Skip to main content
Log in

Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase of Corynebacterium glutamicum is regulated by the global regulator SugR

  • Applied Genetics and Molecular Biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Regulation of expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase essential for glycolysis in Corynebacterium glutamicum was studied. We applied DNA affinity beads to isolate proteins binding to the promoter region of the gapA gene and obtained SugR, which has been shown to be a repressor of pts genes involved in sugar transport system. The results of electrophoretic mobility shift assays revealed that SugR specifically bound to the gapA promoter and the consensus sequence TGTTTG in the promoter region was required for its binding. We examined expression of the gapA gene in a sugR deletion mutant. Effect of mutation in the SugR binding site on gapA-lacZ fusion expression was also examined. These assays revealed that SugR acts as a negative transcriptional regulator of the gapA gene in the absence of sugar, and repression by SugR is alleviated in the presence of sugar, i.e., fructose and sucrose. Fructose-1-phosphate and fructose-1,6-bisphosphate revealed negative effects on binding of SugR to the gapA promoter, indicating that the sugar metabolites are involved in the derepression of gapA expression.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Bott M (2007) Offering surprises: TCA cycle regulation in Corynebacterium glutamicum. Trends Microbiol 15:417–425

    Article  CAS  Google Scholar 

  • Charpentier B, Branlant C (1994) The Escherichia coli gapA gene is transcribed by the vegetative RNA polymerase holoenzyme Eσ70 and by the heat shock RNA polymerase Eσ32. J Bacteriol 176:830–839

    Article  CAS  Google Scholar 

  • Charpentier B, Bardey V, Robas N, Branlant C (1998) The EIIGlc protein is involved in glucose-mediated activation of Escherichia coli gapA and gapB-pgk transcription. J Bacteriol 180:6476–6483

    Article  CAS  Google Scholar 

  • Cramer A, Eikmanns BJ (2007) RamA, the transcriptional regulator of acetate metabolism in Corynebacterium glutamicum, is subject to negative autoregulation. J Mol Microbiol Biotechnol 12:51–59

    Article  CAS  Google Scholar 

  • Cramer A, Gerstmeir R, Schaffer S, Bott M, Eikmanns BJ (2006) Identification of RamA, a novel LuxR-type transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum. J Bacteriol 188:2554–2567

    Article  CAS  Google Scholar 

  • Cramer A, Auchter M, Frunzke J, Bott M, Eikmanns BJ (2007) RamB, the transcriptional regulator of acetate metabolism in Corynebacterium glutamicum, is subject to regulation by RamA and RamB. J Bacteriol 189:1145–1149

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Dominguez H, Lindley ND (1996) Complete sucrose metabolism requires fructose phosphotransferase activity in Corynebacterium glutamicum to ensure phosphorylation of liberated fructose. Appl Environ Microbiol 62:3878–3880

    Article  CAS  Google Scholar 

  • Engels V, Wendisch VF (2007) The DeoR-Type regulator SugR represses expression of ptsG in Corynebacterium glutamicum. J Bacteriol 189:2955–2966

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Gaigalat L, Schlüeter JP, Hartmann M, Mormann S, Tauch A, Püehler A, Kalinowski J (2007) The DeoR-type transcriptional regulator SugR acts as a repressor for genes encoding the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Corynebacterium glutamicum. BMC Mol Biol 8:104

    Article  Google Scholar 

  • Han SO, Inui M, Yukawa H (2007) Expression of Corynebacterium glutamicum glycolytic genes varies with carbon source and growth phase. Microbiology 153:2190–2202

    Article  CAS  Google Scholar 

  • Hayashi M, Mizoguchi H, Shiraishi N, Obayashi M, Nakagawa S, Imai J, Watanabe S, Ota T, Ikeda M (2002) Transcriptome analysis of acetate metabolism in Corynebacterium glutamicum using a newly developed metabolic array. Biosci Biotechnol Biochem 66:1337–1344

    Article  CAS  Google Scholar 

  • Hermann T (2003) Industrial production of amino acids by coryneform bacteria. J Biotechnol 104:155–172

    Article  CAS  Google Scholar 

  • Inui M, Murakami S, Okino S, Kawaguchi H, Vertès AA, Yukawa H (2004a) Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J Mol Microbiol Biotechnol 7:182–196

    Article  CAS  Google Scholar 

  • Inui M, Kawaguchi H, Murakami S, Vertès AA, Yukawa H (2004b) Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. J Mol Microbiol Biotechnol 8:243–254

    Article  Google Scholar 

  • Inui M, Suda M, Okino S, Nonaka H, Puskás LG, Vertès AA, Yukawa H (2007) Transcriptional profiling of Corynebacterium glutamicum metabolism during organic acid production under oxygen deprivation conditions. Microbiology 153:2491–2504

    Article  CAS  Google Scholar 

  • Kim HJ, Kim TH, Kim Y, Lee HS (2004) Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum. J Bacteriol 186:3453–3460

    Article  CAS  Google Scholar 

  • Kinoshita S, Udaka S, Shimono M (1957) Studies on the amino acid fermentation. Part 1. Production of l-glutamic acid by various microorganisms. J Gen Appl Microbiol 3:193–205

    Article  CAS  Google Scholar 

  • Koksharova O, Schubert M, Shestakov S, Cerff R (1998) Genetic and biochemical evidence for distinct key functions of two highly divergent GAPDH genes in catabolic and anabolic carbon flow of the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol 36:183–194

    Article  CAS  Google Scholar 

  • Kormanec J, Lempelová A, Farkašovský M, Homerová D (1995) Cloning, sequencing and expression in Escherichia coli of a Streptomyces aureofaciens gene encoding glyceraldehyde-3-phosphate dehydrogenase. Gene 165:77–80

    Article  CAS  Google Scholar 

  • Krug A, Wendisch VF, Bott M (2005) Identification of AcnR, a TetR-type repressor of the aconitase gene acn in Corynebacterium glutamicum. J Biol Chem 280:585–595

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    CAS  Google Scholar 

  • Ludwig H, Homuth G, Schmalisch M, Dyka FM, 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Martin C, Cerff R (1986) Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba). Eur J Biochem 159:323–331

    Article  CAS  Google Scholar 

  • Martin W, Brinkmann H, Savonna C, Cerff R (1993) Evidence for a chimeric nature of nuclear genomes: eubacterial origin of eukaryotic glyceraldehyde-3-phosphate dehydrogenase genes. PNAS 90:8692–8696

    Article  CAS  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Moon MW et al (2005) Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032. FEMS Microbiol Lett 244:259–266

    Article  CAS  Google Scholar 

  • Muffler A, Bettermann S, Haushalter M, Horlein A, Neveling U, Schramm M, Sorgenfrei O (2002) Genome-wide transcription profiling of Corynebacterium glutamicum after heat shock and during growth on acetate and glucose. J Biotechnol 98:255–268

    Article  CAS  Google Scholar 

  • Okino S, Inui M, Yukawa H (2005) Production of organic acids by Corynebacterium glutamicum under oxygen deprivation. Appl Microbiol Biotechnol 68:475–480

    Article  CAS  Google Scholar 

  • Omumasaba CA, Okai N, Inui M, Yukawa H (2004) Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J Mol Microbiol Biotechnol 8:91–103

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Servant P, Le Coq D, Aymerich S (2005) CcpN (YqzB), a novel regulator for CcpA-independent catabolite repression of Bacillus subtilis gluconeogenic genes. Mol Microbiol 55:1435–1451

    Article  CAS  Google Scholar 

  • Shimada T, Fujita N, Maeda M, Ishihama A (2005) Systematic search for the Cra-binding promoters using genomic SELEX system. Genes Cells 10:907–918

    Article  CAS  Google Scholar 

  • Sprušanský O, Řežuchová B, Homerová D, Kormanec J (2001) Expression of the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase of Streptomyces aureofaciens requires GapR, a member of the AraC/XylS family of transcriptional activators. Microbiology 147:1291–1301

    Article  Google Scholar 

  • Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130

    Article  CAS  Google Scholar 

  • Tanaka Y, Teramoto H, Inui M, Yukawa H (2008) Regulation of expression of general components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) by the global regulator SugR in Corynebacterium glutamicum. Appl Microbiol Biotechnol 78:309–318

    Article  CAS  Google Scholar 

  • Vertès AA, Inui M, Kobayashi M, Kurusu Y, Yukawa H (1993) Presence of mrr- and mcr-like restriction systems in coryneform bacteria. Res Microbiol 144:181–185

    Article  Google Scholar 

  • Wennerhold J, Krug A, Bott M (2005) The AraC-type regulator RipA represses aconitase and other iron proteins from Corynebacterium under iron limitation and is itself repressed by DtxR. J Biol Chem 280:40500–40508

    Article  CAS  Google Scholar 

  • Yukawa H, Omumasaba CA, Nonaka H, Kós P, Okai N, Suzuki N, Suda M, Tsuge Y, Watanabe J, Ikeda Y, Vertès AA, Inui M (2007) Comparative analysis of the Corynebacterium glutamicum group and complete genome sequence of strain R. Microbiology 153:1042–1058

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Crispinus A. Omumasaba (RITE) for critical reading of the manuscript. This work was partially supported by a grant from the New Energy and Industrial Technology Development Organization (NEDO).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Hideaki Yukawa.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Toyoda, K., Teramoto, H., Inui, M. et al. Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase of Corynebacterium glutamicum is regulated by the global regulator SugR. Appl Microbiol Biotechnol 81, 291–301 (2008). https://doi.org/10.1007/s00253-008-1682-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-008-1682-0

Keywords

Navigation