A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice

E thiraj , S. & S uresh , E.R. 1985. A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice. Journal of Applied -Bacteriology 59 , 239–242.
A strain of Leuconostoc oenos was isolated from a blown can of mango juice. Various tests to identify and characterize the bacterium suggested that it could be a strain of L. oenos . This is the first report of L. oenos as a spoilage organism in fruit products other than wine.     

A numerical taxonomic study of Leuconostoc oenos strains from wine

Phenotypic data of 108 tests conducted on 70 malolactic bacteria, including 54 presumptive Leuconostoc oenos strains, five presumptive pediococci isolated from wine and 11 reference strains, were analysed by numerical taxonomic techniques. Using the simple matching coefficient, 58 strains were grouped in six clusters at the 87% S level. Cell wall analysis of the interpeptide bridges and morphology was also used to differentiate between the strains. No L. oenos reference strains were found to group in any cluster. The hypothetical median organism (HMO) of cluster A was related at only the 63% S level to the L. oenos type strain and it is proposed that these strains be regarded as 'L. gracile'. The majority of the L. oenos strains (35) grouped together at above the 91% S level in cluster B, with the HMO of this cluster related at the 75% S level to the L. oenos type strain. Results indicate that the majority of L. oenos strains included in this study are closely related, but more research is needed to justify the separation of these strains into more than one species.     

Occurrence and properties of bacteriophages of Leuconostoc oenos in Australian wines

Bacteriophages specific for Leuconostoc oenos were isolated from four red wines undergoing malolactic fermentation in one winery. Bacteriophages were not found in samples of 16 other wines. The morphology of the phages was examined by electron microscopy. The phages did not lyse all strains of L. oenos, and susceptibility correlated to some extent with the colony morphology of the strain. Phage survived in wines at pH values greater than 3.5 but was inactivated in wines of lower pH and by the addition of sulfur dioxide or bentonite. Phage did not affect the growth of a sensitive strain of L. oenos in filter-sterilized wine.     

Occurrence and properties of bacteriophages of Leuconostoc oenos in Australian wines.

Bacteriophages specific for Leuconostoc oenos were isolated from four red wines undergoing malolactic fermentation in one winery. Bacteriophages were not found in samples of 16 other wines. The morphology of the phages was examined by electron microscopy. The phages did not lyse all strains of L. oenos, and susceptibility correlated to some extent with the colony morphology of the strain. Phage survived in wines at pH values greater than 3.5 but was inactivated in wines of lower pH and by the addition of sulfur dioxide or bentonite. Phage did not affect the growth of a sensitive strain of L. oenos in filter-sterilized wine.     

Using specific polyclonal antibodies to study the malolactic enzyme from Leuconostoc oenos and other lactic acid bacteria

Specific polyclonal antibodies directed against the malolactic enzyme of Leuconostoc oenos were obtained. Despite the homologies between the malolactic enzymes from Leuc. oenos and Lactococcus lactis , no immunological relationship was detected with the L. lactis malolactic enzyme, suggesting differences in their structural organization. The use of the antiserum also demonstrated that the problem of heterologous expression occurring in the recombinant Escherichia coli strain (Labarre et al. 1996a) resulted in a low synthesis of the malolactic enzyme from Leuc. oenos . Moreover, a small amount of the protein was found to be peripherally associated to the membrane of Leuc. oenos.     

Factors affecting the induction of malolactic fermentation in red wines with Leuconostoc oenos

Malolactic fermentation was induced in red wines by inoculation with several strains of Leuconostoc oenos . The progress of Malolactic Fermentation was monitored by following the kinetics of bacterial growth and degradation of malic acid. These kinetics varied significantly depending on the strain of Leuc. oenos inoculated, the strain of Saccharomyces cerevisiae used to conduct the alcoholic fermentation, and the wine properties of pH and concentrations of ethanol and sulphur dioxide. Rapid, predictable malolactic fermentation was achieved by inoculating a high density (> 10⁶ cfu/ml) of Leuc. oenos , whereby malic acid degradation was not connected to the growth of the bacterial cells. Wines after malolactic fermentation were not bacteriologically stable and supported the growth of Leuc. oenos inoculated into the wines.     

Bacteriophages induced by mitomycin C treatment of Leuconostoc oenos strains from Portuguese wines

Twenty-two Leuconostoc oenos strains from Portuguese wines and seven strains from other sources were induced with mitomycin C. Bacteriophages were detected in the supernatants of 19 different cultures. Analysis of their host-range and plating efficiencies suggests that phage typing could be useful for strain identification in Leuc. oenos.     

Plasmids in Leuconostoc oenos

A new procedure was used to isolate 11 plasmids from eight Leuconostoc oenos strains. Plasmid DNA was not detected in 34 other strains of this species. Plasmid sizes ranged from 2.47 to 4.61 kilobase pairs. This is the first report of extrachromosomal elements in L. oenos.     

Application of C Nuclear Magnetic Resonance To Elucidate the Unexpected Biosynthesis of Erythritol by Leuconostoc oenos

Natural-abundance C nuclear magnetic resonance (C-NMR) revealed the production of erythritol and glycerol by nongrowing cells of Leuconostoc oenos metabolizing glucose. The ratio of erythritol to glycerol was strongly influenced by the aeration conditions of the medium. The elucidation of the metabolic pathway responsible for erythritol production was achieved by C-NMR and H-NMR spectroscopy using specifically C-labelled d-glucose. The H-NMR spectrum of the cell supernatant resulting from the metabolism of [2-C]glucose showed that only 75% of the glucose supplied was metabolized heterofermentatively and that the remaining 25% was channelled to the production of erythritol. The synthesis of this polyol resulted from the reduction of the C-4 moiety of the intermediate fructose 6-phosphate. Oxygen has an inhibitory effect on the production of erythritol by L. oenos. Preaeration of a suspension of nongrowing cells of L. oenos resulted in 30% less erythritol and in 70% more glycerol formed during the anaerobic metabolism of glucose. The anaerobic production of erythritol from glucose was also found in growing cultures of L. oenos, although to a smaller extent.     

Characterization of temperate bacteriophages of Leuconostoc oenos and evidence for two prophage attachment sites in the genome of starter strain PSU-1

The close relatedness between 17 Leuconostoc oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. The att B loci were located on the macrorestriction Asc I and Not I fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.     

Isolation and characterization of IS1165, an insertion sequence of Leuconostoc mesenteroides subsp. cremoris and other lactic acid bacteria.

We have cloned and characterized an insertion sequence from Leuconostoc mesenteroides subsp. cremoris strain DB1165. This element, designated IS1165, is 1553 bp, has imperfect inverted repeat ends, contains an open reading frame of 1236 bp, and is not related to any previously described insertion sequence. The copy number of IS1165 varies from 4 to 13 in L. mesenteroides subsp. cremoris strains allowing genetic fingerprinting of strains based on location and number of bands on hybridization. IS1165 or closely related elements have been detected by hybridization in L. lactis, L. oenos, Pediococcus sp., Lactobacillus helveticus, and Lb. casei but not in Lactococcus.     

Inter-strain relationships among wine leuconostocs and their divergence from other Leuconostoc species, as revealed by low frequency restriction fragment analysis of genomic DNA

Thirty Leuconostoc oenos strains, representing 28 different isolates, were distributed into 20 genomic groups according to PFGE patterns of restriction digests. The 8 bp-specific enzymes Sfi I, Not I and Asc I cleaved the Leuc. oenos DNA in a mean of 17, 11 and four fragments respectively and Sma I produced more than 50 fragments per genome. The strain differentiating capacity of the four enzymes was similar; only two related genomic groups failed to be distinguished by Asc I or Not I. Genomic relationships between Leuc. oenos strains were quantified by numerical analysis of Not I and Sfi I banding patterns. More than half of the strains, including the starters ML34 and PSU-1, formed a major cluster. The average size of the Leuc. oenos genome was estimated as 1.86 Mb. Although similar values were obtained for the genomes of Leuc. mesenteroides, Leuc. pseudomesenteroides, Leuc. gelidum and Leuc. citreum, a significant divergence between wine and non-wine species was inferred from comparisons of genome cleavage frequencies, determined with five different enzymes.     

Transposons Tn916 and Tn925 can transfer from Enterococcus faecalis to Leuconostoc oenos

Abstract The streptococcal transposons Tn916 and Tn925 were transferred to several strains of Leuconostoc (Ln.) oenos using the filter mating method. The insertion of both transposons into the chromosome occurred at different sites. Transconjugants of Ln. oenos carrying Tn916 could serve as donors in mating experiments with Lactococcus lactis LM2301. Further analysis of L. lactis LM2301 transconjugants showed that the insertion of the transposon Tn916 into the chromosome was site-specific. These studies establish a basis for the initiation of genetic studies in this Leuconostoc species since there are no efficient conjugal or transformation systems previously described for this microorganism.     

Uniport of anionic citrate and proton consumption in citrate metabolism generates a proton motive force in Leuconostoc oenos.

The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated. Resting cells of L. oenos generate both a membrane potential (delta psi) and a pH gradient (delta pH) upon addition of citrate. After a lag time, the internal alkalinization is followed by a continuous alkalinization of the external medium, demonstrating the involvement of proton-consuming reactions in the metabolic breakdown of citrate. Membrane vesicles of L. oenos were prepared and fused to liposomes containing cytochrome c oxidase to study the mechanism of citrate transport. Citrate uptake in the hybrid membranes is inhibited by a membrane potential of physiological polarity, inside negative, and driven by an inverted membrane potential, inside positive. A pH gradient, inside alkaline, leads to the accumulation of citrate inside the membrane vesicles. Kinetic analysis of delta pH-driven citrate uptake over a range of external pHs suggests that the monovalent anionic species (H2cit-) is the transported particle. Together, the data show that the transport of citrate is an electrogenic process in which H2cit- is translocated across the membrane via a uniport mechanism. Homologous exchange (citrate/citrate) was observed, but no evidence for a heterologous antiport mechanism involving products of citrate metabolism (e.g., acetate and pyruvate) was found. It is concluded that the generation of metabolic energy by citrate utilization in L. oenos is a direct consequence of the uptake of the negatively charged citrate anion, yielding a membrane potential, and from H(+)-consuming reactions involved in subsequent citrate metabolism, yielding a pH gradient. The uptake of citrate is driven by its own concentration gradient, which is maintained by efficient metabolic breakdown (metabolic pull).     

Malolactic fermentation in Chardonnay: growth and sensory effects of commercial strains of Leuconostoc oenos

Samples of fermenting Chardonnay juice were inoculated with five commercial cultures of Leuconostoc oenos to promote malolactic fermentation. Controls were not inoculated with malolactic starter cultures; one was held under the same conditions as the juice inoculated with malolactic starter cultures and the other was held under conditions in which malolactic fermentation was inhibited. Bacterial growth and chemical composition of the wines were monitored for eight weeks after the wines were inoculated with the yeast starter culture. The five strains of L. oenos differed in growth kinetics and rates of malic acid degradation. Significant differences were detected among the finished wines subjected to sensory evaluation.     

Characterization of the lytic activity of bacteriophages of Leuconostoc oenos isolated from wine

Two phage species (sl. ls and lco23) of Leuconostoc oenos , isolated in Switzerland and in Australia, were compared for their ability to inhibit growth of L. oenos in wine. The effect of pH, ethanol, SO₂, temperature and time of infection on phage activity was evaluated in synthetic media and in wine. The phages differed in their response to pH in that the activity of phage sl.ls was highest at low pH (3.5), while that of phage lco23 was highest at a high pH (5.5). The phages were not inhibited by low temperature (15°C) or by 50 mg/l SO_2. Both phages were inhibited by ethanol at a concentration greater than 5% (v/v). In a white and a red wine tested, the red wine partially inhibited and the white wine completely inhibited phage activity. When phage infection at pH 3.5 occurred during the exponential phase of growth, the bacteria outgrew the phage. Phage-resistant mutants developed between 3 and 35 d after phage infection, depending on pH and temperature. Recommendations for the use of starter cultures of L. oenos in the wine industry are given.     

Sugar and organic acid metabolism in mixed cultures of Pediococcus pentosaceus and Leuconostoc oenos isolated from wine

Pediococcus pentosaceus 12p and Leuconostoc oenos X₂L isolated from Argentinian wine were examined for growth and changes in the concentrations of glucose, fructose, sucrose and mannitol and malic, citric, acetic and lactic acids in pure and mixed cultures. In mixed cultures a mutualistic growth response and a change in the balance of end-products of sugar and organic acid metabolism were observed. The production of mannitol and acetic acid was lower while D(-) and L(+) lactic acids were detected in higher levels than in pure cultures. Malic and citric acids were metabolized simultaneously, but the amount of citric acid consumed was lower than in pure culture of Leuc. oenos.     

Common occurrence of plasmid DNA and vancomycin resistance in Leuconostoc spp.

Resistance to vancomycin permitted detection, in a culture of Streptococcus cremoris 290PC, of a contaminant gram-positive coccus. Morphological and physiological characteristics indicated that this bacterium was a strain of Leuconostoc sp., designated PO184. This strain contained four plasmid species, which were distinct from those harbored by S. cremoris 290PC. Antibiotic disk susceptibility tests indicated that Leuconostoc sp. strain PO184 was also resistant to sulfathiazole and trimethoprim and susceptible to 17 other antimicrobials. The MIC of vancomycin for this strain was greater than 2,000 micrograms/ml, and resistance did not depend on drug inactivation. Leuconostoc sp. strain PO184 produced a substance which was inhibitory to S. cremoris U134, but not to S. lactis ATCC 11454. Five other leuconostocs produced substances with antibacterial activity. Of 18 strains of Leuconostoc sp., 14 were resistant to at least 500 micrograms of vancomycin per ml, including four L. oenos strains which harbored no plasmid DNA in the 1- to 76-megadalton range. Twelve Leuconostoc sp. strains contained at least one plasmid species in this mass range. These findings are discussed from the physiological, taxonomical, and ecological standpoints and with regard to their potential applications.     

Pathway and regulation of erythritol formation in Leuconostoc oenos.

It was recently observed that Leuconostoc oenos GM, a wine lactic acid bacterium, produced erythritol anaerobically from glucose but not from fructose or ribose and that this production was almost absent in the presence of O2. In this study, the pathway of formation of erythritol from glucose in L. oenos was shown to involve the isomerization of glucose 6-phosphate to fructose 6-phosphate by a phosphoglucose isomerase, the cleavage of fructose 6-phosphate by a phosphoketolase, the reduction of erythrose 4-phosphate by an erythritol 4-phosphate dehydrogenase and, finally, the hydrolysis of erythritol 4-phosphate to erythritol by a phosphatase. Fructose 6-phosphate phosphoketolase was copurified with xylulose 5-phosphate phosphoketolase, and the activity of the latter was competitively inhibited by fructose 6-phosphate, with a Ki of 26 mM, corresponding to the Km of fructose 6-phosphate phosphoketolase (22 mM). These results suggest that the two phosphoketolase activities are borne by a single enzyme. Extracts of L. oenos were also found to contain NAD(P)H oxidase, which must be largely responsible for the reoxidation of NADPH and NADH in cells incubated in the presence of O2. In cells incubated with glucose, the concentrations of glucose 6-phosphate and of fructose 6-phosphate were higher in the absence of O2 than in its presence, explaining the stimulation by anaerobiosis of erythritol production. The increase in the hexose 6-phosphate concentration is presumably the result of a functional inhibition of glucose 6-phosphate dehydrogenase because of a reduction in the availability of NADP.     

Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria

Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.