Influence of lactose-citrate co-metabolism on the differences of growth and energetics in Leuconostoc lactis, Leuconostoc mesenteroides ssp. mesenteroides and Leuconostoc mesenteroides ssp. cremoris

The biodiversity of growth and energetics in Leuconostoc sp. has been studied in MRS lactose medium with and without citrate. On lactose alone, Ln. lactis has a growth rate double that of Ln. cremoris and Ln. mesenteroides. The pH is a more critical parameter for Ln. mesenteroides than for Ln. lactis or Ln. cremoris; without pH control Ln. mesenteroides is unable to acidify the medium under pH 4.5, while with pH control and as a consequence of a high Y(ATP) its growth is greater than Ln. lactis and Ln. cremoris. In general, lactose-citrate co-metabolism increases the growth rate, the biomass synthesis, the lactose utilisation ratio, and the production of lactate and acetate from lactose catabolism. The combined effect of the pH and the co-metabolism lactose-citrate on the two components of the proton motive force (deltap = deltapsi - ZdeltapH) has been studied using resting-cell experiments. At neutral pH deltap is nearly entirely due to the deltapsi, whereas at acidic pH the deltapH is the major component. On lactose alone, strains have a different aptitude to regulate their intracellular pH value, for Ln. mesenteroides it drastically decreases at acidic pH values (pH, = 5.2 for pH 4), while for Ln. lactis and Ln. cremoris it remains above pH 6. Lactose-citrate co-metabolism allows a better control of pH homeostasis in Ln. mesenteroides, consequently the pHi becomes homogeneous between the three strains studied, for pH 4 it is in an interval of 0.3 pH unit (from pHi = 6.4 to pHi = 6.7). In this metabolic state, and as a consequence of the variation in deltapH, and to some extent in the deltapsi, the difference of deltap between the three strains is restricted to an interval of 20 mV.     

Cloning of the D-lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. cremoris

Summary A genomic library from Leuconostoc mesenteroides subsp. cremoris was screened for D-lactate dehydrogenase activity using a stereospecific lactate detection test on agar plate. Among 3500 clones tested, six positive colonies were found on D-lactate detection plate, displaying significantly higher D-LDH activity than Escherichia coli host strain.     

Characterization of amino acid transport in the dairy strain Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1273

AIMS: To identify and characterize amino acid transport in Leuconostoc mesenteroides. METHODS AND RESULTS: The transport of labelled amino acids was measured in whole cells of Leuc. mesenteroides CNRZ 1273. Systems were operative under physiological conditions of growth, energy dependent and differed from peptide transport. Some of the systems were shared by several amino acids. Kinetic analysis indicated the presence of three transport systems with very high (VH), high (H) and low affinity (H) for the 11 amino acids studied. The K(t) values (micromol l(-1)) ranged from 0.088 to 0.815 (VH), 6-390 (H) and 320-4500 (L) and the V(max) values [nmol s(-1) (g dry weight)(-1)] from 0.015 to 0.8 (VH), 15-95 (H) and 90-470 (L). CONCLUSIONS: The study showed the presence of three transport systems in Leuc. mesenteroides for all amino acids tested, some of them being shared by several amino acids. SIGNIFICANCE AND IMPACT OF THE STUDY. The findings are discussed with reference to the growth of Leuc. mesenteroides in milk as pure or in mixed-strain culture with Lactococcus lactis.     

Optimizing the conditions of dextran synthesis by the bacterium Leuconostoc mesenteroides grown in a molasses-containing medium

Maximal dextran production (54-55 g/l) by the bacterium Leuconostoc mesenteroides strain V-2317D was observed in molasses-containing media in the presence of 17.5% glucose at pHinit 6.75. The beginning of dextran production depended on the amount of inoculate; maximum yield was observed at a shaker rate of 200 rpm. The dextran produced by L. mesenteroides grown in the molasses-containing medium was representative of three fractions differing in the molecular weight and composition: the high- (approximately 54.5%), medium- (approximately 27.9%), and low-molecular-weight (approximately 2.85%) fractions.     

Quantification of the expression of reference and alcohol dehydrogenase genes of some acetic acid bacteria in different growth conditions

Aims:  The aim of this study was to develop a reliable system to analyse the expression of the pyrroloquinoline quinone (PQQ)–alcohol dehydrogenase (ADH) and test its ability to predict the growth and oxidative activity of some acetic acid bacteria (AAB).
Methods and Results:  Specific primers were designed for use in RT-PCR to quantify ADH expression and several housekeeping genes in four species of AAB. 16S rRNA gene was selected as an internal control. The relative expression of adh A was measured in Acetobacter aceti , Acetobacter pasteurianus , Gluconacetobacter hansenii and Gluconobacter oxydans grown in two media that had glucose or ethanol as the carbon source. AAB adh A expression was shown to be related to the two Acetobacter species' ability to oxidise and grow on ethanol, whereas G. oxydans were unable to grow on ethanol and the growth of Ga. hansenii was not related to adh A expression.
Conclusions:  The differential expression of ADH could be a marker to analyse both growth and oxidation ability in some AAB, especially those of the genus Acetobacter .
Significance and Impact of the Study:  Several housekeeping genes were tested in AAB and after growth in different media and it was evident that only the ribosomal coding genes were adequate as reference genes for RT-PCR.     

Cloning of the gene and amino acid sequence for glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides

Amino acid sequencing of glucose 6-phosphate dehydrogenase (Glc6PD) from Leuconostoc mesenteroides yielded sequence for over 75% of the protein. Two oligonucleotides based on the amino acid sequence were used to isolate a partial Glc6PD gene clone (pLmz delta N65), from a pUC9 library, containing 85% of the coding sequence and the 3'-untranslated DNA, but lacking the 5'-noncoding DNA sequence and the portion of the gene encoding the 65 N-terminal amino acids. Attempts to obtain a full-length clone from lambda libraries were unsuccessful, possibly due to restriction of L. mesenteroides DNA by Escherichia coli host cells. The 5'-untranslated DNA was amplified by the polymerase chain reaction and partially sequenced. To obtain unmodified DNA for the gene, oligonucleotides corresponding to the 5'- and 3'-noncoding sequences were used to amplify the gene by the polymerase chain reaction, and a 1.8-kilobase pair fragment was isolated and cloned into pUC19. The recombinant plasmid, pLmz, contains the entire Glc6PD gene and expresses the gene in E. coli. pLmz was sequenced showing that the enzyme consists of 485 amino acids. L. mesenteroides Glc6PD is 31% identical to the human enzyme.     

Identification and characterization of an oligopeptide transport system in Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1463

AIMS: To identify and characterize an oligopeptide transport system in Leuconostoc mesenteroides CNRZ 1473. METHODS AND RESULTS: The uptake of a model substrate was monitored by determining intracellular concentrations of the corresponding amino acids by means of reversed-phase HPLC analysis. The oligopeptide transport system is specific for peptides containing at least four amino acid residues and operative under physiological conditions of growth. It is expressed maximally in the presence of oligopeptides, enhanced in the presence of Mg2+ or Ca2+ ions, and driven by ATP or a related energy-rich phosphorylated intermediate. CONCLUSIONS: The study showed evidence for and characterized the oligopeptide transport system of Leuc. mesenteroides for the first time. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the growth of Leuc. mesenteroides in mixed-strain cultures for the dairy industry.     

Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

1. Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki equals 0.04-0.06 mM. 2. This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+. Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme. 3. Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate. Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme--inhibitor interaction. 4. Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm. Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine. 5. The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole). After protein hydrolysis, N-6-pyridoxyllysine can be identified by paper chromatography. 6. The enzyme is inhibited irreversibly by 1-fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol. At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme. NADP+ AND GLUCOSE 6-PHOSPHATE PROTECT AGAINST INHIBITION BY 1-FLUORO-2,4-DINITROBENZENE. The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups. 7. These studies indicate that there is an essential lysine at the active site of the enzyme. It is suggested that the function of this lysine is to bind glucose 6-phosphate. 8. It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically.     

Influence of growth conditions on production and activity of mesenterocin 5 by a strain of Leuconostoc mesenteroides

The effects of various parameters on production and activity of mesenterocin 5, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides UL5, were investigated. Titres of bacteriocin and minimum inhibitory concentration values were determined by a critical dilution micromethod, using a sensitive strain of Listeria ivanovii as an indicator. Production of the antimicrobial compound was optimal at 37 and 40°C after 9 h of incubation, and was maximized in an aerobic fermentor maintained at pH 5.0. Tween 80 was a major factor in increasing mesenteroxin 5 production and specific production. Large quantities of bacteriocin could be obtained in whey and in whey permeate supplemented with yeast extract in the presence of the surfactant (0.1%). Most of the Listeria strains tested including L. monocytogenes were highly sensitive to the bacteriocin in the pH range 5.5 to 6.0 and at a temperature of 20 to 25°C.     

The citrate metabolic pathway in Leuconostoc mesenteroides: expression, amino acid synthesis, and alpha-ketocarboxylate transport.

Citrate metabolism in Leuconostoc mesenteroides subspecies mesenteroides is associated with the generation of a proton motive force by a secondary mechanism (C. Marty-Teysset, C. Posthuma, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Bacteriol. 178:2178-2185, 1996). The pathway consists of four steps: (i) uptake of citrate, (ii) splitting of citrate into acetate and oxaloacetate, (iii) pyruvate formation by decarboxylation of oxaloacetate, and (iv) reduction of pyruvate to lactate. Studies of citrate uptake and metabolism in resting cells of L. mesenteroides grown in the presence or absence of citrate show that the citrate transporter CitP and citrate lyase are constitutively expressed. On the other hand, oxaloacetate decarboxylase is under stringent control of the citrate in the medium and is not expressed in its absence, thereby blocking the pathway at the level of oxaloacetate. Under those conditions, the pathway is completely directed towards the formation of aspartate, which is formed from oxaloacetate by transaminase activity. The data indicate a role for citrate metabolism in amino acid biosynthesis. Internalized radiolabeled aspartate produced from citrate metabolism could be chased from the cells by addition of the amino acid precursors oxaloacetate, pyruvate, alpha-ketoglutarate, and alpha-ketoisocaproate to the cells, indicating a broad specificity of the transamination reaction. The alpha-ketocarboxylates are readily transported across the cytoplasmic membrane. alpha-Ketoglutarate uptake in resting cells of L. mesenteroides was dependent upon the presence of an energy source and was inhibited by inhibition of the proton motive force generating F(0)F(1) ATPase and by selective dissipation of the membrane potential and the transmembrane pH gradient. It is concluded that in L. mesenteroides alpha-ketoglutarate is transported via a secondary transporter that may be a general alpha-ketocarboxylate carrier.     

Alteration of the growth rate and lag time of Leuconostoc mesenteroides NRRL-B523.

Bacterial profile modification is an important enhanced oil recovery technique used to direct injected water into a reservoir's low permeability zone containing trapped crude oil. During water flooding, the use of bacteria to plug the high permeability water zone and divert flow into the oil-bearing low-permeability zone will have a significant economic impact. However, during the field implementation of bacterial profile modification, the rapid growth of bacteria near the injection well bore may hinder the subsequent injection of growth media so that profile modification of the reservoir occurs only in the immediate vicinity of the well bore. By slowing the growth rate and prolonging the lag phase, the onset of pore-space plugging may be delayed and the biologically active zone extended deep into the reservoir. High substrate loading, high pH values, and the addition of the growth inhibitors sodium dodecylsulfate and sodium benzoate have been used in combination to alter the growth characteristics of Leuconostoc mesenteroides NRRL-B523 grown in batch conditions. The highest sucrose concentration used in these studies, 500 g/L, produced lag times 12-fold greater than the slowest lag times achieved at low sucrose concentrations. When L. mesenteroides was grown in media containing 500 g/L sucrose, an alkaline pH value threshold was found above which bacteria did not grow. At this threshold pH value of 8.1, an average lag time of 200 h was observed. Increasing the concentration of sodium benzoate had no effect on lag time, but reduced the growth rate until the threshold concentration of 0.6%, above which bacteria did not grow. Last, it was found that a solution of 0.075 mM sodium dodecylsulfate in media containing 15 g/L sucrose completely inhibited bacterial growth.