Instability of plasmid-encoded citrate permease in Leuconostoc

M. KIHAL, H. PRÉVOST, M.E. LHOTTE, D.Q. HUANG AND C. DIVIÈS. 1996. The conversion from citrate positive (Cit⁺) to citrate negative (Cit^-) phenotype of six strains of Leuconostoc mesetiteroides was followed during growth in milk and buffered or unbuffered MRS medium at 30 or 37°C. High rate of loss of Cit⁺ phenotype was observed. The Cit^- phenotype was found to be linked to the loss of 22 to 23 kb plasmids. All Cit^- mutants isolated from Leuc. mesenteroides subsp. cremoris 195 reverted spontaneously to the Cit⁺ phenotype. Hybridization experiments using a 0.8 kb fragment of the citP gene of Leuc. mesenteroides showed that all the plasmids which were lost in Cit^- mutants encoded for a citrate permease. However, neither plasmid nor genomic DNA from Leuc. mesenteroides subsp. cremoris 195 hybridized with the citP probe.     

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:     

Specific detection of Leuconostoc mesenteroides subsp. mesenteroides with DNA primers identified by randomly amplified polymorphic DNA analysis

Randomly amplified polymorphic DNA analysis using primer 239 (5' CTGAAGCGGA 3') was performed to characterize Leuconostoc sp. strains. All the strains of Leuconostoc mesenteroides subsp. mesenteroides (with the exception of two strains), two strains formerly identified as L. gelidum, and one strain of Leuconostoc showed a common band at about 1.1 kb. This DNA fragment was cloned and sequenced in order to verify its suitability for identifying L. mesenteroides subsp. mesenteroides strains.     

Differential enumeration of silage inoculants based on utilization of enzymatic activity and antibiotic sensitivity of bacteria

The differences in the composition of culture media inoculated with strains of Leuconostoc oenos were more quantitative than qualitative. Both temperature of incubation and pH significantly affected bacterial growth, the rates of substrate consumption and consequently the amount of metabolites produced. All strains degraded 5 g 1^-1 malate, except one at pH 3.5 and 25°C. Malate was metabolized before glucose except at higher pH (4.0 and 4.5) and temperature (32°C). Citrate was completely metabolized and its consumption rate was pH- and temperature-dependent. Neither acids contributed energy for growth as the Y_glu remained constant in the presence or absence of acids. There was a significant increase in fructose consumed at higher temperature. Also, the final concentration of mannitol was higher but not significantly different. The addition of acids, particularly citrate, significantly repressed mannitol formation.     

The putative immunity protein of the Gram-positive bacteria Leuconostoc mesenteroides is preferentially located in the cytoplasm compartment

Abstract Immunity proteins are thought to protect bacteriocin-producing bacterial strains against the bactericidal effects of their own bacteriocin. The immunity protein which protects the lactic acid bacterium Leuconostoc mesenteroides against mesentericin Y105³⁷ bacteriocin was detected and localized by immunofluorescence and electron microscopy, using antibodies directed against the C-terminal end of the predicted immunity protein. The antibodies recognized the immunity proteins of various strains of Leuconostoc , including Leuconostoc mesenteroides and Leuconostoc gelidum . This study demonstrated that immunity proteins produced by Leuconostoc mesenteroides accumulated in the cytoplasmic compartment of the bacteria. This is in contrast with other known immunity proteins, such as the colicin immunity proteins, which are integral membrane proteins possessing three to four transmembrane domains.     

Glucose metabolism and internal pH of Lactococcus lactis subsp. lactis cells utilizing NMR spectroscopy

The metabolism of glucose was studied in Lactococcus lactis subsp. lactis CNRZ 125 by ¹³C NMR. The initial rate of glucose utilization was higher for exponential phase cells than for stationary phase cells [150 vs 85 nmol g (dry wt)^-1 s^-1]. ³¹P NMR was used to determine changes in glycolytic phosphorylated intermediates (fructose-1,6-diphosphate, dihydroxyacetone phosphate and phosphoglycerate). The internal pHs of L. lactis subsp. lactis CNRZ 141 and CNRZ 125 were also measured by ³¹P NMR as a function of the external pH during growth. When the external pH was 6·8, the internal pHs of strain CNRZ 141 and CNRZ 125 were similar, 7·4. After the external pH had decreased to 5·5, the internal pH of strain CNRZ 141 had declined by 0·6 unit, whereas that of strain CNRZ 125 had decreased by only 0·2 unit of pH.     

Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides.

In Leuconostoc mesenteroides subsp. mesenteroides 19D, citrate is transported by a secondary citrate carrier (CitP). Previous studies of the kinetics and mechanism of CitP performed in membrane vesicles of L. mesenteroides showed that CitP catalyzes divalent citrate HCit2-/H+ symport, indicative of metabolic energy generation by citrate metabolism via a secondary mechanism (C. Marty-Teysset, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Biol. Chem. 270:25370-25376, 1995). This study also revealed an efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate cometabolism, suggesting that under physiological conditions, CitP may function as a precursor/product exchanger rather than a symporter. In this paper, the energetic consequences of citrate metabolism were investigated in resting cells of L. mesenteroides. The generation of metabolic energy in the form of a pH gradient (delta pH) and a membrane potential (delta psi) by citrate metabolism was found to be largely dependent on cometabolism with glucose. Furthermore, in the presence of glucose, the rates of citrate utilization and of pyruvate and lactate production were strongly increased, indicating an enhancement of citrate metabolism by glucose metabolism. The rate of citrate metabolism under these conditions was slowed down by the presence of a membrane potential across the cytoplasmic membrane. The production of D-lactate inside the cell during cometabolism was shown to be responsible for the enhancement of the electrogenic uptake of citrate. Cells loaded with D-lactate generated a delta psi upon dilution in buffer containing citrate, and cells incubated with citrate built up a pH gradient upon addition of D-lactate. The results are consistent with an electrogenic citrate/D-lactate exchange generating in vivo metabolic energy in the form of a proton electrochemical gradient across the membrane. The generation of metabolic energy from citrate metabolism in L. mesenteroides may contribute significantly to the growth advantage observed during cometabolism of citrate and glucose.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.     

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.     

Origin of end-products from the co-metabolism of glucose and citrate by Leuconostoc mesenteroides subsp. cremoris

Summary The addition of citrate to glucose broth led to an increase in specific growth rate and glucose catabolism, but a decrease in molar growth yield from glucose, in Leuconostoc mesenteroides subsp. cremoris. Acetate and formate were produced during the stationary phase of growth. According to the fermentation balance, part of the acetate and lactate came from the pyruvate of citrate metabolism. L. mesenteroides subsp. cremoris incorporated radioactive metabolites from [1,5-¹⁴C] citrate into cell material, primarily into lipids. [U-¹⁴C] Glucose was not incorporated into cell material.     

Utilization of citrate byLeuconostoc mesenteroides subsp.cremoris in continuous culture

Summary Leuconostoc mesenteroides subsp.cremoris was grown in continuous culture in lactose medium with varying citrate concentrations. All citrate (10, 25, 50 and 75 mMol/l) was used and lactose consumption increased with increasing initial citrate concentrations correlate with an increase of dry cell weight. Citrate lead to an increase of acetate and could be a source of ATPvia acetate kinase pathway. For each steady state, Y_ATP values were calculated and were twice greater than the generally accepted value of 10.5. The maintenance energy was calculated it was constant for lactose (2.5 mMol/l.h.) and increased for citrate suggesting a greater requirement of energy for citrate utilization.     

Effect of varying citrate levels on C4 compound formation and on enzyme levels in Leuconostoc mesenteroides subsp. cremoris grown in continuous culture

Summary The effects of citrate on diacetyl, acetoin and 2,3-butylene glycol (2,3-BG) production by Leuconostoc mesenteroides subsp. cremoris grown in continuous culture at pH 5.2 were studied. In glucose alone end-product production agreed with the theoretical stoichiometry. In the presence of citrate, lactate and acetate production was higher than the theoretical stoichiometry from glucose. Lactate production was constant when the initial citrate concentration was increased whereas ethanol production strongly decreased. In the absence of citrate, citrate lyase (CL) exhibited weak activity. Diacetyl reductase (DR) and acetoin reductase (AR) exhibited basal activity. When varying citrate concentrations ranging from 10 to 75 mm were added to glucose broth, DR, AR, lactate dehydrogenase, NADH oxidase and alcohol dehydrogenase decreased as the initial citrate concentration increased suggesting that they were partly repressed by citrate. In contrast, CL increased and the specific citrate utilization rate also increased in the same way, indicating no saturation of the first step of citrate metabolism. Acetate kinase (AK) was slightly higher in the presence of citrate and increased when the initial citrate concentration increased. This result was correlated with an increase of acetate from the acetyl phosphate pathway. More ATP was produced in the presence of citrate, which could explain the increase in biomass formation. Citrate bioconversion into diacetyl, acetoin and 2,3-BG increased as the initial citrate increased. Correspondence to: C. Diviès     

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.     

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     

Effect of some factors on determination of viable microbial cells by peroxyoxalate chemiluminescence method

The effects of physical and chemical factors on the production of H₂O₂ from Escherichia coli cells were studied. When 20 mmol 1^-1 Tris-HCl buffer was used for this purpose the electron transport system (ETS) showed the highest activity at pH 7.6-8.2. KCN promoted the production of H₂O₂ from E. coli cells, and the optimum concentration was changed in different reaction times and pH values. Glucose, 5 mg ml^-1, increased the ETS activity about twofold. The other substrates and surfactants did not increase the chemiluminescence intensity. NaNO₂ and Na₂SO₄ in inorganic salts significantly reduced the ETS activity above 70%. In addition, the optimum temperature for the production of H₂O₂ was 30°C in this study. When glucose (5 mg ml^-1) and KCN (0.2 mmol 1^-1) were added to the reaction buffer containing 0.5 mmol 1^-1 menadione, the detectable minimum cell densities (averages of triplicate assay) of E. coli, Enterobacter cloacae and Serratia marcescens were 5 times 10³ cells ml^-1, 10⁴ cells ml^-1 and 10⁴ cells ml^-1 respectively.     

Exoprotease activity of Leuconostoc oenos in stress conditions

Exoprotease activity during 48 h of total energy and nutrient starvation was examined in Leuconostoc oenos X₂L isolated from wine. Starved cells after 2 h of incubation at 30 °C in citrate buffer, 0.05 mmol 1⁻¹ pH 5, showed greater extracellular proteolytic activity than at the onset of starvation. In the presence of 60 mg l⁻¹ SO₂ and 8% or 12% ethanol, the proteolytic activity was higher ; 10 mmol l⁻¹ Ca²⁺ and Mg²⁺ produced an increase in protease activity during starvation. Glucose and 2-deoxyglucose (2-DOG) were found to repress synthesis by 80% and 100%, respectively. Cyclic adenosine 3'-5'-phosphate increased the exoprotease activity and reverted the repression by glucose and 2-DOG. De novo synthesis of proteins was required for the exoprotease activity by cells submitted to stress conditions. The absence of protease activity in the supernatant fluids from chloramphenicol-treated cells indicated that the activity is a result of deliberate release and not of passive cell lysis.     

Purification and characterization of mitomycin C-induced pectin lyase of Erwinia carotovora subsp. atroseptica strain SCRI 1043

Erwinia carotovora supsp. atroseptica strain SCRI 1043 produces pectin lyase (PNL) which degrades highly methyl-esterified pectin by trans -elimination when induced by DNA damaging agents such as mitomycin C. Purification of the enzyme (66.5-fold) to homogeneity with 42.3% recovery was achieved by cation exchange chromatography on an S-Sepharose fast flow column equilibrated to pH 8.5 with 20 mmol 1^-1 Tris buffer, followed by elution of the bound proteins with a 1 mol^-1 NaCl gradient. SDS-PAGE and IEF-activity staining analyses showed that the mol. wt and pI of the enzymes were 31 kDa and 9.4 respectively and only one isomer was present. The optimum pH and temperature were 8.0 and 35°C respectively, and divalent cations, 1.37 mmol 1^-1 Ca₂₊ and 1.37 mmol 1_-1 Mg₂₊, although not essential, stimulated enzyme activity by about four and six times respectively. The endo mode of action of PNL was determined by viscometry. PNL induction by mitomycin C was determined spectrophotometrically and by ELISA, and was concomitant with bacteriocin production and bacterial cell lysis. The purified enzyme caused rapid maceration of potato tuber discs and IEF-activity staining of PNL in extracts of rotting tuber discs inoculated with strain SCRI 1043 showed that two isoenzymes were present, one corresponding to the enzyme produced in vitro and the other with a slightly higher pI.     

Extracellular material from Leuconostoc mesenteroides subsp. dextranicum mediates the aggregation of lactococcal cells: implications for the isolation of single strains

Cultures of Lactococcus lactis subsp. cremoris , originally derived from mixed cheese starter cultures, were assessed as pure following single colony selection and subculturing, yet nevertheless gave rise, under stress conditions, to an isolate with the ability to ferment citrate. The isolate was characterized with respect to its citrate enzymology and lactose fermentation and was identified as Leuconostoc mesenteroides subsp. dextranicum and assigned the strain number 663. The extracellular material (ECM) from Leuc. mesenteroides subsp. dextranicum 663 was characterized and found to contain carbohydrate, protein and phosphate (30.9, 50.7 and 18.4%, by weight, respectively). Glucose was the most prominent sugar (39% by weight of total carbohydrate) with mannose, galactose and rhamnose being the other major monosaccharides. The ECM protein resolved into a large number of bands on SDS-polyacrylamide gel electrophoresis, the most prominent having molecular masses of 40 and 49 kDa. The ECM from Leuc. mesenteroides subsp. dextranicum 663 caused aggregation of suspensions of lactococcal cells and may facilitate intergeneric interactions and/or co-culture during cheese starter strain isolation.     

Nutrient reserves of Salicornia europaea seeds

Seeds of Salicornia europaea L. were analyzed for their nutrient reserves. The content of potassium and sodium was 216 and 39 mmol (kg dry seeds)^-1, respectively. Calcium and magnesium accounted for 30 and 138 mmol (kg dry seeds)^-1, respectively. Whereas most of the alkali metals were water soluble, the alkaline earth metals were mostly acid soluble. The acid-soluble calcium plus magnesium corresponded well with the acid-soluble phosphate. Chloride was accumulated to a level equivalent to that of sodium. Carbonate was present at a concentration of 9 mmol (kg dry seeds)^-1. Carbohydrates accounted for 93 g (kg dry seeds)^-1, nearly half of which was derived from sucrose. Fructose and glucose were present only in traces. Total nitrogen was determined to be 55 g (kg dry seeds)^-1, 16% of which was diethylether soluble. The remaining nitrogen was separated into 39 g (kg dry seeds)^-1 ethanol-insoluble and 8 g (kg dry seeds)^-1 ethanol-soluble nitrogen. About 10% of the ethanol-soluble nitrogen were derived from amino acids. Total lipid content was about 280 g (kg dry seeds)^-1. The alcoholic component of the storage lipids was glycerol and the glycerides were calculated from gas chromatography to be 66% of the total lipids. About 90% of the fatty acids consisted of unsaturated acids, linoleic and oleic acid, the majority (77%) of which was linoleic acid.     

Leuconostoc mesenteroides SJRP55: a potential probiotic strain isolated from Brazilian water buffalo mozzarella cheese

The probiotic potential of Leuconostoc mesenteroides subsp. mesenteroides SJRP55, isolated from water buffalo mozzarella cheese was evaluated. The microorganism presented resistance to stressful conditions that simulated the gastrointestinal tract, and to the best of our knowledge, Leuconostoc mesenteroides subsp. mesenteroides SJRP55 was the first of this species with the ability to deconjugate bile salts. Tolerance to NaCl was temperature dependent, as well the results obtained by aggregation capacity. The strain presented good adhesion properties, β–galactosidase activity, viability in fermented milk during storage, inactive against Streptococcus thermophilus and sensitive to most of the tested antibiotics. Some analgesic medications inhibited the growth of the strain. Leuconostoc mesenteroides subsp. mesenteroides SJRP55 exhibited in vitro probiotic potential, and it can be better characterized through future in vivo tests. This bacterium presents higher functional properties compared to other studied strains, and therefore, it is a potential candidate for the application as a probiotic strain, which could be used by industries in the manufacture of functional milk-based products.