The Pool of ADP and ATP Regulates Anaerobic Product Formation in Resting Cells of Lactococcus lactis

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P_i was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.     

Effect of temperature and pH on growth and product formation of Lactococcus lactis ssp. lactis ATCC 19435 growing on maltose

Lactococcus lactis ssp. lactis ATCC 19435 is known to produce mixed acids when grown on maltose. A change in fermentation conditions only, elevated temperatures (up to 37 °C) and reduced pH values (down to 5.0) resulted in a shift towards homolactic product formation. This was accompanied by decreased growth rate and cell yield. The results are discussed in terms of redox balance and maintenance, and the regulation of lactate dehydrogenase and pyruvate formate-lyase. Received: 14 December 1998 / Received revision: 12 January 1999 / Accepted: 22 January 1999     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

The pool of ADP and ATP regulates anaerobic product formation in resting cells of Lactococcus lactis

Lactococcus lactis grows homofermentatively on glucose, while its growth on maltose under anaerobic conditions results in mixed acid product formation in which formate, acetate, and ethanol are formed in addition to lactate. Maltose was used as a carbon source to study mixed acid product formation as a function of the growth rate. In batch and nitrogen-limited chemostat cultures mixed acid product formation was shown to be linked to the growth rate, and homolactic fermentation occurred only in resting cells. Two of the four lactococcal strains investigated with maltose, L. lactis 65.1 and MG1363, showed more pronounced mixed acid product formation during growth than L. lactis ATCC 19435 or IL-1403. In resting cell experiments all four strains exhibited homolactic fermentation. In resting cells the intracellular concentrations of ADP, ATP, and fructose 1,6-bisphosphate were increased and the concentration of P(i) was decreased compared with the concentrations in growing cells. Addition of an ionophore (monensin or valinomycin) to resting cultures of L. lactis 65.1 induced mixed acid product formation concomitant with decreases in the ADP, ATP, and fructose 1,6-bisphosphate concentrations. ADP and ATP were shown to inhibit glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase in vitro. Alcohol dehydrogenase was the most sensitive enzyme and was totally inhibited at an adenine nucleotide concentration of 16 mM, which is close to the sum of the intracellular concentrations of ADP and ATP of resting cells. This inhibition of alcohol dehydrogenase might be partially responsible for the homolactic behavior of resting cells. A hypothesis regarding the level of the ATP-ADP pool as a regulating mechanism for the glycolytic flux and product formation in L. lactis is discussed.     

Modeling of growth and lactate fermentation by Lactococcus lactis subsp. lactis biovar. diacetylactis in batch culture

The kinetic behaviour of Lactococcus lactis subsp. lactis biovar. diacetylactis was studied in batch culture under non-limiting conditions that allow high growth and product formation. A model based on laboratory results is proposed for growth and l-lactate fermentation. It shows the necessity for differentiating biomass into three physiological states, two active, X_g (growth + acidification) and X_ng (acidification), and one inactive, X_i. The kinetic theory of the model demonstrates the non-competitive nature of fermentation end-product inhibition on growth and acidification, and describes the passage from one physiological state to another. Satisfying simulations were obtained for batch fermentations, and the use of this type of model for determining and optimizing fermentation parameters is discussed. Correspondence to: C. Diviès     

Formation and Conversion of Oxygen Metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under Different Growth Conditions

A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h⁻¹). Accumulation of H₂O₂ in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H₂O₂ accumulated only temporarily during the lag phase. H₂O₂ is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min⁻¹ mg of protein⁻¹), which reduced oxygen to water, whereby superoxide was formed as a by-product. H₂O₂ originated from the dismutation of superoxide by superoxide dismutase. Although H₂O₂ was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H₂O₂-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H₂O₂ and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect L. lactis strain ATCC 19435 from H₂O₂.     

Banana ripening: implications of changes in glycolytic intermediate concentrations, glycolytic and gluconeogenic carbon flux, and fructose 2,6-bisphosphate concentration

In ripening banana (Musa sp. [AAA group, Cavendish subgroup] cv Valery) fruit, the concentration of glycolytic intermediates increased in response to the rapid conversion of starch to sugars and CO₂. Glucose 6-phosphate (G-6-P), fructose 6-phosphate (Fru 6-P), and pyruvate (Pyr) levels changed in synchrony, increasing to a maximum one day past the peak in ethylene synthesis and declining rapidly thereafter. Fructose 1,6-bisphosphate (Fru 1,6-P₂) and phosphoenolpyruvate (PEP) levels underwent changes dissimilar to those of G 6-P, Fru 6-P, and Pyr, indicating that carbon was regulated at the PEP/Pyr and Fru 6-P/Fru 1,6-P₂ interconversion sites. During the climacteric respiratory rise, gluconeogenic carbon flux increased 50- to 100-fold while glycolytic carbon flux increased only 4- to 5-fold. After the climacteric peak in CO₂ production, gluconeogenic carbon flux dropped dramatically while glycolytic carbon flux remained elevated. The steady-state fructose 2,6-bisphosphate (Fru 2,6-P₂) concentration decreased to ½ that of preclimacteric fruit during the period coinciding with the rapid increase in gluconeogenesis. Fru 2,6-P₂ concentration increased thereafter as glycolytic carbon flux increased relative to gluconeogenic carbon flux. It appears likely that the initial increase in respiration in ripening banana fruit is due to the rapid influx of carbon into the cytosol as starch is degraded. As starch reserves are depleted and the levels of intermediates decline, the continued enhancement of respiration may, in part, be maintained by an increased steady-state Fru 2,6-P₂ concentration acting to promote glycolytic carbon flux at the step responsible for the interconversion of Fru 6-P and Fru 1,6-P₂.     

β-Glucose-1-Phosphate, a Possible Mediator for Polysaccharide Formation in Maltose-Assimilating Lactococcus lactis

Homolactic fermentation of glucose and heterolactic fermentation of maltose with Lactococcus lactis 65.1 were confirmed. When moles of glucose were compared, the uptake rates of the two carbon sources were similar. The intracellular concentration of fructose-1,6-diphosphate (FDP) in maltose-assimilating cells was half of that in glucose-assimilating cells. Similarly, formation of FDP and lactate from maltose by extracts of maltose-grown cells was half of that formed from glucose by extracts of glucose-grown cells, indicating a difference in the utilization of the two carbon sources for energy metabolism. Concentrations of adenine nucleotides were similar in both types of cells. Glucose-1-phosphate was found in extracts of maltose-grown cells given maltose and, in addition, an inducible and low β-specific phosphoglucomutase activity was observed. β-Glucose-1-phosphate was not metabolized by cell extracts to either FDP or lactate, suggesting an alternative metabolic route. The amount of [¹⁴C]maltose incorporated into the cell material of maltose-grown cells was four times greater than that of [¹⁴C]glucose incorporated into the cell material of glucose-grown cells. The intracellular concentration of UTP was lower in maltose-assimilating cells than in glucose-assimilating cells. Cells grown on maltose were more spherical and less fragile than cells grown on glucose.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Evaluation of acceptor selectivity of Lactococcus lactis ssp. lactis trehalose 6-phosphate phosphorylase in the reverse phosphorolysis and synthesis of a new sugar phosphate

Trehalose 6-phosphate phosphorylase (TrePP), a member of glycoside hydrolase family 65, catalyzes the reversible phosphorolysis of trehalose 6-phosphate (Tre6P) with inversion of the anomeric configuration to produce β-d-glucose 1-phosphate (β-Glc1P) and d-glucose 6-phosphate (Glc6P). TrePP in Lactococcus lactis ssp. lactis (LlTrePP) is, alongside the phosphotransferase system, involved in the metabolism of trehalose. In this study, recombinant LlTrePP was produced and characterized. It showed its highest reverse phosphorolytic activity at pH 4.8 and 40°C, and was stable in the pH range 5.0–8.0 and at up to 30°C. Kinetic analyses indicated that reverse phosphorolysis of Tre6P proceeded through a sequential bi bi mechanism involving the formation of a ternary complex of the enzyme, β-Glc1P, and Glc6P. Suitable acceptor substrates were Glc6P, and, at a low level, d-mannose 6-phosphate (Man6P). From β-Glc1P and Man6P, a novel sugar phosphate, α-d-Glcp-(1?1)-α-d-Manp6P, was synthesized with 51% yield.     

Coarse control of sucrose-phosphate synthase in leaves: Alterations of the kinetic properties in response to the rate of photosynthesis and the accumulation of sucrose

It has been investigated whether diurnal rhythms of sucrose-phosphate synthase (SPS) are involved in controlling the rate of photosynthetic sucrose synthesis. Extracts were prepared from spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.) leaves and assayed for enzyme activity. The activity of SPS increased in parallel with a rising rate of photosynthesis, and was increased by feeding mannose and decreased by supplying inorganic phosphate. In leaf material where sucrose had accumulated during the photoperiod or when sucrose was supplied exogenously, SPS activity decreased. During a diurnal rhythm, SPS activity increased after illumination, declined gradually during the light period, decreased further after darkening and then recovered gradually during the night. These changes did not involve an alteration of the maximal activity, but were caused by changes in the kinetic properties, revealed as a change in sensitivity to inhibition by inorganic phosphate. In experiments which modelled the response of SPS to changing metabolite concentrations, it was shown that these alterations of kinetic properties would strongly modify the activity of SPS in vivo. It is proposed that SPS can exist in kinetically distinct forms in vivo, and that the distribution between these forms can be rapidly altered. As the rate of photosynthesis increases there is an activation of SPS, which may be directly or indirectly linked to changes in the availability of P_i. This activation can be modified by factors related to the accumulation of sucrose. Under normal conditions there is a balance between these factors, and the leaf contains a mixture of the different forms of SPS.Abbreviations Chl chlorophyll - Frul,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Fru1,6bisPase fructose-1,6-bisphosphatase - Fru6P 2kinase fructose-6-phosphate, 2kinase - Fru2,6bisPase fructose-2,6-bisphosphatase - Glc6P glucose-6-phosphate - P_j inorganic phosphate - SPS sucrose-phosphate synthase - UDPGLc uridine 5-diphosphate glucose     

Influence of the phosphorus and nitrogen source on nisin production in Lactococcus lactis subsp. lactis batch fermentations using a complex medium

The influence of different phosphorus and nitrogen sources on Lactococcus lactis subsp. lactis NIZO 22186 growth and nisin production was studied in batch fermentations using a complex medium. KH₂PO₄ was found to be the best phosphorus source for nisin production. Increasing initial phosphate concentrations from 0 to 5% KH₂PO₄ exerted a double effect, creating favourable pH conditions and particularly stimulating the nisin production levels, which were highest at 5% KH₂PO₄. Up to now, no such high initial phosphate concentrations have been reported for the production of other antibiotics or bacteriocins. Nisin, a lanthionine-containing peptide antibiotic with bacteriocin properties, clearly behaved as a primary metabolite, since its formation was linked with active growth and was not suppressed by phosphate concentrations up to 5%. A complex medium supplemented with cotton seed meal as nitrogen source also gave very high nisin yields. Correspondence to: L. De Vuyst     

Phosphorus-31 NMR studies of maltose and glucose metabolism in Streptococcus lactis

Summary Streptococcus lactis ferments glucose in a homolactic fashion but a heterolactic fermentation pattern is observed when it is grown on maltose. Using in vivo phosphorus-31 and carbon-13 NMR studies of glucose-metabolizing cells we confirmed that fructosediphosphate (FDP) is the major glycolytic intermediate and that the production of lactate causes major changes both in the intra- and extracellular pH values. Starved cells contain mainly 3-phosphoglycerate (3-PGA) and some phosphoenolpyruvate (PEP). Metabolism of maltose also brings about major changes in pH, but it was unclear from the poorly resolved in vivo spectra if FDP was the main glycolytic intermediate present. This question was further studied by analyzing perchloric acid extracts by phosphorus-31 NMR. These studies showed that glucose-metabolizing cells have higher levels of FDP and lower levels of inorganic phosphate (P _i ) than maltose-metabolizing cells. 3-PGA always remained present in the latter cells suggesting that these exist in a semi-starved state which is probably the reason for their heterolactic fermentation pattern. In the course of these studies we also examined the effects of the inhibitors 2-deoxyglucose, fluoride and iodoacetate. We could demonstrate that by judicious choice of carbon sources and inhibitors one could completely reduce the intracellular P _i pool. This suggests that one should be able to regulate the shift from heterolactic to homolactic fermentation, as P _i is considered to be the most potent inhibitor of pyruvate kinase in these cells.Sponsored by grants from the Swedish Natural Sciences Research Council (NFR) (to BHH and HJV), the Biotechnology Research Foundation (SBF) (to BHH) and the Canadian Natural Sciences Research Council (NSERC) (to HJV)     

Cross-Feeding of Lactate Between Streptococcus lactis and Bacteroides sp. Isolated from Termite Hindguts

Streptococcus lactis and Bacteroides sp., isolated from hindguts of Reticulitermes flavipes termites, were grown anaerobically in monoculture and coculture. When grown in a glucose medium, S. lactis monoculture produced lactate as the major fermentation product, with small amounts of formate, acetate, ethanol, and CO₂. In coculture, glucose was completely consumed during growth of S. lactis. Lactate, produced by S. lactis, then supported much of the growth of Bacteroides and was fermented to propionate, acetate, and CO₂. Small amounts of succinate were formed during growth of Bacteroides in the coculture, but little change in the formate or ethanol concentration was observed. Monoculture growth of Bacteroides in a tryptone-yeast extract medium revealed that incorporation of 20 to 40 mM lactate increased cell yields and production of organic acids. However, initial lactate concentrations greater than 40 mM suppressed not only growth of Bacteroides but also acidic product formation. Results suggest that cross-feeding of lactate between streptococci and bacteroides constitutes one aspect of the overall hindgut fermentation in termites.     

Two tandem promoters to increase gene expression in Lactococcus lactis

Two plasmids, pPAH and pAH, containing a staphylokinase variant gene (sakXH) under the control of two tandem promoters (P₃₂-P_lacA) or promoter P_lacA alone were constructed and introduced into Lactococcus lactis MG5267. The expression of sakXH in the strain MG5267(pPAH) was approximately twice as high as that in the strain MG5267(pAH), according to the formation of fibrinolytic halos on fibrinolytic plates detected at the same conditions, indicating that the two tandem promoters were stronger than one alone. Difference between the expressions of sakXH under the inducible and non-inducible conditions suggested that P_lacA retained its feature as an inducible promoter when fused to promoter P₃₂.     

Simultaneous enzymatic wheat starch saccharification and fermentation to lactic acid by Lactococcus lactis

Simultaneous saccharification of starch from whole-wheat flour and fermentation to lactic acid (SSF) was investigated. For saccharification the commercial enzyme mixture SAN Super 240 L, having α-amylase, amyloglucosidase and protease activity, was used, and Lactococcus lactis ssp. lactis ATCC 19435 was used for the fermentation. SSF was studied at flour concentrations corresponding to starch concentrations of 90 g/l and 180 g/l and SAN Super concentrations between 3 μl/g and 8 μl/g starch. Kinetic models, developed for the saccharification and fermentation, respectively, were used for simulation and data from SSF experiments were used for model verification. The model simulated SSF when sufficient amounts of nutrients were available during fermentation. This was achieved with high wheat flour concentrations or with addition of yeast extract or amino acids. Nutrient release was dependent on the level of enzyme activity. Received: 26 January 1999 / Accepted: 20 February 1999     

L-lactate production from xylose employingLactococcus lactis IO-1

Summary The growth, substrate utilisation and L-lactate production ofLactococcus lactis IO-1 were examined on xylose, and glucose and xylose media. The yield of lactate on xylose was 0.47 g lactate/g xylose at an initial xylose concentration of 51.2 g/l and the _max was 0.72 h^–1. Xylose cultures were more susceptible to lactate inhibition than were glucose cultures but showed similar kinetic behaviour. The organism was capable of complete sugar utilisation when grown on a mixture of 20 g/l xylose and 20 g/l glucose and synthesised 0.66 g lactate/g sugar.     

The physiology of Lactococcus lactis subsp. lactis biovar. diacetylactis immobilized in hollow-fibre bioreactors: glucose,lactose and citrate metabolism at high cell densities

Lactococcus lactis subsp. lactis biovar. diacetylactis was selected to study the physiological influences of immobilization and growth to high cell densities. Cells were cultivated on glucose or lactose medium in the presence and absence of citrate. With excess glucose the cells produced mainly lactate as the fermentation product (homofermentative) providing that not all of the substrate was consumed. The population so cultivated was exposed to extreme gradients of pH and lactate concentrations. When the glucose concentration was reduced the population showed a mixed product profile with half of the glucose being fermented to lactate, the remainder to formate, acetate, ethanol and 2,3-butanediol. Inclusion of citrate in the medium shifted the population to homofermentation, with respect to the amount of glucose or lactose consumed. The citrate was metabolized via the pyruvate-formate lyase and -acetolactate synthase routes. The pH of the medium was shown to strongly influence the product profile from citrate, presumably by affecting the activity of the key enzymes of pyruvate metabolism. The lactococci immobilized at high cell densities show product profiles typical of carbohydrate limitation at low dilution rates. Correspondence to: M. R. Smith