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PubMed1993

Phylogenetic analysis of some leuconostocs and related organisms as determined from large-subunit rRNA gene sequences: assessment of congruence of small- and large-subunit rRNA derived trees.

The large-subunit ribosomal RNA gene sequences of Leuconostoc carnosum, Leuc. mesenteroides, Leuc. oenos, Leuc. paramesenteroides and Lactobacillus confusus were determined by direct sequencing of enzymatically-amplified DNA. Comparative analysis of the large-subunit rDNA sequences revealed that the leuconostocs form three distinct lines of descent: Leuconostoc sensu stricto, Leuc. paramesenteroides group of organisms, and Leuc. oenos. The distance matrix tree constructed from the large-subunit sequence data was in excellent agreement with that derived from small-subunit sequences, and demonstrates the 'synchrony' of the two chronometers. The results of the present and earlier sequence analyses indicate the taxonomy of the genus Leuconostoc is in need of revision, and that the Leuc. paramesenteroides group of organisms and the species Leuc. oenos represent distinct phylogenetic units, separate from Leuconostoc sensu stricto.

Base Sequence↗
PubMed2006

Structural and functional features of fructansucrases present in Leuconostoc mesenteroides ATCC 8293.

Glycosyltransferases produced by Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 (equivalent to NRRL B-1118) were identified. Two glucansucrases and one fructansucrases were observed in batch culture while levC and levL genes, corresponding to two fructansucrases, were isolated from information obtained from the released draft sequence of this Leuconostoc strain genome and cloned in Escherichia coli. The recombinant enzymes were shown to be fructansucrases producing a polymer identified by NMR as levan, confirming our recent report stating that these are also mosaic levansucrases bearing structural features of glucansucrases in the amino and carboxy terminal regions, as is also the case of inulosucrase (IslA) from Leuconostoc citreum CW28 and levansucrase (LevS) from L. mesenteroides NRRL B-512F. The recombinant levansucrase LevC was purified and characterized in terms of pH, temperature, and kinetic properties. The enzyme exhibits Michaelis-Menten kinetic properties with a K(m) = 27.3 mM and a k(cat) = 282.9 s(-1). This levansucrase behaves mainly as a transferase as only 30% of the substrate is hydrolyzed in a wide range of sucrose concentrations, with higher hydrolytic activities at low substrate concentrations. With this report we experimentally confirm the unusual structural pattern displayed by fructansucrases present in Leuconostoc species that group as a novel sub family of fructansucrases.

Amino Acid Sequence↗
PubMedDate not supplied

Chemical, microbial and sensory changes during the anaerobic cold storage of beef inoculated with a homofermentative Lactobacillus sp. or a Leuconostoc sp.

Slices of beef were inoculated with about 3.5 log cfu/cm2 of Lactobacillus sp. 93 SMRICC 235 (homofermentative) or Leuconostoc sp. 89 SMRICC 189 and stored in 5% CO2 + 95% N2 at 4 degrees C. The microbial, chemical (glucose, L-lactate, D-lactate, acetate, formate, ethanol, H2S) and sensory changes of the beef slices were studied. For beef inoculated with Lactobacillus sp. 93 the flavour score started to decrease when the maximum bacterial count was reached. Leuconostoc sp. 89 caused a rapid decrease in the flavour score before reaching the maximum bacterial count. Concentrations of acetate and D-lactate increased while glucose and L-lactate decreased in beef slices inoculated with Lactobacillus sp. 93. In the presence of Leuconostoc sp. 89 ethanol and D-lactate increased while glucose decreased. Lactobacillus sp. 93 formed the highest level of H2S, and a sulphurous off-odour was noted only in the presence of this strain. D-Lactate and acetate indicated high numbers of Lactobacillus sp. 93 on the meat surface, while D-lactate and ethanol indicated high numbers of Leuconostoc sp. 89. More studies are needed in order to correlate levels of D-lactate, acetate and ethanol with sensory changes.

Acetates↗
PubMed2005

pIH01, a small cryptic plasmid from Leuconostoc citreum IH3.

A small cryptic plasmid pIH01 from Leuconostoc citreum IH3 was characterized. This 1.8-kb sized plasmid contains single open reading frame that encodes a RepC class protein (342 amino acids) and a conserved pT181-type double strand origin, suggesting a rolling circle replication mode. This putative replicase protein shows the highest similarity to a replicase from pFR18 plasmid of Leuconostoc mesenteroides FR52 (64% identity), one of the pT181-type rolling circle plasmid family and contains a strictly conserved RepC-type active site sequence of pT181 family. A shuttle vector that was developed on the basis of this cryptic plasmid by insertion of both erythromycin resistance gene (ermC) from pE194 and Escherichia coli ColE1 origin was able to transform Leuconostoc strains, Lactobacillus plantarum, and Lactococcus lactis. Therefore, pIH01 derivative plasmids might be useful for the manipulation of Leuconostoc strains.

Amino Acid Sequence↗
PubMed2003

Novel Leuconostoc citreum starter culture system for the fermentation of kimchi, a fermented cabbage product.

To determine the dominant microorganisms involved in kimchi fermentation and to examine their effect on kimchi fermentation, we randomly isolated and characterized 120 lactic acid bacteria from kimchi during a 5-day fermentation at 15 degrees C. Leuconostoc citreum was dominant during the early and mid-phases of kimchi fermentation whereas Lactobacillus sake/Lactobacillus curvatus or Lactobacillus brevis were found during later stages. Eighty-two out of 120 isolates (68%) were identified as Leuconostoc citreum by means of a polyphasic method, including 16S rDNA sequencing and DNA/DNA hybridization. A few Weissella confusa-like strains were also isolated during the mid-phase of the fermentation. Strain IH22, one of the Leuconostoc citreum isolates from kimchi, was used as an additive to evaluate growth and acid production in kimchi fermentation. This strain was consistently over 95% of the population in IH22-treated kimchi over a 5-day fermentation, while heterogeneous lactic acid bacteria were observed in the control kimchi. The pH in IH22-treated kimchi dropped rapidly but was stably maintained for 5 days, compared to its slow and prolonged decrease in the control kimchi. These results indicate that Leuconostoc citreum IH22 dominates over and retards the growth of other lactic acid bacteria in kimchi, suggesting it can be used as a bacterial starter culture to maintain the quality of kimchi for prolonged periods.

Brassica↗
PubMed1999

Partial characterization and cloning of leuconocin J, a bacteriocin produced by Leuconostoc sp. J2 isolated from the Korean fermented vegetable Kimchi.

Leuconostoc sp. J2, isolated from naturally fermented Kimchi, produced a bacteriocin which was named leuconocin J. This bacteriocin exhibited an inhibitory activity against several lactic acid bacteria and some food-borne pathogens. The antimicrobial substance was secreted into the medium during the late log phase. It appears to be proteinaceous since its activity was completely inactivated by a range of proteolytic enzymes, and it was also relatively heat-stable. The bacteriocin was partially purified by ammonium sulphate precipitation, following dialysis. The apparent molecular mass of partially purified bacteriocin, as indicated by activity detection after Tricine-SDS-PAGE, was 2.5-3.5 kDa. Leuconostoc sp. J2 plasmid DNA digested by EcoRI was cloned into pUC118 and transformed into Escherichia coli DH5 alpha. Phenotypic expression of the bacteriocin production was detected in transformants harboring pULBJ5.5. Finally, Southern blotting with the 2.3 kb insert as a probe against plasmid digests of Leuconostoc sp. J2 revealed that the cloned foreign DNA originated from Leuconostoc sp. J2.

Bacteriocins↗
PubMed2003

Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rDNA-based techniques.

Ribosomal DNA-based techniques including the analysis of profiles generated by ISR amplification, ISR restriction and ARDRA have been evaluated as molecular tools for identifying Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. They have been applied for the molecular characterization of 91 strains with the following identities: eight Carnobacterium including the eight type species of the genus; 61 Lactobacillus including 40 type strains out of 45 species, 13 Leuconostoc, out of them 11 are type strains and three are subspecies of Lc. mesenteroides; and nine strains representing the six species of genus Pediococcus. The genetic relationship displayed between these species by rrn-based profiles is sustained by their phylogenetic relationships and can therefore be considered useful for taxonomic purposes. Profiles obtained by ISR amplification allowed identification at genus level of Carnobacterium and Leuconostoc, and even at species level in genus Carnobacterium. Genera Lactobacillus and Pediococcus could not be distinguished from each other by applying this technique. The Lactobacillus species analysed here (45) were differentiated using ARDRA-DdeI and ISR-DdeI profiles, sequentially, and Pediococcus species by ISR-DdeI profiles. It was necessary to combine profiles generated by restriction of ISR-DdeI, ARDRA-DdeI and ARDRA-HaeIII in order to complete the identification of Leuconostoc species.

Bacterial Typing Techniques↗
PubMed1999

Leuconostoc species: a case-cluster hospital infection.

Leuconostoc species are members of the Streptococcacae family. They are generally regarded as non-pathogenic culture contaminants and are thought to be an uncommon cause of infection. We present a study of a case-cluster nosocomial infection due to Leuconostoc spp. Three patients were hospitalized at the time of the infection with significant underlying diseases and all had a compromised skin and mucous barriers. Two had received previous antibiotic therapy. This report highlights the importance of Leuconostoc spp. as an emerging pathogen, even though the modes of transmission and reservoirs of Leuconostoc spp. are as yet unknown.

Anti-Bacterial Agents↗
PubMed1996

16S-23S rDNA intergenic sequences indicate that Leuconostoc oenos is phylogenetically homogeneous.

The study of the intra-specific phylogenetic structure of Leuconostoc oenos is essential to understand the participation of several strains in malo-lactic fermentation (MLF). RFLP of the PCR-amplified 16S-23S rDNA intergenic spacer region (ISR) was performed in Leuc. oenos and other related species. The RFLP patterns with seven endonucleases were identical for the 37 Leuc. oenos strains, but differed from those obtained for all other species tested. This method could provide an invaluable insight for molecular identification of the wine leuconostocs. The RFLP relationships of members of the genera Leuconostoc and Weissella were highly similar to those previously reported by 16S and 23S rRNA sequencing studies. The 16S-23S rDNA ISR was sequenced in five strains of Leuc. oenos. A single tRNA(Ala) was detected. The ISR sequence seems to be identical in the two rRNA (rrn) operons found in Leuc. oenos and no significant sequence variation was observed between strains that revealed relative differences as previously shown by PFGE. Results from the present study demonstrated that Leuc. oenos is phylogenetically a very homogeneous species (according to DNA-DNA hybridization studies) and sustain that this species is different from the genus Leuconostoc. The extremely conserved ISR of these organisms suggests that Leus. oenos strains currently isolated and characterized must have spread with the transfer of viticulture rather than coming from indigenous populations.

Base Sequence↗
PubMed2003

Leuconostoc inhae sp. nov., a lactic acid bacterium isolated from kimchi.

Six strains of a hitherto unknown bacterium isolated from kimchi, a fermented vegetable food produced in Korea, were characterized by using phenotypic methods, phylogenetic analysis and DNA-DNA hybridization. The novel strains were gram-positive, non-spore-forming, heterofermentative and spherical or lenticular lactic acid bacteria. Comparative 16S rRNA gene sequencing and DNA relatedness demonstrated that the unknown strains represented a novel clade within the genus Leuconostoc and were close to, but distinct from, Leuconostoc gelidum. The unknown strains were clearly distinguished from all described members of the genus Leuconostoc by using RFLP pattems of genus-specific 16S rRNA gene PCR products with a single endonuclease, BsmAI. Based on the polyphasic evidence, the unknown isolates are classified as Leuconostoc inhae sp. nov. The type strain is strain IH003T (= KCTC 3774T = DSM 15101T).

Base Sequence↗
PubMed1993

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

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 100 degrees C (15 min), but it had a greater stability at pH 4.5 than at pH 7.0 after 6 h at 80 degrees C. 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.

Bacteriocins↗
PubMed2004

Simultaneous detection of Carnobacterium and Leuconostoc in meat products by multiplex PCR.

AIMS: To develop a multiplex PCR approach for simultaneous detection of Leuconostoc and Carnobacterium and its validation in meat products. METHODS AND RESULTS: Two multiplex PCR assays were developed using newly designed 16S rDNA-directed primers adapted to the current taxonomic situation of genera Leuconostoc and Carnobacterium that allow: (i) simultaneous detection of both genera, and members of the nonmotile species of genus Carnobacterium and (ii) identification in a single assay of the nonmotile species C. divergens, C. maltaromicum and C. gallinarum. Sensitivity values of 10(3) and 10(4) CFU g(-1) were determined for multiplex PCR detection of Carnobacterium and Leuconostoc, respectively, following artificially inoculated meat trials. In addition, both multiplex PCR assays were validated in 14 naturally contaminated samples covering nine types of meat products. Results obtained by colony identification were confirmed by PCR detection. CONCLUSIONS: The methods described in this study provide a rapid and reliable tool for PCR detection of Carnobacterium and Leuconostoc, in meat products, and for colony identification. SIGNIFICANCE AND IMPACT OF THE STUDY: This multiplex PCR approach will help in the analysis of the spoilage microbiota of refrigerated vacuum-packaged meat product in order to determine the appropriate preservation method.

Animals↗
PubMed1995

Detection of dairy Leuconostoc strains using the polymerase chain reaction.

This paper reports the design of a Leuconostoc-specific oligonucleotide based on 16S rRNA sequence data. When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc-specific PCR product of approximately 470 bp was produced. The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control. The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures. The Leuconostoc-specific oligonucleotide was used also as a specific hybridization probe.

Base Sequence↗
PubMed1994

[Microbiological and clinical studies of vancomycin resistant Leuconostoc spp. and Pediococcus spp. isolated from septicemia patients].

We described three septicemia cases in which blood cultures yielded gram-positive cocci identified as Leuconostoc spp. and Pediococcus spp. Patients were three male adults aged 63 to 71 years with severe underlying diseases, pancreatic cancer, esophageal cancer and diabetes mellitus with chronic renal failure. They had fever and chills at the onsets of septicemia with acute obstructive suppurative cholangitis, acute pneumonia, and infection complicated with invasion sites of esophageal cancer contagious to bronchus and subcutaneous tissue. Blood cultures yielded catalase and oxidase negative highly vancomycin-resistant (MIC: 1024 micrograms/ml <) gram-positive cocci showing alpha or gamma hemolysis on blood agar plates. Two cases were polymicrobial infections. In one case with esophageal cancer, clinical symptoms persisted after the start of antimicrobial chemotherapy and the patient died 10 days later associated with complications of esophageal cancer. Leuconostoc lactis, Leuconostoc mesenteroides subsp. dextranicum, and Pediococcus acidilactici wee identified by physiological reactions. These strains were also highly resistant to teicoplanin and fosfomycin, and tolerant to all rested beta-lactams such as benzylpenicillin. This is the first report in Japan to our knowledge on the identification of Leuconostoc spp. and Pediococcus spp. isolated from human infectious diseases.

Aged↗
PubMed1988

Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp.

Transfer of the broad-host-range resistance plasmids pIP501 and pAM beta 1 from Streptococcus faecalis to Leuconostoc dextranicum and Leuconostoc cremoris occurred between cells that were immobilized on nitrocellulose filters in the presence of DNase. Transfer of pIP501 to Leuconostoc spp. also occurred when Streptococcus sanguis and Streptococcus lactis were used as donors. In addition, transfer of pIP501 and pAM beta 1 was observed from L. cremoris and L. dextranicum transconjugants to S. sanguis and S. faecalis. Expression of the pAM beta 1 erythromycin and pIP501 erythromycin and chloramphenicol resistance determinants was essentially equivalent in donors and transconjugants. Frequencies of transfer generally ranged from 10(-4) to 10(-7) transconjugants per input donor cell. Intrageneric transfer of pIP501 and pAM beta 1 occurred between L. cremoris and L. dextranicum strains in the same approximate range. These data further extend the host range of pIP501 and pAM beta 1 and demonstrate another example of gene transfer in the genus Leuconostoc.

Chloramphenicol↗
PubMed1998

Mechanism of the citrate transporters in carbohydrate and citrate cometabolism in Lactococcus and Leuconostoc species.

Citrate metabolism in the lactic acid bacterium Leuconostoc mesenteroides generates an electrochemical proton gradient across the membrane 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). Reports on the energetics of citrate metabolism in the related organism Lactococcus lactis are contradictory, and this study was performed to clarify this issue. Cloning of the membrane potential-generating citrate transporter (CitP) of Leuconostoc mesenteroides revealed an amino acid sequence that is almost identical to the known sequence of the CitP of Lactococcus lactis. The cloned gene was expressed in a Lactococcus lactis Cit- strain, and the gene product was functionally characterized in membrane vesicles. Uptake of citrate was counteracted by the membrane potential, and the transporter efficiently catalyzed heterologous citrate-lactate exchange. These properties are essential for generation of a membrane potential under physiological conditions and show that the Leuconostoc CitP retains its properties when it is embedded in the cytoplasmic membrane of Lactococcus lactis. Furthermore, using the same criteria and experimental approach, we demonstrated that the endogenous CitP of Lactococcus lactis has the same properties, showing that the few differences in the amino acid sequences of the CitPs of members of the two genera do not result in different catalytic mechanisms. The results strongly suggest that the energetics of citrate degradation in Lactococcus lactis and Leuconostoc mesenteroides are the same; i.e., citrate metabolism in Lactococcus lactis is a proton motive force-generating process.

Amino Acid Sequence↗
PubMed1993

Identification of Leuconostoc spp. by analysis of soluble whole-cell protein patterns.

Leuconostoc spp. share several physiologic characteristics, which sometimes makes it difficult to identify these organisms to the species level. We developed a system, based on the patterns of soluble whole-cell proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that was able to discriminate between the six Leuconostoc spp. that are capable of growth at 37 degrees C. Nine unidentified Leuconostoc-like bacterial isolates that were included in the study did not fit any of the protein profiles of the type strains and may represent new Leuconostoc spp.

Bacterial Proteins↗
PubMed1997

Leuconostoc spp. septicaemia in a child with short bowel syndrome.

Septicaemia caused by the vancomycin-resistant Gram-positive bacteria Leuconostoc spp. is uncommon. We report a case of Leuconostoc spp. septicaemia in a child with short bowel syndrome fed through a central venous catheter and a gastrostomy. Leuconostoc spp. were isolated from several blood cultures. Despite several courses of antibiotics the fever continued and her condition deteriorated. After removal of the thrombotized central venous catheter her condition improved. Leuconostoc spp. was isolated from the thrombotic masses.

Bacteremia↗