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1. Koga  T, Nakamura  K, Shirokane  Y, Mizusawa  K, Kitao  S, Kikuchi  M,     ( 1991 )

Purification and some properties of sucrose phosphorylase from Leuconostoc mesenteroides.

Agricultural and biological chemistry 55 (7)
PMID : 1368718  :  
Abstract >>
Sucrose phosphorylase (EC 2.4.1.7) was purified to homogeneity from Leuconostoc mesenteroides cells with a specific activity of 173.8 units per mg protein by ammonium sulfate fractionation, anion exchange HPLC on TSKgel DEAE-5PW, and hydrophobic HPLC on TSKgel Ether-5PW. The purified enzyme was an acidic protein having an isoelectric point of pH 4.6 and s0(20),W of 4.34S. The molecular weight of this enzyme was estimated to be 56,400 by sedimentation equilibrium, 55,000 by SDS-polyacrylamide gel electrophoresis, and HPLC gel filtration on TSKgel G3000SW, suggesting that the enzyme is a monomeric protein. With regard to molecular weight, amino acid composition, and N-terminal amino acid sequence of 30 residues, this enzyme is close to the glucosyltransferase A of Streptococcus mutans.
KeywordMeSH Terms
2. Lee  WT, Levy  HR,     ( 1992 )

Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction.

Protein science : a publication of the Protein Society 1 (3)
PMID : 1304341  :   DOI  :   10.1002/pro.5560010304     PMC  :   PMC2142207    
Abstract >>
Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) was isolated in high yield and purified to homogeneity from a newly constructed strain of Escherichia coli which lacks its own glucose 6-phosphate dehydrogenase gene. Lys-21 is one of two lysyl residues in the enzyme previously modified by the affinity labels pyridoxal 5'-phosphate and pyridoxal 5'-diphosphate-5'-adenosine, which are competitive inhibitors of the enzyme with respect to glucose 6-phosphate (LaDine, J.R., Carlow, D., Lee, W.T., Cross, R.L., Flynn, T.G., & Levy, H.R., 1991, J. Biol. Chem. 266, 5558-5562). K21R and K21Q mutants of the enzyme were purified to homogeneity and characterized kinetically to determine the function of Lys-21. Both mutant enzymes showed increased Km-values for glucose 6-phosphate compared to wild-type enzyme: 1.4-fold (NAD-linked reaction) and 2.1-fold (NADP-linked reaction) for the K21R enzyme, and 36-fold (NAD-linked reaction) and 53-fold (NADP-linked reaction) for the K21Q enzyme. The Km for NADP+ was unchanged in both mutant enzymes. The Km for NAD+ was increased 1.5- and 3.2-fold, compared to the wild-type enzyme, in the K21R and K21Q enzymes, respectively. For the K21R enzyme the kcat for the NAD- and NADP-linked reactions was unchanged. The kcat for the K21Q enzyme was increased in the NAD-linked reaction by 26% and decreased by 30% in the NADP-linked reaction from the values for the wild-type enzyme. The data are consistent with Lys-21 participating in the binding of the phosphate group of the substrate to the enzyme via charge-charge interaction.
KeywordMeSH Terms
Lysine
3. Johansen  E, Kibenich  A,     ( 1992 )

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

Plasmid 27 (3)
PMID : 1325060  :  
Abstract >>
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.
KeywordMeSH Terms
DNA Transposable Elements
4. Neubauer  H, Bauché  A, Mollet  B,     ( 2003 )

Molecular characterization and expression analysis of the dextransucrase DsrD of Leuconostoc mesenteroides Lcc4 in homologous and heterologous Lactococcus lactis cultures.

Microbiology (Reading, England) 149 (Pt 4)
PMID : 12686639  :   DOI  :   10.1099/mic.0.26029-0    
Abstract >>
The gene encoding the dextransucrase DsrD from the industrial strain Leuconostoc mesenteroides Lcc4 was isolated by PCR using degenerate primers recognizing conserved regions present in other dextransucrase-encoding genes from Leuconostoc spp. and Southern blot analyses on total genomic DNA. N-terminal sequence analysis of the active protein recovered in the culture showed that the secreted protein of 165 kDa is devoid of a 42 aa prepeptide which is removed post-translationally, most likely by signal peptidase cleavage. Primer extension and Northern blot analysis identified a monocistronic dsrD mRNA with two transcription initiation sites. Expression of the dextransucrase DsrD was investigated in pH-controlled fed-batch cultures via Northern blot analysis and enzyme activity measurement during the experiments. Sucrose levels of 20 g l(-1) were shown to induce the DsrD biosynthesis around 10-fold. The combination of pH-controlled fed-batch fermentation and Northern analysis clearly showed that dsrD expression was related to the growth of the bacteria. dsrD was transferred to and expressed in Lactococcus lactis MG1363. Controlled fed-batch cultures revealed that active dextransucrase was produced and secreted by the recombinant L. lactis strain. The expression was independent of sucrose levels. These results show that dextransucrase can be efficiently expressed and secreted in a non-Leuconostoc, heterologous host and is able to drive dextran synthesis.
KeywordMeSH Terms
5. Morse  R, O'Hanlon  K, Collins  MD,     ( 2002 )

Phylogenetic, amino acid content and indel analyses of the beta subunit of DNA-dependent RNA polymerase of gram-positive and gram-negative bacteria.

International journal of systematic and evolutionary microbiology 52 (Pt 5)
PMID : 12361249  :   DOI  :   10.1099/00207713-52-5-1477    
Abstract >>
In this study, we have sequenced the rpoB gene, encoding the beta subunit of DNA-dependent RNA polymerase, from a selection of gram-positive and gram-negative bacteria. The presence of insertions and deletions (indels) in the beta subunit separate the gram-positive and gram-negative bacteria from each other and support the division of the gram-positive organisms into two clades based on DNA G+C content. Phylogenetic and amino acid content analyses further separate the clostridia from bacilli, leuconostocs, listeriae and relatives, forming an early branch after the common gram-positive ancestor. The occurrence in the beta subunit of Asn-Ala at positions 471-472 in Porphyromonas cangingivalis and Asn at position 372 in Weissella paramesenteroides are postulated to be the cause of the natural rifampicin resistance of these species.
KeywordMeSH Terms
6. Bozonnet  S, Dols-Laffargue  M, Fabre  E, Pizzut  S, Remaud-Simeon  M, Monsan  P, Willemot  RM,     ( 2002 )

Molecular characterization of DSR-E, an alpha-1,2 linkage-synthesizing dextransucrase with two catalytic domains.

Journal of bacteriology 184 (20)
PMID : 12270834  :   DOI  :   10.1128/jb.184.20.5753-5761.2002     PMC  :   PMC139595    
Abstract >>
A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of alpha-1,6 and alpha-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and alpha-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.
KeywordMeSH Terms
7. Aarnikunnas  J, Rönnholm  K, Palva  A,     ( 2002 )

The mannitol dehydrogenase gene (mdh) from Leuconostoc mesenteroides is distinct from other known bacterial mdh genes.

Applied microbiology and biotechnology 59 (6)
PMID : 12226722  :   DOI  :   10.1007/s00253-002-1070-0    
Abstract >>
The N-terminal amino acid sequences of the intact protein and three tryptic peptides from a 41 kDa protein purified from a commercial mannitol dehydrogenase (MDH) enzyme preparation of Leuconostoc mesenteroides ATCC-9135 were determined. Oligonucleotides deduced from these peptide sequences were used to isolate the putative mdh gene from L. mesenteroides using the Vectorette system. Nucleotide sequence analysis revealed an open reading frame (ORF1) of 1,014 bp encoding a putative MDH protein of 338 amino acids, and another open reading frame (ORF2) encoding an unknown protein of 245 amino acids. In Northern blots, a transcript of approximately 2.2-kb was detected with an mdh-specific probe. Mapping of the 5'-end of the 2.2-kb transcript indicated that mdh was the first gene of the operon. After fusion of six histidine codons to the 3'-end of the mdh gene and expression in Escherichia coli M15, active MDH was isolated using HisTrap purification. The overexpressed enzyme showed high specificity for mannitol and fructose. In dot blot hybridisation, the L. mesenteroides mdh-specific probe bound strongly to chromosomal DNA of Leuconostoc pseudomesenteroides and weakly to DNA of some heterofermentative Lactobacillus strains, whereas no hybridisation signals were obtained with DNA derived from strains carrying characterised mdh genes. Furthermore, the amino acid sequence similarity between L. mesenteroides MDH and other known MDHs was very low, suggesting that MDHs from heterofermentative lactic acid bacteria form a structurally and functionally separate enzyme group. Interestingly, L. mesenteroides MDH shared significant sequence similarity with the medium-chain dehydrogenase/reductase protein family.
KeywordMeSH Terms
8. Fimland  G, Sletten  K, Nissen-Meyer  J,     ( 2002 )

The complete amino acid sequence of the pediocin-like antimicrobial peptide leucocin C.

Biochemical and biophysical research communications 295 (4)
PMID : 12127968  :   DOI  :   10.1016/s0006-291x(02)00769-6    
Abstract >>
The pediocin-like antimicrobial peptide leucocin C produced by a strain of Leuconostoc mesenteroides has been purified using a recently developed rapid two-step procedure. The complete and corrected amino acid sequence of the peptide has been determined by Edman degradation of the intact peptide and a C-terminal fragment generated by cleavage with Asp-N endoprotease. Leucocin C contained 43 residues with the following sequence: KNYGNGVHCTKKGCSVDWGYAWTNIANNSVMNGLTGGNAGWHN. The molecular weight of leucocin C as determined by mass spectrometry was 4595, which is consistent with the theoretical molecular weight of 4596 calculated from the sequence. Moreover, the molecular weights of the two fragments generated by cleavage with Asp-N were also consistent with the determined sequence.
KeywordMeSH Terms
9. Naylor  CE, Gover  S, Basak  AK, Cosgrove  MS, Levy  HR, Adams  MJ,     ( 2001 )

NADP+ and NAD+ binding to the dual coenzyme specific enzyme Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: different interdomain hinge angles are seen in different binary and ternary complexes.

Acta crystallographica. Section D, Biological crystallography 57 (Pt 5)
PMID : 11320304  :   DOI  :   10.1107/s0907444901003420    
Abstract >>
The reduced coenzymes NADH and NADPH only differ by one phosphate, but in the cell NADH provides reducing power for catabolism while NADPH is utilized in biosynthetic pathways. Enzymes almost invariably discriminate between the coenzymes, but glucose 6-phosphate dehydrogenase (G6PD) from Leuconostoc mesenteroides is rare in being functionally dual specific. In order to elucidate the coenzyme selectivity, the structures of NADP(+)- and NAD(+)-complexed L. mesenteroides G6PD have been determined including data to 2.2 and 2.5 A resolution, respectively, and compared with unliganded G6PD crystallized in the same space groups. Coenzyme binding is also compared with that in a ternary complex of a mutant in which Asp177 in the active site has been mutated to asparagine. There are no gross structural differences between the complexes. In both binary complexes, the enzyme interdomain hinge angle has opened. NADP(+) binds to the furthest open form; of the residues within the coenzyme domain, only Arg46 moves, interacting with the 2'-phosphate and adenine. NAD(+) is less well defined in the binding site; smaller hinge opening is seen but larger local changes: Arg46 is displaced, Thr14 bonds the 3'-hydroxyl and Gln47 bonds the 2'-hydroxyl. In the ternary complex, the hinge angle has closed; only the adenine nucleotide is ordered in the binding site. Arg46 again provides most binding interactions.
KeywordMeSH Terms
10. Cosgrove  MS, Gover  S, Naylor  CE, Vandeputte-Rutten  L, Adams  MJ, Levy  HR,     ( 2000 )

An examination of the role of asp-177 in the His-Asp catalytic dyad of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: X-ray structure and pH dependence of kinetic parameters of the D177N mutant enzyme.

Biochemistry 39 (49)
PMID : 11106478  :   DOI  :   10.1021/bi0014608    
Abstract >>
The role of Asp-177 in the His-Asp catalytic dyad of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides has been investigated by a structural and functional characterization of the D177N mutant enzyme. Its three-dimensional structure has been determined by X-ray cryocrystallography in the presence of NAD(+) and in the presence of glucose 6-phosphate plus NADPH. The structure of a glucose 6-phosphate complex of a mutant (Q365C) with normal enzyme activity has also been determined and substrate binding compared. To understand the effect of Asp-177 on the ionization properties of the catalytic base His-240, the pH dependence of kinetic parameters has been determined for the D177N mutant and compared to that of the wild-type enzyme. The structures give details of glucose 6-phosphate binding and show that replacement of the Asp-177 of the catalytic dyad with asparagine does not affect the overall structure of glucose 6-phosphate dehydrogenase. Additionally, the evidence suggests that the productive tautomer of His-240 in the D177N mutant enzyme is stabilized by a hydrogen bond with Asn-177; hence, the mutation does not affect tautomer stabilization. We conclude, therefore, that the absence of a negatively charged aspartate at 177 accounts for the decrease in catalytic activity at pH 7.8. Structural analysis suggests that the pH dependence of the kinetic parameters of D177N glucose 6-phosphate dehydrogenase results from an ionized water molecule replacing the missing negative charge of the mutated Asp-177 at high pH. Glucose 6-phosphate binding orders and orients His-178 in the D177N-glucose 6-phosphate-NADPH ternary complex and appears to be necessary to form this water-binding site.
KeywordMeSH Terms
Aspartic Acid
Catalytic Domain
11. Vought  V, Ciccone  T, Davino  MH, Fairbairn  L, Lin  Y, Cosgrove  MS, Adams  MJ, Levy  HR,     ( 2000 )

Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase.

Biochemistry 39 (49)
PMID : 11106479  :   DOI  :   10.1021/bi0014610    
Abstract >>
The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state.
KeywordMeSH Terms
12. Mizuno  K, Funane  K,     ( 2000 )

Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F.

Bioscience, biotechnology, and biochemistry 64 (1)
PMID : 10705445  :  
Abstract >>
A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of Leuconostoc mesenteroides NRRL B-512F dextransucrase-like gene. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-512F, dextransucrase genes of strain NRRL B-1299 and streptococcal glucosyltransferase genes, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. dsrT mRNA was produced in this strain L. mesenteroides when cells were grown in a sucrose medum, but at a level of 20% of that of dsrS mRNA. The molecular weight of the dsrT gene product was 150,000 by SDS-PAGE. The product did not synthesize dextran, but had weak sucrose cleaving activity. The insertion of five nucleotides at the putative deletion point in dsrT resulted in an enzyme with a molecular weight of 210,000 and with dextransucrase activity.
KeywordMeSH Terms
13. López-Munguía  A, Remaud-Simeon  M, Quirasco  M,     ( 1999 )

Induction and transcription studies of the dextransucrase gene in Leuconostoc mesenteroides NRRL B-512F.

Applied and environmental microbiology 65 (12)
PMID : 10584010  :   PMC  :   PMC91750    
Abstract >>
Dextransucrase production by Leuconostoc mesenteroides NRRL B-512F in media containing carbon sources other than sucrose is reported for the first time. Dextransucrases were analyzed by gel electrophoresis and by an in situ activity assay. Their polymers and acceptor reaction products were also compared by (13)C nuclear magnetic resonance and high-performance liquid chromatography techniques, respectively. From these analyses, it was found that, independently of the carbon source, L. mesenteroides NRRL B-512F produced dextransucrases of the same size and product specificity. The 5' ends of dextransucrase mRNAs isolated from cells grown under different culture conditions were identical. Based on this evidence, we conclude that dextransucrases obtained from cells grown on the various carbon sources result from the transcription of the same gene. The control of expression occurs at this level. The low dextransucrase yields from cultures in D-glucose or D-fructose and the enhancement of dextransucrase gene transcription in the presence of sucrose suggest that an activating phenomenon may be involved in the expression mechanism. Dextransucrase mRNA has a size of approximately 4.8 kb, indicating that the gene is located in a monocistronic operon. The transcription start point was localized 34 bp upstream from the ATG start codon. The -10 and -35 sequences found, TATAAT and TTTACA, were highly homologous to the only glycosyltransferase promoter sequence reported for lactic acid bacteria.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Transcription, Genetic
14. Remaud-Simeon  M, Pizzut  S, Argüello-Morales  MA,     ( 2000 )

Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355.

FEMS microbiology letters 182 (1)
PMID : 10612736  :   DOI  :   10.1111/j.1574-6968.2000.tb08878.x    
Abstract >>
The gene encoding alternansucrase (ASR) from Leuconostoc mesenteroides NRRL B-1355, an original sucrose glucosyltransferase (GTF) specific to alternating alpha-1,3 and alpha-1,6 glucosidic bond synthesis, was cloned, sequenced and expressed into Escherichia coli. Recombinant enzyme catalyzed oligoalternan synthesis from sucrose and maltose acceptor. From sequence comparison, it appears that ASR possesses the same domains as those described for GTFs specific to either contiguous alpha-1,3 osidic bond or contiguous alpha-1,6 osidic bond synthesis. However, the variable region and the glucan binding domain are longer than in other GTFs (by 100 and 200 amino acids respectively). The N-catalytic domain which presents 49% identity with the other GTFs from L. mesenteroides possesses the three determinants potentially involved in the glucosyl enzyme formation.
KeywordMeSH Terms
Glycosyltransferases
Sequence Analysis, DNA
15. Berjeaud  JM, Héchard  Y,     ( 1999 )

Characterization of the mesB gene and expression of bacteriocins by Leuconostoc mesenteroides Y105.

Current microbiology 39 (5)
PMID : 10489435  :  
Abstract >>
Leuconostoc mesenteroides Y105, previously described for production of mesentericin Y105, an anti-Listeria bacteriocin, was shown to secrete a second bacteriocin. The latter was purified, and its molecular mass of 3446 Da, obtained by mass spectrometric analysis, indicates that this bacteriocin should be identical to mesenterocin 52B [Revol-Junelles et al., Lett Appl Microbiol 23:120, 1996]. This second bacteriocin produced by L. mesenteroides Y105 was named mesentericin B105. Its structural gene, mesB, was then localized by a reverse genetic approach, cloned, and sequenced. MesB was found on the pHY30 plasmid, next to mesY gene clusters. Curing experiments led to isolation of two L. mesenteroides Y105 derivatives, named L. mesenteroides Y29 and Y30. The latter had lost pHY30 plasmid, encoding bacteriocin determinants, therefore explaining its phenotype (MesY-, MesB-). On the contrary, Y29 derivative still harbors the pHY30 but did not produce any bacteriocin. Thus, its phenotype could likely result from a point mutation within a gene, probably encoding a protein involved in production of both mesentericin Y105 and mesentericin B105.
KeywordMeSH Terms
Genes, Bacterial
16. Scheirlinck  I, Van der Meulen  R, Van Schoor  A, Vancanneyt  M, De Vuyst  L, Vandamme  P, Huys  G,     ( 2007 )

Influence of geographical origin and flour type on diversity of lactic acid bacteria in traditional Belgian sourdoughs.

Applied and environmental microbiology 73 (19)
PMID : 17675431  :   DOI  :   10.1128/AEM.00894-07     PMC  :   PMC2075033    
Abstract >>
A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.
KeywordMeSH Terms
Bacterial Typing Techniques
Fermentation
Genetic Variation
17. Van der Meulen  R, Grosu-Tudor  S, Mozzi  F, Vaningelgem  F, Zamfir  M, de Valdez  GF, De Vuyst  L,     ( 2007 )

Screening of lactic acid bacteria isolates from dairy and cereal products for exopolysaccharide production and genes involved.

International journal of food microbiology 118 (3)
PMID : 17716765  :   DOI  :   10.1016/j.ijfoodmicro.2007.07.014    
Abstract >>
A total of 174 lactic acid bacteria (LAB) strains isolated from dairy and cereal products were screened for the production of exopolysaccharides (EPS). Therefore, a rapid screening method was developed based on ultrafiltration and gel permeation chromatography. Furthermore, a screening through the polymerase chain reaction (PCR) was performed with primer pairs targeting different genes involved in EPS production. Nine isolates produced a homopolysaccharide of the glucan type, whereas only one strain produced a heteropolysaccharide. The production of a glucan by a strain of Lactococcus lactis and the production of a heteropolysaccharide by a strain of Lactobacillus curvatus are reported for the first time. The PCR screening revealed many positive strains. For three of the ten EPS-producing strains, no corresponding genes could be detected. Furthermore, a lot of strains possessed one or more eps genes but did not produce an EPS. Therefore, a screening on the molecular level should always be accompanied by another screening method that is able to distinguish true EPS producer strains from non-producing ones. Statistical analysis did not reveal any relationship between the type and origin of the strains, the presence or absence of a capsular polysaccharide or EPS, and the presence or absence of eps genes.
KeywordMeSH Terms
18. Jeong  SJ, Park  JY, Lee  HJ, Kim  JH,     ( 2007 )

Characterization of pFMBL1, a small cryptic plasmid isolated from Leuconostoc mesenteroides SY2.

Plasmid 57 (3)
PMID : 17084452  :   DOI  :   10.1016/j.plasmid.2006.09.003    
Abstract >>
A 4661bp cryptic plasmid, pFMBL1, was isolated from Leuconostoc mesenteroides SY2, an isolate from Kimchi, and characterized. Nucleotide sequence analysis revealed two open reading frames, orf1 and orf2. orf2 was 453bp in size and its translation product had 58% identity with a putative protein possibly involved in the replication of pTXL1, a cryptic plasmid from L. mesenteroides ssp. mesenteroides Y110. RNA transcript from orf2 was detected but not from orf1 or intergenic region. Minimum 3.5kb fragment encompassing orf1 and orf2 was required for the replication of pFMBL1 and employed for the construction of Escherichia coli-Leuconostoc shuttle vector, pSJ33E. L. mesenteroides SY1 (another Kimchi isolate), Leuconostoc ssp., and Lactobacillus brevis were successfully transformed with pSJ33E, and the transformation efficiencies were ranged between 1.1x10(1) and 4x10(5)transformants/microg DNA. No single-stranded DNA intermediate was detected from L. mesenteroides SY1 cells harboring pSJ33E, indicating that pFMBL1 probably replicated via theta-type mechanism. pSJ33E was stably maintained in L. mesenteroides SY1 in the absence of erythromycin (Em, 5 microg/ml) and after 1 month of daily subculturing in MRS broth without selective pressure, three percent of cells still retained pSJ33E.
KeywordMeSH Terms
Plasmids
19. Lee  JH, Kang  HK, Moon  YH, Cho  DL, Kim  D, Choe  JY, Honzatko  R, Robyt  JF,     ( 2006 )

Cloning, expression and characterization of an extracellular enolase from Leuconostoc mesenteroides.

FEMS microbiology letters 259 (2)
PMID : 16734786  :   DOI  :   10.1111/j.1574-6968.2006.00274.x    
Abstract >>
Enolase on the surface of streptococci putatively facilitates pathogenic invasion of the host organisms. The related Leuconostoc mesenteroides 512FMCM is nonpathogenic, but it too has an extracellular enolase. Purified isolates of extracellular dextransucrase from cultures of L. mesenteroides contain minute amounts of enolase, which separate as small crystals. Expression of L. mesenteroides enolase in Escherichia coli provides a protein (calculated subunit mass of 47 546 Da) catalyzing the conversion of 2-phsopho-D-glycerate to phosphoenolpyruvate. The pH optimum is 6.8, with Km and kcat values of 2.61 mM and 27.5 s(-1), respectively. At phosphate concentrations of 1 mM and below, fluoride is a noncompetitive inhibitor with respect to 2-phospho-D-glycerate, but in the presence of 20 mM phosphate, fluoride becomes a competitive inhibitor. Recombinant enolase significantly inhibits the activity of purified dextransucrase, and does not bind human plasminogen. Results here suggest that in some organisms enolase may participate in protein interactions that have no direct relevance to pathogenic invasion.
KeywordMeSH Terms
20. Morales-Arrieta  S, Rodríguez  ME, Segovia  L, López-Munguía  A, Olvera-Carranza  C,     ( 2006 )

Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B-512 F.

Gene 376 (1)
PMID : 16632262  :   DOI  :   10.1016/j.gene.2006.02.007    
Abstract >>
A Leuconostoc mesenteroides NRRL B-512 F levansucrase gene, (levS), was isolated, sequenced and cloned in Escherichia coli. The recombinant enzyme was shown to be a fructosyltransferase producing a polymer identified by (13)C-NMR as levan. Based on sequence analysis, we found that this levansucrase is a mosaic protein, bearing structural features of glucosyltransferases in the amino and carboxy terminal regions similarly to inulosucrase from Leuconostoc citreum. The phylogenetic analysis of the C-terminal region domain of levansucrases from L. mesenteroides demonstrates that they group together into a novel putative sub-family of genes and evolved long before all other glucosyltransferases, while their catalytic domain structure is species related.
KeywordMeSH Terms
Phylogeny
21. Renouf  V, Claisse  O, Lonvaud-Funel  A,     ( 2006 )

rpoB gene: a target for identification of LAB cocci by PCR-DGGE and melting curves analyses in real time PCR.

Journal of microbiological methods 67 (1)
PMID : 16626824  :   DOI  :   10.1016/j.mimet.2006.03.008    
Abstract >>
Lactic acid bacteria (LAB) are essential in the quality of many fermented beverages like beer, cider and wine. In the two later cases, they convert malic acid into lactic acid during the malolactic fermentation. After fermentation, microbial stabilization is needed to prevent the development of spoilage bacteria species. Among them, cocci lead to different alterations: Pediococcus sp., and some strains of Leuconostoc mesenteroides and Oenococcus oeni can produce exopolysaccharides which modify wine viscosity and lead to ropiness. They also can produce acetic acid, biogenic amine, ethyl carbamate and volatile phenols. Therefore detection and identification are crucial. Results of phenotypic tests and DNA-DNA probes are not accurate enough. 16S RNA gene which is currently used for bacterial species identification presents intraspecies heterogeneity. The rpoB gene is an alternative to this limitation. However previous PCR targeting partial sequence of rpoB gene could not delimit cocci species. Therefore we compared the rpoB gene sequence of the six main cocci species found in fermented beverages: P. damnosus, P. dextrinicus, P. parvulus, P. pentosaceus, L. mesenteroides and O. oeni. The most discriminating partial sequence of the rpoB gene was chosen for designing primers. By PCR-DGGE the reliability of these primers was verified. It was controlled in a mixture of several cocci and other lactic acid bacteria (Lactobacillus sp.). Then we adapted the primers and the PCR conditions in order to achieve the identification of cocci species by real time PCR program including the fluorescent dye SYBR Green I, which gives faster results. PCR melt curves were established and a specific T(m) was attributed to each species.
KeywordMeSH Terms
Genes, Bacterial
22. Koo  OK, Jeong  DW, Lee  JM, Kim  MJ, Lee  JH, Chang  HC, Kim  JH, Lee  HJ,     ( 2005 )

Cloning and characterization of the bifunctional alcohol/acetaldehyde dehydrogenase gene (adhE) in Leuconostoc mesenteroides isolated from kimchi.

Biotechnology letters 27 (7)
PMID : 15928858  :   DOI  :   10.1007/s10529-005-2541-z    
Abstract >>
A bifunctional alcohol/acetaldehyde dehydrogenase (AdhE) gene (adhE) was cloned from Leuconostoc mesenteroides C7 (LMC7), which is the dominant lactic acid bacterium produced during heterofermentation of kimchi. The nucleotide sequence of the DNA fragment containing putative adhE, which is 2685 bp long and encodes an 886 amino acid polypeptide, exhibits 99% homology with Leu. mesenteroides sp. cremoris. The deduced AdhE comprises two conserved domains: alcohol dehydrogenase (Adh) and acetaldehyde dehydrogenase (Aldh). Moreover, two NAD-binding sites were observed, based on the presence of the GXGXXG motif. A pADHE containing the adhE gene expressed AdhE at the translational level in Escherichia coli BL21, which was at a higher level than in E. coli DH5alpha and E. coli JM109. The AdhE of LMC7 showed Adh and Aldh activities that, when expressed in E. coli. BL21, were 7.5 and 5.7 U mg(-1) , respectively.
KeywordMeSH Terms
23. Lonvaud-Funel  A, de Saad  AM,     ( 1982 )

Purification and Properties of a Malolactic Enzyme from a Strain of Leuconostoc mesenteroides Isolated from Grapes.

Applied and environmental microbiology 43 (2)
PMID : 16345941  :   PMC  :   PMC241831    
Abstract >>
An enzymatic complex able to transform l-malate to l-lactate was obtained from a Leuconostoc mesenteroides strain isolated from grapes. The molecular weight was about 235,000, the isoelectric point was at pH 4.35, and the optimal pH for activity was 5.75. The malolactic activity followed a sequential pattern concerning the involved substrates. At pH values substantially different from the optimum, a positive cooperativity between malate molecules was observed. Oxamate, fructose-1, 6-diphosphate, and l-lactate acted as noncompetitive inhibitors, whereas succinate, citrate, and tartrate isomers produced a competitive inhibition.
KeywordMeSH Terms
24. Kang  HK, Kim  SH, Park  JY, Jin  XJ, Oh  DK, Kang  SS, Kim  D,     ( 2005 )

Cloning and characterization of a dextranase gene from Lipomyces starkeyi and its expression in Saccharomyces cerevisiae.

Yeast (Chichester, England) 22 (15)
PMID : 16278932  :   DOI  :   10.1002/yea.1311    
Abstract >>
A dextranase-encoding cDNA from L. starkeyi KSM22 was isolated and characterized. The 2052 bp cDNA fragment (lsd1) harbouring the dextranase gene exhibited one open reading frame (ORF) composed of 1824 bp flanked by a 41 bp 5'-UTR and a 184 bp 3'-UTR, including a 27 bp poly(A) tail. The lsd1 gene contains no introns. The open reading frame encodes a 608 amino acid polypeptide (LSD1) with a 67.6 kDa predicted molecular mass. There was a 77% deduced amino acid sequence identity between the LSD1 dextranase and the dextranase from Penicillium minioluteum. The primary structure of LSD1 dextranase exhibits distant similarity with the enzymes of the glycosyl hydrolase family 49 that comprises Penicillium dextranase. The optimum pH of LSD1 was 6.0 and the optimum temperature was 37 degrees C. LSD1 dextranase activity was substantially abolished by exposure to 1 mM Hg2+, Ag3+ and Mn2+. LSD1 exhibited high hydrolysing activity towards dextran (100%), soluble starch (22%) and mutan (8%).
KeywordMeSH Terms
Cloning, Molecular
25. Duwat  P, Ehrlich  SD, Gruss  A,     ( 1992 )

A general method for cloning recA genes of gram-positive bacteria by polymerase chain reaction.

Journal of bacteriology 174 (15)
PMID : 1629178  :   DOI  :   10.1128/jb.174.15.5171-5175.1992     PMC  :   PMC206342    
Abstract >>
An internal fragment of the recA gene from eight gram-positive organisms has been amplified by using degenerate primers in a polymerase chain reaction. The internal 348- or 360-bp recA DNA segments from Bacillus subtilis, Clostridium acetobutylicum, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesanteroides, Listeria monocytogenes, Staphylococcus aureus, and Streptococcus salivarus subsp. thermophilus were amplified, cloned, and sequenced. The G + C contents of the DNA from these species range from 28 to 52%. The sequences of the bacterial recA genes show strong relatedness. This method is particularly useful for the recovery of the recA genes of gram-positive bacteria and avoids the difficulties of using a genetic complementation test for cloning.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
Polymerase Chain Reaction
26. de Las Rivas  B, Marcobal  Ae, Gómez  A, Muñoz  R,     ( 2005 )

Characterization of ISLpl4, a functional insertion sequence in Lactobacillus plantarum.

Gene 363 (N/A)
PMID : 16278055  :   DOI  :   10.1016/j.gene.2005.09.005    
Abstract >>
A Lactobacillus plantarum strain, CECT 4645, was found to have insertions of a sequence (985 bp in length) at least in eight loci in its genome. The prototype copy (Lp1) of this insertion sequence (named ISLpl4) has one open reading frame encoding a putative protein that is 292 amino acids in length with significant levels of similarity with IS982 family transposases. Perfect 16-bp inverted repeats were found at its termini. Upon transposition, generates 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. The ISLpl4 pattern changed over many generations on the CECT 4645 strain. This finding strongly supports our hypothesis that ISLpl4 is a functional element in L. plantarum. Some of these elements may be cryptic, since point mutation or 1-nucleotide deletions were found in their transposase encoding genes. ISLpl4 copies have been detected in Leuconostoc mesenteroides, Oenococcus oeni, and Lactobacillus sakei. An ISLpl4 copy of O. oeni contained a +1 nucleotide insertion on its transposase encoding gene and, by using an experimental system, we were able to demonstrate that this specific sequence originates a +1 programmed translational frameshifting. Although the frameshifting process reported here operates at a low rate, this description might represent the first case of a functional +1 frameshifting among IS.
KeywordMeSH Terms
Genes, Bacterial
27. Lee  JM, Jeong  DW, Koo  OK, Kim  OK, Kim  MJ, Lee  JH, Chang  HC, Kim  JH, Lee  HJ,     ( 2005 )

Cloning and characterization of the gene encoding phosphoketolase in Leuconostoc mesenteroides isolated from kimchi.

Biotechnology letters 27 (12)
PMID : 16086247  :   DOI  :   10.1007/s10529-005-6718-2    
Abstract >>
The gene encoding phosphoketolase, which is 2749 bp long and contains 814 amino acid polypeptides with a total molecular mass of 91.9 kDa, was cloned from Leuconostoc mesenteroides C7 (LMC7) and expressed in Escherichia coli. It exhibited a homology of >58% with phosphoketolases from other lactic acid bacteria. The phosphoketolase of LMC7 belongs to the xylulose 5-phosphate (X5P)/fructose 6-phosphate (F6P) phosphoketolase (Xfp) family, which is an enzyme with dual specificity for X5P and F6P. The members of this family contain typical thiamin pyrophosphate (TPP) binding sites as reported for other TPP-dependent enzymes, and several highly conserved regions as signature patterns for phosphoketolases. The plasmid pGPK containing the Xfp gene (xfp) exhibits phosphoketolase activity in E. coli. The specific activities of the enzyme from E. coli BL21 and E. coli EC101 harboring xfp were 0.28 and 0.14 units/mg, respectively. They both exhibited a 1.5-fold increase in the production of acetic acid from acetyl phosphate compared with their corresponding original strain.
KeywordMeSH Terms
28. Kang  HK, Seo  MY, Seo  ES, Kim  D, Chung  SY, Kimura  A, Day  DF, Robyt  JF,     ( 2005 )

Cloning and expression of levansucrase from Leuconostoc mesenteroides B-512 FMC in Escherichia coli.

Biochimica et biophysica acta 1727 (1)
PMID : 15652153  :   DOI  :   10.1016/j.bbaexp.2004.10.012    
Abstract >>
Leuconostoc mesenteroides B-512 FMC produces dextran and levan using sucrose. Because of the industrial importance of dextrans and oligosaccharides synthesized by dextransucrase (one of glycansucrases from L. mesenteroides), much is known about the dextransucrase, including expression and regulation of gene. However, no detailed report about levansucrase, another industrially important glycansucrase from L. mesenteroides, and its gene was available. In this paper, we report the first-time isolation and molecular characterization of a L. mesenteroides levansucrase gene (m1ft). The gene m1ft is composed of 1272-bp nucleotides and codes for a protein of 424 amino acid residues with calculated molecular mass of 47.1 kDa. The purified protein was estimated to be about 51.7 kDa including a His-tag based on SDS-PAGE. It showed an activity band at 103 kDa on a non-denaturing SDS-PAGE, indicating a dimeric form of the active M1FT. M1FT levan structure was confirmed by NMR and dot blot analysis with an anti-levan-antibody. M1FT converted 150 mM sucrose to levan (18%), 1-kestose (17%), nystose (11%) and 1,1,1-kestopentaose (7%) with the liberation of glucose. The M1FT enzyme produced erlose [O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->2)-beta-D-fructofuranoside] as an acceptor product with maltose. The optimum temperature and pH of this enzyme for levan formation were 30 degrees C and pH 6.2, respectively. M1FT levansucrase activity was completely abolished by 1 mM Hg2+ or Ag2+. The Km and Vmax values for levansucrase were calculated to be 26.6 mM and 126.6 micromol min-1 mg-1.
KeywordMeSH Terms
29. Héchard  Y, Dérijard  B, Letellier  F, Cenatiempo  Y,     ( 1992 )

Characterization and purification of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides.

Journal of general microbiology 138 (12)
PMID : 1487737  :   DOI  :   10.1099/00221287-138-12-2725    
Abstract >>
A Leuconostoc mesenteroides ssp. mesenteroides was isolated from goat's milk on the basis of its ability to inhibit the growth of Listeria monocytogenes. The antimicrobial effect was due to the presence in the culture medium of a compound, named mesentericin Y105, excreted by the Leuconostoc mesenteroides Y105. The compound displayed known features of bacteriocins from lactic acid bacteria. It appeared as a proteinaceous molecule exhibiting a narrow inhibitory spectrum limited to genus Listeria. The apparent relative molecular mass, as indicated by activity detection after SDS-PAGE, was 2.5-3.0 kDa. The bacteriocin was purified to homogeneity by a simple three-step procedure: a crude supernatant obtained from an early-stationary-phase culture in a defined medium was subjected to affinity chromatography on a blue agarose column, followed by ultrafiltration through a 5 kDa cut-off membrane, and finally by reverse-phase HPLC on a C4 column. Microsequencing of the pure bacteriocin and of tryptic fragments showed that mesentericin Y105 is a 36 amino acid polypeptide whose primary structure is close to that of leucocin A-UAL 187, which contains an extra residue at the C-terminus and displays only two differences in the overlapping sequence. However, unlike leucocin A-UAL 187, mesentericin Y105 displayed a bactericidal mode of action.
KeywordMeSH Terms
30. Lee  WT, Flynn  TG, Lyons  C, Levy  HR,     ( 1991 )

Cloning of the gene and amino acid sequence for glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides.

The Journal of biological chemistry 266 (20)
PMID : 2071589  :  
Abstract >>
Amino acid sequencing of glucose 6-phosphate dehydrogenase (Glc6PD) from Leuconostoc mesenteroides yielded sequence for over 75% of the protein. Two oligonucleotides based on the amino acid sequence were used to isolate a partial Glc6PD gene clone (pLmz delta N65), from a pUC9 library, containing 85% of the coding sequence and the 3'-untranslated DNA, but lacking the 5'-noncoding DNA sequence and the portion of the gene encoding the 65 N-terminal amino acids. Attempts to obtain a full-length clone from lambda libraries were unsuccessful, possibly due to restriction of L. mesenteroides DNA by Escherichia coli host cells. The 5'-untranslated DNA was amplified by the polymerase chain reaction and partially sequenced. To obtain unmodified DNA for the gene, oligonucleotides corresponding to the 5'- and 3'-noncoding sequences were used to amplify the gene by the polymerase chain reaction, and a 1.8-kilobase pair fragment was isolated and cloned into pUC19. The recombinant plasmid, pLmz, contains the entire Glc6PD gene and expresses the gene in E. coli. pLmz was sequenced showing that the enzyme consists of 485 amino acids. L. mesenteroides Glc6PD is 31% identical to the human enzyme.
KeywordMeSH Terms
Genes, Bacterial
31. Araque  I, Gil  J, Carreté  R, Bordons  A, Reguant  C,     ( 2009 )

Detection of arc genes related with the ethyl carbamate precursors in wine lactic acid bacteria.

Journal of agricultural and food chemistry 57 (5)
PMID : 19219988  :   DOI  :   10.1021/jf803421w    
Abstract >>
Trace amounts of the carcinogen ethyl carbamate can appear in wine by the reaction of ethanol with compounds such as citrulline and carbamyl phosphate, which are produced from arginine degradation by some wine lactic acid bacteria (LAB). In this work, the presence of arc genes for the arginine-deiminase pathway was studied in several strains of different species of LAB. Their ability to degrade arginine was also studied. To detect the presence of arc genes, degenerate primers were designed from the alignment of protein sequences in already sequenced LAB. The usefulness of these degenerate primers has been proven by sequencing some of the amplified PCR fragments and searching for homologies with published sequences of the same species and related ones. Correlation was found between the presence of genes and the ability to degrade arginine. Degrading strains included all heterofermentative lactobacilli, Oenococcus oeni , Pediococcus pentosaceus , and some strains of Leuconostoc mesenteroides and Lactobacillus plantarum .
KeywordMeSH Terms
32. Kang  HK, Kim  YM, Kim  DM,     ( 2008 )

Functional, genetic, and bioinformatic characterization of dextransucrase (DSRBCB4) gene in Leuconostoc mesenteroides B-1299CB4.

Journal of microbiology and biotechnology 18 (6)
PMID : 18600046  :  
Abstract >>
A gene encoding a dextransucrase (dsrBCB4) that synthesizes only alpha-1,6-linked dextran was cloned from Leuconostoc mesenteroides B-1299CB4. The coding region consisted of an open reading frame (ORF) of 4,395 bp that coded a 1,465-amino-acids protein with a molecular mass 163,581 Da. The expressed recombinant DSRBCB4 (rDSRBCB4) synthesized oligosaccharides in the presence maltose or isomaltose as an acceptor, plus the products included alpha-1,6-linked glucosyl residues in addition to the maltosyl or isomaltosyl residue. Alignments of the amino acid sequence of DSRBCB4 with glucansucrases from Streptococcus and Leuconostoc identified conserved amino acid residues in the catalytic core that are critical for enzyme activity. The mutants D530N, E568Q, and D641N displayed a 98- to 10,000-fold reduction of total enzyme activity.
KeywordMeSH Terms
33. Sarwat  F, Ul Qader  SA, Aman  A, Ahmed  N,     ( 2008 )

Production & characterization of a unique dextran from an indigenous Leuconostoc mesenteroides CMG713.

International journal of biological sciences 4 (6)
PMID : 18953402  :   DOI  :   10.7150/ijbs.4.379     PMC  :   PMC2567811    
Abstract >>
On the basis of high enzyme activity a newly isolated strain of L. mesenteroides CMG713 was selected for dextran production. For maximum yield of dextran, effects of various parameters such as pH, temperature, sucrose concentration and incubation period were studied. L. mesenteroides CMG713 produced maximum dextran after 20 hours of incubation at 30 masculineC with 15% sucrose at pH 7.0. The molecular mass distribution of dextran produced by this strain showed that its molecular mass was about 2.0 million Da. Dextran analysis by (13)C-NMR spectrometry showed no signals corresponding to any other linkages except alpha-(1-->6) glycosidic linkage in the main chain, which has not been reported before. Physico-chemical properties of this unique dextran were also studied. These optimised conditions could be used for the commercial production of this unique high molecular weight dextran, which have significant industrial perspectives.
KeywordMeSH Terms
Dextran
L. mesenteroides
NMR
34. Zhang  H, Hu  Y, Zhu  C, Zhu  B, Wang  Y,     ( 2008 )

Cloning, sequencing and expression of a dextransucrase gene (dexYG) from Leuconostoc mesenteroides.

Biotechnology letters 30 (8)
PMID : 18414801  :   DOI  :   10.1007/s10529-008-9711-8    
Abstract >>
The gene dexYG encoding the dextransucrase from an industrial strain of Leuconostoc mesenteroides 0326 was isolated by PCR. The nucleotide sequence of the dexYG gene consists of an open reading frame (ORF) of 4,584 bp, coding for a 1,527 aa protein with a Mr of 170 kDa. The results were analysed by a BLAST similarity search of the GenBank database, which revealed the amino acid sequence was similiar to dsrD derived from L. mesenteroides Lcc4. The dexYG gene was subcloned into the plasmid pET28a(+) and was expressed in E. coli BL21 (DE3) by IPTG induction. The pH value was one of the main reasons which caused the degradation of enzyme activity in the later stage of induction. The highest activity was reached 36 U/ml after 5 h induction in medium at pH 6.0. Biotransformation yield of the enzyme reached 65% and the molecular weight of transformed dextran was more than 68 kDa in 2 h.
KeywordMeSH Terms
Sequence Analysis, DNA
35. Endo  A, Okada  S,     ( 2008 )

Reclassification of the genus Leuconostoc and proposals of Fructobacillus fructosus gen. nov., comb. nov., Fructobacillus durionis comb. nov., Fructobacillus ficulneus comb. nov. and Fructobacillus pseudoficulneus comb. nov.

International journal of systematic and evolutionary microbiology 58 (Pt 9)
PMID : 18768629  :   DOI  :   10.1099/ijs.0.65609-0    
Abstract >>
A taxonomic study was made of the genus Leuconostoc. The species in the genus were divided into three subclusters by phylogenetic analysis based on the 16S rRNA gene sequences. The three subclusters were the Leuconostoc mesenteroides subcluster (comprising L. carnosum, L. citreum, L. gasicomitatum, L. gelidum, L. inhae, L. kimchii, L. lactis, L. mesenteroides and L. pseudomesenteroides), the L. fructosum subcluster (L. durionis, L. ficulneum, L. fructosum and L. pseudoficulneum) and the L. fallax subcluster (L. fallax). Phylogenetic trees based on the sequences of the 16S-23S rRNA gene intergenic spacer region, the rpoC gene or the recA gene indicated a good correlation with the phylogenetic tree based on 16S rRNA gene sequences. The species in the L. fructosum subcluster were morphologically distinguishable from the species in the L. mesenteroides subcluster and L. fallax as species in the L. fructosum subcluster had rod-shaped cells. In addition, the four species in the L. fructosum subcluster needed an electron acceptor for the dissimilation of d-glucose and produced acetic acid from d-glucose rather than ethanol. On the basis of evidence presented in this study, it is proposed that the four species in the L. fructosum subcluster, Leuconostoc durionis, Leuconostoc ficulneum, Leuconostoc fructosum and Leuconostoc pseudoficulneum, should be transferred to a novel genus, Fructobacillus gen. nov., as Fructobacillus durionis comb. nov. (type strain D-24(T)=LMG 22556(T)=CCUG 49949(T)), Fructobacillus ficulneus comb. nov. (type strain FS-1(T)=DSM 13613(T)=JCM 12225(T)), Fructobacillus fructosus comb. nov. (type strain IFO 3516(T)=DSM 20349(T)=JCM 1119(T)=NRIC 1058(T)) and Fructobacillus pseudoficulneus comb. nov. (type strain LC-51(T)=DSM 15468(T)=CECT 5759(T)). The type species of the genus Fructobacillus is Fructobacillus fructosus gen. nov., comb. nov.. No significant physiological and biochemical differences were found between the species in the L. mesenteroides subcluster and L. fallax in the present study and thus L. fallax remains as a member of the genus Leuconostoc.
KeywordMeSH Terms
36. Olvera  C, Fernández-Vázquez  JL, Ledezma-Candanoza  L, López-Munguía  A,     ( 2007 )

Role of the C-terminal region of dextransucrase from Leuconostoc mesenteroides IBT-PQ in cell anchoring.

Microbiology (Reading, England) 153 (Pt 12)
PMID : 18048914  :   DOI  :   10.1099/mic.0.2007/008854-0    
Abstract >>
dsrP, a gene that encodes a cell-associated dextransucrase produced by Leuconostoc mesenteroides IBT-PQ, was isolated, sequenced and expressed in Escherichia coli. From sequence analysis, seven repeat units in the N-terminal region were found, as well as five cell wall binding repeats in the C-terminal region. A model of the C-terminal domain of dextransucrase was built based on the solenoid structure of the cell wall binding domain already described in LytA. By experiments involving direct interactions of the enzyme with L. mesenteroides cells, as well as among the cells and the single C-terminal domain expressed in E. coli, evidence was obtained concerning the anchoring function of this region in cell-associated dextransucrase, a function which may be independent of its capacity to bind dextran.
KeywordMeSH Terms
37. Lee  JH, Moon  YH, Kim  N, Kim  YM, Kang  HK, Jung  JY, Abada  E, Kang  SS, Kim  D,     ( 2008 )

Cloning and expression of the sucrose phosphorylase gene from Leuconostoc mesenteroides in Escherichia coli.

Biotechnology letters 30 (4)
PMID : 18038113  :   DOI  :   10.1007/s10529-007-9608-y    
Abstract >>
The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K (m) of 3 mM with sucrose as a substrate; optimum activity was at 37 degrees C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP).
KeywordMeSH Terms
38. Flórez  AB, Campedelli  I, Delgado  S, Alegría  ?, Salvetti  E, Felis  GE, Mayo  B, Torriani  S,     ( 2016 )

Antibiotic Susceptibility Profiles of Dairy Leuconostoc, Analysis of the Genetic Basis of Atypical Resistances and Transfer of Genes In Vitro and in a Food Matrix.

PloS one 11 (1)
PMID : 26726815  :   DOI  :   10.1371/journal.pone.0145203     PMC  :   PMC4699710    
Abstract >>
In spite of a global concern on the transfer of antibiotic resistances (AR) via the food chain, limited information exists on this issue in species of Leuconostoc and Weissella, adjunct cultures used as aroma producers in fermented foods. In this work, the minimum inhibitory concentration was determined for 16 antibiotics in 34 strains of dairy origin, belonging to Leuconostoc mesenteroides (18), Leuconostoc citreum (11), Leuconostoc lactis (2), Weissella hellenica (2), and Leuconostoc carnosum (1). Atypical resistances were found for kanamycin (17 strains), tetracycline and chloramphenicol (two strains each), and erythromycin, clindamycin, virginiamycin, ciprofloxacin, and rifampicin (one strain each). Surprisingly, L. mesenteroides subsp. mesenteroides LbE16, showed resistance to four antibiotics, kanamycin, streptomycin, tetracycline and virginiamycin. PCR analysis identified tet(S) as responsible for tetracycline resistance in LbE16, but no gene was detected in a second tetracycline-resistant strain, L. mesenteroides subsp. cremoris LbT16. In Leuconostoc mesenteroides subsp. dextranicum LbE15, erythromycin and clindamycin resistant, an erm(B) gene was amplified. Hybridization experiments proved erm(B) and tet(S) to be associated to a plasmid of ?35 kbp and to the chromosome of LbE15 and LbE16, respectively. The complete genome sequence of LbE15 and LbE16 was used to get further insights on the makeup and genetic organization of AR genes. Genome analysis confirmed the presence and location of erm(B) and tet(S), but genes providing tetracycline resistance in LbT16 were again not identified. In the genome of the multi-resistant strain LbE16, genes that might be involved in aminoglycoside (aadE, aphA-3, sat4) and virginiamycin [vat(E)] resistance were further found. The erm(B) gene but not tet(S) was transferred from Leuconostoc to Enterococcus faecalis both under laboratory conditions and in cheese. This study contributes to the characterization of AR in the Leuconostoc-Weissella group, provides evidence of the genetic basis of atypical resistances, and demonstrates the inter-species transfer of erythromycin resistance.
KeywordMeSH Terms
Dairying
39. Brison  Y, Malbert  Y, Czaplicki  G, Mourey  L, Remaud-Simeon  M, Tranier  S,     ( 2016 )

Structural Insights into the Carbohydrate Binding Ability of an �\-(1��2) Branching Sucrase from Glycoside Hydrolase Family 70.

The Journal of biological chemistry 291 (14)
PMID : 26865636  :   DOI  :   10.1074/jbc.M115.688796     PMC  :   PMC4817182    
Abstract >>
The �\-(1��2) branching sucrase �GN123-GBD-CD2 is a transglucosylase belonging to glycoside hydrolase family 70 (GH70) that catalyzes the transfer ofd-glucosyl units from sucroseto dextrans or gluco-oligosaccharides via the formation of �\-(1��2) glucosidic linkages. The first structures of �GN123-GBD-CD2 in complex withd-glucose, isomaltosyl, or isomaltotriosyl residues were solved. The glucose complex revealed three glucose-binding sites in the catalytic gorge and six additional binding sites at the surface of domains B, IV, and V. Soaking with isomaltotriose or gluco-oligosaccharides led to structures in which isomaltosyl or isomaltotriosyl residues were found in glucan binding pockets located in domain V. One aromatic residue is systematically identified at the bottom of these pockets in stacking interaction with one glucosyl moiety. The carbohydrate is also maintained by a network of hydrogen bonds and van der Waals interactions. The sequence of these binding pockets is conserved and repeatedly present in domain V of several GH70 glucansucrases known to bind �\-glucans. These findings provide the first structural evidence of the molecular interaction occurring between isomalto-oligosaccharides and domain V of the GH70 enzymes.
KeywordMeSH Terms
alpha-1,2 branching sucrase
carbohydrate-binding protein
crystal structure
enzyme
family GH70
glucan-binding domain
glucansucrase
glycoside hydrolase
oligosaccharide
α-glucan
40. Mu  F, Masuda  Y, Zendo  T, Ono  H, Kitagawa  H, Ito  H, Nakayama  J, Sonomoto  K,     ( 2014 )

Biological function of a DUF95 superfamily protein involved in the biosynthesis of a circular bacteriocin, leucocyclicin Q.

Journal of bioscience and bioengineering 117 (2)
PMID : 23906710  :   DOI  :   10.1016/j.jbiosc.2013.06.023    
Abstract >>
Biological functions of a DUF95 superfamily protein in the biosynthesis gene cluster of a novel circular bacteriocin, leucocyclicin Q (LcyQ), were characterized in this paper. Sequence analysis and database search of the regions flanking the LcyQ structural gene lcyQ revealed four open reading frames (lcyR, lcyB, lcyC, and lcyD) related to bacteriocin biosynthesis. LcyD shares some similarity to the DUF95 superfamily proteins, often found in the biosynthetic gene clusters of circular bacteriocins. Mass spectrometry analysis showed accumulation of active mature LcyQ inside lcyD knockout cells. Heterologous expression of lcyD demonstrated that it confers robust immunity against LcyQ. Peptide release/binding assay revealed that the immunity could be attributed to the secretion of LcyQ to the cell exterior. Thus, the DUF95 superfamily protein has a dual function in the biosynthesis of LcyQ, as an immunity-associated transporter and as a secretion-aiding agent. Accumulation of mature LcyQ inside the cell in lcyD knockout strains, further implied that cyclization occurs within the cell. To the best of our knowledge, this is the first report on LcyQ cyclization inside the cell and the dual role of a DUF95 superfamily protein in circular bacteriocin biosynthesis.
KeywordMeSH Terms
Circular bacteriocin
Cyclization
Immunity
Leucocyclicin Q
Secretion
41. Palomba  S, Cavella  S, Torrieri  E, Piccolo  A, Mazzei  P, Blaiotta  G, Ventorino  V, Pepe  O,     ( 2012 )

Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture.

Applied and environmental microbiology 78 (8)
PMID : 22307283  :   DOI  :   10.1128/AEM.07302-11     PMC  :   PMC3318808    
Abstract >>
After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an �\-(1��6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30�XC with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives.
KeywordMeSH Terms
Food Microbiology
42. Brison  Y, Pijning  T, Malbert  Y, Fabre  ?, Mourey  L, Morel  S, Potocki-Véronèse  G, Monsan  P, Tranier  S, Remaud-Siméon  M, Dijkstra  BW,     ( 2012 )

Functional and structural characterization of �\-(1->2) branching sucrase derived from DSR-E glucansucrase.

The Journal of biological chemistry 287 (11)
PMID : 22262856  :   DOI  :   10.1074/jbc.M111.305078     PMC  :   PMC3318707    
Abstract >>
�GN(123)-glucan-binding domain-catalytic domain 2 (�GN(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays �\-(1��2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of �\-(1��2) linkages. The crystal structure of the apo �\-(1��2) branching sucrase �GN(123)-GBD-CD2 was solved at 1.90 ? resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-�GN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-�GN and �GN(123)-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to �GN(123)-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-�GN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to �GN(123)-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of �\-(1��2) branches.
KeywordMeSH Terms
Protein Folding
43.     ( 1996 )

Identification and sequence analysis of IS1297, an ISS1-like insertion sequence in a Leuconostoc strain.

Gene 174 (2)
PMID : 8890744  :   DOI  :   10.1016/0378-1119(96)00091-1    
Abstract >>
The insertion sequence (IS) ISS1 from Lactococcus lactis was amplified from lactococcal genomic DNA using a primer to the 18-bp inverted repeat sequence. The amplified product hybridized to a single EcoRI fragment in a total genomic DNA digest of Leuconostoc mesenteroides ssp. dextranicum NZDRI 2218. The DNA sequence of this ISS1-like element (IS1297) and the Le. mesenteroides sequences flanking the IS were determined and compared with other iso-ISS1 elements. No direct repeats were found immediately flanking IS1297; however, direct repeats were present approximately 60 bp on either side of the insertion site. IS1297 contained a major open reading frame (ORF) of 681 bp, encoding a putative 226-amino-acid protein with 96.5% homology to the presumed transposase of ISS1. An overlapping ORF of 174 bp in the same orientation was also present. A putative ORF in the opposite orientation to the transposase ORF, which has been shown in some iso-ISS1 elements, was not present in IS1297. IS1297 was shown to hybridize with other dairy Leuconostoc strains. This is the first sequence of an ISS1-like element from a genus other than Lactococcus; however, IS1297 has close similarity to the lactococcal iso-ISS1 elements, especially the iso-ISS1 element from the lactose plasmid, pTD1.
KeywordMeSH Terms
44.     ( 1996 )

Cloning and sequencing of a gene coding for a novel dextransucrase from Leuconostoc mesenteroides NRRL B-1299 synthesizing only alpha (1-6) and alpha (1-3) linkages.

Gene 182 (1��2��)
PMID : 8982063  :   DOI  :   10.1016/s0378-1119(96)00443-x    
Abstract >>
The coding region for a Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrA) was isolated and sequenced. Using a pair of primers designed on the basis of two highly conserved amino-acid (aa) sequences in L. mesenteroides NRRL B-512F dextransucrase and streptococcal glucosyltransferases (GTFs), a fragment of dsrA was amplified by the polymerase chain reaction (PCR). This PCR product was used as an hybridization probe to isolate a 1.8-kb fragment identified as the central region of dsrA. Cleavage by Sac I of this fragment allowed two probes to be obtained to isolate the 5' and the 3' ends of dsrA. The nucleotide sequence of the dsrA gene was determined and found to consist of an open reading frame (ORF) of 4870 base pairs (bp) coding for a 1290-aa protein with an M(r) of 145590. The aa sequence exhibited a high similarity with other GTFs. The two domains previously described in GTFs are conserved in DSRA: an N-terminal conserved domain and a C-terminal domain composed of a series of repeats. Surprisingly, the expected signal peptide was not detected. The entire gene was reconstructed and the activity of DSRA was investigated. The dextran produced appeared to be composed of 85% alpha (1-6) and 15% alpha (1-3) linkages and the oligosaccharides synthesized in the presence of maltose were mainly composed of alpha (1-6) linkages. This enzyme is a novel dextransucrase from L. mesenteroides NRRL B-1299 producing no alpha (1-2) linkages and is the first glucosyltransferase having no signal peptide described.
KeywordMeSH Terms
45.     ( 1996 )

Purification and N-terminal amino acid sequence of dextranicin 24, a bacteriocin of Leuconostoc sp.

Current microbiology 33 (2)
PMID : 8662187  :  
Abstract >>
Leuconostoc mesenteroides subsp. dextranicum strain J24 synthesized a bacteriocin named Dextranicin 24 (Dex-24), which inhibited only other Leuconostoc sp. strains. It was purified by a two-step procedure from the fraction of the bacteriocin bound to the producer cells at the end of the growth: desorption form the cells at acidic pH, followed by reserve phase HPLC. The N-terminal sequence of Dex-24 was the following: NH2(-) K G V L G W L S M A S S A L T G P Q Q . . .
KeywordMeSH Terms
46. Rowland  P, Basak  AK, Gover  S, Levy  HR, Adams  MJ,     ( 1994 )

The three-dimensional structure of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides refined at 2.0 A resolution.

Structure (London, England : 1993) 2 (11)
PMID : 7881907  :  
Abstract >>
Glucose 6-phosphate dehydrogenase (G6PD) is the first enzyme of the pentose phosphate pathway. Normally the pathway is synthetic and NADP-dependent, but the Gram-positive bacterium Leuconostoc mesenteroides, which does not have a complete glycolytic pathway, also uses the oxidative enzymes of the pentose phosphate pathway for catabolic reactions, and selects either NAD or NADP depending on the demands for catabolic or anabolic metabolism. The structure of G6PD has been determined and refined to 2.0 A resolution. The enzyme is a dimer, each subunit consisting of two domains. The smaller domain is a classic dinucleotide-binding fold, while the larger one is a new beta+ alpha fold, not previously seen, with a predominantly antiparallel nine-stranded beta-sheet. There are significant structural differences in the coenzyme-binding domains of the two subunits, caused by Pro 149 which is cis in one subunit and trans in the other. The structure has allowed us to propose the location of the active site and the coenzyme-binding site, and suggests the role of many of the residues conserved between species. We propose that the conserved Arg46 would interact with both the adenine ring and the 2'-phosphate of NADP. Gln47, which is not conserved, may contribute to the change from NADP to dual coenzyme specificity. His178, in a nine-residue peptide conserved for all known sequences, binds a phosphate in the active site pocket. His240 is the most likely candidate for the base to oxidize the 1-hydroxyl group of the glucose 6-phosphate substrate.
KeywordMeSH Terms
47. Elisha  BG, Courvalin  P,     ( 1995 )

Analysis of genes encoding D-alanine:D-alanine ligase-related enzymes in Leuconostoc mesenteroides and Lactobacillus spp.

Gene 152 (1)
PMID : 7828933  :   DOI  :   10.1016/0378-1119(94)00692-l    
Abstract >>
Degenerate oligodeoxyribonucleotides complementary to sequences encoding conserved amino acid (aa) motifs in D-alanine:D-alanine ligases (Ddl) were used to amplify approx. 600-bp fragments from glycopeptide-resistant strains of Leuconostoc mesenteroides (Lm), Lactobacillus plantarum, La. salivarius and La. confusus, and from a susceptible strain of La. leichmannii. Comparison of the deduced aa sequences of the PCR products revealed that the Ddl-related enzymes of resistant Lm and Lactobacillus spp. are more akin to each other (47-63% aa identity) than to that of susceptible La. leichmannii (33-37% aa identity), indicating that the Ddl-related enzymes in these intrinsically resistant species of Gram+ bacteria exhibit structural differences with those in susceptible species. The Ddl-related enzymes, VanA and VanB, implicated in acquired resistance to glycopeptides in enterococci, were not closely related to their counterparts in Lm and Lactobacillus spp., as they displayed only 26-32% aa identity.
KeywordMeSH Terms
48. Fremaux  C, Héchard  Y, Cenatiempo  Y,     ( 1995 )

Mesentericin Y105 gene clusters in Leuconostoc mesenteroides Y105.

Microbiology (Reading, England) 141 (Pt 7) (N/A)
PMID : 7551032  :   DOI  :   10.1099/13500872-141-7-1637    
Abstract >>
Because of their potential usefulness as natural food preservatives, increased interest has focused on bacteriocins from lactic acid bacteria. Mesentericin Y105 is a small non-lantibiotic bacteriocin (class II) encoded within a 35 kb plasmid from Leuconostoc mesenteroides Y105 and it is active against Listeria monocytogenes. Using reverse genetic methodologies, an 8 kb DraII fragment has been cloned that contains the mesentericin Y105 structural gene, mesY, which encodes a precursor of the bacteriocin with a 24 amino acid N-terminal extension ending with a Gly-Gly motif upstream of the cleavage site, which is typical of class II bacteriocins. Four other putative genes are associated with mesY within two divergent putative operons. In addition to mesY, the first putative operon is predicted to encode a protein, similar to that encoded by ORF2 in the leucocin A operon, whose function remains to be elucidated. The second putative operon contains three ORFs, two of which, mesD and mesE, encode proteins that resemble ATP-dependent transporters and accessory factors, respectively. For three other class II bacteriocin systems (lactococcin A, pediocin PA-1, colicin V), these proteins have been shown to be involved in bacteriocin secretion independently of the general sec-dependent secretion pathway. The last putative gene (mesC) does not resemble any previously characterized gene. Results concerning the heterologous expression of the cloned mesY in Lactobacillus johnsonii NCK64 suggest that the maturation and secretion functions dedicated to lactacin F (another class II bacteriocin) are efficient for mesentericin Y105 as well. This characteristic may be of great interest in the development of industrial fermentation starters producing multiple bactericidal activities.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
49. Funane  K, Yamada  M, Shiraiwa  M, Takahara  H, Yamamoto  N, Ichishima  E, Kobayashi  M,     ( 1995 )

Aggregated form of dextransucrases from Leuconostoc mesenteroides NRRL B-512F and its constitutive mutant.

Bioscience, biotechnology, and biochemistry 59 (5)
PMID : 7540436  :   DOI  :   10.1271/bbb.59.776    
Abstract >>
Purified dextransucrases [EC 2.4.1.5], DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively. DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively. Although the sugar contents of the purified enzymes were different, their molecular masses by SDS-PAGE were all 170 kDa. DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V0) on Sepharose 6B, while the DSM-G was eluted at 1.2 x V0 volume. On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively. The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.
KeywordMeSH Terms
50. Olive  C, Geroch  ME, Levy  HR,     ( 1971 )

Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides. Kinetic studies.

The Journal of biological chemistry 246 (7)
PMID : 4396688  :  
Abstract >>
N/A
KeywordMeSH Terms
Glucosephosphate Dehydrogenase
51. Bhadbhade  MM, Adams  MJ, Flynn  TG, Levy  HR,     ( 1987 )

Sequence identity between a lysine-containing peptide from Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and an active site peptide from human erythrocyte glucose-6-phosphate dehydrogenase.

FEBS letters 211 (2)
PMID : 3100332  :   DOI  :   10.1016/0014-5793(87)81445-x    
Abstract >>
Peptides recently isolated and sequenced from a bacterial (Leuconostoc mesenteroides) glucose-6-phosphate dehydrogenase are remarkably homologous to an active site region of the human erythrocyte enzyme, although the enzymes differ in their overall amino acid composition and kinetic properties. The computer program ALIGN, used to determine the best alignment between the two enzyme sequences, gives match-scores which are statistically highly significant.
KeywordMeSH Terms
52. Jadaun  JS, Narnoliya  LK, Agarwal  N, Singh  SP,     ( 2019 )

Catalytic biosynthesis of levan and short-chain fructooligosaccharides from sucrose-containing feedstocks by employing the levansucrase from Leuconostoc mesenteroides MTCC10508.

International journal of biological macromolecules 127 (N/A)
PMID : 30659880  :   DOI  :   10.1016/j.ijbiomac.2019.01.070    
Abstract >>
Levansucrase gene (LmLEVS) was cloned from Leuconostoc mesenteroides MTCC 10508. The heterologous expression and purification of the truncated (TrLmLEVS) gene, lacking the N-terminal signal peptide, was performed in Escherichia coli. The recombinant enzyme (TrLmLEVS) was physico-kinetically characterized using sucrose as substrate. TrLmLEVS exhibited the maximum activity at pH 6 and temperature 30 �XC. Thin layer chromatography and high performance liquid chromatography analyses unveiled the biosynthesis of fructooligosaccharides and levan by TrLmLEVS using sucrose as substrate. The catalytically synthesized polymer was characterized by Fourier-Transform Infrared Spectroscopy and Nuclear Magnetic Resonance analyses, confirming it as levan. TrLmLEVS was capable of catalyzing the transformation of raffinose-derived molecules, besides sucrose, into fructans. Further, TrLmLEVS was employed for the genesis of non-digestible fructans from sucrose-containing feedstocks like table sugar, jaggery, cane molasses, and sweet sorghum juice. The results suggest that Leu. mesenteroides MTCC 10508 levansucrase is a potential candidate for the production of levan-type biomolecules in plant-based food products.
KeywordMeSH Terms
Fructooligosaccharides
Leuconostoc mesenteroides
Levan
Levansucrase
Prebiotic
Raffinose
53.     ( 1998 )

Sequence and structural relationships of leucocins A-, B- and C-TA33a from Leuconostoc mesenteroides TA33a.

Microbiology (Reading, England) 144 (Pt 5) (N/A)
PMID : 9611809  :   DOI  :   10.1099/00221287-144-5-1343    
Abstract >>
Amino acid sequences of two of the three bacteriocins from Leuconostoc mesenteroides TA33a were determined and their sequence-structure relationships investigated. Leucocin B-TA33a consists of 31 amino acid residues, with a molecular mass of 3466 Da. Leucocin B-TA33a does not belong to the pediocin family of bacteriocins, but shares 62% homology with mesenterocin 52B. A partial sequence of 36 amino acids of leucocin C-TA33a (4598 Da) was determined. Leucocin C-TA33a belongs to the class II bacteriocins having the consensus YGNGV motif. The third bacteriocin, leucocin A-TA33a, is identical to leucocin A-UAL 187. Circular dichroism spectra of the leucocins in aqueous solution and micellar SDS indicated that they undergo a structural transition when in a membrane-mimicking environment. Theoretical predictions from circular dichroism data suggest that leucocins A-, B- and C-TA33a adopt a beta-structure (48%) in membrane-mimicking environments. Sequence alignments and secondary structure predictions for the N-terminus of leucocins A- and C-TA33a predicted that Cys-9 and Cys-14 are connected by a disulfide bridge and form two beta-strands.
KeywordMeSH Terms
54.     ( 2012 )

Characterization of glycosyltransferase activity of wild-type Leuconostoc mesenteroides strains from Bulgarian fermented vegetables.

Applied biochemistry and biotechnology 168 (3)
PMID : 22932848  :   DOI  :   10.1007/s12010-012-9812-7    
Abstract >>
Glycosyltransferase activity of 13 Leuconostoc mesenteroides strains isolated from Bulgarian fermented vegetables was investigated. All the strains displayed a mucoid phenotype on sucrose-containing agar media. Strains were characterized according to carbohydrate fermentation, species-specific multiple PCR using several primers, repetitive element-PCR fingerprinting using (GTG)(5) primers and glycosyltransferase activity. Level of activity and cellular localization (soluble or cell-associated) were variable among strains. Precipitation of exopolysaccharides produced from sucrose by the soluble fractions from these strains allowed recovery of only glucans and further characterization by (1)H and (13)C NMR analysis and enzymatic digestion with dextranase revealed dextran production. However, levans could be detected in presence of raffinose as fructosyl donor. Both fructosyltransferase and glucosyltransferase encoding genes were detected by PCR and both active enzymes were detected after functional characterization by SDS-PAGE electrophoresis and in situ polymer production after incubation with sucrose. This work therefore showed that concomitant production of glucosyltransferase and fructosyltransferase is widespread in L. mesenteroides strains.
KeywordMeSH Terms
55.     ( 1998 )

Glucan binding regions of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

Bioscience, biotechnology, and biochemistry 62 (1)
PMID : 9501523  :  
Abstract >>
We isolated glucan-binding peptides of a dextransucrase from Leuconostoc mesenteroides B-512F. The dextransucrase was bound to DEAE-Sephadex A-50, Sephadex G-100, and mutan from Streptococcus mutans. Mild trypsin digestion dissociated the enzyme and glucan binding. In the presence of ammonium sulfate, several peptides were bound to glucan after trypsin digestion. Four main mutan-binding peptides were obtained by this method, and those amino acid sequences were analyzed. One of them was identical with the dextran-binding peptide that contains lysine, which was previously isolated by differential chemical modification with o-phthalaldehyde. We also found mutan-binding peptides in sucrose- and dextran-binding regions and a lysine-rich region. Also, there was a peptide similar in sequence to glucan-binding A-repeat of streptococcal glucosyltransferases.
KeywordMeSH Terms
56.     ( 1998 )

On the mechanism of the reaction catalyzed by glucose 6-phosphate dehydrogenase.

Biochemistry 37 (9)
PMID : 9485426  :   DOI  :   10.1021/bi972069y    
Abstract >>
The catalytic mechanism of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides was investigated by replacing three amino acids, His-240, Asp-177, and His 178, with asparagine, using site-directed mutagenesis. Each of the mutant enzymes was purified to homogeneity and characterized by substrate binding studies and steady-state kinetic analyses. The three-dimensional structure of the H240N glucose 6-phosphate dehydrogenase was determined at 2.5 A resolution. The results support a mechanism in which His-240 acts as the general base that abstracts the proton from the C1-hydroxyl group of glucose 6-phosphate, and the carboxylate group of Asp-177 stabilizes the positive charge that forms on His-240 in the transition state. The results also confirm the postulated role of His-178 in binding the phosphate moiety of glucose 6-phosphate.
KeywordMeSH Terms
57.     ( 1998 )

Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster.

Journal of bacteriology 180 (3)
PMID : 9457870  :   PMC  :   PMC106934    
Abstract >>
A citrate lyase (EC 4.1.3.6) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the beta subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the alpha subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris. This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD, citE, and citF, encoding, respectively, the three citrate lyase subunits gamma (acyl carrier protein [ACP]), beta (citryl-S-ACP lyase; EC 4.1.3.34), and alpha (citrate:acetyl-ACP transferase; EC 2.8.3.10). The gene (citC) encoding the citrate lyase ligase (EC 6.2.1.22) was localized in the region upstream of citD. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebsiella pneumoniae and Haemophilus influenzae. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the citCDEFG gene cluster in Escherichia coli led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.
KeywordMeSH Terms
Bacterial Proteins

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