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.     

Leuconostoc mesenteroides subsp. mesenteroides FR52 synthesizes two distinct bacteriocins

A.M. REVOL-JUNELLES, R. MATHIS, F. KRIER, Y. FLEURY, A. DELFOUR AND G. LEFEBVRE. 1996. Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, was purified from producing cells by the adsorption-desorption method, combined with reverse-phase high-performance liquid chromatography. The elution profile revealed the presence o two inhibitory peaks of activity, each displaying different inhibitory spectra. Mesenterocin 52A possessed a broad inhibitory spectrum, including anti- Listeria activity, while Mesenterocin 52B was only active against Leuconostoc spp. The amino acid sequence and M_r of Mesenterocin 52A appeared identical to the previously described Mesentericing Y105. In contrast, Mesenerocin 52B possessed a M_r of 3446 Da, corresponding to 32 amino acids and a sequence that shared no homology with known bacteriocins:     

Kinases in Leuconostoc mesenteroides

Enzyme extracts of Leuconostoc mesenteroides were found to contain at least four separate kinases: one active with glucose, glucosamine, and N-acetylglucosamine; one active with fructose and mannose; and two active with gluconate, one constitutive and one inducible. The molecular sizes of all the kinases, estimated from sucrose gradient centrifugation, are about the same, 37,000 to 50,000 daltons, except the constitutive gluconate kinase, which is about 100,000 daltons. Apparent Michaelis constants were calculated for all of the substrates mentioned. The kinases are separable on triethylaminoethyl cellulose.     

Characterization of a citrate-negative mutant ofLeuconostoc mesenteroides subsp.mesenteroides: metabolic and plasmidic properties

Summary Comparison of the parental strain of the Leuconostoc mesenteroides subsp. mesenteroides (19D) and its citrate-negative mutant, which has lost a 22-kb plasmid, has confirmed the energetic role of citrate. Fermentation balance analysis showed that citrate led to a change in heterolactic fermentation from glucose. High levels of enzyme activity in both mutant and parental strains were found for NADH oxidase, lactate dehydrogenase, acetate kinase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, although NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase were partly repressed by citrate. All these enzymes studied were not plasmid linked. In the parental strain, citrate lyase was induced by citrate. No citrate lyase activity was found in the citrate-negative mutant grown in presence of citrate, but this does not provide evidence that citrate lyase is linked to the 22-kb plasmid. Offprint requests to: C. Diviès     

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.     

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.     

Molecular cloning and characterization of a novel glucansucrase from Leuconostoc mesenteroides subsp. mesenteroides LM34

Leuconostoc mesenteroides LM34 was isolated from kimchi, a traditional fermented Korean food. L. mesenteroides LM34 produced extracellular glucansucrase (DSRLM34), which is responsible for the synthesis of soluble glucan using sucrose. The DSRLM34 gene consists of a 4,503 bp open reading frame (ORF) and encodes an enzyme of 1,500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (98%) to that of glucansucrase of Lactobacillus lactis. The gene was over-expressed in Escherichia coli strain and the recombinant enzyme (rDSRLM34) was purified. Both DSRLM34 and rDSRLM34 synthesized glucan mainly containing α-1, 6 glucosidic linkage and branched α-1, 3 glucosidic linkages. The enzyme exhibited optimum activity at 30°C and pH 5.0. DSRLM34 has promising potential as a thickening agent in sucrose-supplemented milk.     

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.     

Citrate metabolism in 16 Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum strains

Citrate metabolism was studied in non-growing cells of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum with respect to energetics, formation of degradation products and stoichiometry. The use of selective ionophores and uncoupler showed that citrate utilization was coupled to the proton motive force generated by ATP hydrolysis. Differences in citrate metabolism observed in 20 Leuconostoc strains were related to strains but not to the species or subspecies studied. Citrate metabolism was stimulated by glucose up to a concentration of 25 mmol 1^-1 and decreased at higher concentrations. The main degradation products resulting from the co-metabolism of citrate (10 mmol 1^-1) and glucose (2 mmol 1^-1) were acetate, lactate and pyruvate. Only four Leuconostoc strains produced low levels of acetoin and diacetyl. No strains produced ethanol or acetaldehyde. Citrate degradation ability was stable for at least 130 generations in 81% of the Leuconostoc strains.     

Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides ssp. mesenteroides FR 52

F. MATHIEU, I.S. SUWANDHI, N. REKHIF, J.B. MILLIERE AND G. LEFEBVRE. 1993. One hundred and sixty-five isolates of Leuconostoc spp. were tested for bacteriocin production. Only one strain, Leuc. mesenteroides ssp. mesenteroides FR 52, isolated from a raw milk, produced a bacteriocin which was named Mesenterocin 52. This bacteriocin inhibited other Leuconostoc strains and several strains of Enterococcus and Listeria spp. No activity was found against lactococci and lactobacilli. The antibacterial spectrum differed from that of previously described Leuconostoc bacteriocins. Mesenterocin 52 was secreted into the medium during the growth phase. It was inactivated with protease treatments. At pH 7.0 it had a relative stability after heating at 100C (15 min), but it had a greater stability at pH 4.5 than at pH 7.0 after 6 h at 80C. The apparent molecular mass was estimated to be less than 10 kDa by ultrafiltration. Mesenterocin 52 showed a bactericidal effect on Leuconostoc paramesenteroides DSM 20288.     

Complete genome sequence of Leuconostoc mesenteroides subsp. mesenteroides strain J18, isolated from kimchi

Leuconostoc mesenteroides subsp. mesenteroides is one of the most predominant lactic acid bacterial groups during kimchi fermentation. Here, we report the complete genome sequence of L. mesenteroides subsp. mesenteroides J18, which was isolated from kimchi. The genome of the strain consists of a 1,896,561-bp chromosome and five plasmids.     

Acetoin production in growing Leuconostoc mesenteroides

Leuconostoc mesenteroides NCDO 518, provided with oxygen and pyruvate, preferentially used oxygen as accessory electron acceptor and converted pyruvate to acetoin. With glucose, 5.6 mM, as sole energy source only small amounts of acetoin were formed (0.08–0.21 mM). With glucose, 5.6 mM, and pyruvate, 20 mM, substantial amounts of acetoin were produced in growing, aerated cultures at pH 5 (2.8 mM, equivalent to 0.5 mol [mol glucose fermented]^–1). On exhaustion of glucose, growth ceased but metabolism of pyruvate continued with the formation of acetate and a little acetoin. In aerated cultures at pH 6 the general pattern was similar to that at pH 5 but less acetoin (0.6 mM) was formed during the growth phase and, after the exhaustion of glucose, pyruvate was converted very slowly to acetate only. Leuc. mesenteroides did not grow with pyruvate as sole energy source.     

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.     

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997