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.     

Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.

Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative delta psi and is stimulated by an inside positive delta psi. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.     

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.     

The generation of metabolic energy by solute transport

Secondary metabolic-energy-generating systems generate a proton motive force (pmf) or a sodium ion motive force (smf) by a process that involves the action of secondary transporters. The (electro)chemical gradient of the solute(s) is converted into the electrochemical gradient of protons or sodium ions. The most straightforward systems are the excretion systems by which a metabolic end product is excreted out of the cell in symport with protons or sodium ions (energy recycling). Similarly, solutes that were accumulated and stored in the cell under conditions of abundant energy supply may be excreted again in symport with protons when conditions become worse (energy storage). In fermentative bacteria, a proton motive force is generated by fermentation of weak acids, such as malate and citrate. The two components of the pmf, the membrane potential and the pH gradient, are generated in separate steps. The weak acid is taken up by a secondary transporter either in exchange with a fermentation product (precursor/product exchange) or by a uniporter mechanism. In both cases, net negative charge is translocated into the cell, thereby generating a membrane potential. Decarboxylation reactions in the metabolic breakdown of the weak acid consume cytoplasmic protons, thereby generating a pH gradient across the membrane. In this review, several examples of these different types of secondary metabolic energy generation will be discussed.     

Microbial transport: Adaptations to natural environments

The cytoplasmic membrane of bacteria is the matrix for metabolic energy transducing processes such as proton motive force generation and solute transport. Passive permeation of protons across the cytoplasmic membrane is a crucial determinant in the proton motive generating capacity of the organisms. Adaptations of the membrane composition are needed to restrict the proton permeation rates especially at higher temperatures. Thermophilic bacteria cannot sufficiently restrict this proton permeation at their growth temperature and have to rely on the much␣lower permeation of Na + to generate a sodium motive force for driving metabolic energy-dependent membrane processes. Specific transport systems mediate passage across the membrane at physiological rates of all compounds needed for growth and metabolism and of all end products of metabolism. Some of transport systems, the secondary transporters, transduce one form of electrochemical energy into another form. These transporters can play crucial roles in the generation of metabolic energy. This is especially so in anaerobes such as Lactic Acid Bacteria which live under energy-limited conditions. Several transport systems are specifically aimed at the generation of metabolic energy during periods of energy-limitation. In their natural environment bacteria are also often exposed to cytotoxic compounds, including antibiotics. Many bacteria can respond to this live-threatening condition by overexpressing powerful drug-extruding multidrug resistance systems.     

The cell membrane and the struggle for life of lactic acid bacteria

The major life-threatening event for lactic acid bacteria (LAB) in their natural environment is the depletion of their energy sources and LAB can survive such conditions only for a short period of time. During periods of starvation LAB can exploit optimally the potential energy sources in their environment usually by applying proton motive force generating membrane transport systems. These systems include in addition to the proton translocating F_oF₁-ATPase: a respiratory chain when hemin is present in the medium, electrogenic solute uptake and excretion systems, electrogenic lactate/proton symport and precursor/ product exchange systems. Most of these metabolic energy-generating systems offer as additional bonus the prevention of a lethal decrease of the internal and external pH. LAB have limited biosynthetic capacities and rely heavily on the presence of essential components such as sources of amino acids in their environment. The uptake of amino acids requires a major fraction of the available metabolic energy of LAB. The metabolic energy cost of amino acid uptake can be reduced drastically by accumulating oligopeptides instead of the individual amino acids and by proton motive force-generating efflux of excessively accumulated amino acids. Other life-threatening conditions that LAB encounter in their environment are rapid changes in the osmolality and the exposure to cytotoxic compounds, including antibiotics. LAB respond to osmotic upshock or downshock by accumulating or releasing rapidly osmolytes such as glycine-betaine. The life-threatening presence of cytotoxic compounds, including antibiotics, is effectively counteracted by powerful drug extruding multidrug resistance systems. The number and variety of defense mechanisms in LAB is surprisingly high. Most defense mechanisms operate in the cytoplasmic membrane to control the internal environment and the energetic status of LAB. Annotation of the functions of the genes in the genomes of LAB will undoubtely reveal additional defense mechanisms.     

Energy transduction in lactic acid bacteria

Abstract: In the discovery of some general principles of energy transduction, lactic acid bacteria have played an important role. In this review, the energy transducing processes of lactic acid bacteria are discussed with the emphasis on the major developments of the past 5 years. This work not only includes the biochemistry of the enzymes and the bioenergetics of the processes, but also the genetics of the genes encoding the energy transducing proteins. The progress in the area of carbohydrate transport and metabolism is presented first. Sugar translocation involving ATP-driven transport, ion-linked cotransport, heterologous exchange and group translocation are discussed. The coupling of precursor uptake to product product excretion and the linkage of antiport mechanisms to the deiminase pathways of lactic acid bacteria is dealt with in the second section. The third topic relates to metabolic energy conservation by chemiosmotic processes. There is increasing evidence that precursor/product exchange in combination with precursor decarboxylation allows bacteria to generate additional metabolic energy. In the final section transport of nutrients and ions as well as mechanisms to excrete undesirable (toxic) compounds from the cells are discussed.     

Serine metabolism in legume nodules: Purification and properties of phosphoserine aminotransferase

Plant cells excrete protons via an electrogenic proton pump, the K⁺-stimulated, Mg²⁺-dependent H⁺-ATPasc, located on the cytoplasmic side of the plasma membrane. Plasma membrane redox reactions are also coupled to proton excretion. Various inhibitors were used on carrot ( Daucus carola L.) cells in an attempt to distinguish between the two processes. Inhibitors of electron transport reactions in the plasma membrane (chloroquine, 8-hydroxyquinolinc, 4,7-dichloroquinolinc and retinoic acid) inhibited ferricyanide-induced proton excretion by 37–100%, while they inhibited potassium ferricyanide reduction, a measure of plasma membrane redox activity, by 42–100%. The above-mentioned quinolines and retinoic acid inhibited cell growth by 49–98%, with the exception of chloroquine, which stimulated carrot cell growth by 36%.     

Effect of the methylation of uracil and/or glycine on their mutual interaction

In order to simulate the hydrogen bonding and proton transfer (PT) in protein-DNA/RNA interactions, a series of simplified models were employed and investigated in the gas phase. These models included various neutral, anionic and cationic glycine-uracil dimers, and their methylated derivatives generated by the mono- or dimethylation of glycine and/or uracil moieties of the dimer. The results reveal that the only process that can occur in the neutral complexes is a double-PT process leading to proton exchange between the two moieties (i.e., point mutation). The first methyl substitute can reduce the activation energy of the PT process and thus promote the isomerization of the two moieties; further methylation can reduce the isomerization in only some of the cases. In the anionic complexes, only the one-way PT (i.e., amino acid → nucleic acid base) process is energetically favorable, and this PT process is an interesting barrier-free one (BFPT), with the attached electron locating itself at the base moiety. Methylation will disfavor BFPT, but it cannot alter the nature of BFPT. In the cationic complexes, three different PT processes can occur. These processes can transform mutually by adjusting either or both of the methylated sites and methyl number, indicating that the methylation can regulate the dynamics of these PT processes.     

Antioxidant Properties of Lactic Acid Bacteria

Microbiology - Lactic acid bacteria (LAB) are widely used in fermentation processes for the preparation of various foodstuffs, including dairy, meat and vegetable products. In the course of...     

Use of inexpensive nitrogen sources and starch for L(+) lactic acid production in anaerobic submerged fermentation

L(+) Lactic acid fermentation was studied by Lactobacillus amylophilus GV6 under the influence of inexpensive nitrogen sources (red lentil-RL, and Baker's yeast cells-YC) and starch by response surface methodology (RSM). Central composite rotatable design (CCRD) was employed to determine maximum lactic acid production at optimum values for process variables RL, YC and incubation period (IP) and a satisfactory fit model was realized. Lactic acid production was significantly affected by RL and IP interactions as well as by independent variables RL and YC. Maximum lactic acid production of 13.5 g/15.2g starch was obtained with RL 0.8%, YC 1% and IP of 48 h, with 92% lactic acid yield efficiency (g lactic acid produced/g substrate utilized) and 40% increase (from 50 g to 92 g/100 g starch utilized) in lactic acid production. This is the first report on response optimization in direct fermentation of starch to lactic acid using inexpensive nitrogen sources substituting peptone and yeast extract in anaerobic submerged fermentation by amylolytic lactic acid bacteria (LAB).     

Identification, purification, and reconstitution of OxlT, the oxalate: formate antiport protein of Oxalobacter formigenes.

We had proposed earlier that the anaerobe Oxalobacter formigenes sustains a proton-motive force by exploiting a secondary carrier rather than a primary proton pump. In this view, a carrier protein would catalyze the exchange of extracellular oxalate, a divalent anion, and intracellular formate, the monovalent product of oxalate decarboxylation. Such an electrogenic exchange develops an internally negative membrane potential, and since the decarboxylation reaction consumes an internal proton, the combined activity of the carrier and the soluble decarboxylase would constitute an "indirect" proton pump with a stoichiometry of 1H+ per turnover. This model is now verified by identification and purification of OxlT, the protein responsible for the anion exchange reaction. Membranes of O. formigenes were solubilized at pH 7 with 1.25% octyl glucoside in 20 mM 3-(N-morpholino)propanesulfonic acid/K, in the presence of 0.4% Escherichia coli phospholipids and with 20% glucerol present as the osmolyte stabilant. Rapid methods for reconstitution were developed to monitor the distribution of OxlT during biochemical fractionation, allowing its purification by sequential anion and cation exchange chromatography. OxlT proved to be a single hydrophobic polypeptide, of 38 kDa mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a turnover number estimated as at least 1000/s. The properties of OxlT point to an indirect proton pump as the mechanism by which a proton-motive force arises in O. formigenes, and one may reasonably argue that indirect proton pumps take part in bacterial events such as acetogenesis, malolactate fermentation, and perhaps methanogenesis.     

Comparative assessment of fermentation techniques useful in the processing of ogi

Ogiis processed traditionally by the use of uncontrolled spontaneous fermentation of maize, sorghum and millet. In this study, traditionally applied spontaneous fermentation was compared with accelerated batch fermentation (or back-slopping) and the use of starter cultures to initiate fermentation. Lactic acid bacteria populations comprised 95 of the total viable bacteria and remained prominent throughout the fermentations, while number of moulds and coliform bacteria declined as the fermentation progressed. The fermentation method involving the application of starter culture helps most to control the prevalence of coliforms and moulds. Lactic acid bacteria, such as Lactococcus raffinolactis, Pediococcussp.,Pediococcus pentosaceus, Lactobacillus plantarum, Lb. suebicus and Lb. brevis,were isolated at different processing stages of ogi using accelerated batch fermentation (back-slopping) technique. Highest increase in acidity was observed immediately after wet-milling and sieving fermenting maize grains at 28 and 48 h. Sharp increases in the reducing sugar levels were noted between 24 and 28 h of fermentations during wet-milling and sieving processes.     

Membrane H Conductance of Clostridium thermoaceticum and Clostridium acetobutylicum: Evidence for Electrogenic Na/H Antiport in Clostridium thermoaceticum

H conductance in de-energized cells of Clostridium thermoaceticum and Clostridium acetobutylicum was determined from the rate of realkalinization of the medium after an acid pulse. In both organisms, cell membrane proton permeability was increased by fermentation end products and ionophores. In C. thermoaceticum, H conductance was increased by Na ions compared with K as counterions. In these cells, addition of Na, but not K, elicited efflux of H; H efflux was stimulated by SCN and decreased by various ionophores. We concluded that C. thermoaceticum possesses an electrogenic Na/H antiporter. In contrast, C. acetobutylicum cells did not have an electrogenic Na/H antiporter.     

Towards lactic acid bacteria-based biorefineries

Lactic acid bacteria (LAB) have long been used in industrial applications mainly as starters for food fermentation or as biocontrol agents or as probiotics. However, LAB possess several characteristics that render them among the most promising candidates for use in future biorefineries in converting plant-derived biomass—either from dedicated crops or from municipal/industrial solid wastes—into biofuels and high value-added products. Lactic acid, their main fermentation product, is an attractive building block extensively used by the chemical industry, owing to the potential for production of polylactides as biodegradable and biocompatible plastic alternative to polymers derived from petrochemicals. LA is but one of many high-value compounds which can be produced by LAB fermentation, which also include biofuels such as ethanol and butanol, biodegradable plastic polymers, exopolysaccharides, antimicrobial agents, health-promoting substances and nutraceuticals. Furthermore, several LAB strains have ascertained probiotic properties, and their biomass can be considered a high-value product. The present contribution aims to provide an extensive overview of the main industrial applications of LAB and future perspectives concerning their utilization in biorefineries. Strategies will be described in detail for developing LAB strains with broader substrate metabolic capacity for fermentation of cheaper biomass.     

The mechanism of the tyrosine transporter TyrP supports a proton motive tyrosine decarboxylation pathway in Lactobacillus brevis

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.     

Lactic acid production from lignocellulose-derived sugars using lactic acid bacteria: Overview and limits

Lactic acid is an industrially important product with a large and rapidly expanding market due to its attractive and valuable multi-function properties. The economics of lactic acid production by fermentation is dependent on many factors, of which the cost of the raw materials is very significant. It is very expensive when sugars, e.g., glucose, sucrose, starch, etc., are used as the feedstock for lactic acid production. Therefore, lignocellulosic biomass is a promising feedstock for lactic acid production considering its great availability, sustainability, and low cost compared to refined sugars. Despite these advantages, the commercial use of lignocellulose for lactic acid production is still problematic. This review describes the “conventional” processes for producing lactic acid from lignocellulosic materials with lactic acid bacteria. These processes include: pretreatment of the biomass, enzyme hydrolysis to obtain fermentable sugars, fermentation technologies, and separation and purification of lactic acid. In addition, the difficulties associated with using this biomass for lactic acid production are especially introduced and several key properties that should be targeted for low-cost and advanced fermentation processes are pointed out. We also discuss the metabolism of lignocellulose-derived sugars by lactic acid bacteria.     

Lactic acid production from lignocellulose-derived sugars using lactic acid bacteria: overview and limits

Lactic acid is an industrially important product with a large and rapidly expanding market due to its attractive and valuable multi-function properties. The economics of lactic acid production by fermentation is dependent on many factors, of which the cost of the raw materials is very significant. It is very expensive when sugars, e.g., glucose, sucrose, starch, etc., are used as the feedstock for lactic acid production. Therefore, lignocellulosic biomass is a promising feedstock for lactic acid production considering its great availability, sustainability, and low cost compared to refined sugars. Despite these advantages, the commercial use of lignocellulose for lactic acid production is still problematic. This review describes the "conventional" processes for producing lactic acid from lignocellulosic materials with lactic acid bacteria. These processes include: pretreatment of the biomass, enzyme hydrolysis to obtain fermentable sugars, fermentation technologies, and separation and purification of lactic acid. In addition, the difficulties associated with using this biomass for lactic acid production are especially introduced and several key properties that should be targeted for low-cost and advanced fermentation processes are pointed out. We also discuss the metabolism of lignocellulose-derived sugars by lactic acid bacteria.     

Dual Role for the Tyrosine Decarboxylation Pathway in Enterococcus faecium E17: Response to an Acid Challenge and Generation of a Proton Motive Force

In this work we investigated the role of the tyrosine decarboxylation pathway in the response of Enterococcus faecium E17 cells to an acid challenge. It was found that 91% of the cells were able to remain viable in the presence of tyrosine when they were incubated for 3 h in a complex medium at pH 2.5. This effect was shown to be related to the tyrosine decarboxylation pathway. Therefore, the role of tyrosine decarboxylation in pH homeostasis was studied. The membrane potential and pH gradient, the parameters that compose the proton motive force (PMF), were measured at different pHs (pH 4.5 to 7). We obtained evidence showing that the tyrosine decarboxylation pathway generates a PMF composed of a pH gradient formed due to proton consumption in the decarboxylation reaction and by a membrane potential which results from electrogenic transport of tyrosine in exchange for the corresponding biogenic amine tyramine. The properties of the tyrosine transporter were also studied in this work by using whole cells and right-side-out vesicles. The results showed that the transporter catalyzes homologous tyrosine/tyrosine antiport, as well as electrogenic heterologous tyrosine-tyramine exchange. The tyrosine transporter had properties of a typical precursor-product exchanger operating in a proton motive decarboxylation pathway. Therefore, the tyrosine decarboxylation pathway contributes to an acid response mechanism in E. faecium E17. This decarboxylation pathway gives the strain a competitive advantage in nutrient-depleted conditions, as well as in harsh acidic environments, and a better chance of survival, which contributes to higher cell counts in food fermentation products.     

Real-time study of the effect of different stress factors on lactic acid bacteria by electrochemical optical waveguide lightmode spectroscopy

Lactic acid bacteria play an important role in the fermentation of different food products. During the fermentation processes, lactobacilli are confronted with many inhibitor factors. These factors by themselves or in combination can influence the growth of lactic acid bacteria and their acidification capacity. The subject of our study was to monitor with a newly developed biosensing technique how the different chemical stress factors influence the survival of lactic acid bacteria. Electrochemical optical waveguide lightmode spectroscopy combines evanescent-field optical sensing with electrochemical control of surface adsorption processes. For optical sensing, a layer of indium tin oxide served as a high refractive index waveguide and as a conductive electrode, as well. Lactobacillus plantarum 2142 suspended in Jerusalem artichoke syrup was used in the experiments. Electrochemical optical waveguide lightmode spectroscopy measurements were undertaken by using OW 2,400c indium tin oxide coated waveguide sensors (MicroVacuum, Budapest, Hungary) and were performed in a flow-injection analyzer system. The bacterial cells were adsorbed in native form without any chemical binding on the surface of the sensor by ensuring polarizing potential (1V) and were exposed to different concentration of acetic acid/Jerusalem artichoke syrup, lactic acid/Jerusalem artichoke syrup and hydrogen peroxide/Jerusalem artichoke syrup solution for 1h, respectively, and the effect on bacteria cells was monitored. Results were compared to the traditional micro-assay method, and it can be assumed that after further investigations this new technique could be used in real-time application.