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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.

Journal Article↗

Molecular diversity of leuconostoc mesenteroides and leuconostoc citreum isolated from traditional french cheeses as revealed by RAPD fingerprinting, 16S rDNA sequencing and 16S rDNA fragment amplification.

For a long time, the identification of the Leuconostoc species has been limited by a lack of accurate biochemical and physiological tests. Here, we use a combination of RAPD, 16S rDNA sequencing, and 16S rDNA fragment amplification with specific primers to classify different leuconostocs at the species and strain level. We analysed the molecular diversity of a collection of 221 strains mainly isolated from traditional French cheeses. The majority of the strains were classified as Leuconostoc mesenteroides (83.7%) or Leuconostoc citreum (14%) using molecular techniques. Despite their presence in French cheeses, the role of L. citreum in traditional technologies has not been determined, probably because of the lack of strain identification criteria. Only one strain of Leuconostoc lactis and Leuconostoc fallax were identified in this collection, and no Weissella paramesenteroides strain was found. However, dextran negative variants of L. mesenteroides, phenotypically misclassified as W. paramesenteroides, were present. The molecular techniques used did not allow us to separate strains of the three L. mesenteroides subspecies (mesenteroides, dextranicum and cremoris). In accordance with previously published results, our findings suggest that these subspecies may be classified as biovars. Correlation found between phenotypes dextranicum and mesenteroides of L. mesenteroides and cheese technology characteristics suggests that certain strains may be better adapted to particular technological environments.

Cheese↗

Leuconostoc ficulneum sp. nov., a novel lactic acid bacterium isolated from a ripe fig, and reclassification of Lactobacillus fructosus as Leuconostoc fructosum comb. nov.

An isolate, designated strain FS-1T, was recovered from a ripe fig. Phylogenetic analysis of the 16S rRNA genes and DNA-DNA reassociation values showed that the organism represented a novel species of the genus Leuconostoc closely related to Lactobacillus fructosus. The novel isolate could be distinguished from the type strain of Lactobacillus fructosus by the fatty acid composition and several phenotypic and growth characteristics. In strain FS-1T, 18:1 delta9 (18:1omega9c) was present in relatively large amounts whilst, in Lactobacillus fructosus, this fatty acid was a minor component. Strain FS-1T and Lactobacillus fructosus produced acid in API 50CHL microtubes from glucose, fructose and mannitol within 48 h, whereas only strain FS-1T also fermented trehalose, gluconate, turanose and sucrose after 48 h. Other differences in acid production from carbohydrates also distinguished strain FS-1T from Lactobacillus fructosus. Both organisms were heterofermentative with fructose as a substrate and fermented glucose only in the presence of fructose, as determined by nuclear magnetic resonance studies. Strain FS-1T was catalase-positive. On the basis of the phylogenetic analysis, DNA-DNA reassociation values, physiological and biochemical characteristics and fatty acid composition, the name Leuconostoc ficulneum is proposed for the novel species represented by strain FS-1T, and it is proposed that Lactobacillus fructosus be reclassified in the genus Leuconostoc as Leuconostoc fructosum comb. nov.

Carbohydrate Metabolism↗

A phylogenetic analysis of an atypical leuconostoc: description of Leuconostoc fallax sp. nov.

The 16S rRNA sequence of an unknown leuconostoc originally isolated from sauerkraut was investigated by reverse transcription. A comparison of the sequence with those from other lactic acid bacteria revealed the unknown organism represents a new albeit peripheral line within the genus Leuconostoc sensu stricto. A new species, Leuconostoc fallax, is proposed for this organism. The type strain is DSM 20189.

Base Sequence↗

Reclassification of Leuconostoc argentinum as a later synonym of Leuconostoc lactis.

Leuconostoc argentinum, Leuconostoc lactis and ten related strains from Romanian dairy products formed a single cluster, clearly separated from other Leuconostoc species, after numerical analysis of repetitive extragenic palindromic-PCR patterns, whole-cell protein profiles (SDS-PAGE) and fluorescent amplified fragment length polymorphism (FAFLP) band patterns. 16S rRNA gene sequence analysis confirmed a very high similarity between both type strains and representative dairy isolates (>99.6 %). DNA-DNA hybridization experiments revealed high relatedness values between the type strains of L. argentinum and L. lactis and between these strains and representative Romanian strains. These data and the lack of phenotypic distinctive characteristics demonstrate that L. argentinum and L. lactis are synonymous.

Bacterial Proteins↗

Taxonomic studies on some leuconostoc-like organisms from fermented sausages: description of a new genus Weissella for the Leuconostoc paramesenteroides group of species.

Taxonomic studies were performed on some unknown Leuconostoc-like organisms from fermented Greek sausage. Comparative 16S rRNA sequence analysis showed the unidentified organisms represent a new line within the Leuconostoc paramesenteroides group of species. On the basis of the results of this and earlier phylogenetic investigations, it is proposed that Leuconostoc paramesenteroides and related species be reclassified in a new genus Weissella. In addition a new species, Weissella hellenica, is proposed for the isolates from fermented sausage.

Base Sequence↗

Cell envelope analysis of insensitive, susceptible or resistant strains of Leuconostoc and Weissella genus to Leuconostoc mesenteroides FR 52 bacteriocins.

Mesenterocins 52A and 52B belong to class II of lactic acid bacteria bacteriocins. To study susceptibility, insensitivity and resistance to these mesenterocins, four wild-type bacterial strains and four resistant strains, all from Leuconostoc or Weissella genus, were compared. Several cell envelope features were investigated: susceptibilities to antibiotics and to lysozyme, cell morphology and membrane phospholipids contents. The strain insensitive to the two mesenterocins appeared to be resistant to lysozyme and exhibited the highest resistance to antibiotics. Resistant strains displayed cell morphology modifications, several increases in antibiotic resistance and modifications in lysozyme susceptibility. Moreover, mesenterocin 52A-resistant strains displayed modifications in their membrane phospholipids, leading to a more cationic membrane. Insensitivity and resistance of Leuconostoc or Weissella strains seem to be due to various minor modifications of the membrane and/or of the cell wall.

Anti-Bacterial Agents↗

Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated the lactic acid bacterium population associated with strong slime formation in an acetic-acid herring preserve.

Spoilage characterised by strong slime and gas formation affected some manufacture lots of an acetic-acid Baltic herring (Culpea haerengus membras) preserve after few weeks of storage at 0-6 degrees C. The product consisted of herring filets in acetic acid marinade containing sugar, salt, allspice and carrot slices. Microbiological analyses of the spoiled product showed high lactic acid bacterium (LAB) levels ranging from 4.5x10(8) to 2.4x10(9) CFU/g. Yeasts were not detected in any of the herring samples. Since LAB contaminants are seldom associated with fresh fish, LAB populations associated with marinade ingredients (carrots, allspice) were also analyzed. The highest LAB levels exceeding 10(7) CFU/g were detected in equilibrium modified atmosphere packaged baby carrots whereas the levels detected in the allspice samples did not exceed 4.3x10(5). A total of 176 randomly selected LAB isolates originating from herring, carrot and allspice samples were further identified to species level using a 16 and 23S rRNA gene RFLP (ribotyping) database. Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated both in the spoiled herring and carrot samples. These species are heterofermentative-producing CO(2) from glucose and they also produce dextran from sucrose. Inoculation of some commercial-herring products with spoilage-associated L. gelidum and L. gasicomitatum strains verified that these strains have the capability of producing slime and gas in herring preserves although slime formation was not as strong as in the original samples. Since L. gelidum and L. gasicomitatum strains were commonly detected in carrots, carrot slices used for the fish marinade were considered to be the probable source of these specific spoilage organisms.

Animals↗

Variations in the membrane fatty acid composition of resistant or susceptible Leuconostoc or Weissella strains in the presence or absence of Mesenterocin 52A and Mesenterocin 52B produced by Leuconostoc mesenteroides subsp. mesenteroides FR52.

Mesenterocins 52A (Mes52A) and 52B (Mes52B) are antimicrobial peptides produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52. Mes52A is a class IIa bacteriocin of lactic acid bacteria with a broad spectrum of activity. Mes52B is an atypical class II bacteriocin with a narrow spectrum of activity. Four Leuconostoc and Weissella wild-type strains were selected for their susceptibility or insensitivity to these mesenterocins. Four strains resistant to Mes52A or Mes52B were generated from the three susceptible wild-type strains by increasing bacteriocin concentrations in culture media. These resistant strains were at least 30 times more resistant than the wild-type strains. No cross-resistance to Mes52A and Mes52B was observed in these strains. No significant differences in membrane fatty acid composition were observed among the three susceptible wild-type strains and the four resistant strains cultured in MRS broth. Thus, the mesenterocin resistance is unlikely to be due to changes in membrane fatty acid composition. When cultured with Mes52A or Mes52B, the membranes of insensitive and resistant strains contained more saturated fatty acids (1 to 10% more) and less unsaturated fatty acids (3 to 6% less), resulting in a more rigid membrane. Thus, the presence of mesenterocin in the culture media of insensitive or resistant strains induced a significant increase in saturated fatty acid contents and a decrease in unsaturated fatty acid contents. Weissella paramesenteroides DSM 20288BR, resistant to Mes52B, responded atypically, probably due to the production of an inhibitor.

Bacteriocins↗

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.

Alanine↗

Occurrence of Leuconostoc mesenteroides and leuconostoc-like organisms in Lagos, Nigeria.

A total of 91 catalase--negative Gram-positive coccal isolates obtained from 245 clinical specimens in Lagos were characterized. Ten (11.0%) of the isolates were vancomycin resistant, they fermented glucose, sucrose, fructose, lactose, mannose, mannitol, ribose, salicin, sorbitol, arabinose and xylose with acid production. One of the isolates produced in addition gas inclusive and ethanol, thus identified as Leuconostoc mesenteroides. The ten vancomycin-resistant Gram-positive coccal organisms (VRGPC) showed variable sensitivity patterns to penicillin, tetracycline, erythromycin, ampicillin, streptomycin, chloramphenicol, cloxacillin and co-trimoxazole. The possible role of Leuconostoc spp. and VRGPC in clinical infections in hospital setting is still to be defined.

Bacterial Typing Techniques↗

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.

Base Sequence↗

Multiplex PCR-based detection and identification of Leuconostoc species.

A multiplex polymerase chain reaction (PCR) assay has been developed for rapid and reliable identification of Leuconostoc species, by using species-specific primers targeted to the genes encoding 16S rRNA. This assay can detect and differentiate Leuconostoc species from mixed populations in natural sources as well as from pure cultures, within 3 h. This assay system consists of a total of 10 primers, two primers from each target species, and comprises two multiplex PCR reactions: one reaction for Leuconostoc carnosum, Leuconostoc citreum and Leuconostoc mesenteroides, and another reaction for Leuconostoc gelidum and Leuconostoc lactis. This multiplex PCR assay was used to identify 31 Leuconostoc strains isolated from kimchi, a fermented-cabbage product, and the results showed perfect correlation with the results of a polyphasic method, including 16S rDNA sequencing and DNA-DNA hybridization. In addition, this assay enables simultaneous detection of the above-mentioned Leuconostoc species when chromosomal DNA from these Leuconostoc species was mixed. Thus, these results suggest that this multiplex PCR is a rapid and reliable method for identification of Leuconostoc species in pure cultures or in mixed populations.

Brassica↗

Development and use of a selective medium for isolation of Leuconostoc spp. from vegetables and dairy products.

A selective medium (LUSM medium) for the isolation of Leuconostoc spp. was developed. This medium contained 1.0% glucose, 1.0% Bacto Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25% sodium acetate, 0.1% (vol/vol) Tween 80, 15% tomato juice, 30 micrograms of vancomycin (Sigma) per ml, 0.20 microgram of tetracycline (Serva) per ml, 0.5 mg of cysteine hydrochloride per ml, and 1.5% agar (Difco). LUSM medium was used successfully for isolation and enumeration of Leuconostoc spp. in dairy products and vegetables. Of 116 colony isolates obtained from fresh raw milk, curdled milk, or various vegetables, 115 were identified as members of the genus Leuconostoc. A total of 89 of these isolates were identified to species; 13.5% of the isolates were Leuconostoc cremoris, 7.9% were Leuconostoc mesenteroides subsp. mesenteroides, 11.2% were Leuconostoc mesenteroides subsp. dextranicum, 16.9% were Leuconostoc mesenteroides subsp. paramesenteroides, 10.1% were leuconostoc lactis, and 40.4% were Leuconostoc oenos. When we compared the counts obtained for two Leuconostoc strains, Leuconostoc dextranicum 181 and L. cremoris JLL8, on MRS agar and LUSM medium, we found no significant difference between the values obtained on the two media.

Bacteriological Techniques↗

Bacteriocins produced by Leuconostoc species.

Leuconostoc spp. are lactic acid bacteria that are commonly associated with foods and that are used as starter bacteria in some dairy fermentations. Lactic acid bacteria are inhibitory to other bacteria because of pH, organic acids, hydrogen peroxide, and other chemicals produced during their growth, including bacteriocins. Bacteriocin production by Leuconostoc spp. was first observed in the 1950s, but only since 1984, when antagonistic activity of Leuconostoc spp. was reported, have more extensive studies of bacteriocins produced by Leuconostoc spp. been conducted, including mesentericin Y105, produced by Leuconostoc mesenteroides spp. mesenteroides; leucocin A-UAL 187, produced by Leuconostoc gelidum; carnosin 44A, produced by Leuconostoc carnosum; and leuconocin S, produced by Leuconostoc paramesenteroides. Bacteriocins produced by leuconostocs may or may not be active against other lactic acid bacteria, but all include Listeria in their activity spectra. Mesentericin Y105 is reported to be exclusively active against Listeria spp. The amino acid sequences for leucocin A and mesentericin Y105 have been determined. Despite considerable differences in antibacterial spectra, only two amino acids differ between these bacteriocins. The prevalence of leuconostocs in many adventitious fermentations of food and the use of leuconostocs as starter bacteria in controlled fermentations make the bacteriocins produced by these bacteria of interest as possible food preservatives by addition of the bacteriocin or its producer organism to foods.

Amino Acid Sequence↗

Studies on the large subunit rRNA genes and their flanking regions of Leuconostocs.

The 16S-23S (spacer-1) and 23S-5S (spacer-2) rRNA intergenic spacer regions of Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc mesenteroides subsp. dextranicum, and Leuconostoc mesenteroides subsp. cremoris were amplified by polymerase chain reactions and sequenced. The 23S rRNA genes of Leuconostoc lactis, Leuconostoc mesenteroides, and Leuconostoc mesenteroides subsp. dextranicum were also sequenced. The RNase III-like and RNase E processing sites, as well as putative antitermination signals, were identified within the spacer regions. A single tRNA(Ala) gene without the 3'-terminal CCA sequence was found in spacer-1 regions. Secondary structure models are proposed showing interactions between the two spacer regions of leuconostocs. For all strains studied, spacer-1 and spacer-2 were highly conserved and therefore could not be directly used for strain typing. Sequence information on 23S rRNA genes from Leuconostoc species allowed the determination of regions that can be used as targets for diagnostic probes and amplification primers. Secondary structures of variable helical elements of leuconostocs 23S rRNA were constructed and their primary structures were compared with those of several Gram-positive bacteria with low G+C contents. Comparative analysis revealed that restriction analysis of 23S rRNA variable regions appeared to be sufficient for the search for species-specific signatures. Our experimental observations revealed that one form of the rRNA operons was present in leuconostocs. We have also demonstrated the direct linkage between the three species of rRNA genes, which are organized as follows: 5'-16S rRNA-spacer-1-tRNA(Ala)-23S rRNA-spacer-2-5S rRNA-3'.

Base Sequence↗