Advertisement

Applied Microbiology and Biotechnology

, Volume 82, Issue 6, pp 1115–1122 | Cite as

Multiple control of the acetate pathway in Lactococcus lactis under aeration by catabolite repression and metabolites

  • Felix Lopez de FelipeEmail author
  • Philippe Gaudu
Genomics and Proteomics

Abstract

To explore the factors controlling metabolite formation under aeration in Lactococcus lactis, metabolic patterns, enzymatic activities, and transcriptional profiles of genes involved in the aerobic pathway for acetate anabolism were compared between a parental L. lactis strain and its NADH-oxidase-overproducer derivative. Deregulated catabolite repression mutans in the ccpA or pstH genes, encoding CcpA or its co-activator HPr, respectively, were compared with a parental strain, as well. Although the NADH-oxidase activity was derepressed in ccpA, but not in the pstH background, a mixed fermentation was displayed by either mutant, with a higher acetate production in the pstH variant. Moreover, transcription of genes encoding phosphotransacetylase and acetate kinase were derepressed, and the corresponding enzymatic activities increased, in both catabolite repression mutants. These results and the dependence on carbon source for acetate production in the NADH-oxidase-overproducer support the conclusion that catabolite repression, rather than NADH oxidation, plays a critical role to control acetate production. Furthermore, fructose 1,6-bisphosphate influenced the in vitro phosphotransacetylase and acetate kinase activities, while the former was sensitive to diacetyl. Our study strongly supports the model that, under aerobic conditions, the homolactic fermentation in L. lactis MG1363 is maintained by CcpA-mediated repression of mixed acid fermentation.

Keywords

Lactococcus lactis Aerobic conditions Catabolite repression CcpA Regulation NADH-oxidase 

Notes

Acknowledgments

We thank Dr J. Deutscher for providing us the strain pstH1 mutant (LIG101 strain) and M.-A. Petit for helpful discussion. F.L.F would like to acknowledge grant no. PR2004-0289 from the Secretaría de Estado de Educación y Universidades of Spain to promote the mobility of senior researchers.

References

  1. Andersen HW, Pedersen MB, Hammer K, Jensen PR (2001) Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis. Eur J Biochem 268:6379–6389CrossRefGoogle Scholar
  2. Charrier V, Buckley E, Parsonage D, Galinier A, Darbon E, Jaquinod M, Forest E, Deutscher J, Claiborne 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 272:14166–14174CrossRefGoogle Scholar
  3. Cocaign-Bousquet M, Garrigues C, Loubiere P, Lindley ND (1996) Physiology of pyruvate metabolism in Lactococcus lactis. Antonie Van Leeuwenhoek 70:253–267CrossRefGoogle Scholar
  4. Deutscher J, Kuster E, Bergstedt U, Charrier V, Hillen W (1995) Protein kinase-dependent HPr/CcpA interaction links glycolytic activity to carbon catabolite repression in gram-positive bacteria. Mol Microbiol 15:1049–1053CrossRefGoogle Scholar
  5. Even S, Garrigues C, Loubiere P, Lindley ND, Cocaign-Bousquet M (1999) Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity. Metab Eng 1:198–205CrossRefGoogle Scholar
  6. Even S, Lindley ND, Cocaign-Bousquet M (2001) Molecular physiology of sugar catabolism in Lactococcus lactis IL1403. J Bacteriol 183:3817–3824CrossRefGoogle Scholar
  7. Garrigues C, Loubiere P, Lindley ND, Cocaign-Bousquet M (1997) Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD + ratio. J Bacteriol 179:5282–5287CrossRefGoogle Scholar
  8. Gaudu P, Lamberet G, Poncet S, Gruss A (2003) CcpA regulation of aerobic and respiration growth in Lactococcus lactis. Mol Microbiol 50:183–192CrossRefGoogle Scholar
  9. Hoefnagel MH, Starrenburg MJ, Martens DE, Hugenholtz J, Kleerebezem M, Van Swam II, Bongers R, Westerhoff HV, Snoep JL (2002) Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis. Microbiology 148:1003–1013CrossRefGoogle Scholar
  10. Hugenholtz J, Starrenburg MJ (1992) Diacetyl production by different strains of Lactococcus lactis subsp. lactis var diacetyllactis and Leuconostoc spp. Appl Environ Biotechnol 38:17–22CrossRefGoogle Scholar
  11. Iyer PP, Ferry JG (2001) Role of arginines in coenzyme A binding and catalysis by the phosphotransacetylase from Methanosarcina thermophila. J Bacteriol 183:4244–4250CrossRefGoogle Scholar
  12. Jay JM (1982) Antimicrobial properties of diacetyl. Appl Environ Microbiol 44:525–532CrossRefGoogle Scholar
  13. Jensen NB, Melchiorsen CR, Jokumsen KV, Villadsen J (2001) Metabolic behavior of Lactococcus lactis MG1363 in microaerobic continuous cultivation at a low dilution rate. Appl Environ Microbiol 67:2677–2682CrossRefGoogle Scholar
  14. Kuipers OP, Beerthuyzen MM, Siezen RJ, De Vos WM (1993) Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis: requirement of expression of the nisA and nisI genes for development of immunity. Eur J Biochem 216:281–291CrossRefGoogle Scholar
  15. Lopez de Felipe F, Starrenburg M, Hugenholtz J (1997) The role of NADH-oxidation in acetoin and diacetyl production from glucose in Lactococcus lactis susp. lactis MG1363. FEMS Microbiol Lett 156:15–19CrossRefGoogle Scholar
  16. Lopez de Felipe F, Kleerebezem M, de Vos WM, Hugenholtz J (1998) Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. J Bacteriol 180:3804–3808CrossRefGoogle Scholar
  17. Loubiere P, Cocaign-Bousquet M, Matos J, Goma G, Lindley ND (1997) Influence of end-products inhibition and nutrient limitations on the growth of Lactococcus lactis subsp. lactis. J Appl Microbiol 85:95–100CrossRefGoogle Scholar
  18. Luesink EJ, van Herpen RE, Grossiord BP, de Kuipers OP, Vos WM (1998) Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Mol Microbiol 30:789–798CrossRefGoogle Scholar
  19. Monedero V, Kuipers OP, Jamet E, Deutscher J (2001) Regulatory functions of serine-46-phosphorylated HPr in Lactococcus lactis. J Bacteriol 183:3391–3398CrossRefGoogle Scholar
  20. Neves AR, Ramos A, Costa H, van Swam II II, Hugenholtz J, Kleerebezem M, de Vos WM, Santos H (2002a) Effect of different NADH oxidase levels on glucose metabolism by Lactococcus lactis: kinetics of intracellular metabolite pools determined by in vivo nuclear magnetic resonance. Appl Environ Microbiol 68:6332–6342CrossRefGoogle Scholar
  21. Neves AR, Ventura R, Mansour N, Shearman C, Gasson MJ, Maycock C, Ramos A, Santos H (2002b) Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD(+) and NADH pools determined in vivo by 13C NMR. J Biol Chem 277:28088–28098CrossRefGoogle Scholar
  22. Pederersen MB, Iversen SL, Sørensen KI, Johansen E (2005) The long and winding road from the research laboratory to industrial applications of lactic acid bacteria. FEMS Microbiol Rev 29:611–24CrossRefGoogle Scholar
  23. Pullan LM, Igarashi P, Noltmann EA (1983) Arginine-specific modification of rabbit muscle phosphoglucose isomerase: differences in the inactivation by phenylglyoxal and butanedione and in the protection by substrate analogs. Arch Biochem Biophys 221:489–498CrossRefGoogle Scholar
  24. Ramos A, Neves AR, Ventura R, Maycock C, Lopez P, Santos H (2004) Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis. Microbiology 150:1103–1111CrossRefGoogle Scholar
  25. Raya R, Bardowski J, Andersen P, Ehrlich S, Chopin A (1998) Multiple transcriptional control of the Lactococcus lactis trp operon. J Bacteriol 10:3174–3180CrossRefGoogle Scholar
  26. Saier MJ, Chauvaux S, Cook G, Deutscher J, Paulsen I, Reizer J, Ye J (1996) Catabolite repression and inducer control in Gram-positive bacteria. Microbiology 142:217–230CrossRefGoogle Scholar
  27. Simon D, Chopin A (1988) Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70:559–566CrossRefGoogle Scholar
  28. Snoep JL, Teixeira de Mattos MJ, Starrenburg MJ, Hugenholtz J (1992) Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis. J Bacteriol 174:4838–4841CrossRefGoogle Scholar
  29. Solem C, Koebmann BJ, Jensen PR (2003) Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis MG1363. J Bacteriol 185:1564–1571CrossRefGoogle Scholar
  30. Thomas TD, Ellwood DC, Longyear VM (1979) Change from homo- to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures. J Bacteriol 138:109–117CrossRefGoogle Scholar
  31. Thomas TD, Turner KW, Crow VL (1980) Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation. J Bacteriol 144:672–682CrossRefGoogle Scholar
  32. Thompson J, Torchia D (1984) Use of 31P nuclear magnetic resonance spectroscopy and 14C fluorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis. J Bacteriol 158:791–800CrossRefGoogle Scholar
  33. van Niel EW, Palmfeldt J, Martin R, Paese M, Hahn-Hagerdal B (2004) Reappraisal of the regulation of lactococcal L-lactate dehydrogenase. Appl Environ Microbiol 70:1843–1846CrossRefGoogle Scholar
  34. Zomer AL, Buist G, Larsen R, Kok J, Kuipers OP (2007) Time-resolved determination of the CcpA regulon of Lactococcus lactis subsp. cremoris MG1363. J Bacteriol 189:1366–1381CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.Instituto del FríoConsejo Superior de Investigaciones Científicas (CSIC)MadridSpain
  2. 2.Unité Bactéries lactiques et Opportunistes (UBLO)Institut National de la Recherche AgronomiqueJouy en JosasFrance

Personalised recommendations