Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides.

In Leuconostoc mesenteroides subsp. mesenteroides 19D, citrate is transported by a secondary citrate carrier (CitP). Previous studies of the kinetics and mechanism of CitP performed in membrane vesicles of L. mesenteroides showed that CitP catalyzes divalent citrate HCit2-/H+ symport, indicative of metabolic energy generation by citrate metabolism via a secondary mechanism (C. Marty-Teysset, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Biol. Chem. 270:25370-25376, 1995). This study also revealed an efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate cometabolism, suggesting that under physiological conditions, CitP may function as a precursor/product exchanger rather than a symporter. In this paper, the energetic consequences of citrate metabolism were investigated in resting cells of L. mesenteroides. The generation of metabolic energy in the form of a pH gradient (delta pH) and a membrane potential (delta psi) by citrate metabolism was found to be largely dependent on cometabolism with glucose. Furthermore, in the presence of glucose, the rates of citrate utilization and of pyruvate and lactate production were strongly increased, indicating an enhancement of citrate metabolism by glucose metabolism. The rate of citrate metabolism under these conditions was slowed down by the presence of a membrane potential across the cytoplasmic membrane. The production of D-lactate inside the cell during cometabolism was shown to be responsible for the enhancement of the electrogenic uptake of citrate. Cells loaded with D-lactate generated a delta psi upon dilution in buffer containing citrate, and cells incubated with citrate built up a pH gradient upon addition of D-lactate. The results are consistent with an electrogenic citrate/D-lactate exchange generating in vivo metabolic energy in the form of a proton electrochemical gradient across the membrane. The generation of metabolic energy from citrate metabolism in L. mesenteroides may contribute significantly to the growth advantage observed during cometabolism of citrate and glucose.     

Generation of a proton motive force by the anaerobic oxalate-degrading bacterium Oxalobacter formigenes.

The generation of transmembrane ion gradients by Oxalobacter formigenes cells metabolizing oxalate was studied. The magnitudes of both the transmembrane electrical potential (delta psi) and the pH gradient (internal alkaline) decreased with increasing external pH; quantitatively, the delta psi was the most important component of the proton motive force. As the extracellular pH of metabolizing cells was increased, intracellular pH increased and remained alkaline relative to the external pH, indicating that O. formigenes possesses a limited capacity to regulate internal pH. The generation of a delta psi by concentrated suspensions of O. formigenes cells was inhibited by the K+ ionophore valinomycin and the protonophore carbonyl cyanide-m-chlorophenylhydrazone, but not by the Na+ ionophore monensin. The H+ ATPase inhibitor N,N'-dicyclohexyl-carbodiimide inhibited oxalate catabolism but did not dissipate the delta psi. The results support the concept that energy from oxalate metabolism by O. formigenes is conserved not as a sodium ion gradient but rather, at least partially, as a transmembrane hydrogen ion gradient produced during the electrogenic exchange of substrate (oxalate) and product (formate) and from internal proton consumption during oxalate decarboxylation.     

Uniport of anionic citrate and proton consumption in citrate metabolism generates a proton motive force in Leuconostoc oenos.

The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated. Resting cells of L. oenos generate both a membrane potential (delta psi) and a pH gradient (delta pH) upon addition of citrate. After a lag time, the internal alkalinization is followed by a continuous alkalinization of the external medium, demonstrating the involvement of proton-consuming reactions in the metabolic breakdown of citrate. Membrane vesicles of L. oenos were prepared and fused to liposomes containing cytochrome c oxidase to study the mechanism of citrate transport. Citrate uptake in the hybrid membranes is inhibited by a membrane potential of physiological polarity, inside negative, and driven by an inverted membrane potential, inside positive. A pH gradient, inside alkaline, leads to the accumulation of citrate inside the membrane vesicles. Kinetic analysis of delta pH-driven citrate uptake over a range of external pHs suggests that the monovalent anionic species (H2cit-) is the transported particle. Together, the data show that the transport of citrate is an electrogenic process in which H2cit- is translocated across the membrane via a uniport mechanism. Homologous exchange (citrate/citrate) was observed, but no evidence for a heterologous antiport mechanism involving products of citrate metabolism (e.g., acetate and pyruvate) was found. It is concluded that the generation of metabolic energy by citrate utilization in L. oenos is a direct consequence of the uptake of the negatively charged citrate anion, yielding a membrane potential, and from H(+)-consuming reactions involved in subsequent citrate metabolism, yielding a pH gradient. The uptake of citrate is driven by its own concentration gradient, which is maintained by efficient metabolic breakdown (metabolic pull).     

The role of transport processes in survival of lactic acid bacteria, Energy transduction and multidrug resistance

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural an d functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist.     

Histamine-producing pathway encoded on an unstable plasmid in Lactobacillus hilgardii 0006

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.     

Role of scalar protons in metabolic energy generation in lactic acid bacteria

Lactic acid bacteria are able to generate a protonmotive force across the cytoplasmic membrane by various metabolic conversions without involvement of substrate level phosphorylation or proton pump activity. Weak acids like malate and citrate are taken up in an electrogenic process in which net negative charge is translocated into the cell thereby generating a membrane potential. The uptake is either an exchange process with a metabolic end-product (precursor/ product exchange) or a uniporter mechanism. Subsequent metabolism of the internalized substrate drives uptake and results in the generation of a pH gradient due to the consumption of scalar protons. The generation of the membrane potential and the pH gradient involve separate steps in the pathway. Here it is shown that they are nevertheless coupled. Analysis of the pH gradient that is formed during malolactic fermentation and citrate fermentation shows that a pH gradient, inside alkaline, is formed only when the uptake system forms a membrane potential, inside negative. These secondary metabolic energy generating systems form a pmf that consists of both a membrane potential and a pH gradient, just like primary proton pumps do. It is concluded that the generation of a pH gradient, inside alkaline, upon the addition of a weak acid to cells is diagnostic for an electrogenic uptake mechanism translocating negative charge with the weak acid.     

Isotopic microassay of histamine, histidine, histidine decarboxylase and histamine methyltransferase in brain tissue

Abstract— Microassays are described for histamine, histidine, and the activities of the enzymes histidine decarboxylase (EC 4.1.1.22) and histamine niethyltransferase (EC 2.1.1.8) in brain tissue. The enzymic-isotopic microassay for histamine is based on the methylation of tissue histamine by added histamine methyl-transferase and [¹⁴C]- or [³H]-labelled S-adenosyl-l -methionine. In a double-isotopic form of the assay, a tracer of [³H]histamine is employed along with [¹⁴C]S-adenosyl-l -methionine, and the ratio [¹⁴C]:[³H] reflects the amount of histamine in the sample. Because the methylation of histamine is uniform in brain samples studied, a single isotopic assay with [³H]S-adenosyl-l -methionine as the methyl donor is possible and increases sensitivity, so that 10 pg of tissue histamine can be estimated reliably. The assay for histidine involves decarboxylation of histidine by a bacterial histidine decarboxylase and measurement of the histamine formed by the enzymicisotopic procedure. In the histidine decarboxylase assay, histamine synthesized from added histidine is measured. The assay for histamine methyltransferase involves measuring the formation of [¹⁴C]methylhistamine with [¹⁴C]S-adenosyl-l -methionine serving as the methyl donor.     

Ion-dependent generation of the electrochemical proton gradient delta muH+ in reconstituted plasma membrane vesicles from the yeast Metschnikowia reukaufii

Plasma membrane vesicles were reconstituted by freezing and thawing of purified plasma membrane fraction from the yeast Metschnikowia reukaufii and phosphatidylcholine (type II-S from Sigma). The reconstituted plasma membrane vesicles generated a proton gradient (acidic inside) upon addition of ATP in presence of alkali cations. delta pH generation was most efficient when K+ was present both outside and inside the plasma membrane vesicles. Both ATPase activity and proton translocation in plasma membrane vesicles were inhibited by orthovanadate (50% inhibition at 100 microM). Plasma membrane vesicles reconstituted without added phosphatidylcholine generated in addition to delta pH, also an electrical potential difference delta psi (inside positive). Delta psi generation exhibited no K+ specificity. 50 microM dicyclohexylcarbodiimide inhibited completely delta psi generation whereas the K+-channel blocker quinine (5 microM) caused an 8-fold increase of delta psi. The proton gradient was much less affected by the agents. Taking into account the K+-dependent stimulation of the plasma membrane ATPase of M. reukaufii, these results further support the conclusion that the ATPase operates as a partially electrogenic H+/K+ exchanger, as was also suggested for other yeast plasma membrane ATPases.     

Electrogenic glutamine uptake by Peptostreptococcus anaerobius and generation of a transmembrane potential.

Peptostreptococcus anaerobius converted glutamine stoichiometrically to ammonia and pyroglutamic acid, and the Eadie-Hofstee plot of glutamine transport was biphasic. High-affinity, sodium-dependent glutamine transport (affinity constant [Kt] of 1.5 microM) could be driven by the chemical gradient of sodium, and more than 20 mM sodium was required for half-maximal velocity. High-affinity glutamine transport was not stimulated or inhibited by a membrane potential (delta psi). Low-affinity glutamine transport had a rate which was directly proportional to the external glutamine concentration, required less than 100 microM sodium, and was inhibited strongly by a delta psi. Cells which were treated with N,N-dicyclohexylcarbodiimide to inhibit the F1F0 ATPase still generated a delta psi but did so only if the external glutamine concentration was greater than 15 mM. Low-affinity glutamine uptake could not be saturated by as much as 200 mM glutamine, but glutamine-1 accounts for only a small fraction of the total glutamine at physiological pH values (pH 6 to 7). On the basis of these results, it appeared that the low-affinity glutamine transport was an electrogenic mechanism which was converting a chemical gradient of glutamine-1 into a delta psi. Other mechanisms of delta psi generation (electrogenic glutamine-pyroglutamate or -ammonium exchange) could not be demonstrated.     

Histamine-Producing Pathway Encoded on an Unstable Plasmid in Lactobacillus hilgardii 0006

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.     

Functional comparison of DCCD-sensitive Na+/H+ antiporter in Halobacterium halobium with monensin

Membrane vesicles from Halobacterium halobium create a large, inside negative membrane potential (delta psi) and small, inside alkaline pH gradient (delta pH) by illumination in 3 M NaCl. delta psi was the major component of a proton electrochemical potential (delta microH+) over a pH range from 5 to 8. After DCCD treatment of the vesicles, delta psi was replaced by delta pH due to the inhibition of the intrinsic delta pH----delta psi transformation process: delta psi formation in light is markedly retarded and an inversely large delta pH is established at these pHs. DCCD-caused changes in delta psi and delta pH were completely restored to the control level by the addition of monensin, an electroneutral Na+/H+ exchanger. The ratio of DCCD-caused change in delta pH and delta psi was identical to that of monensin-recovered delta psi and delta pH. The delta psi/delta pH ratio was approximately 0.8, that is, 100 mV of delta pH was transformed into 78 mV of delta psi. The present results indicate that the intrinsic activity of the DCCD-sensitive delta pH----delta psi transformation is mediated by an electroneutral Na+/H+ exchange.     

Improved acid stress survival of Lactococcus lactis expressing the histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524

Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 5-6.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (ΔΨ) and/or the pH gradient (ΔpH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ΔΨ and ΔpH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate.     

Malolactic fermentation: electrogenic malate uptake and malate/lactate antiport generate metabolic energy.

The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generated by the metabolism of a glycolytic substrate. Malolactic fermentation results in the synthesis of ATP which is inhibited by the ionophore nigericin and the F0F1-ATPase inhibitor N,N-dicyclohexylcarbodiimide. Since substrate-level phosphorylation does not occur during malolactic fermentation, the generation of metabolic energy must originate from the uptake of L-malate and/or excretion of L-lactate. The initiation of malolactic fermentation is stimulated by the presence of L-lactate intracellularly, suggesting that L-malate is exchanged for L-lactate. Direct evidence for heterologous L-malate/L-lactate (and homologous L-malate/L-malate) antiport has been obtained with membrane vesicles of an L. lactis mutant deficient in malolactic enzyme. In membrane vesicles fused with liposomes, L-malate efflux and L-malate/L-lactate antiport are stimulated by a membrane potential (inside negative), indicating that net negative charge is moved to the outside in the efflux and antiport reaction. In membrane vesicles fused with liposomes in which cytochrome c oxidase was incorporated as a proton motive force-generating mechanism, transport of L-malate can be driven by a pH gradient alone, i.e., in the absence of L-lactate as countersubstrate. A membrane potential (inside negative) inhibits uptake of L-malate, indicating that L-malate is transported an an electronegative monoanionic species (or dianionic species together with a proton). The experiments described suggest that the generation of metabolic energy during malolactic fermentation arises from electrogenic malate/lactate antiport and electrogenic malate uptake (in combination with outward diffusion of lactic acid), together with proton consumption as result of decarboxylation of L-malate. The net energy gain would be equivalent to one proton translocated form the inside to the outside per L-malate metabolized.     

Effects of potassium ions on proton motive force in Rhodobacter sphaeroides.

The proton motive force (PMF) was determined in Rhodobacter sphaeroides under anaerobic conditions in the dark and under aerobic-dark and anaerobic-light conditions. Anaerobically in the dark in potassium phosphate buffer, the PMF at pH 6 was -20 mV and was composed of an electrical potential (delta psi) only. At pH 7.9 the PMF was composed of a high delta psi of -98 mV and was partially compensated by a reversed pH gradient (delta pH) of +37 mV. ATPase inhibitors did not affect the delta psi, which was most likely the result of a K+ diffusion potential. Under energized conditions in the presence of K+ the delta psi depolarized due to electrogenic K+ uptake. This led to the generation of a delta pH (inside alkaline) in the external pH range of 6 to 8. This delta pH was dependent on the K+ concentration and was maximal at external K+ concentrations larger than 1.2 mM. In energized cells in 50 mM KPi buffer containing 5 mM MgSO4, a delta pH (inside alkaline) was present at external pHs from pH 6 to 8. As a result the overall magnitude of the PMF at various external pHs remained constant at -130 mV, which was significantly higher than the PMF under anaerobic-dark conditions. In the absence of K+, in 50 mM NaPi buffer containing 5 mM MgSO4, no depolarization of the delta psi was found and the PMF was composed of a large delta psi and a small delta pH. The delta pH became even reversed (inside acidic) at alkaline pHs (pH>7.3), resulting in a lowering of the PMF. These results demonstrate that in R. sphaeroides K+ uptake is essential for the generation of a delta pH and plays a central role in the regulation of the internal pH.     

On the mechanism of sodium ion translocation by methylmalonyl-CoA decarboxylase from Veillonella alcalescens

Veillonella alcalescens during lactate degradation developed an Na+ concentration gradient with 7-8 times higher external than internal Na+ concentrations in the logarithmic growth phase. The gradient declined to a factor of 1.9 in the late stationary phase. Methylmalonyl-CoA decarboxylase reconstituted into proteoliposomes performed an active electrogenic Na+ transport, creating delta psi of 60 mV, delta pNa+ of 50 mV, and delta mu Na+ of 110 mV. In the initial phase of the transport, the decarboxylase catalyzed the uptake of 2 Na+ ions malonyl-CoA molecule decarboxylated. During further development of the electrochemical Na+ gradient, this ratio gradually declined to zero, when decarboxylation continued without further increase of the internal Na+ concentration. The rate of malonyl-CoA decarboxylation declined initially during development of the membrane potential, but remained unchanged later on. Monensin abolished the Na+ gradient and increased the malonyl-CoA decarboxylation rate 2.8-fold. On dissipating the membrane potential with valinomycin, the internal Na+ concentration reached three times higher values than in its absence, and the decarboxylation rate increased 2.8-fold. Methylmalonyl-CoA decarboxylase catalyzed an exchange of internal and external Na+ ions in addition to net Na+ accumulation. The initial rate of Na+ influx was double that of malonyl-CoA decarboxylation. In the following, both rates decreased about twofold in parallel to values which remained constant during further development of the electrochemical Na+ gradient. Thus, Na+ influx and malonyl-CoA decarboxylation follow a stoichiometry of approximately 2:1, independent of the magnitude of the electrochemical Na+ gradient and are thus highly coupled events.     

Histamine formation in rat brain in vivo: effects of histidine loads

Abstract— Administration of l -histidine at the rate of 500 mg/kg induced an increase of nearly 50 per cent in the level of histamine in rat brain which lasted several hours. The augmentation of histamine level was not significant 3 h after lower doses or after d -histidine α-methyl DOPA and Ro 4-4602 neither affected the cerebral level of histamine nor its elevation induced by l -histidine. Brocresine, a known histidine decarboxylase inhibitor not only prevented the effect of histidine load but also induced a prompt fall in the amine level. These results confirm those from earlier experiments in vitro indicating that histamine synthesis in rat brain depends on a specific decarboxylase (EC 4 , 1.1.22) which is not normally saturated by the endogenous level of its substrate. When histamine levels were enhanced by histidine treatment, histidine decarboxylase activity, as evaluated on hypothalamus homogenates, was significantly reduced; intracisternal administration of cycloheximide, an inhibitor of protein synthesis, had similar effects. On the other hand, enzyme activity was not altered by the addition of histamine to hypothalamus homogenates. These results are compatible with the existence of a regulation mechanism of histidine decarboxylase involving repression by its end-product.     

Isotope effect studies of the pyruvate-dependent histidine decarboxylase from Lactobacillus 30a

The decarboxylation of histidine by the pyruvate-dependent histidine decarboxylase of Lactobacillus 30a shows a carbon isotope effect of k12/k13 = 1.0334 +/- 0.0005 and a nitrogen isotope effect k14/k15 = 0.9799 +/- 0.0006 at pH 4.8, 37 degrees C. The carbon isotope effect is slightly increased by deuteriation of the substrate and slightly decreased in D2O. The observed nitrogen isotope effect indicates that the imine nitrogen in the substrate-Schiff base intermediate complex is ordinarily protonated, and the pH dependence of the carbon isotope effect indicates that both protonated and unprotonated forms of this intermediate are capable of undergoing decarboxylation. As with the pyridoxal 5'-phosphate dependent enzyme, Schiff base formation and decarboxylation are jointly rate-limiting, with the intermediate histidine-pyruvate Schiff base showing a decarboxylation/Schiff base hydrolysis ratio of 0.5-1.0 at pH 4.8. The decarboxylation transition state is more reactant-like for the pyruvate-dependent enzyme than for the pyridoxal 5'-phosphate dependent enzyme. These studies find no particular energetic or catalytic advantage to the use of pyridoxal 5'-phosphate over covalently bound pyruvate in catalysis of the decarboxylation of histidine.     

ADI pathway and histidine decarboxylation are reciprocally regulated in Lactobacillus hilgardii ISE 5211: proteomic evidence

Amine production by amino acid decarboxylation is a common feature that is used by lactic acid bacteria (LAB) to complement lactic fermentation, since it is coupled with a proton-extruding antiport system which leads to both metabolic energy production and the attenuation of intracellular acidity. Analogous roles are played in LAB by both malolactic fermentation (MLF) and the arginine deiminase (ADI) pathway. The present investigation was aimed at establishing reciprocal interactions between amino acid decarboxylation and the two above mentioned routes. The analyses were carried out on a Lactobacillus hilgardii strain (ISE 5211) that is able to decarboxylate histidine to histamine, which had previously been isolated from wine and whose complete genome is still unknown. The 2DE proteomic approach, followed by MALDI TOF–TOF and De Novo Sequencing, was used to study the protein expression levels. The experimental evidence has indicated that malate does not influence histidine decarboxylase (HDC) biosynthesis and that histidine does not affect the malolactic enzyme level. However, the expression of the ADI route enzymes, arginine deiminase and ornithine transcarbamylase, is down-regulated by histidine: this biosynthetic repression is more important (4-fold) in cultures that are not supplemented with arginine, but is also significant (2-fold) in an arginine supplemented medium that normally induces the ADI pathway. On the other hand, arginine partially represses HDC expression, but only when histidine and arginine are both present in the culture medium. This proteomic study has also pointed out a down-regulation exerted by histidine over sugar metabolism enzymes and a GroEL stress protein. These data, together with the reciprocal antagonism between arginine deimination and histidine decarboxylation, offer clue keys to the understanding of the accumulation of lactate, amine, ammonia and ethylcarbamate in wine, with consequent implications on different health risk controls.     

Histamine, histidine, and growth-phase mediated regulation of the histidine decarboxylase gene in lactic acid bacteria isolated from wine

Fermented foods are frequently contaminated by histamine that is generated by microorganisms with histidine decarboxylase activity. The ingestion of large amounts of histamine can cause serious toxicological problems in humans. A study of the effects of histamine, histidine, and growth phase on histamine production by lactic acid bacteria isolated from wine is reported here. With northern blots and specific activity analysis, we observed that histidine induces the expression of the histidine decarboxylase gene (hdc) and that histamine causes a decrease in the expression of this gene. The expression of hdc is also mediated by the bacterial growth phase. Histidine and histamine do not affect histidine decarboxylase activity, whereas pyridoxal 5'-phosphate does. Data on histamine-producing lactic acid bacteria isolated from wine should contribute to the prevention of histamine formation during winemaking and storage.     

Energy dependence and functional reconstitution of the gamma-aminobutyric acid carrier from synaptic vesicles.

The energy dependence of gamma-aminobutyric acid (GABA) uptake was characterized in rat brain synaptic vesicles and in proteoliposomes reconstituted with a new procedure from vesicular detergent extracts. The proteoliposomes displayed high ATP-dependent GABA uptake activity with properties virtually identical to those of intact vesicles. GABA uptake was similar at chloride concentrations of 0 and 150 mM, i.e. conditions under which either the membrane potential (delta psi) or the pH difference (delta pH) predominates. Delta psi was gradually dissipated by increasing the concentration of SCN-. GABA uptake was reduced by 10 mM SCN-, showing less sensitivity to delta psi reduction than glutamate uptake but more than dopamine uptake. Dissipation of delta pH with NH+4 abolished GABA uptake at pH 7.3, whereas no significant inhibition occurred at pH 6.5. In contrast, dopamine uptake was inhibited more strongly, even at pH 6.5, and glutamate uptake was not reduced in either condition. We conclude that GABA uptake is driven by both components of the proton electrochemical gradient, delta pH and delta psi, and that this is different from the uptake of both dopamine and glutamate, which is more strongly dependent on delta pH and delta psi, respectively. Thus, our data suggest that GABA uptake is electrogenic and occurs in exchange for protons.