Genome sequence of Weissella confusa LBAE C39-2, isolated from a wheat sourdough

Weissella confusa is a rod-shaped heterofermentative lactic acid bacterium from the family of Leuconostocaceae. Here we report the draft genome sequence of the strain W. confusa LBAE C39-2 isolated from a traditional French wheat sourdough.     

Behavior of psychrotrophic lactic acid bacteria isolated from spoiling cooked meat products

Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10 degrees C. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10 degrees C. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4 degrees C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10(8) CFU/g at 10 degrees C after 7 to 12 days.     

NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa

Dextrans are the main exopolysaccharides produced by Leuconostoc species. Other dextran-producing lactic acid bacteria include Streptococci, Lactobacilli, and Weissella species. Commercial production and structural analysis has focused mainly on dextrans from Leuconostoc species, particularly on Leuconostoc mesenteroides strains. In this study, we used NMR spectroscopy techniques to analyze the structures of dextrans produced by Leuconostoc citreum E497 and Weissella confusa E392. The dextrans were compared to that of L. mesenteroides B512F produced under the same conditions. Generally, W. confusa E392 showed better growth and produced more EPS than did L. citreum E497 and L. mesenteroides B512F. Both L. citreum E497 and W. confusa E392 produced a class 1 dextran. Dextran from L. citreum E497 contained about 11% alpha-(1-->2) and about 3.5% alpha-(1-->3)-linked branches whereas dextran from W. confusa E392 was linear with only a few (2.7%) alpha-(1-->3)-linked branches. Dextran from W. confusa E392 was found to be more linear than that of L. mesenteroides B512F, which, according to the present study, contained about 4.1% alpha-(1-->3)-linked branches. Functionality, whether physiological or technological, depends on the structure of the polysaccharide. Dextran from L. citreum E497 may be useful as a source of prebiotic gluco-oligosaccharides with alpha-(1-->2)-linked branches, whereas W. confusa E392 could be a suitable alternative to widely used L. mesenteroides B512F in the production of linear dextran.     

Complete genome sequence of Leuconostoc citreum KM20

Leuconostoc citreum is one of the most prevalent lactic acid bacteria during the manufacturing process of kimchi, the best-known Korean traditional dish. We have determined the complete genome sequence of L. citreum KM20. It consists of a 1.80-Mb chromosome and four circular plasmids and reveals genes likely involved in kimchi fermentation and its probiotic effects.     

Mannitol production by Leuconostoc citreum KACC 91348P isolated from Kimchi

Leuconostoc genus, which comprise heterofermentative lactic acid bacteria, reduces fructose to mannitol by recycling intracellular NADH. To evaluate the mannitol productivities of different Leuconostoc species, 5 stock cultures and 4 newly isolated strains were cultivated in MRS and simplified media containing glucose and fructose (1:2 ratio). Among them, L. citreum KACC 91348P, which was isolated from kimchi, showed superior result in cell growth rate, mannitol production rate, and yield in both media. The optimal condition for mannitol production of this strain was pH 6.5 and 30°C. When L. citreum KACC was cultured in simplified medium in a 2 l batch fermenter under optimal conditions, the maximum volumetric productivity was 14.83 g·l(-1)h(-1) and overall yield was 86.6%. This strain is a novel and efficient mannitol producer originated from foods to be used for fermentation of fructose-containing foods.     

Biofilm formation by strains of <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> and <Emphasis Type="Italic">L. mesenteroides</Emphasis>

Although biofilms produced by various Leuconostoc sp. are economically important as contaminants of sugar processing plants, very few studies are available on these systems. Twelve strains of Leuconostoc citreum and L. mesenteroides that produce a variety of extracellular glucans were compared for their capacity to produce biofilms. 16s rRNA sequence analysis was used to confirm the species identity of these strains, which included four isolates of L. mesenteroides, five isolates of L. citreum, and three glucansucrase mutants of L. citreum strain NRRL B-1355. Strains identified as L. mesenteroides produce glucans that are generally similar to commercial dextran. Nevertheless, these strains differed widely in their capacity to form biofilms, with densities ranging from 2.7 to 6.1 log cfu/cm(2). L. citreum strains and their derivatives produce a variety of glucans. These strains exhibited biofilm densities ranging from 2.5 to 5.9 log cfu/cm(2). Thus, biofilm-forming capacity varied widely on a strain-specific basis in both species. The types of polysaccharides produced did not appear to affect the ability to form biofilms.     

Identification of Mur, an atypical peptidoglycan hydrolase derived from Leuconostoc citreum

A gene encoding a protein homologous to known bacterial N-acetyl-muramidases has been cloned from Leuconostoc citreum by a PCR-based approach. The encoded protein, Mur, consists of 209 amino acid residues with a calculated molecular mass of 23,821 Da including a 31-amino-acid putative signal peptide. In contrast to most of the other known peptidoglycan hydrolases, L. citreum Mur protein does not contain amino acid repeats involved in cell wall binding. The purified L. citreum Mur protein was shown to exhibit peptidoglycan-hydrolyzing activity by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An active chimeric protein was constructed by fusion of L. citreum Mur to the C-terminal repeat-containing domain (cA) of AcmA, the major autolysin of Lactococcus lactis. Expression of the Mur-cA fusion protein was able to complement an acmA mutation in L. lactis; normal cell separation after cell division was restored by Mur-cA expression.     

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.     

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.     

Leuconostoc citreum HJ-P4 (KACC 91035) regulates immunoglobulin E in an ovalbumin-induced allergy model and induces interleukin-12 through nuclear factor-kappa B and p38/c-Jun N-terminal kinases signaling in macrophages

Leuconostoc citreum ( L. citreum ) HJ-P4 (KACC 91035) is one of the major predominant species in kimchi fermentation in Korea. The purpose of the present study was to test the immunomodulatory capacity of L. citreum to modulate the IgE-mediated allergic response and to examine the involvement of NF-κB and MAPK in IL-12 production in macrophages. Balb/c mice were sensitized with OVA/alum and oral administration of L. citreum to the mice began before or after the OVA sensitization. Protein and mRNA expression of Th1 cytokines in splenocytes by L. citreum in vitro was measured. The role of NF-κB and MAPK such as p38, ERK1/2 and JNK in L. citreum -induced IL-12 was investigated in peritoneal macrophages and RAW264.7 cell lines. L. citreum inhibited the serum levels of total IgE, IgG1 and IgG2a altogether and increased OVA-specific IFN-γ production in splenocytes from pre- and post-sensitized animals. However, the downregulation of IL-4 and IL-5 production was observed only in the pre-sensitization group. The ability of L. citreum to stimulate IFN-γ was dependent on its induction of IL-12. NF-κB, p38 and JNK were mainly involved in L. citreum -induced IL-12 production. In conclusion, the current study demonstrated that L. citreum is able to regulate serum IgE generation at the induction and effector phases of allergic response through overall control over antibody production and that its involvement of IL-12 production was mediated through NF-κB and p38/JNK. Taken together, the use of L. citreum can be useful in preventing the development and progression of IgE production.     

Biodiversity of lactic acid bacteria in Moroccan soft white cheese (Jben)

The bacterial diversity occurring in traditional Moroccan soft white cheese, produced in eight different regions in Morocco, was studied. A total of 164 lactic acid bacteria were isolated, purified and identified by whole-cell protein fingerprinting and rep-PCR genomic fingerprinting. The majority of the strains belonged to the genera Lactobacillus, Lactococcus, Leuconostoc and Enterococcus. Sixteen species were identified: Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, Lactococcus garvieae, Lactococcus raffinolactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc citreum, Eterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus saccharominimus and Streptococcus sp.     

Genome sequence of a food spoilage lactic acid bacterium, Leuconostoc gasicomitatum LMG 18811T, in association with specific spoilage reactions

Leuconostoc gasicomitatum is a psychrotrophic lactic acid bacterium causing spoilage of cold-stored, modified-atmosphere-packaged (MAP), nutrient-rich foods. Its role has been verified by challenge tests in gas and slime formation, development of pungent acidic and buttery off odors, and greening of beef. MAP meats have especially been prone to L. gasicomitatum spoilage. In addition, spoilage of vacuum-packaged vegetable sausages and marinated herring has been reported. The genomic sequencing project of L. gasicomitatum LMG 18811T was prompted by a need to understand the growth and spoilage potentials of L. gasicomitatum, to study its phylogeny, and to be able to knock out and overexpress the genes. Comparative genomic analysis was done within L. gasicomitatum LMG 18811T and the three fully assembled Leuconostoc genomes (those of Leuconostoc mesenteroides, Leuconostoc citreum, and Leuconostoc kimchii) available. The genome of L. gasicomitatum LMG 18811T is plasmid-free and contains a 1,954,080-bp circular chromosome with an average GC content of 36.7%. It includes genes for the phosphoketolase pathway and alternative pathways for pyruvate utilization. As interesting features associated with the growth and spoilage potential, LMG 18811T possesses utilization strategies for ribose, external nucleotides, nucleosides, and nucleobases and it has a functional electron transport chain requiring only externally supplied heme for respiration. In respect of the documented specific spoilage reactions, the pathways/genes associated with a buttery off odor, meat greening, and slime formation were recognized. Unexpectedly, genes associated with platelet binding and collagen adhesion were detected, but their functionality and role in food spoilage and processing environment contamination need further study.     

Large increase in Leuconostoc citreum KM20 dextransucrase activity achieved by changing the strain/inducer combination in an E. coli expression system

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction (OD???) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of 17 degrees C. These results may effectively apply to the heterologous expression of other large DexT genes.     

Synbiotic synthesis of oligosaccharides during milk fermentation by addition of leuconostoc starter and sugars

Synthesis of oligosaccharides during milk fermentation was attempted by inoculating Leuconostoc citreum with Lactobacillus casei, Lb. delbrueckii subsp. bulgaricus, and Streptococcus thermophilus as starters. Dextransucrase of Ln. citreum worked as a catalyst for the transglycosylation reaction of sugars; sucrose was added as the glucose donor, and lactose or maltose acted as the acceptor compound for the reaction. When 4% sucrose was added in milk, glucosyl-lactose was synthesized (about 1%, w/v) after 1-2 days of fermentation at 15 or 25 degrees C. Alternatively, when sucrose and maltose (2% each, w/v) were added, panose (about 1%, w/v) and other isomaltooligosaccharides were made in a day at 15-35 degrees C. Growth patterns of lactobacilli and streptococci starters were not affected by the coculture of leuconostoc starter, but the rate of acid synthesis was slightly slowed at every temperature. Addition of sugars in milk did not give any adverse effect on the lactate fermentation. Accordingly, the use of leuconostoc starter and addition of sugars in milk allowed the production of oligosaccharides-containing fermented milk, and application of this method will facilitate the extensive development of synbiotic lactate foods.     

DNA Fingerprinting of Lactic Acid Bacteria in Sauerkraut Fermentations

Previous studies using traditional biochemical identification methods to study the ecology of commercial sauerkraut fermentations revealed that four species of lactic acid bacteria, Leuconostoc mesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, and Lactobacillus brevis, were the primary microorganisms in these fermentations. In this study, 686 isolates were collected from four commercial fermentations and analyzed by DNA fingerprinting. The results indicate that the species of lactic acid bacteria present in sauerkraut fermentations are more diverse than previously reported and include Leuconostoc citreum, Leuconostoc argentinum, Lactobacillus paraplantarum, Lactobacillus coryniformis, and Weissella sp. The newly identified species Leuconostoc fallax was also found. Unexpectedly, only two isolates of P. pentosaceus and 15 isolates of L. brevis were recovered during this study. A better understanding of the microbiota may aid in the development of low-salt fermentations, which may have altered microflora and altered sensory characteristics.     

A new vector, pGID052, for genetic transfer in Oenococcus oeni

Despite the large number of techniques available for the transformation of bacteria, several species are still resistant to the introduction of foreign DNA. Oenococcus oeni are among the organisms that are particularly refractory to transformation. However, conjugal experiments from Lactococcus lactis to O. oeni with a new plasmid, pGID052, were performed via mobilization with success. This plasmid, a derivative of pORI19, encompasses: (i) the oriT of pIP501, which permitted the transfer to O. oeni, (ii) the replication genes of a native Leuconostoc citreum plasmid. Frequencies of 10(-7) conjugants per recipient were found. The transfer did not affect the structure of this low-copy-number plasmid. Moreover, pGID052 seems segregationally stable and could be used in the future as an expression vector.     

Complete genome sequence of Leuconostoc kimchii strain C2, isolated from Kimchi

Leuconostoc kimchii strain C2 was isolated from fermented kimchi in Korea. Here we announce the complete genome sequence of Leuconostoc kimchii strain C2, consisting of a 1,877,174-bp chromosome with a G+C content of 37.9% and no plasmid and describe major findings from its annotation.     

L.SP.HXQ001菌对家免抗氧化能力的影响

目的：探讨乳酸细菌中的明串珠对家兔抗氧化能力的影响。方法：检测健康家兔口服L.sp.HXQ001菌液前后,血清丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-px）水平及红细胞超氧化物歧化酶（SOD）总活性。结果： L.sp.HXQ001菌及其发酵产物可明显提高GSH-ox、SOD活性,降低血清MDA水平,并维持较长时间。结论：L.sp.HXQ001菌及其发酵产物有提高家兔抗氧化能力的作用,有望成为新的微生态调节剂。     

Characterization of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA

Of 215 leuconostocs isolated from field grass, natural whey cultures and water-buffalo milk, 178 were identified as Leuconostoc mesenteroides ssp. mesenteroides while 37 strains could not be identified Biochemical characterization allowed seven groups to be defined. Representative strains of each group and different habitat and nine reference strains were selected for further analyses. Protein profiles appeared suitable for species discrimination, but did not differentiate between the three subspecies of Leuc. mesenteroides. The technique also showed some differences among equivocal strains. DNA fingerprinting for most strains of Leuc. mesenteroides ssp. mesenteroides examined showed a different restriction pattern from that of the type strain. Ribotyping was not useful for discriminating species and subspecies of the genus Leuconostoc: Leuc. mesenteroides ssp. mesenteroides and ssp. dextranicum showed the same ribopattern as Leuc. lactis while Leuc. mesenteroides ssp. cremoris exhibited a pattern distinct from all the other species examined. On the basis of ARDRA-PCR, two main groups could be distinguished: the larger group included Leuc. mesenteroides, Leuc. lactis, Leuc. pseudomesenteroides and some unidentifiable strains; the second one included Leuc. citreum, Leuc. fallax, Weissella paramesenteroides and some unidentified strains.