Abstract
In this study we analyzed under various pH conditions including low pH, the effects of l-malic acid and citric acid, combined or not, on the growth, the proton motive force components and the transcription level of selected genes of the heterolactic bacterium Oenococcus oeni. It is shown here that l-malate enhanced the growth yield at pH equal or below 4.5 while the presence of citrate in media led to a complete and unexpected inhibition of the growth at pH 3.2. Nevertheless, whatever the growth conditions, both l-malate and citrate participated in the enhancement of the transmembrane pH gradient, whereas the membrane potential decreased with the pH. These results suggested that it was not citrate that was directly responsible for the inhibition observed in cultures done at low pH, but probably its end products. This was confirmed since, in media containing l-malate, the addition of acetate substantially impaired the growth rate of the bacterium and slightly the membrane potential and pH gradient. Finally, study of the expression of genes involved in the metabolism of organic acids showed that at pH 4.5 and 3.2 the presence of l-malate led to an increased amount of mRNA of mleP encoding a malate transporter.
This is a preview of subscription content, log in via an institution to check access.
References
Bandell M, Ansanay V, Rachidi N, Dequin S, Lolkema JS (1997) Membrane potential-generating malate (MleP) and citrate (CitP) transporters of lactic acid bacteria are homologous proteins. Substrate specificity of the 2-hydroxycarboxylate transporter family. J Biol Chem 272:18140–18146
Bandell M, Lhotte ME, Marty-Teysset C, Veyrat A, Prevost H, Divies C, Konings WN, Lolkema JS (1998) Mechanism of the citrate transporters in carbohydrate and citrate cometabolism in Lactococcus and Leuconostoc species. Appl Environ Microbiol 64:1594–1600
Bennik MH, Verheul A, Abee T, Naaktgeboren-Stoffels G, Gorris LG, Smid EJ (1997) Interactions of nisin and pediocin PA-1 with closely related lactic acid bacteria that manifest over 100-fold differences in bacteriocin sensitivity. Appl Environ Microbiol 63:3628–3636
Booth IR (1985) Regulation of cytoplasmic pH in bacteria. Microbiol Rev 49:359–378
Breeuwer JA, Abee T (2004) Assessment of the membrane potential, intracellular pH and respiration of bacteria employing fluorescence techniques. In: Molecular microbial ecology manual, 2nd edn, pp 1563–1580
Breeuwer P, Drocourt JL, Rombouts FM, Abee T (1996) A novel method for continuous determination of the intracellular pH in bacteria with the internally conjugated fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester. Appl Environ Microbiol 62:178–183
Busch W, Saier MH Jr (2004) The IUBMB-endorsed transporter classification system. Mol Biotechnol 27:253–262
Caspritz G, Radler F (1983) Malolactic enzyme of Lactobacillus plantarum. Purification, properties, and distribution among bacteria. J Biol Chem 258:4907–4910
Cavin JF, Prevost H, Lin J, Schmitt P, Divies C (1989) Medium for screening Leuconostoc oenos strains defective in malolactic fermentation. Appl Environ Microbiol 55:751–753
Cogan TM (1987) Co-metabolism of citrate and glucose by Leuconostoc spp. Effects on growth, substrates and products. J Appl Bacteriol 63:551–558
Desroche N, Beltramo C, Guzzo J (2005) Determination of an internal control to apply reverse transcription quantitative PCR to study stress response in the lactic acid bacterium Oenococcus oeni. J Microbiol Methods 60:325–333
Garcia-Quintans N, Magni C, de Mendoza D, Lopez P (1998) The citrate transport system of Lactococcus lactis subsp. lactis biovar diacetylactis is induced by acid stress. Appl Environ Microbiol 64:850–857
Henick-Kling T (1986) Growth and metabolism of Leuconostoc oenos and Lactobacillus plantarum in wine. In: PhD thesis, University of Adelaïde, Adelaïde
Henick-Kling T (1995) Control of malo-lactic fermentation in wine : energetics, flavour modification and methods of starter culture preparation. J Appl Bacteriol 79:29S-37S
Konings WN (2002) The cell membrane and the struggle for life of lactic acid bacteria. Antonie Van Leeuwenhoek 82:3–27
Kunkee RE (1991) Some roles of malic acid in the malolactic fermentation in wine making. FEMS Microbiol Lett 88:55–71
Labarre C, Divies C, Guzzo J (1996a) Genetic organization of the mle locus and identification of a mleR-like gene from Leuconostoc oenos. Appl Environ Microbiol 62:4493–4498
Labarre C, Guzzo J, Cavin JF, Divies C (1996b) Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos. Appl Environ Microbiol 62:1274–1282
Liu SQ (2002) A review: malolactic fermentation in wine—beyond deacidification. J Appl Microbiol 92:589–601
Lolkema JS, Poolman B, Konings WN (1995) Role of scalar protons in metabolic energy generation in lactic acid bacteria. J Bioenerg Biomembr 27:467–473
Lonvaud-Funel A (1986) Recherches sur les bactéries lactiques du vin. Fonctions métaboliques, croissance, génétique plasmidique. In: PhD Thesis, Université Bordeaux II, Institut d’Oenologie, Bordeaux, France
Lonvaud-Funel A (1999) Lactic acid bacteria in the quality improvement and depreciation of wine. Antonie Van Leeuwenhoek 76:317–331
Magni C, de Mendoza D, Konings WN, Lolkema JS (1999) Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH. J Bacteriol 181:1451–1457
Martin M, Magni C, Lopez P, de Mendoza D (2000) Transcriptional control of the citrate-inducible citMCDEFGRP operon, encoding genes involved in citrate fermentation in Leuconostoc paramesenteroides. J Bacteriol 182:3904–3912
Martin MG, Sender PD, Peiru S, de Mendoza D, Magni C (2004) Acid-inducible transcription of the operon encoding the citrate lyase complex of Lactococcus lactis biovar diacetylactis CRL264. J Bacteriol 186:5649–5660
Marty-Teysset C, Lolkema JS, Schmitt P, Divies C, Konings WN (1995) Membrane potential-generating transport of citrate and malate catalyzed by CitP of Leuconostoc mesenteroides. J Biol Chem 270:25370–25376
Marty-Teysset C, Lolkema JS, Schmitt P, Divies C, Konings WN (1996a) The citrate metabolic pathway in Leuconostoc mesenteroides: expression, amino acid synthesis, and alpha-ketocarboxylate transport. J Bacteriol 178:6209–6215
Marty-Teysset C, Posthuma C, Lolkema JS, Schmitt P, Divies C, Konings WN (1996b) Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides. J Bacteriol 178:2178–2185
Miller JH (1959) Use of dinitrosalycilic acid reagent for determination of reducing sugar. Anal Chem 31:426–428
Mills DA, Rawsthorne H, Parker C, Tamir D, Makarova K (2005) Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking. FEMS Microbiol Rev 29:465–475
Miranda M, Ramos A, Veiga-da-Cunha M, Loureiro-Dias MC, Santos H (1997) Biochemical basis for glucose-induced inhibition of malolactic fermentation in Leuconostoc oenos. J Bacteriol 179:5347–5354
Ramos A, Santos H (1996) Citrate and sugar co-fermentation in Leuconosctoc oenos, a 13C nuclear magnetic resonance study. Appl Environ Microbiol 62:2577–2585
Ramos A, Poolman B, Santos H, Lolkema JS, Konings WN (1994) Uniport of anionic citrate and proton consumption in citrate metabolism generates a proton motive force in Leuconostoc oenos. J Bacteriol 176:4899–4905
Ramos A, Lolkema JS, Konings WN, Santos H (1995) Enzyme basis for pH regulation of citrate and pyruvate metabolism by Leuconostoc oenos. Appl Environ Microbiol 61:1303–1310
Richter H, Hamann I, Unden G (2003) Use of the mannitol pathway in fructose fermentation of Oenococcus oeni due to limiting redox regeneration capacity of the ethanol pathway. Arch Microbiol 179:227–233
Saguir FM, Manca de Nadra MC (1996) Organic acid metabolism under different glucose concentrations of Leuconostoc oenos from wine. J Appl Bacteriol 81:393–397
Saguir FM, Manca de Nadra MC (2002) Effect of L-malic and citric acids metabolism on the essential amino acid requirements for Oenococcus oeni growth. J Appl Microbiol 93:295–301
Saier MH Jr (2000) A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev 64:354–411
Salema M, Poolman B, Lolkema JS, Dias MC, Konings WN (1994) Uniport of monoanionic L-malate in membrane vesicles from Leuconostoc oenos. Eur J Biochem 225:289–295
Salema M, Capucho I, Poolman B, San Romao MV, Dias MC (1996a) In vitro reassembly of the malolactic fermentation pathway of Leuconostoc oenos (Oenococcus oeni). J Bacteriol 178:5537–5539
Salema M, Lolkema JS, San Romao MV, Lourero Dias MC (1996b) The proton motive force generated in Leuconostoc oenos by L-malate fermentation. J Bacteriol 178:3127–3132
Salou P, Loubiere P, Pareilleux A (1994) Growth and energetics of Leuconostoc oenos during cometabolism of glucose with citrate or fructose. Appl Environ Microbiol 60:1459–1466
Schmitt P, Divies C, Merlot C (1990) Utilization of citrate by Leuconostoc mesenteroides subsp. cremoris in continuous culture. Biotechnol Lett 12:127–130
Sobczak I, Lolkema JS (2005) The 2-hydroxycarboxylate transporter family: physiology, structure, and mechanism. Microbiol Mol Biol Rev 69:665–695
Starrenburg MJ, Hugenholtz J (1991) Citrate Fermentation by Lactococcus and Leuconostoc spp. Appl Environ Microbiol 57:3535–3540
Versari A, Parpinello GP, Cattaneo M (1999) Leuconostoc oenos and malolactic fermentation in wine: a review. J Ind Microbiol Biotechnol 23:447–455
Zaunmuller T, Eichert M, Richter H, Unden G (2006) Variations in the energy metabolism of biotechnologically relevant heterofermentative lactic acid bacteria during growth on sugars and organic acids. Appl Microbiol Biotechnol 72:421–429
Acknowledgments
We thank Julian and Tina Coles for their reading of the English manuscript. We also thank Nicolas Desroche and Charlotte Beltramo for helpful discussion on Real-Time PCR experiments. This work was supported by the European Commission, contract number QLKI-CT-2002-02388. Daniel M. Linares was the recipient of a fellowship from the Spanish Ministry of Science and Technology.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Augagneur, Y., Ritt, JF., Linares, D.M. et al. Dual effect of organic acids as a function of external pH in Oenococcus oeni . Arch Microbiol 188, 147–157 (2007). https://doi.org/10.1007/s00203-007-0230-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00203-007-0230-0