids. Microbiology 143, 489–498.[CrossRef]
    [Google Scholar]
  17. Dunn, M. F. ( 1998; ). Tricarboxylic acid cycle and anaplerotic enzymes in rhizobia. FEMS Microbiol Rev 22, 105–123.[CrossRef]
    [Google Scholar]
  18. Ferrari, F. A., Ferrari, E. & Hoch, J. A. ( 1982; ). Chromosomal location of a Bacillus subtilis DNA fragment uniquely transcribed by sigma-28-containing RNA polymerase. J Bacteriol 152, 780–785.
    [Google Scholar]
  19. Fillinger, S., Boschi-Muller, S., Azza, S., Dervyn, E., Branlant, G. & Aymerich, S. ( 2000; ). Two glyceraldehyde-3-phosphate dehydrogenases with opposite physiological roles in a nonphotosynthetic bacterium. J Biol Chem 275, 14031–14037.[CrossRef]
    [Google Scholar]
  20. Fujita, Y. & Freese, E. ( 1981; ). Isolation and properties of a Bacillus subtilis mutant unable to produce fructose-bisphosphatase. J Bacteriol 145, 760–767.
    [Google Scholar]
  21. Golby, P., Davies, S., Kelly, D. J., Guest, J. R. & Andrews, S. C. ( 1999; ). Identification and characterization of a two-component sensor-kinase and response-regulator system (DcuS-DcuR) controlling gene expression in response to C4-dicarboxylates in Escherichia coli. J Bacteriol 181, 1238–1248.
    [Google Scholar]
  22. Groisillier, A. & Lonvaud-Funel, A. ( 1999; ). Comparison of partial malolactic enzyme gene sequences for phylogenetic analysis of some lactic acid bacteria species and relationships with the malic enzyme. Int J Syst Bacteriol 49, 1417–1428.[CrossRef]
    [Google Scholar]
  23. Guérout-Fleury, A. M., Frandsen, N. & Stragier, P. ( 1996; ). Plasmids for ectopic integration in Bacillus subtilis. Gene 180, 57–61.[CrossRef]
    [Google Scholar]
  24. Hansen, E. J. & Juni, E. ( 1974; ). Two routes for synthesis of phosphoenolpyruvate from C4-dicarboxylic acids in Escherichia coli. Biochem Biophys Res Commun 59, 1204–1210.[CrossRef]
    [Google Scholar]
  25. Hansen, E. J. & Juni, E. ( 1975; ). Isolation of mutants of Escherichia coli lacking NAD- and NADP-linked malic enzyme. Biochem Biophys Res Commun 65, 559–566.[CrossRef]
    [Google Scholar]
  26. Ito, M., Guffanti, A. A., Wang, W. & Krulwich, T. A. ( 2000; ). Effects of nonpolar mutations in each of the seven Bacillus subtilis mrp genes suggest complex interactions among the gene products in support of Na+ and alkali but not cholate resistance. J Bacteriol 182, 5663–5670.[CrossRef]
    [Google Scholar]
  27. Kaspar, S. & Bott, M. ( 2002; ). The sensor kinase CitA (DpiB) of Escherichia coli functions as a high-affinity citrate receptor. Arch Microbiol 177, 313–321.[CrossRef]
    [Google Scholar]
  28. Kaspar, S., Perozzo, R., Reinelt, S., Meyer, M., Pfister, K., Scapozza, L. & Bott, M. ( 1999; ). The periplasmic domain of the histidine autokinase CitA functions as a highly specific citrate receptor. Mol Microbiol 33, 858–872.[CrossRef]
    [Google Scholar]
  29. Kawai, S., Suzuki, H., Yamamoto, K., Inui, M., Yukawa, H. & Kumagai, H. ( 1996; ). Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene. Appl Environ Microbiol 62, 2692–2700.
    [Google Scholar]
  30. Knichel, W. & Radler, F. ( 1982; ). d-Malic enzyme of Pseudomonas fluorescens. Eur J Biochem 123, 547–552.
    [Google Scholar]
  31. Kobayashi, K., Doi, S., Negoro, S., Urabe, I. & Okada, H. ( 1989; ). Structure and properties of malic enzyme from Bacillus stearothermophilus. J Biol Chem 264, 3200–3205.
    [Google Scholar]
  32. Kobayashi, K., Ogura, M., Yamaguchi, H., Yoshida, K., Ogasawara, N., Tanaka, T. & Fujita, Y. ( 2001; ). Comprehensive DNA microarray analysis of Bacillus subtilis two-component regulatory systems. J Bacteriol 183, 7365–7370.[CrossRef]
    [Google Scholar]
  33. Lamed, R. & Zeikus, J. G. ( 1981; ). Thermostable, ammonium-activated malic enzyme of Clostridium thermocellum. Biochim Biophys Acta 660, 251–255.[CrossRef]
    [Google Scholar]
  34. 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]
  35. Ludwig, H., Rebhan, N., Blencke, H. M., Merzbacher, M. & Stülke, J. ( 2002; ). Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA-mediated regulation. Mol Microbiol 45, 543–553.[CrossRef]
    [Google Scholar]
  36. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  37. Mitsch, M. J., Voegele, R. T., Cowie, A., Osteras, M. & Finan, T. M. ( 1998; ). Chimeric structure of the NAD(P)+- and NADP+-dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti. J Biol Chem 273, 9330–9336.[CrossRef]
    [Google Scholar]
  38. Moriya, S., Tsujikawa, E., Hassan, A. K., Asai, K., Kodama, T. & Ogasawara, N. ( 1998; ). A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition. Mol Microbiol 29, 179–187.[CrossRef]
    [Google Scholar]
  39. Murai, T., Tokushige, M., Nagai, J. & Katsuki, H. ( 1971; ). Physiological functions of NAD- and NADP-linked malic enzymes in Escherichia coli. Biochem Biophys Res Commun 43, 875–881.[CrossRef]
    [Google Scholar]
  40. Ogura, M., Yamaguchi, H., Yoshida, K., Fujita, Y. & Tanaka, T. ( 2001; ). DNA microarray analysis of Bacillus subtilis DegU, ComA and PhoP regulons: an approach to comprehensive analysis of B. subtilis two-component regulatory systems. Nucleic Acids Res 29, 3804–3813.[CrossRef]
    [Google Scholar]
  41. Ogura, M., Yamaguchi, H., Kobayashi, K., Ogasawara, N., Fujita, Y. & Tanaka, T. ( 2002; ). Whole-genome analysis of genes regulated by the Bacillus subtilis competence transcription factor ComK. J Bacteriol 184, 2344–2351.[CrossRef]
    [Google Scholar]
  42. Perego, M. & Hoch, J. A. ( 2002; ). Two-component systems, phosphorelays, and regulation of their activities by phosphatases. In Bacillus subtilis and its Closest Relatives: from Genes to Cells, pp. 423–481. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC: American Society for Microbiology.
  43. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  44. Steinmetz, M. & Richter, R. ( 1994; ). Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome. J Bacteriol 176, 1761–1763.
    [Google Scholar]
  45. Stols, L. & Donnelly, M. I. ( 1997; ). Production of succinic acid through overexpression of NAD+-dependent malic enzyme in an Escherichia coli mutant. Appl Environ Microbiol 63, 2695–2701.
    [Google Scholar]
  46. Tanaka, K., Kobayashi, K. & Ogasawara, N. ( 2003; ). The Bacillus subtilis YufLM two-component system regulates the expression of the malate transporters MaeN (YufR) and YflS, and is essential for utilization of malate in minimal medium. Microbiology 149, 2317–2329.[CrossRef]
    [Google Scholar]
  47. Vagner, V., Dervyn, E. & Ehrlich, S. D. ( 1998; ). A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144, 3097–3104.[CrossRef]
    [Google Scholar]
  48. van der Rest, M. E., Frank, C. & Molenaar, D. ( 2000; ). Functions of the membrane-associated and cytoplasmic malate dehydrogenases in the citric acid cycle of Escherichia coli. J Bacteriol 182, 6892–6899.[CrossRef]
    [Google Scholar]
  49. Voegele, R. T., Mitsch, M. J. & Finan, T. M. ( 1999; ). Characterization of two members of a novel malic enzyme class. Biochim Biophys Acta 1432, 275–285.[CrossRef]
    [Google Scholar]
  50. Wei, Y., Guffanti, A. A., Ito, M. & Krulwich, T. A. ( 2000; ). Bacillus subtilis YqkI is a novel malic/Na+-lactate antiporter that enhances growth on malate at low protonmotive force. J Biol Chem 275, 30287–30292.[CrossRef]
    [Google Scholar]
  51. Yamamoto, H., Murata, M. & Sekiguchi, J. ( 2000; ). The CitST two-component system regulates the expression of the Mg-citrate transporter in Bacillus subtilis. Mol Microbiol 37, 898–912.[CrossRef]
    [Google Scholar]
  52. Yoshida, K., Aoyama, D., Ishio, I., Shibayama, T. & Fujita, Y. ( 1997; ). Organization and transcription of the myo-inositol operon, iol, of Bacillus subtilis. J Bacteriol 179, 4591–4598.
    [Google Scholar]
  53. Yoshida, K., Kobayashi, K., Miwa, Y. & 9 other authors ( 2001; ). Combined transcriptome and proteome analysis as a powerful approach to study genes under glucose repression in Bacillus subtilis. Nucleic Acids Res 29, 683–692.[CrossRef]
    [Google Scholar]
  54. Zientz, E., Bongaerts, J. & Unden, G. ( 1998; ). Fumarate regulation of gene expression in Escherichia coli by the DcuSR (dcuSR genes) two-component regulatory system. J Bacteriol 180, 5421–5425.
    [Google Scholar]
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Table of oligonucleotides used in the study.

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Figureshowing purified His6-YufM protein (lane 1) and total soluble fraction of the overproducing strain induced (lane 2) by addition of IPTG.

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