Effects of pH and trace minerals on long-term starvation of Leuconostoc mesenteroides

Laboratory experiments have definitively shown that exopolymer-producing bacteria have the potential to modify the flow of fluids in oil reservoirs to enhance oil production. Once injected into the reservoir, they will be subjected to a wide range of pH values and to starvation resulting from nutrient depletion. For successful field implementation it is necessary to have a fundamental understanding of these effects on the viability of bacteria. This paper addresses the effects of pH and trace minerals on cell viability of Leuconostoc mesenteroides during carbon source depletion. Two different carbon sources were used to grow cells before transferring the cells to starvation conditions: sucrose and a combination of glucose and fructose. These substrates were chosen because L. mesenteroides produces a significant amount of water-insoluble exopolymers (dextran) under sucrose-fed conditions, which may enhance cell survival under harsh conditions. The effects of dextran on the cell viability were tested at different pH values with and without trace minerals. The rate of cell death followed an exponential-decay law for different values of the solution pH. The optimal solution pH for survival was pH 5, whereas cells died rapidly at pH 3 and below and at pH 13 and above. The sucrose-fed cells showed a greater viability than cells fed glucose and fructose for all pH ranges tested. The results indicated that water-insoluble exopolymers help cells survive for longer periods of time under starvation conditions. The effects of trace minerals on cell culturability were tested at two pH values, 4.5 and 7. For both cases, cells showed a greater culturability (smaller decay rate constant) in the presence of trace minerals than without trace minerals. It was also found that the effects of trace minerals on cell culturability were greater for glucose-fructose-fed cells than for sucrose-fed cells. The Michaelis pH function theory was used for comparing the relationships between the cell decay rate and pH.     

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.     

Cell wall constituents of Leuconostoc citrovorum and Leuconostoc mesenteroides

The cell wall constituents of Leuconostoc citrovorum 8082, L. mesenteroides 10830a, and L. mesenteroides 11449 have been ascertained. All three strains contained glycerol. Glucose and rhamnose were the major reducing sugar constituents. Alanine, glutamic acid, lysine, glucosamine, and muramic acid were the principal amino acids and amino sugars in all three strains. In addition, strain 10830a contained l-serine as a major cell wall component. Quantitative amino acid analyses indicate that glutamic acid, lysine, glucosamine, muramic acid, and serine may be present in the cell walls in equimolar amounts and that alanine is present in three to four times these quantities. The similarities and differences between the cell wall constituents of the leuconostocs and those of the lactobacilli and streptococci are discussed.     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

pH Homeostasis and Citric Acid Utilization: Differences Between Leuconostoc mesenteroides and Lactococcus lactis

This study presents the effects of citric acid and extracellular pH (pH_e) on the intracellular pH (pH_i) of wild-type and citrate negative variants (cit⁻) Leuconostoc mesenteroides subsp. mesenteroides (Ln. mesenteroides M) and Lactococcus lactis subsp. lactis bv. diacetylactis (L. lactis LD). A recent method using a pH-sensitive fluorescent indicator carboxyfluorescein succinimidyl ester (cFSE) was adapted to measure the pH_i of these two lactic acid bacteria in resting cells. Energized cells with 10 mM lactose of Ln. mesenteroides M and L. lactis LD modified their pH gradient (ΔpH) in the same manner; when the pH_e was decreased from 7 to 4, the pH_i decreased from 7 to about 5. The adjunction of 10 mM citric acid had no effect on the pH_i of wild-type and cit(−) variant of L. lactis LD, nor on the pH_i of Ln. mesenteroides cit(−) variant. Nevertheless, in Ln. mesenteroides M wild-type, citric acid utilization increased the pH_i, which was maintained at about 6.5–7.0 when the pH_e was decreased from 7 to 4. It could be concluded that citric acid allows the maintenance of pH homeostasis in Leuconostoc mesenteroides. Received: 7 March 1997 / Accepted: 14 April 1997     

Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293

Aims: To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent‐cell preparation and electroporation conditions were optimized. Methods and Results: Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 cells were sequentially treated with penicillin G and lysozyme, and the plasmid pLeuCM was subsequently transformed into the cells. Our results demonstrated that transformation efficiencies were significantly increased (100‐fold), and increased electric field strength also contributed to enhance transformation efficiency. Maximum transformation efficiency (1 × 10⁴ or more transformants per μg DNA) was achieved when cells were grown in De Man, Rogosa, Sharpe (MRS) media containing 0·25 mol l^?1 sucrose and 0·8 μg ml^?1 penicillin G, followed by treatment with 600 U ml^?1 lysozyme and electroporation at a field strength of 10 kV cm^?1. When this protocol was used to transform pLeuCM into Leuc. mesenteroides, Leuconostoc gelidum, Leuconostoc fallax and Leuconostoc argentinun, successful transformations were obtained in all cases. Furthermore, this procedure was applicable to species belonging to other genera, including Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa. Conclusions: The results demonstrate that the transformation efficiency for Leuconostoc spp. could be increased via optimization of the entire electroporation procedures. Significance and Impact of the Study: These optimized conditions can be used for the extensive genetic study and the metabolic engineering of not only Leuconostoc spp. but also different species of lactic acid bacteria.     

Peptide utilization by Lactococcus lactis and Leuconostoc mesenteroides

To explain the competition for nitrogenous nutrients observed in mixed strain cultures of Lactococcus lactis and Leuconostoc mesenteroides, the utilization of peptides as a source of essential amino acids for growth in a chemically defined medium was compared in 12 strains of dairy origin. Both species were multiple amino acid auxotrophs and harboured a large set of intracellular peptidases. Lactococcus lactis can use a wide variety of peptides up to 13 amino acid residues whereas Leuc. mesenteroides assimilated only shorter peptides containing up to seven amino acids. Growth was limited by the transport of peptides and not by their hydrolysis. The nutritional value of peptides varied with the strains and the composition of the peptides, L. lactis being advantaged over Leuc. mesenteroides.     

Proteolytic processing of dextransucrase of Leuconostoc mesenteroides

Various dextransucrase molecular mass forms found in enzyme preparations may sometimes be products of proteolytic activity. Extracellular protease in Leuconostoc mesenteroides strains NRRL B-512F and B-512FMC dextransucrase preparations was identified. Protease had a molecular mass of 30 kDa and was the predominant form derived from a high molecular mass precursor. The production and activity of protease in culture medium was strongly dependent on pH. When L. mesenteroides dextransucrase (173 kDa) was hydrolyzed by protease, at pH 7 and 37 degrees C, various dextransucrase forms with molecular masses as low as 120 kDa conserving dextransucrase activity were obtained.     

Acid Tolerance of Leuconostoc mesenteroides and Lactobacillus plantarum

In this study, we determined the internal cellular pH response of Leuconostoc mesenteroides and Lactobacillus plantarum to the external pH created by the microorganisms themselves or by lactic or acetic acids and their salts added to the growth medium. Growth of Leuconostoc mesenteroides stopped when its internal pH reached 5.4 to 5.7, and growth of L. plantarum stopped when its internal pH reached 4.6 to 4.8. Variation in growth medium composition or pH did not alter the growth-limiting internal pH reached by these microorganisms. L. plantarum maintained its pH gradient in the presence of either 160 mM sodium acetate or sodium lactate down to an external pH of 3.0 with either acid. In contrast, the DeltapH of Leuconostoc mesenteroides was zero at pH 4.0 with acetate and 5.0 with lactate. No differences were found between d-(-)- and l-(+)-lactic acid for the limiting internal pH for growth of either microorganism. The comparatively low growth-limiting internal pH and ability to maintain a pH gradient at high organic acid concentration may contribute to the ability of L. plantarum to terminate vegetable fermentations.     

Optimization of Protoplast Formation and Regeneration in Leuconostoc mesenteroides

Conditions are reported for efficient protoplast formation and regeneration in four strains of Leuconostoc mesenteroides. Protoplasts were produced from each strain at frequencies greater than 99%, although the rate of their production was variable from strain to strain. Bovine serum albumin and Mg²⁺ were required for maximal regeneration, while the presence of Ca²⁺ was inhibitory. Regeneration frequencies of 16% could be obtained with strain ATCC 10830. This frequency was four- to eightfold higher than the frequencies of the other strains examined.     

Purification and properties of DL-lactate dehydrogenase from Leuconostoc mesenteroides

A dl-lactate dehydrogenase from the bacterium, Leuconostoc mesenteroides, has been purified and characterized with respect to amino acid composition, molecular weight, and kinetic properties. The turnover number of the enzyme was 1.7 × 10⁵ moles DPNH/mole enzyme/min for the most active of three preparations. On the basis of a sedimentation constant of 3.52 S and a diffusion constant of 5.0 × 10^?7 cm²/ ml, the molecular weight of the enzyme was determined to be approximately 64,000. Similar values were derived from sedimentation equilibrium data. The enzyme exhibits typical Michaelis-Menten kinetics except when lactate is the variable substrate. In this case, double reciprocal plots of activity versus substrate concentration are curved upward, suggesting that lactate either activates or stabilizes a more active form of the enzyme.