α-Acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions (original) (raw)
Related papers
Applied and Environmental Microbiology, 2008
Lactococcus lactis subsp. lactis bv. diacetylactis strains are aroma-producing organisms used in starter cultures for the elaboration of dairy products. This species is essentially a fermentative microorganism, which cometabolizes glucose and citrate to yield aroma compounds through the diacetyl/acetoin biosynthetic pathway. Our previous results have shown that under acidic growth Lactococcus bv. diacetylactis CRL264 expresses coordinately the genes responsible for citrate transport and its conversion into pyruvate. In the present work the impact of acidic growth on glucose, citrate, and pyruvate metabolism of Lactococcus bv. diacetylactis CRL264 has been investigated by proteomic analysis. The results indicated that acid growth triggers the conversion of citrate, but not glucose, into alpha-acetolactate via pyruvate. Moreover, they showed that low pH has no influence on levels of lactate dehydrogenase and pyruvate dehydrogenase. Therefore, the influence of external pH on regulation of the diacetyl/acetoin biosynthetic pathway in Lactococcus bv. diacetylactis CRL264 has been analyzed at the transcriptional level. Expression of the als, aldB, aldC, and butBA genes encoding the enzymes involved in conversion of pyruvate into aroma compounds has been investigated by primer extension, reverse transcription-PCR analysis, and transcriptional fusions. The results support that this biosynthetic pathway is induced at the transcriptional level by acidic growth conditions, presumably contributing to lactococcal pH homeostasis by synthesis of neutral compounds and by decreasing levels of pyruvate.
International Journal of Food Microbiology, 2013
Lactococcus lactis subsp. lactis biovar diacetylactis strains are used in the dairy industry for generating acetoin and notably diacetyl which imparts a high level of buttery flavor notes. A collection of domesticated and environmental strains was screened for the production of diacetyl or acetoin (D/ A), and citrate fermentation. Unexpectedly, both domesticated and environmental strains produced D/A. Domesticated strains belonging to the currently named "biovar diacetylactis" metabolized citrate and produced large amounts of D/A during early growth. They harbored the citP plasmid gene encoding citrate permease and a chromosomal region citM-citI-citCDEFXG involved in citrate metabolism. In these strains, citrate consumption was identified as the major determinant of aroma production. Environmental strains, specifically UCMA5716 and A12, produced as much D/A as the CitP + strains, though at slightly lower rates. UCMA5716 was found to contain the citM-citI-citCDEFXG cluster but not the citP gene. A12 had neither. In these strains, production rate of D/A was linearly correlated with pyruvate synthesis rate. However, the correlation factor was strain-dependent, suggesting different modes of regulation for pyruvate rerouting towards fermentation end-products and flavors. This work highlights the genetic and metabolic differences between environmental and domesticated strains. The introduction of environmental strains into industrial processes could considerably increase the diversity of starters, enhancing the delivery of new technological properties.
Applied Microbiology and Biotechnology, 2003
Co-fermentation of glucose and citrate by Lactococcus lactis diacetylactis: quantification of the relative metabolic rates by isotopic analysis at natural abundance Abstract The simultaneous catabolism of citrate and glucose by growing Lactococcus lactis subsp. lactis biovar. diacetylactis to obtain energy was followed quantitatively, using a non-enrichment isotopic technique. Both citrate and glucose are precursors of pyruvate, which may either be reduced to lactate, the principle product that accumulates, or be converted to diacetyl and acetoin. Under suitable conditions, both routes regenerate NAD + . Until recently, however, the quantitative relationships between the two substrates and these three products were poorly defined. It was recently shown, by exploiting differences in natural abundance 13 C/ 12 C ratios in the two substrates, that there is no metabolic separation of the catabolism of these two carbon sources. In this study, it is shown that the relative consumption rates change throughout the growth phase, citrate being preferentially metabolised at the onset of a culture of energy-depleted cells, with a subsequent evolution towards a metabolism dominated by glucose consumption. Additionally, it is shown that the relative consumption rates are influenced by environmental factors, notably initial pH and temperature. Schematic pathway showing the metabolic relationships between glucose, citrate, lactic acid, acetate, diacetyl and acetoin. AALDC a-Acetolactate decarboxylase, ALS a-acetolactate synthase, DDH diacetyl reductase, NON-ENZ non-enzymatic
Le Lait, 1998
Citrate metabolism performed by a number of lactic acid bacteria yields volatile compounds, such as diacetyl and acetaldehyde, which are important for flavour development in fermented milk products. Citrate uptake in Lactococcus laetis subsp. laetis biovar diacetylactis and Leuconostoc mesenteroides subsp. mesenteroides is catalyzed by a secondary carrier, the citrate permease P (CitP). The presence of CitP is essential for citrate utilization, since in its absence no citrate metabolism is observed although ail enzymes involved in conversion of citrate are present inside the cells. In this review the genetic organization of the plasmid encoding the citrate transport system of lactococci is described, the posttranscriptional regulation of the citQRP operon is presented and the influence of external pH on citP transcription, citrate uptake and cometabolism of citrate and glucose is discussed. These last studies reveal a novel molecular mechanism that improves the adaptation of L. diacetylactis to acidic pH. © Inra/Elsevier, Paris. Lactococcus lactis subsp. lactis biovar diacetylactis / citrate transport 1 regulation of gene expression 1 lactic acid bacteria 1 acidic stress Résumé-Régulation de 1'expression du système de transport du citrate chez Lactococcus lactis subsp.lactis biovar diacetylactis. Le métabolisme du citrate chez certaines bactéries lactiques se traduit par la synthèse de composés d'arôme tels que le di acétyle et l'acétaldéhyde. Ces composés contribuent à l'élaboration de la flaveur de certains aliments issus de la fermentation lactique. Le transport du citrate chez Lactococcus lactis subsp. lactis biovar diacetylactis et Leuconostoc mesenteroides subsp mesenteroides est catalysé par un transporteur secondaire, la citrate perméase P. Cette perméase est essentielle pour l'utilisation du citrate. En son absence le citrate n'est pas métabolisé, bien que toutes les enzymes impliquées dans sa conversion soient présentes au niveau de la cellule. Dans cette revue, nous décrivons l'organisation génétique du plasmide codant pour le transport du citrate chez L. diacetylactis, la régulation post-transcriptionnelle de l'opéron citQRP et l'influence du pH extracellulaire sur la transcription du gène citP. Par ailleurs, l'étude du transport du citrate et du cométabolisme citrate-glucose a révélé un nouveau mécanisme moléculaire permettant une meilleure adaptation de L. diacetylaetis à des pH acides.
1999
The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the ␣-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp. lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV-or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucineresistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains.
Journal of Bacteriology, 2004
Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex. In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region. Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization. The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb. This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG). We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs. Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI. This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions.
Journal of Applied Microbiology, 2007
Aims: Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions. Methods and Results: A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant. This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. lactis. Conclusions: The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions. Significance and impact of the study: This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.
Citrate metabolism in lactic acid bacteria
FEMS Microbiology Reviews, 1993
Citrate metabolism plays an important role in many food fermentations involving lactic acid bacteria. Since citrate is a highly oxidized substrate, no reducing equivalents are produced during its degradation, resulting in the formation of metabolic end products other than lactic acid. Some of these end products, such as diacetyl and acetaldehyde, have very distinct aroma properties and contribute significantly to the quality of the fermented foods. In this review the metabolic pathways involved in product formation from citrate are described, the bioenergetic consequences of this metabolism for the lactic acid bacteria are discussed and detailed information on some key enzymes in the citrate metabolism is presented. The combined knowledge is used for devising strategies to avoid, control or improve product formation from citrate.