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References

  1. Arndt A, Auchter M, Ishige T, Wendisch VF, Eikmanns BJ. 2008. Ethanol catabolism in Corynebacterium glutamicum. J. Mol. Microbiol. Biotechnol. 15: 222-233.
    Pubmed CrossRef
  2. Arndt A, Eikmanns BJ. 2007. The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to carbon catabolite repression. J. Bacteriol. 189: 7408-7416.
    Pubmed CrossRef
  3. Auchter M, Arndt A, Eikmanns BJ. 2009. Dual transcriptional control of the acetaldehyde dehydrogenase gene ald of Corynebacterium glutamicum by RamA and RamB. J. Biotechnol. 140: 84-91.
    Pubmed CrossRef
  4. Bernt E, Gutmann I. 1974. Aethanol, pp. 1545-1548. In Bergmeyer HU (ed.), Methoden der Enzymatischen Analyse. Verlag Chemie, Weinheim.
    Pubmed CrossRef
  5. Cha PH, P ark SY, Moon MW, Subhadra B, Oh TK, K im E, Kim JF, Lee JK. 2010. Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032. Appl. Microbiol. Biotechnol. 85: 1061-1068.
    Pubmed CrossRef
  6. Claes WA, Pühler A, Kalinowski J. 2002. Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2methylcitrate cycle. J. Bacteriol. 184: 2728-2739.
    CrossRef
  7. Cocaign M, Monnet C, Lindley ND. 1993. Batch kinetics of Corynebacterium glutamicum during growth on various carbon substrates: use of substrate mixtures to localize metabolic bottlenecks. Appl. Microbiol. Biotechnol. 40: 526-530.
    CrossRef
  8. Dominguez H, Cocaign-Bousquet M, Lindley ND. 1993. Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 47: 600-603.
    CrossRef
  9. Eggeling L, Bott M. 2005. Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton, USA.
  10. Eggeling L and Reyes O. 2004. Experiments, pp. 535-566. In Eggeling L, Bott M (ed.), Handbook of Corynebacterium glutamicum, Taylor & Francis, Boca Raton, FL.
    Pubmed CrossRef
  11. Engels V, Wendisch VF. 2007. The DeoR-type regulator SugR represses expression of ptsG in Corynebacterium glutamicum. J. Bacteriol. 189: 2955-2966.
    Pubmed CrossRef
  12. Frunzke J, Engels V, Hasenbein S, Gätgens C, Bott M. 2008. Co-ordinated regulation of gluconate catabolism and glucose uptake in Corynebacterium glutamicum by two functionally equivalent transcriptional regulators, GntR1 and GntR2. Mol. Microbiol. 67: 305-322.
    Pubmed CrossRef
  13. Gerstmeir R, Cramer A, Dangel P, Schaffer S, Eikmanns BJ. 2004. RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum. J. Bacteriol. 186: 2798-2809.
    CrossRef
  14. Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580.
    Pubmed CrossRef
  15. Jungwirth B, Emer D, Brune I, Hansmeier N, Pühler A, Eikmanns BJ, Tauch A. 2008. Triple transcriptional control of the resuscitation promoting factor 2 (rpf2) gene of Corynebacterium glutamicum by the regulators of acetate metabolism RamA and RamB and the cAMP-dependent regulator GlxR. FEMS Microbiol. Lett. 281: 190-197.
    Pubmed CrossRef
  16. 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.
    Pubmed CrossRef
  17. Kohl TA, Baumbach J, Jungwirth B, Pühler A, Tauch A. 2008. The GlxR regulon of the amino acid producer Corynebacterium glutamicum: in silico and in vitro detection of DNA binding sites of a global transcription regulator. J. Biotechnol. 135: 340-350.
    Pubmed CrossRef
  18. Kohl TA, Tauch A. 2009. The GlxR regulon of the amino acid producer Corynebacterium glutamicum: detection of the corynebacterial core regulon and integration into the transcriptional regulatory network model. J. Biotechnol. 143:239-246.
    Pubmed CrossRef
  19. Kotrbova-Kozak A, Kotrba P, Inui M, Sajdok J, Yukawa H. 2007. Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum R. Appl. Microbiol. Biotechnol. 76: 1347-1356.
    CrossRef
  20. Lee HW, Pan JG, Lebeault JM. 1998. Enhanced l-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose. Appl. Microbiol. Biotechnol. 49: 915.
    Pubmed CrossRef
  21. Merkens H, Beckers G, Wirtz A, Burkovski A. 2005. Vanillate metabolism in Corynebacterium glutamicum. Curr. Microbiol. 51: 59-65.
    CrossRef
  22. Mori A, Shiio I. 1987. Phosphoenol pyruvate: sugar phosphotransferase systems and sugar metabolism in Brevibacterium flavum. Agric. Biol. Chem. 51: 2671-2678.
    Pubmed CrossRef
  23. Netzer R, Krause M, Rittmann D, Peters-Wendisch PG, Eggeling L, Wendisch VF, Sahm H. 2004. Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis. Arch. Microbiol. 182: 354-363.
    Pubmed
  24. Parche S, Burkovski A, Sprenger GA, Weil B, Krämer R, Titgemeyer F. 2001. Corynebacterium glutamicum: a dissection of the PTS. J. Mol. Microbiol. Biotechnol. 3: 423-428.
  25. Park SD, Lee SN, Park IH, Choi JS, Jeong WK, Kim YH, Lee HS. 2004. Isolation and characterization of transcriptional elements from Corynebacterium glutamicum. J. Microbiol. Biotechnol. 14: 789-795.
    Pubmed CrossRef
  26. Park SY, Moon MW, Subhadra B, Lee JK. 2010. Functional characterization of the glxR deletion mutant of Corynebacterium glutamicum ATCC 13032: involvement of GlxR in acetate metabolism and carbon catabolite repression. FEMS Microbiol. Lett. 304: 107-115.
  27. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    Pubmed CrossRef
  28. Schreiner ND, Fiur D, Holátko J. 2005. E1 enzyme of the pyruvate dehydrogenase complex in Corynebacterium glutamicum:molecular analysis of the gene and phylogenetic aspects. J. Bacteriol. 187: 6005-6018.
    CrossRef
  29. Shaw WV. 1975. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 43:737-755.
    Pubmed CrossRef
  30. Stansen C, Uy D, Delaunay S, Eggeling L, Goergen JL, Wendisch VF. 2005. Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperaturetriggered glutamate production. Appl. Environ. Microbiol. 71:5920-5928.
    Pubmed CrossRef
  31. 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.
    Pubmed CrossRef
  32. Tauch A, Kirchner O, Löffler B, Götker S, Pühler A, Kalinowski J. 2002. Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. Curr. Microbiol. 45: 362-367.
    Pubmed CrossRef
  33. Wendisch VF, de Graaf AA, Sahm H, Eikmanns BJ. 2000. Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose. J. Bacteriol. 182: 3088-3096.

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Article

Research article

J. Microbiol. Biotechnol. 2013; 23(12): 1683-1690

Published online December 28, 2013 https://doi.org/10.4014/jmb.1310.10031

Copyright © The Korean Society for Microbiology and Biotechnology.

Elucidation of the Regulation of Ethanol Catabolic Genes and ptsG Using a glxR and Adenylate Cyclase Gene (cyaB) Deletion Mutants of Corynebacterium glutamicum ATCC 13032

Bindu Subhadra 1 and Jung-Kee Lee 1*

Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea

Received: October 10, 2013; Accepted: October 13, 2013

Abstract

The cyclic AMP receptor protein (CRP) homolog, GlxR, controls the expression of several
genes involved in the regulation of diverse physiological processes in Corynebacterium
glutamicum. In silico analysis has revealed the presence of glxR binding sites upstream of genes
adhA, ald, and ptsG, encoding glucose-specific phosphotransferase system protein, alcohol
dehydrogenase (ADH), and acetaldehyde dehydrogenase (ALDH), respectively. However, the
involvement of the GlxR-cAMP complex on the expression of these genes has been explored
only in vitro. In this study, the expressions of ptsG, adhA, and ald were analyzed in detail using
an adenylate cyclase gene (cyaB) deletion mutant and glxR deletion mutant. The specific
activities of ADH and ALDH were increased in both the mutants in glucose and glucose plus
ethanol media, in contrast to the wild type. In accordance, the promoter activities of adhA and
ald were derepressed in the cyaB mutant, indicating that glxR acts as a repressor of adhA.
Similarly, both the mutants exhibited derepression of ptsG regardless of the carbon source.
These results confirm the involvement of GlxR on the expression of important carbon
metabolic genes; adhA, ald, and ptsG.

Keywords: Corynebacterium glutamicum, GlxR, cyaB, adhA, ald, ptsG

References

  1. Arndt A, Auchter M, Ishige T, Wendisch VF, Eikmanns BJ. 2008. Ethanol catabolism in Corynebacterium glutamicum. J. Mol. Microbiol. Biotechnol. 15: 222-233.
    Pubmed CrossRef
  2. Arndt A, Eikmanns BJ. 2007. The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to carbon catabolite repression. J. Bacteriol. 189: 7408-7416.
    Pubmed CrossRef
  3. Auchter M, Arndt A, Eikmanns BJ. 2009. Dual transcriptional control of the acetaldehyde dehydrogenase gene ald of Corynebacterium glutamicum by RamA and RamB. J. Biotechnol. 140: 84-91.
    Pubmed CrossRef
  4. Bernt E, Gutmann I. 1974. Aethanol, pp. 1545-1548. In Bergmeyer HU (ed.), Methoden der Enzymatischen Analyse. Verlag Chemie, Weinheim.
    Pubmed CrossRef
  5. Cha PH, P ark SY, Moon MW, Subhadra B, Oh TK, K im E, Kim JF, Lee JK. 2010. Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032. Appl. Microbiol. Biotechnol. 85: 1061-1068.
    Pubmed CrossRef
  6. Claes WA, Pühler A, Kalinowski J. 2002. Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2methylcitrate cycle. J. Bacteriol. 184: 2728-2739.
    CrossRef
  7. Cocaign M, Monnet C, Lindley ND. 1993. Batch kinetics of Corynebacterium glutamicum during growth on various carbon substrates: use of substrate mixtures to localize metabolic bottlenecks. Appl. Microbiol. Biotechnol. 40: 526-530.
    CrossRef
  8. Dominguez H, Cocaign-Bousquet M, Lindley ND. 1993. Simultaneous consumption of glucose and fructose from sugar mixtures during batch growth of Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 47: 600-603.
    CrossRef
  9. Eggeling L, Bott M. 2005. Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton, USA.
  10. Eggeling L and Reyes O. 2004. Experiments, pp. 535-566. In Eggeling L, Bott M (ed.), Handbook of Corynebacterium glutamicum, Taylor & Francis, Boca Raton, FL.
    Pubmed CrossRef
  11. Engels V, Wendisch VF. 2007. The DeoR-type regulator SugR represses expression of ptsG in Corynebacterium glutamicum. J. Bacteriol. 189: 2955-2966.
    Pubmed CrossRef
  12. Frunzke J, Engels V, Hasenbein S, Gätgens C, Bott M. 2008. Co-ordinated regulation of gluconate catabolism and glucose uptake in Corynebacterium glutamicum by two functionally equivalent transcriptional regulators, GntR1 and GntR2. Mol. Microbiol. 67: 305-322.
    Pubmed CrossRef
  13. Gerstmeir R, Cramer A, Dangel P, Schaffer S, Eikmanns BJ. 2004. RamB, a novel transcriptional regulator of genes involved in acetate metabolism of Corynebacterium glutamicum. J. Bacteriol. 186: 2798-2809.
    CrossRef
  14. Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580.
    Pubmed CrossRef
  15. Jungwirth B, Emer D, Brune I, Hansmeier N, Pühler A, Eikmanns BJ, Tauch A. 2008. Triple transcriptional control of the resuscitation promoting factor 2 (rpf2) gene of Corynebacterium glutamicum by the regulators of acetate metabolism RamA and RamB and the cAMP-dependent regulator GlxR. FEMS Microbiol. Lett. 281: 190-197.
    Pubmed CrossRef
  16. 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.
    Pubmed CrossRef
  17. Kohl TA, Baumbach J, Jungwirth B, Pühler A, Tauch A. 2008. The GlxR regulon of the amino acid producer Corynebacterium glutamicum: in silico and in vitro detection of DNA binding sites of a global transcription regulator. J. Biotechnol. 135: 340-350.
    Pubmed CrossRef
  18. Kohl TA, Tauch A. 2009. The GlxR regulon of the amino acid producer Corynebacterium glutamicum: detection of the corynebacterial core regulon and integration into the transcriptional regulatory network model. J. Biotechnol. 143:239-246.
    Pubmed CrossRef
  19. Kotrbova-Kozak A, Kotrba P, Inui M, Sajdok J, Yukawa H. 2007. Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum R. Appl. Microbiol. Biotechnol. 76: 1347-1356.
    CrossRef
  20. Lee HW, Pan JG, Lebeault JM. 1998. Enhanced l-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose. Appl. Microbiol. Biotechnol. 49: 915.
    Pubmed CrossRef
  21. Merkens H, Beckers G, Wirtz A, Burkovski A. 2005. Vanillate metabolism in Corynebacterium glutamicum. Curr. Microbiol. 51: 59-65.
    CrossRef
  22. Mori A, Shiio I. 1987. Phosphoenol pyruvate: sugar phosphotransferase systems and sugar metabolism in Brevibacterium flavum. Agric. Biol. Chem. 51: 2671-2678.
    Pubmed CrossRef
  23. Netzer R, Krause M, Rittmann D, Peters-Wendisch PG, Eggeling L, Wendisch VF, Sahm H. 2004. Roles of pyruvate kinase and malic enzyme in Corynebacterium glutamicum for growth on carbon sources requiring gluconeogenesis. Arch. Microbiol. 182: 354-363.
    Pubmed
  24. Parche S, Burkovski A, Sprenger GA, Weil B, Krämer R, Titgemeyer F. 2001. Corynebacterium glutamicum: a dissection of the PTS. J. Mol. Microbiol. Biotechnol. 3: 423-428.
  25. Park SD, Lee SN, Park IH, Choi JS, Jeong WK, Kim YH, Lee HS. 2004. Isolation and characterization of transcriptional elements from Corynebacterium glutamicum. J. Microbiol. Biotechnol. 14: 789-795.
    Pubmed CrossRef
  26. Park SY, Moon MW, Subhadra B, Lee JK. 2010. Functional characterization of the glxR deletion mutant of Corynebacterium glutamicum ATCC 13032: involvement of GlxR in acetate metabolism and carbon catabolite repression. FEMS Microbiol. Lett. 304: 107-115.
  27. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    Pubmed CrossRef
  28. Schreiner ND, Fiur D, Holátko J. 2005. E1 enzyme of the pyruvate dehydrogenase complex in Corynebacterium glutamicum:molecular analysis of the gene and phylogenetic aspects. J. Bacteriol. 187: 6005-6018.
    CrossRef
  29. Shaw WV. 1975. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 43:737-755.
    Pubmed CrossRef
  30. Stansen C, Uy D, Delaunay S, Eggeling L, Goergen JL, Wendisch VF. 2005. Characterization of a Corynebacterium glutamicum lactate utilization operon induced during temperaturetriggered glutamate production. Appl. Environ. Microbiol. 71:5920-5928.
    Pubmed CrossRef
  31. 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.
    Pubmed CrossRef
  32. Tauch A, Kirchner O, Löffler B, Götker S, Pühler A, Kalinowski J. 2002. Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. Curr. Microbiol. 45: 362-367.
    Pubmed CrossRef
  33. Wendisch VF, de Graaf AA, Sahm H, Eikmanns BJ. 2000. Quantitative determination of metabolic fluxes during coutilization of two carbon sources: comparative analyses with Corynebacterium glutamicum during growth on acetate and/or glucose. J. Bacteriol. 182: 3088-3096.