Leuconostoc

Biochim Biophys Acta, 2005 Jan 21, 1727(1), 5 - 15 Epub 2005 Jan 04.
Cloning and expression of levansucrase from Leuconostoc mesenteroides B-512 FMC in Escherichia coli; Kang HK et al.; 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 Hg(2+) or Ag(2+) . The K(m) and V(max) values for levansucrase were calculated to be 26.6 mM and 126.6 mumol min(-1) mg(-1).

Appl Environ Microbiol, 2005 Jan, 71(1), 554 - 7
Purification and Characterization of a Novel Class IIa Bacteriocin, Piscicocin CS526, from Surimi-Associated Carnobacterium piscicola CS526; Yamazaki K et al.; The bacteriocin piscicocin CS526 was inactivated by proteolytic enzymes, was stable at 100 degrees C for 30 min, had a pH range of 2 to 8, and was active against Enterococcus, Listeria, Pediococcus, and Leuconostoc . The N-terminal sequence was YGNGL, not the YGNGV consensus motif common in class IIa bacteriocins (alternate residues underlined) . The molecular mass of piscicocin CS526, which had a bactericidal mode of action, was approximately 4,430 Da.

J Appl Microbiol, 2005, 98(1), 33 - 42
In vitro and in situ growth characteristics and behaviour of spoilage organisms associated with anaerobically stored cooked meat products; Vermeiren L et al.; Abstract l . vermeiren, f . devlieghere, v . de graef and j . debevere . 2004.Aims: Understanding spoilage caused by different types of spoilage organisms, associated with vacuum-packaged sliced cooked meat products (CMP) . Methods and Results: First, strains were characterized in a broth at 7 degrees C under anaerobic conditions to compare their growth rate, acidifying character and metabolite production under conditions simulating refrigerated vacuum-packaged conditions . Brochotrix thermosphacta grew faster than the lactic acid bacteria (LAB) . Within the group of the LAB, all strains grew fast except Leuconostoc mesenteroides subsp . dextranicum and Leuconostoc carnosum . Secondly, the organisms were inoculated on a model cooked ham to better understand the relationship between spoilage, microbial growth, pH, metabolite production and accompanying sensory changes . Most rapidly growing strains were Leuc . mesenteroides subsp . mesenteroides followed by B . thermosphacta, while Leuc . mesenteroides subsp . dextranicum and Leuc . carnosum grew very slowly compared with the other LAB . Brochotrix thermosphacta caused sensory deviations at a lower cell number compared with the LAB . The related pH changes, metabolite production and sensory perception are presented . Conclusions: In this pure culture study, B . thermosphacta and Leuc . mesenteroides subsp . mesenteroides had the highest potential to cause rapid spoilage on CMP . Significance and Impact of the Study: A systematic study on the behaviour of spoilage organisms on a model cooked ham to establish the relationship between microbial growth, pH, metabolite formation and organoleptic deviations.

J Bacteriol, 2005 Jan, 187(1), 296 - 303
Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-1,2 branched dextran; Fabre E et al.; The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD) . From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains . The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor . To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli . Gene products were then small-scale purified to isolate the various corresponding enzymes . Dextran and oligosaccharide syntheses were performed . Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages . On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages . Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages . This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages . This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.

Biotechnol Bioeng, 2005 Jan 20, 89(2), 206 - 18
Production and secretion of recombinant Leuconostoc mesenteroides dextransucrase DsrS in Bacillus megaterium; Malten M et al.; Leuconostoc mesenteroides dextransucrase DsrS was recombinantly produced in Bacillus megaterium and exported into the growth medium . For this purpose a plasmid-based xylose-inducible gene expression system was optimized via introduction of a multiple cloning site and an encoded optimal B . megaterium ribosome binding site . A cre mediating glucose-dependent catabolite repression was removed . Recombinant DsrS was found in the cytoplasm and exported via its native leader sequence into the growth medium . Elimination of the extracellular protease NprM increased extracellular DsrS concentrations by a factor of 4 and stabilized the recombinant protein for up to 12 h . Cultivation in a semi-defined medium resulted in a further doubling of extracellular DsrS concentration up to an activity of 65 Units/L . To develop an industrial process a high cell density cultivation of B . megaterium was established yielding cell dry weights of up to 80 g/L . After induction of dsrS expression high specific (362 Units/g) and volumetric (28,600 Units/L) activities of dextran free DsrS were measured . However, using high cell density cultivation, most DsrS was found cell-associated indicating current limitations of the production process . A protease accessibility assay identified the major limitation of DsrS production at the level of protein folding . Intracellular misfolding of DsrS hampered DsrS export via the SEC pathway at high cell densities . The subsequent use of a semi-defined mineral medium and the induction of DsrS production at lower cell densities increased protein export efficiency remarkably, but also led to extracellular DsrS aggregation . Further optimization strategies for the production of recombinant DsrS in B . megaterium are discussed . (c) 2004 Wiley Periodicals, Inc.

J Bacteriol, 2004 Dec, 186(24), 8301 - 8
Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides; Shah DS et al.; Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine . In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD) . Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity . Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei . Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine) . A microplate binding assay was developed to measure the binding affinity of recombinant GBDs . GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan) . Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases . Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.

FEMS Microbiol Lett, 2004 Dec 1, 241(1), 49 - 55
Cell envelope analysis of insensitive, susceptible or resistant strains of Leuconostoc and Weissella genus to Leuconostoc mesenteroides FR 52 bacteriocins; Limonet M et al.; Mesenterocins 52A and 52B belong to class II of lactic acid bacteria bacteriocins . To study susceptibility, insensitivity and resistance to these mesenterocins, four wild-type bacterial strains and four resistant strains, all from Leuconostoc or Weissella genus, were compared . Several cell envelope features were investigated: susceptibilities to antibiotics and to lysozyme, cell morphology and membrane phospholipids contents . The strain insensitive to the two mesenterocins appeared to be resistant to lysozyme and exhibited the highest resistance to antibiotics . Resistant strains displayed cell morphology modifications, several increases in antibiotic resistance and modifications in lysozyme susceptibility . Moreover, mesenterocin 52A-resistant strains displayed modifications in their membrane phospholipids, leading to a more cationic membrane . Insensitivity and resistance of Leuconostoc or Weissella strains seem to be due to various minor modifications of the membrane and/or of the cell wall.

Biotechnol Bioeng, 2005 Jan 5, 89(1), 96 - 101
Growth of Leuconostoc mesenteroides NRRL-B523 in an alkaline medium: suboptimal pH growth inhibition of a lactic acid bacterium; Wolf BF et al.; Bacterial profile modification (BPM), a form of tertiary oil recovery, diverts water from the water-flooded high-permeability zone into the oil-bearing low-permeability zone . During field use, exopolymer-producing bacteria plug the high-permeability zone only in the immediate vicinity of the injection point (the near-well bore region) . For effective BPM the plug must penetrate far into the formation . Slowing the specific growth rate, lengthening the lag phase, and slowing the polymerization rate are techniques that can prolong the onset of biopolymer gelation and extend the depth of the biological plug . In batch experiments, the growth of Leuconostoc mesenteroides NRRL-B523 was inhibited by the synergistic effects of high substrate loading and an alkaline pH . Exponential growth was delayed up to 190 h . It was observed that cell division was significantly retarded until the medium pH, reduced by the acid byproducts of fermentation, reached a critical value of 6.79 +/- 0.06 . A mathematical model was developed to describe the relationship between specific growth rate, lag time, and medium pH . (c) 2004 Wiley Periodicals, Inc.

J Biotechnol, 2004 Nov 9, 114(3), 255 - 67
Design of immobilised dextransucrase for fluidised bed application; Berensmeier S et al.; Immobilisation of dextransucrase from Leuconostoc mesenteroides NRRL B-512F in alginate is optimised for applications in a fluidised bed reactor with high concentrated sugar solutions, in order to allow a continuous formation of defined oligosaccharides as prebiotic isomalto-oligosaccharides . Efficient design of fluidised bed immobilised biocatalyst in high density solutions requires particles with elevated density, high effectiveness and both thermal and mechanical stability . Inert silica flour/sand (Mikrosil 300) as supplement turned out to be best suited for increasing the density up to 1400 kg m(-3) of the alginate beads and generating a stable expanded bed without diffusional restrictions . Kinetic investigations demonstrate that low effectiveness of immobilised enzyme due to close association to dextranpolymers (dextran content of enzyme preparation >90%) is compensated by reducing the particle size and/or by decreasing the dextran content . A low dextran content (5%) is sufficient to immobilise and stabilise the enzyme, thus diffusional limitation is reduced essentially while operational stability is maintained . Fluidisation behaviour and bed expansion proved to be appropriate for the intended application . Both calculated and measured expansion coefficients showed good agreement for different conditions.

Biotechnol Prog, 2004 Sep-Oct, 20(5), 1550 - 4
Modified oligosaccharides as potential dental plaque control materials; Seo ES et al.; Metabolic acids produced by oral pathogens demineralize tooth surfaces, leading to dental caries . Glucosyltransferases are the key factor in this process . We synthesized various modified oligosaccharides and tested them for their inhibitory effects on glucosyltransferase activity . Oligosaccharides were produced using a mixed-culture fermentation of Lipomyces starkeyi and Leuconostoc mesenteroides and then further modified as iron- and sulfate-oligosaccharides . Iron- and sulfate-oligosaccharides reduced glucosyltransferase activity of Streptococci from 17% to 43% and prevented the formation of insoluble biomass on the surface of glass vials or stainless steel wires in the presence of sucrose . They also reduced the growth and acid productions of oral pathogens including S . mutans, S . sobrinus, Eikenella corrodens, Prevotella intermedia, and Actinobacillus actinomycetemcmitans.

Biotechnol Prog, 2004 Sep-Oct, 20(5), 1414 - 20
Immobilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F on Eupergit C supports; Gomez de Segura A et al.; Dextransucrase from Leuconostoc mesenteroides B-512F was immobilized on epoxy-activated acrylic polymers with different textural properties (Eupergit C and Eupergit C 250L) . Prior to immobilization, dextransucrase was treated with dextranase to remove the dextran layer covering the enzyme surface, thus increasing the accessibility of its reactive groups to the epoxide centers of the support . Elimination of 99% of the initial carbohydrate content was determined by the anthrone method . To prevent enzyme inactivation, the immobilization was carried out at pH 5.4, at which the coupling to the support took place through the carboxylic groups of the enzyme . The effects of the amount (mg) of dextransucrase added per gram of support (from 0.2:1 to 30:1), temperature and contact time were studied . Maximum activity recovery of 22% was achieved using Eupergit C 250L . Using this macroporous support, the maximum specific activity (710 U/g biocatalyst) was significantly higher than that obtained with the less porous Eupergit C (226 U/g biocatalyst) . The dextransucrase immobilized on Eupergit C 250L showed similar optimal temperature (30 degrees C) and pH (5-6) compared with the native enzyme . In contrast, a notable stabilization effect at 30 degrees C was observed as a consequence of immobilization . After a fast partial inactivation, the dextransucrase immobilized on Eupergit C 250L maintained more than 40% of the initial activity over the following 2 days . The features of this immobilized system are very attractive for its application in batch and fixed-bed bioreactors.

Biosci Biotechnol Biochem, 2004 Sep, 68(9), 1912 - 20
Construction of chimeric glucansucrases for analyzing substrate-binding regions that affect the structure of glucan products; Funane K et al.; A gene that encodes dextransucrase S (dsrS) from Leuconostoc mesenteroides NRRL B-512F encodes a glucansucrase dextransucrase S (DSRS) which mainly produces water-soluble glucan (dextran), while the dsrT5 gene derived from dsrT of the B-512F strain encodes an enzyme dextransucrase T5 (DSRT5), which mainly produces water-insoluble glucan . Tyr340-Asn510 of DSRS and Tyr307-Asn477 of DSRT5 (Site 1), Lys696-Gly768 of DSRS and Lys668-Gly740 of DSRT5 (Site 2), and Asn917-Lys1131 of DSRS and Asn904-Lys1118 of DSRT5 (Site 3) were exchanged and six different chimeric enzymes were constructed . Water-soluble glucan produced by recombinant DSRS was composed of 64% 6-linked glucopyranoside (Glcp), 9% 3,6-linked Glcp, and 13% 4-linked Glcp . Water-insoluble glucan produced by recombinant DSRT5 was composed of 47% 6-linked Glcp and 43% 3-linked Glcp . All of the chimeric enzymes produced glucans different from the ones produced by their parental enzymes . Some of the glucans produced by chimeric enzymes were extremely changed . The Site 1 chimeric enzyme of DSRS (STS1) produced water-soluble glucan composed mostly of 6-linked Glcp . That of DSRT5 (TST1) produced water-insoluble glucan composed mostly of 4-linked Glcp . The Site 3 chimeric enzyme of DSRS (STS3) produced mainly water-insoluble glucan, DSRT5 (TST3) produced mainly water-soluble glucans, and all of the glucan fractions consisted of 3-Glcp, 4-Glcp, and 6-Glcp . The amounts of the three linkages in the water-soluble glucan produced by TST3 were about 1:1:1 . Site 1 was assumed to be important for making or avoiding making alpha-1,4 linkages, while Site 3 was assumed to be important for determining the kinds of glucosyl linkages made.

J Agric Food Chem, 2004 Sep 8, 52(18), 5583 - 7
Detection and differentiation of several food-spoilage lactic acid bacteria by multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence; Garcia-Canas V et al.; In this work, a complete analytical procedure is investigated to differentiate several food-spoilage lactic acid bacteria . To do that, a method involving multiplex Polymerase Chain Reaction (PCR), capillary gel electrophoresis (CGE), and laser-induced fluorescence (LIF) is developed . The PCR-CGE-LIF protocol allows the simultaneous detection and differentiation of the genera Leuconostoc and Carnobacterium, the nonmotile group of species within the genus Carnobacterium, and the three species of the group individually (C . divergens, C . gallinarum, and C . maltaromicum) . The capability of this approach is clearly illustrated through the sensitive and efficient analysis of the two closest amplicons, with sizes equal to 397 and 412 bp, showing very different yields in all of the amplification reactions tested . These two fragments, which could not be resolved by agarose gel electrophoresis (AGE), are clearly distinguishable by CGE-LIF even when very different areas for both peaks are obtained . The PCR-CGE-LIF method also allows the sensitive detection of these bacteria, demonstrating both a significant resolution improvement compared with traditional AGE and the usefulness of this approach to solve real-life analytical challenges . Good reproducibility of the CGE-LIF procedure is shown for the analysis of multiplex PCR samples with percent relative standard deviation values for migration times and corrected peak areas as low as 0.80 and 6.50 for the same sample and three different days (n = 12), respectively.

J Food Prot, 2004 Aug, 67(8), 1719 - 24
Dextran from Leuconostoc mesenteroides augments immunostimulatory effects by the introduction of phosphate groups; Sato T et al.; The immunological effects of phosphorylated dextran (in which phosphate groups were chemically introduced) on murine splenocytes were examined . When dextran produced by Leuconostoc mesenteroides was phosphorylated by a reaction with polyphosphoric acid in formamide solution for 48 h, the degree of phosphorylation of dextran was maximal . The highest phosphorus content (1.7%, wt/wt) was observed in 40 kDa of dextran . The mitogenic response of murine splenocytes was enhanced by the phosphorylated dextran, but its activity was not related to its molecular weight . A strong response was detected at a concentration of 10 to 500 microg/ml, and the highest activity was obtained 48 h after stimulation . Phosphorylated dextran was characterized as a B-cell-specific mitogen . The expressions of CD86 on CD8alpha- CD11c- and CD8alpha- CD11c+ cells were augmented by phosphorylated dextran . The levels of mRNA expression of gamma interferon and interleukin-10 on murine splenocytes were also increased by the stimulation . These results demonstrate that dextran exerts immunostimulation by the introduction of phosphate groups.

Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 305 - 8
Dextransucrase mutants of Leuconostoc mesenteroides BI-08 strain; Iliev I et al.; We have isolated mutants constitutive for dextransucrase from Leuconostoc mesenteroides--BI-08 using ethyl methane sulfonate . The mutants produced mainly extracellular dexrtansucrase on glucose media with higher activity (3.5-4 times) than what the parental strain produced on glucose and 2 times more than what the parental strain produced on sucrose . Based on endo-dextranase from Penicillium hydrolysis, mutant BI-08 dextransucrases produced slightly different dextrans when they were elaborated on a glucose medium and on a sucrose medium Differences in viscosity, water solubility, susceptibility to endo-dextranase hydrolysis dextrans elaborated by different mutants grown on glucose media and sucrose media were found . SDS-PAGE analysis shows that the molecular masses of the dextransucrases from the studing mutants were 130-180 kDa.

Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 255 - 62
Application of NAD-dependent polyol dehydrogenases for enzymatic mannitol/sorbitol production with coenzyme regeneration; Parmentier S et al.; D-Mannitol and D-sorbitol were produced enzymatically from D-fructose using NAD-dependent polyol dehydrogenases . For the production of D-mannitol the Leuconostoc mesenteroides mannitol dehydrogenase could be used . Gluconobacter oxydans cell extract contained however both mannitol and sorbitol dehydrogenase . When this cell extract was used, the reduction of D-fructose resulted in a mixture of D-sorbitol and D-mannitol . To determine the optimal bioconversion conditions the polyol dehydrogenases were characterized towards pH- and temperature-optimum and -stability . As a compromise between enzyme activity and stability, the bioconversion reactions were performed at pH 6.5 and 25 degrees C . Since the polyol dehydrogenases are NADH-dependent, an efficient coenzyme regeneration was needed . Regeneration of NADH was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2.

Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 235 - 40
Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation; Kaup B et al.; A whole-cell biotransformation system for the conversion of D-fructose to D-mannitol was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle . First, the mdh gene encoding for the mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH) was expressed, effecting a strong catalytic activity of a NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant E . coli strain but not enabling whole cells of the strain to produce D-mannitol from D-fructose . To provide a source for reduction equivalents needed for D-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH) encoding formate dehydrogenase was functionally co-expressed . FDH generates NADH used for D-fructose reduction by dehydrogenation of formate to carbon dioxide . These recombinant E . coli cells were able to form D-mannitol from D-fructose in a low but significant quantity (15 mM) . The introduction of a further gene, encoding for the glucose facilitator protein of Zymomonas mobilis (GLF) enabled the cells to efficiently take up D-fructose into the cells, without simultaneous phosphorylation . Resting cells of this E . coli strain (3 g cell dry weight/l) produced 216 mM D-mannitol in 17 hours . Biotransformations conducted under pH-control by formic acid addition yielded D-mannitol at a concentration of 362 mM within 8 hours . The yield Y(D-mannitol D-fructose) was 84 {mol%} . These results show that the recombinant strain of E . coli can be utilized as an efficient biocatalyst for D-mannitol formation.

Biotechnol Lett, 2004 Jul, 26(14), 1119 - 24
Transformation of alternan-producing strains of Leuconostoc by electroporation; Leathers TD et al.; Alternan-producing Leuconostoc mesenteroides strain NRRL B-1355 and its glucansucrase-negative derivative NRRL B-21414 were transformed by electroporation using four Gram positive-Gram negative shuttle vectors . Optimal conditions were 400 Omega and 10 kV cm(-1), resulting in transformation efficiencies of up to 3.5 x 10(4) per microg DNA . Relatively low copy numbers and native plasmids made it difficult to visualize the introduced plasmids on ethidium bromide-stained gels and, in some cases, on blot hybridizations . However, PCR analysis indicated that 95% of putative transformants carried plasmid sequences . Direct colony PCR was shown to work well for this system and also for transformants of L . mesenteroides subsp . cremoris.

J Appl Microbiol, 2004, 97(2), 384 - 94
Simultaneous detection of Carnobacterium and Leuconostoc in meat products by multiplex PCR; Macian MC et al.; AIMS: To develop a multiplex PCR approach for simultaneous detection of Leuconostoc and Carnobacterium and its validation in meat products . METHODS AND RESULTS: Two multiplex PCR assays were developed using newly designed 16S rDNA-directed primers adapted to the current taxonomic situation of genera Leuconostoc and Carnobacterium that allow: (i) simultaneous detection of both genera, and members of the nonmotile species of genus Carnobacterium and (ii) identification in a single assay of the nonmotile species C . divergens, C . maltaromicum and C . gallinarum . Sensitivity values of 10(3) and 10(4) CFU g(-1) were determined for multiplex PCR detection of Carnobacterium and Leuconostoc, respectively, following artificially inoculated meat trials . In addition, both multiplex PCR assays were validated in 14 naturally contaminated samples covering nine types of meat products . Results obtained by colony identification were confirmed by PCR detection . CONCLUSIONS: The methods described in this study provide a rapid and reliable tool for PCR detection of Carnobacterium and Leuconostoc, in meat products, and for colony identification . SIGNIFICANCE AND IMPACT OF THE STUDY: This multiplex PCR approach will help in the analysis of the spoilage microbiota of refrigerated vacuum-packaged meat product in order to determine the appropriate preservation method.

Carbohydr Res, 2004 Jun 1, 339(8), 1517 - 29
Enzymatic synthesis of two salicin analogues by reaction of salicyl alcohol with Bacillus macerans cyclomaltodextrin glucanyltransferase and Leuconostoc mesenteroides B-742CB dextransucrase; Yoon SH et al.; Beta-Salicin is a naturally occurring glycoside found in the bark of poplar and willow trees . Ancient man used it as an analgesic and antipyretic . It has a D-glucopyranose unit attached by a beta-linkage to the phenolic hydroxyl of salicyl alcohol . Two new salicin analogues have been enzymatically synthesized by transglycosylation reactions: (a) by the reaction of Bacillus macerans cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and salicyl alcohol, followed by reactions with alpha amylase and glucoamylase to give D-glucopyranose attached by an alpha-linkage to the phenolic hydroxyl of salicyl alcohol as the major product, alpha-salicin; and (b) by the reaction of Leuconostoc mesenteroides B-742CB dextransucrase with sucrose and salicyl alcohol, followed by reactions with dextranase and glucoamylase to give alpha-d-glucopyranose attached to the primary alcohol hydroxyl of salicyl alcohol as the major product, alpha-isosalicin .

Int J Food Microbiol, 2004 Jun 15, 93(3), 287 - 96
Use of RAPD-PCR as a method to follow the progress of starter cultures in sauerkraut fermentation; Plengvidhya V et al.; DNA fingerprinting methods were used to follow the progress of unmarked starter cultures in laboratory sauerkraut fermentations (1.2 and 13 l) . Random prime PCR (RAPD-PCR) was used for strain-specific identification of Leuconostoc mesenteroides cultures . A comparative analysis of RAPD banding patterns for fermentation isolates and starter cultures was carried out using both genetically marked and unmarked cultures . While some variation in the RAPD patterns was observed, the results showed that the starter cultures dominated the fermentation during early heterofermentative stage for up to 5 days after the start of fermentation . Results from marked and unmarked starter cultures were confirmed by intergenic transcribed spacer (ITS)-PCR, and strain identify was confirmed by pulse field gel electrophoresis (PFGE) patterns . The results demonstrate the utility of RAPD to follow the progression of unmarked starter cultures of L . mesenteroides in sauerkraut fermentations.

Int J Food Microbiol, 2004 Apr 15, 92(2), 129 - 40
The antilisterial effect of Leuconostoc carnosum 4010 and leucocins 4010 in the presence of sodium chloride and sodium nitrite examined in a structured gelatin system; Hornbaek T et al.; To further enhance biopreservation of meat products, the antilisterial effect of the newly described protective culture Leuconostoc carnosum 4010 and its bacteriocins, leucocins 4010, was examined in the presence of sodium chloride and sodium nitrite in a solid matrix using a structured gelatin system . Interaction between Listeria monocytogenes 4140 and Leuc . carnosum 4010 or the leucocins 4010-resistant mutant L . monocytogenes 4140P showed that the inhibitory effect of Leuc . carnosum 4010 in the gelatin system was caused by the production and activity of leucocins 4010 . The presence of sodium chloride (2.5% w/v) and sodium nitrite (60 mg/l) reduced the antilisterial effect of Leuc . carnosum 4010 in the structured gel system compared to the use of Leuc . carnosum 4010 alone . Investigations carried out at 10 degrees C showed that the lag phase of L . monocytogenes 4140 in the presence of Leuc . carnosum 4010 was reduced from 71 to 58 h by the addition of sodium chloride and to 40 h by the addition of sodium nitrite . Addition of sodium chloride increased the maximum specific growth rate of L . monocytogenes 4140 in the presence of Leuc . carnosum 4010 from 0.02 to 0.06 h(-1), whereas no change was observed by the addition of sodium nitrite . Compared to the antilisterial effect of leucocins 4010 alone, the addition of sodium chloride (2.5%, w/v) decreased the antilisterial effect at high concentrations of leucocins 4010 (5.3 and 10.6 AU/ml) as measured after 11 days of incubation at 10 degrees C . In gels with added leucocins 4010, the most pronounced reduction in growth of L . monocytogenes 4140 was observed at the highest concentration of leucocins 4010 (10.6 AU/ml) together with sodium nitrite (60 mg/l) . More detailed information on the lag phase and the maximum specific growth rate of single colonies of L . monocytogenes 4140 in the presence of leucocins 4010 was obtained using microscopy and image analysis . No pronounced difference in the growth of single colonies was observed in the gel system . Real-time measurements of colony growth at 10 degrees C in the gelatin matrix showed that the growth inhibiting effect of leucocins 4010, including a longer lag phase as well as a lower maximum specific growth rate for L . monocytogenes 4010, was negated in the presence of 2.5% (w/v) sodium chloride.

Carbohydr Res, 2004 Apr 28, 339(6), 1029 - 34
Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity; Devulapalle KS et al.; Mutans streptococci are oral bacteria with a key role in the initiation of dental caries, because their glucosyltransferases synthesize polysaccharides from sucrose that allow them to colonize the tooth surface . Among the strategies to prevent dental caries that are being investigated are (1) the inhibition of bacterial growth of mutans streptococci or (2) the inhibition of glucosyltransferases involved in polysaccharide formation . Pure fatty acid esters of sucrose, maltose and maltotriose were synthesized by an enzyme-catalyzed process and tested as inhibitors of two glucosyltransferases of great homology, those from Streptococcus sobrinus and Leuconostoc mesenteroides NRRL B-512F . In spite of having their nonreducing end glucose blocked at 6-OH, they did not inhibit dextran synthesis . However, their effect on the growth of S . sobrinus in the solid and liquid phase was notable . 6-O-Lauroylsucrose, 6'-O-lauroylmaltose and 6"-O-lauroylmaltotriose at 100 microg/mL showed complete inhibition of S . sobrinus in agar plates . Consequently, these nontoxic derivatives are very promising for inclusion in oral-hygiene products aimed at disrupting plaque formation and preventing caries.

Curr Microbiol, 2004 Mar, 48(3), 204 - 7
Synergistic mode of action of mesenterocins 52A and 52B produced by Leuconostoc mesenteroides subsp . mesenteroides FR 52; Limonet M et al.; Few studies have been published on the effects of two bacteriocins combinations and particularly on combinations of two bacteriocins with different structures produced by the same strain . In this work, the actions of mesenterocin 52A (class IIa) and mesenterocin 52B (class II), produced by Leuconostoc mesenteroides subsp . mesenteroides FR 52, were studied on strains susceptible to only one bacteriocin or to both . In broth, combination of mesenterocins enhanced the adaptation time of the strain susceptible to the both mesenterocins (48 h vs 17 h with only one bacteriocin) . In agar medium, mesenterocins displayed, as expected, a synergistic effect on this strain (FIC(index) < 1), but also on the two strains susceptible to only one mesenterocin . This original result was probably due to membrane composition modifications induced by the mesenterocin that enhanced bacteriocin action . Thus, this hurdle technique seems to be interesting in food preservation in terms of minimizing bacteriocin concentrations.

FEMS Microbiol Lett, 2004 Mar 12, 232(1), 15 - 22
Differences in mesentericin secretion systems from two Leuconostoc strains; Aucher W et al.; Leuconostoc mesenteroides Y105 and L . mesenteroides FR52 produce both mesentericin Y105 and B105, in equal amounts . The mesentericin operons of L . mesenteroides FR52 and Y105 which are involved in mesentericin Y105 and B105 production, were both sequenced and compared . Differences were limited to the two genes, mesD and mesE, which encode the dedicated transport system of mesentericin Y105 . Analysis of mesentericin non-producing mutants and complementation experiments demonstrated that the major role of the membrane fusion protein, MesE, was in bacteriocin secretion for both strains . Moreover, the secretion machinery MesDE was demonstrated to be capable of transportation and maturation of the two pre-bacteriocins, mesentericin Y105 and B105 . We also demonstrate that although MesDEs from strains Y105 and FR52 have significant sequence differences, both transporters were capable of assuring secretion of either bacteriocin.

Electrophoresis, 2004 Mar, 25(6), 861 - 9
Capillary electrophoresis analysis of glucooligosaccharide regioisomers; Joucla G et al.; Complex gluco-oligosaccharide mixtures of two regioisomer series were successfully separated by CE . The gluco-oligosaccharide series were synthesized, employing a dextransucrase from Leuconostoc mesenteroides NRRL B-512F, by successive glucopyranosyl transfers from sucrose to the acceptor glucose or maltose . The glucosyl transfer to both acceptors, occurring through the formation of alpha1-->6 linkages, differed for the two series only in the glucosidic bond to the reducing end namely alpha1-->6 or alpha1-->4 bond for glucose or maltose acceptor, respectively . Thus, the combination of the two series results in mixed pairs of gluco-oligosaccharide regioisomers with different degrees of polymerization (DP) . These regioisomer series were first derivatized by reductive amination with 9-aminopyrene-1,4,6-trisulfonate (APTS) . Under acidic conditions using triethyl ammonium acetate as electrolyte, the APTS-gluco-oligosaccharides of each series were separated enabling unambiguous size determination by coupling CE to electrospray-mass spectrometry . However, neither these acidic conditions nor alkaline buffer systems could be adapted for the separation of the gluco-oligosaccharide regioisomers arising from the two combined series . By contrast, increased resolution was observed in an alkaline borate buffer, using differential complexation of the regioisomers with the borate anions . Such conditions were also successfully applied to the separation of glucodisaccharide regioisomers composed of alpha1-->2, alpha1-->3, alpha1-->4, and alpha1-->6 linkages commonly synthesized by glucansucrase enzymes.

Appl Environ Microbiol, 2004 Feb, 70(2), 1116 - 22
Evaluation of structural changes induced by high hydrostatic pressure in Leuconostoc mesenteroides; Kaletunc G et al.; Scanning electron microcopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC) were used to evaluate structural changes in Leuconostoc mesenteroides cells as a function of high-hydrostatic-pressure treatment . This bacterium usually grows in chains of cells, which were increasingly dechained at elevated pressures . High-pressure treatments at 250 and 500 MPa also caused changes in the external surface and internal structure of cells . Dechaining and blister formation on the surface of cells increased with pressure, as observed in SEM micrographs . TEM studies showed that cytoplasmic components of the cells were affected by high-pressure treatment . DSC studies of whole cells showed increasing denaturation of ribosomes with pressure, in keeping with dense compacted regions in the cytoplasm of pressure-treated cells observed in TEM micrographs . Apparent reduction of intact ribosomes observed in DSC thermograms was related to the reduction in number of viable cells . The results indicate that inactivation of L . mesenteroides cells is mainly due to ribosomal denaturation observed as a reduction of the corresponding peak in DSC thermograms and condensed interior regions of cytoplasm in TEM micrographs.

Int J Food Microbiol, 2004 Jan 15, 90(2), 207 - 18
Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated the lactic acid bacterium population associated with strong slime formation in an acetic-acid herring preserve; Lyhs U et al.; Spoilage characterised by strong slime and gas formation affected some manufacture lots of an acetic-acid Baltic herring (Culpea haerengus membras) preserve after few weeks of storage at 0-6 degrees C . The product consisted of herring filets in acetic acid marinade containing sugar, salt, allspice and carrot slices . Microbiological analyses of the spoiled product showed high lactic acid bacterium (LAB) levels ranging from 4.5x10(8) to 2.4x10(9) CFU/g . Yeasts were not detected in any of the herring samples . Since LAB contaminants are seldom associated with fresh fish, LAB populations associated with marinade ingredients (carrots, allspice) were also analyzed . The highest LAB levels exceeding 10(7) CFU/g were detected in equilibrium modified atmosphere packaged baby carrots whereas the levels detected in the allspice samples did not exceed 4.3x10(5) . A total of 176 randomly selected LAB isolates originating from herring, carrot and allspice samples were further identified to species level using a 16 and 23S rRNA gene RFLP (ribotyping) database . Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated both in the spoiled herring and carrot samples . These species are heterofermentative-producing CO(2) from glucose and they also produce dextran from sucrose . Inoculation of some commercial-herring products with spoilage-associated L . gelidum and L . gasicomitatum strains verified that these strains have the capability of producing slime and gas in herring preserves although slime formation was not as strong as in the original samples . Since L . gelidum and L . gasicomitatum strains were commonly detected in carrots, carrot slices used for the fish marinade were considered to be the probable source of these specific spoilage organisms.

Bioconjug Chem, 2003 Nov-Dec, 14(6), 1148 - 55
Multivalent conjugates of poly-gamma-D-glutamic acid from Bacillus licheniformis with antibody F(ab') and glycopeptide ligands; Prodhomme EJ et al.; Poly-gamma-D-glutamic acid from Bacillus licheniformis is a water-soluble, nontoxic, nonimmunogenic exopolymer . Using synthetic linkers, the alpha-carboxylate side chains of PGA were conjugated to an exposed thiol side chain of an antibody F(ab') fragment, Mc109F4 . Analysis of the PGA-Mc109F4 conjugate by gel filtration HPLC revealed a mixture of multivalent conjugates . The PGA-Mc109F4 conjugate retained biological activity, but showed a lower binding affinity to target BCL3B3 cells than free Mc109F4 F(ab')(2) by flow cytometry, and a lower efficacy for BCL3B3 growth inhibition than free Mc109F4 F(ab')(2) . PGA was also conjugated with the free amino group of glycopeptide antibiotic vancomycin . The PGA-vancomycin conjugate showed slightly lower antibacterial activity than free vancomycin versus susceptible Bacillus subtilis, but slightly higher activity versus intrinsically resistant Leuconostoc mesenteroides.

Appl Microbiol Biotechnol, 2004 Apr, 64(3), 333 - 9 Epub 2003 Oct 28.
Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation; Kaup B et al.; A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle . First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant E . coli strain . By contrast whole cells of the strain were unable to produce D-mannitol from D-fructose . To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed . FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide . These recombinant E . coli cells were able to form D-mannitol from D-fructose in a low but significant quantity (15 mM) . The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up D-fructose, without simultaneous phosphorylation . Resting cells of this E . coli strain (3 g cell dry weight/l) produced 216 mM D-mannitol in 17 h . Due to equimolar formation of sodium hydroxide during NAD(+)-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h . Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h . The yield Y(D-mannitol/D-fructose) was 84 mol% . These results show that the recombinant strain of E . coli can be utilized as an efficient biocatalyst for d-mannitol formation.

Anal Chem, 2003 Aug 1, 75(15), 3898 - 901
Amperometric sensing at high temperature with a "wired" thermostable glucose-6-phosphate dehydrogenase from Aquifex aeolicus; Iyer R et al.; An amperometric enzyme sensor capable of operating at high temperatures was developed by utilizing a "wired" thermostable glucose-6-phosphate dehydrogenase (tG6PDH) from the hyperthermophilic bacterium Aquifex aeolicus . The response of the system was monitored through detection of the product of the enzymatic reaction, NADH, which was electrocatalytically reoxidized to NAD by a thermostable redox mediator, osmium (1,10-phenanthroline-5,6-dione)2-poly(4-vinylpyridine), at Eapp = +150 mVvs Ag/AgCl/KClsat . The enzyme was "wired" onto the surface of graphite electrodes by using an epoxy-based poly(ethylene glycol) diglycidyl ether cross-linker . The stability of the sensor at higher temperatures clearly surpassed the conventional system utilizing a mesophilic G6PDH (mG6PDH) from Leuconostoc mesenteroides . The mG6PDH-based system lost 26% of its response after 20 min at 50 degrees C . The response of the tG6PDH-based system remained unchanged under the same conditions . The tG6PDH-based system demonstrated excellent stability up to a temperature of 83 degrees C.

Biotechnol Adv, 1989, 7(3), 333 - 60
Inducing malolactic fermentation in wines; Edwards CG et al.; Malolactic fermentation (MLF) in wine can be accomplished by relying on the natural microflora or by inducing through inoculation of a specific strain(s) of malolactic bacteria, primarily strains of Leuconostoc oenos . Problems with inducing MLF include intrinsic factors of the grape must such as pH, presence of sulfur dioxide, and ethanol in addition to antagonism of malolactic bacteria by wine yeast . Current methods and new technology to improve the predictability of MLF are discussed.

Bioprocess Biosyst Eng, 2003 Nov, 26(1), 57 - 62 Epub 2003 Sep 20.
Effect of phosphate concentration on the production of dextransucrase by Leuconostoc mesenteroides NRRL B512F; Rodrigues S et al.; Leuconostoc mesenteroides NRRL B512F is the main strain used in industrial fermentations to produce dextransucrase and dextran . This process has been studied since the Second World War, when it was used as blood plasma expander . A study about the effect of phosphate concentration on cell propagation in a semicontinuous shake-flask culture is described in this work . Dextransucrase is obtained by fermentation of the Leuconostoc mesenteroides NRRL B512F in the presence of sucrose as substrate, a nitrogen source (corn liquor or yeast extract) and minerals . Phosphate is currently used in order to buffer the culture medium . Cell propagation can be done through a repeated batch culture, where dilution in a fresh medium is made with relatively short periods . The standard medium for dextransucrase production is prepared using 0.1 M of K(2)HPO(4) . In this work the level of phosphate was increased to 0.3 M, and an increase on biomass and on the enzyme activity was found when phosphate enriched medium was used . Higher phosphate buffer concentration was also able to keep the pH values above 5.0 during the entire process, avoiding enzyme denaturation.

J Microbiol Methods, 2003 Oct, 55(1), 295 - 302
A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis; Jang J et al.; A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method was developed to detect and identify typical Leuconostoc species . This method utilises a set of specific primers for amplification of the 16S rDNA region of typical Leuconostoc species . All Leuconostoc-type strains, all Leuconostoc isolates from kimchi, Korea's traditional, fermented vegetable product, and strains from closely related genera were examined to verify the identification by this method . The primers resulted in amplification only for nine typical Leuconostoc spp., but not for any other genera tested . The size of the amplified products was 976 bp and the amplicons of the different species could be differentiated from each other with MseI, HaeIII and Tsp509I endonucleases, except for the species Leuconostoc argentinum and Leuconostoc lactis, which were indistinguishable . A PCR-RFLP method for the typical Leuconostoc species was optimized to identify a large number of isolates from fermented vegetable product . This PCR-RFLP method enables the rapid and reliable identification of Leuconostoc species and to distinguish them from the other phylogenetically related lactic acid bacteria in food samples.

Biotechnol Appl Biochem, 2003 Dec, 38(Pt 3), 267 - 9
Optimization of dextran production by Leuconostoc mesenteroides NRRL B-512 using cheap and local sources of carbohydrate and nitrogen; Behravan J et al.; Using pure components for the fermentation medium in dextran production imposes high costs on the industry . In the present study, the economic production of dextran using local and cheap sources of carbohydrate and nitrogen was investigated . Different concentrations of molasses and wheat-bran extract, after filtration, steam sterilization and pH adjustment, were inoculated with a fresh suspension of Leuconostoc mesenteroides . Cultures were incubated, and then diluted with an equal volume of ethanol . The bacteria were pelleted, and an aliquot of the supernatant was diluted with ethanol and dextran was precipitated . The supernatant was removed and the precipitate was dissolved in a minimal volume of water . Activated charcoal was added and the solution was boiled . The solution was filtered and protein impurities removed by 2-methylbutan-2-ol/chloroform extraction . Dextran was again precipitated with cold ethanol as described above, and the precipitate was dried in a desiccator . Optimum conditions and composition of culture media for dextran production using sugar-beet molasses and wheat bran were determined . The best results were obtained when 20% (w/v) molasses and 15% (w/v) wheat bran were used . The optimal initial pH for dextran production was 7.5.

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1123 - 6
Leuconostoc inhae sp . nov., a lactic acid bacterium isolated from kimchi; Kim B et al.; Six strains of a hitherto unknown bacterium isolated from kimchi, a fermented vegetable food produced in Korea, were characterized by using phenotypic methods, phylogenetic analysis and DNA-DNA hybridization . The novel strains were gram-positive, non-spore-forming, heterofermentative and spherical or lenticular lactic acid bacteria . Comparative 16S rRNA gene sequencing and DNA relatedness demonstrated that the unknown strains represented a novel clade within the genus Leuconostoc and were close to, but distinct from, Leuconostoc gelidum . The unknown strains were clearly distinguished from all described members of the genus Leuconostoc by using RFLP pattems of genus-specific 16S rRNA gene PCR products with a single endonuclease, BsmAI . Based on the polyphasic evidence, the unknown isolates are classified as Leuconostoc inhae sp . nov . The type strain is strain IH003T (= KCTC 3774T = DSM 15101T).

J Appl Microbiol, 2003, 95(2), 242 - 9
Application of Leuconostoc carnosum for biopreservation of cooked meat products; Jacobsen T et al.; AIMS: To optimize the practical use of the bacteriocin producing Leuconostoc carnosum 4010 in order to inhibit the growth of Listeria monocytogenes in sliced meat products . METHODS AND RESULTS: Four different methods for biopreservation using the partially purified bacteriocin or the living culture of Leuc . carnosum 4010 were evaluated . The methods using the living protective culture added to the sliced gas packed meat product were more effective in preventing growth of L . monocytogenes than the use of the partially purified leucocins 4010 or bacteriocin produced during fermentation before heat treatment of the saveloy . The application method giving the highest reduction in L . monocytogenes used nozzles for sprinkling the protective culture on all surfaces of each slice of the meat product . In the control samples without the protective culture, L . monocytogenes grew to ca . 107 CFU g(-1), whereas for the application method using nozzles for distributing the protective culture, counts of L . monocytogenes never exceeded 10 CFU g(-1) during 4 weeks of storage at 10 degrees C . CONCLUSIONS: The live cells of the bacteriocin producing Leuc . carnosum 4010 was the most efficient method as it inhibited the growth of L . monocytogenes in cooked, sliced and gas packed saveloy stored at 5 and 10 degrees C for 4 weeks . SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that biopreservation with lactic acid bacteria is a suitable alternative to chemical preservatives . An even distribution of the protective culture was found to be essential for the efficacy of the protective culture in pilot plant trials.

Biotechnol Prog, 2003 May-Jun, 19(3), 815 - 21
Scale-up of a new bacterial mannitol production process; von Weymarn FN et al.; D-Mannitol is a sugar alcohol with applications in chemistry, food and pharmaceutical industries, and medicine . Commercially, mannitol is produced by catalytic hydrogenation . Although this process is widely used, it is not optimal for mannitol production . New processes, including chemical, enzymatic, and microbial processes, are frequently developed and evaluated against the existing hydrogenation processes . In earlier papers, we have described the identification of a food-grade lactic acid bacterium strain, Leuconostoc mesenteroides ATCC-9135, with efficient mannitol production capabilities and the development and optimization of a new bioprocess in which the strain was applied . The new bioprocess is simple . It requires a reduced bioreactor with the following features: sterilization, pH and T control (at mild conditions), and slow mixing . The contamination risk of the new bioprocess is low, and the downstream processing protocol comprises simple, widely used unit operations: evaporation, crystallization, crystal separation, and drying . On a 2-L laboratory scale, high mannitol yields from fructose (93-97%) and volumetric mannitol productivities (>20 g L(-1) h(-1)) were achieved . In this paper, the scalability of the new bioprocess was tested on a small pilot scale (100 L) . In the pilot plant, production levels were achieved similar to those in the laboratory . Also, high-purity mannitol crystals were obtained at similar yield levels . The results presented in this paper indicate that the new bioprocess can easily be scaled-up to an industrial scale and that the production levels achieved with it are comparable to the catalytic hydrogenation processes.

Int J Food Microbiol, 2003 Jul 15, 84(1), 117 - 23
Screening of biogenic amine production by lactic acid bacteria isolated from grape must and wine; Moreno-Arribas MV et al.; The potential to produce the biogenic amines tyramine, histamine and putrescine, was investigated for lactic acid bacteria (LAB) of various origin, including commercial malolactic starter cultures, type strains and 78 strains isolated from Spanish grape must and wine . The presence of biogenic amines in a decarboxylase synthetic broth was determined by reverse-phase high performance liquid chromatography (RP-HPLC) . Tyramine was the main amine formed by the LAB strains investigated . Leuconostoc strains were the most intensive tyramine formers . No potential to form biogenic amines was observed in Oenococcus oeni strains . Two strains of Latobacillus buchneri were associated with putrescine formation . None of the lactic acid bacteria produced histamine . According to these in vitro results, the commercial starter bacteria analyzed did not produce histamine, tyramine and putrescine.

J Bacteriol, 2003 Jun, 185(12), 3606 - 12
Molecular characterization of inulosucrase from Leuconostoc citreum: a fructosyltransferase within a glucosyltransferase; Olivares-Illana V et al.; The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli . The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme . Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355 . From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase . Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E . coli and retained their ability to synthesize inulin but lost thermal stability . This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.

Carbohydr Res, 2003 May 23, 338(11), 1183 - 9
Dextran molecular size and degree of branching as a function of sucrose concentration, pH, and temperature of reaction of Leuconostoc mesenteroides B-512FMCM dextransucrase; Kim D et al.; Reactions of Leuconostoc mesenteroides B-512FMCM dextransucrase with increasing concentrations of sucrose, from 0.1 to 4.0 M, gave a decreasing amount of high-molecular weight dextran (HMWD) (>10(6) Da) with a concomitant increase in low-molecular weight dextran (LMWD) (<10(5) Da) . At 0.1 M sucrose, pH 5.5, and 28 degrees C, 99.8% of the dextran had a MW>10(6) Da and at 4.0 M sucrose, 69.9% had a MW<10(5) Da and 30.1% had a MW>10(6) Da, giving a bimodal distribution . The degree of branching increased from 5% for 0.1 M sucrose to 16.6% for 4.0 M sucrose . The temperature had very little effect on the size of the dextran, which was >10(6) Da, but it had a significant effect on the degree of branching, which was 4.8% at 4 degrees C and increased to 14.7% at 45 degrees C . Both the molecular weight (MW) and the degree of branching were not significantly affected by different pH values between 4.5 and 6.0.

Antibiot Khimioter, 2003, 48(1), 18 - 22
{Antimicrobial activity of Laetiporus sulphureus strains grown in submerged culture}; Ershova EIu et al.; Cultural conditions for growth and fruit body formation were elaborated to four strains of Laetiporus sulphureus isolated from nature . All strains demonstrated antimicrobial activity against a wide spectrum of gram-positive and gram-negative bacteria during agar and submerged cultivation including methicillin-resistant strain of Staphylococcus aureus (MRSA) and glycopeptide-resistant strain of Leuconostoc mesenteroides . Antifungal activity was not found . The level of antimicrobial activity during submerged cultivation reached maximum after seven days of growth on specific medium with soybean meal and corn liquid; the next four weeks its increasing was not so manifested . Antimicrobial activity correlated with orange pigment secretion and cultural liquid acidification to pH 2.0-2.8 that indicates on acid nature of synthesized products.

Int J Food Microbiol, 2003 Jun 15, 83(2), 171 - 84
Leuconostoc carnosum 4010 has the potential for use as a protective culture for vacuum-packed meats: culture isolation, bacteriocin identification, and meat application experiments; Budde BB et al.; A new culture, Leuconostoc carnosum 4010, for biopreservation of vacuum-packed meats is described . The culture originated from bacteriocin-producing lactic acid bacteria (LAB) naturally present in vacuum-packed meat products . Approximately, 72,000 colonies were isolated from 48 different vacuum-packed meat products and examined for antibacterial activity . Bacteriocin-producing colonies were isolated from 46% of the packages examined . Leuc . carnosum was the predominant bacteriocin-producing strain and Leuc . carnosum 4010 was selected for further experiments because it showed strong antilisterial activity without producing any undesirable flavour components in meat products . For identification of the bacteriocins produced, partial purification was carried out by ammonium sulphate precipitation, dialysis, and cation exchange chromatography . SDS-PAGE analysis revealed two bands with inhibitory activity corresponding to molecular sizes of 4.6 and 5.3 kDa . N-terminal amino acid sequencing showed that Leuc . carnosum 4010 produced two bacteriocins highly similar or identical to leucocin A and leucocin C . Application experiments showed that the addition of 10(7) cfu/g Leuc . carnosum 4010 to a vacuum-packaged meat sausage immediately reduced the number of viable Listeria monocytogenes cells to a level below the detection limit and no increase of L . monocytogenes was observed during storage at 5 degrees C for 21 days . The results presented demonstrate that Leuc . carnosum 4010 is suitable as a new protective culture for cold-stored, cooked, sliced, and vacuum-packed meat products.

Carbohydr Res, 2003 Apr 22, 338(9), 855 - 64
Glucosylation of alpha-butyl- and alpha-octyl-D-glucopyranosides by dextransucrase and alternansucrase from Leuconostoc mesenteroides; Richard G et al.; For the first time, glucosylation of alpha-butyl- and alpha-octylglucopyranoside was achieved using dextransucrase (DS) of various specificities, and alternansucrase (AS) from Leuconostoc mesenteroides . All the glucansucrases (GS) tested used alpha-butylglucopyranoside as acceptor; in particular, DS produced alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside and alpha-D-glucopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1-->6)-O-butyl-alpha-D-glucopyranoside . In contrast, alpha-octylglucopyranoside was glucosylated only by AS which was shown to be the most efficient catalyst . The conversion rates, obtained with this enzyme at sucrose to acceptor molar ratio of 2:1 reached 81 and 61% for alpha-butylglucopyranoside and alpha-octylglucopyranoside, respectively . Analyses obtained from liquid chromatography coupled with mass spectrometry revealed that different series of alpha-alkylpolyglucopyranosides regioisomers of increasing polymerization degree can be formed depending on the specificity of the catalyst.

Minerva Pediatr, 2003 Feb, 55(1), 83 - 6
Leuconostoc bacteremia in a healthy infant; Casanova-Roman M et al.; Infections by Leuconostoc species bacteria are uncommon, and usually affect patients with an underlying disease, or those fitted with a venous catheter or subjects previously treated with vancomycin . The most common clinical presentation is fever secondary to a central venous line infection . We report a case of Leuconostoc sp . bacteremia in an otherwise apparently healthy 2.5 month-old infant . The patient was successfully treated with cefotaxime . Leuconostoc sp . is an emerging pathogen that should be considered in the differential diagnosis of vancomycin-resistant Gram-positive bacteremia.

J Ind Microbiol Biotechnol, 2003 Feb, 30(2), 114 - 7 Epub 2003 Jan 11.
Purification of alternanase by affinity chromatography; Ahlgren JA et al.; The enzyme alternanase, produced by Bacillus sp . NRRL B-21195, hydrolyzes alternan, a polysaccharide produced by certain strains of Leuconostoc mesenteroides that consists of glucose linked by alternating alpha(1-->6), alpha(1-->3) linkages . The main product of enzymatic hydrolysis by alternanase is a novel cyclic tetrasaccharide of glucose that also has alternating linkages between the glucose moieties . An improved purification scheme for alternanase has been developed that incorporates the use of isomaltosyl units linked to agarose for selectively binding the alternanase enzyme . Bound enzyme was eluted with 0.5 M sodium chloride and was nearly pure after this procedure . When followed by preparative isoelectric focusing, a single band of 117 kDa was measured when the purified protein was analyzed by HPLC size-exclusion chromatography/multiangle light scattering . The purification procedure can be scaled to permit large quantities of enzyme to be purified in high (36%) yield.

Arch Microbiol, 2003 Jan-Feb, 179(2), 101 - 7 Epub 2003 Jan 11.
A zinc-containing mannitol-2-dehydrogenase from Leuconostoc pseudomesenteroides ATCC 12291: purification of the enzyme and cloning of the gene; Hahn G et al.; Mannitol-2-dehydrogenase (EC 1.1.1.67) of Leuconostoc pseudomesenteroides ATCC 12291 catalyzing the NADH-dependent reduction of d-fructose to d-mannitol was purified to homogeneity . Native mannitol-2-dehydrogenase has a molecular mass of 155 kDa as determined by gel filtration chromatography . In SDS-PAGE, a single band appeared corresponding to a molecular mass of 43 kDa which indicated that the enzyme was composed of four identical subunits . Enzyme activity was completely inhibited by EDTA and could be restored by zinc ions, but not by Mn(2+) or Mg(2+) which demonstrated that zinc is a cofactor . Purified mannitol-2-dehydrogenase exhibited a maximal specific activity of 400 micromol fructose reduced min(-1) x (mg protein)(-1), using NADH as electron donor . The enzyme showed a high substrate specificity for d-fructose and d-mannitol, however it accepted NADPH as a cofactor with 32% activity ( V(max)) relative to NADPH (100%) . The mdh gene, encoding mannitol-2-dehydrogenase, was identified by hybridization with a degenerate gene probe complementary to the nucleotide sequence encoding the first eight N-terminal amino acids of the enzyme . The mdh gene was cloned on a 4.2-kb DNA fragment, subcloned, and expressed in Escherichia coli . Sequencing of the gene revealed an open reading frame of 1017 bp, encoding a protein of 338 amino acids with a predicted molecular mass of 36.0 kDa . Plasmid-encoded mdh was functionally expressed, with 70 U/mg of cell-free protein in E . coli . Multiple sequence alignments showed that mannitol-2-dehydrogenase was affiliated with members of the Zn(2+)-containing medium-chain alcohol/polyol dehydrogenase/reductase protein family (MDR).

J Appl Microbiol, 2003, 94(2), 280 - 8
The response of Leuconostoc mesenteroides to low external oxidoreduction potential generated by hydrogen gas; Bourel G et al.; AIMS: The physiological consequences of low external oxidoreduction potential in Leuconostoc mesenteroides were investigated . METHODS AND RESULTS: Leuconostoc mesenteroides was grown under two initial oxidoreduction potential conditions (Eh7: +200 mV and -400 mV) using nitrogen and hydrogen as reducing agents . Growth was affected by Eh7; the lag phase increased from 1 h at an initial Eh7 of +200 mV to 6 h at an initial Eh7 of -400 mV; the maximum specific growth rate at -400 mV was 68% of the one observed at +200 mV . The NADH/NAD+ ratio and (NADH + NAD+) pool were independent of the external Eh7 . CONCLUSIONS: This study shows that changing the external oxidoreduction potential from +200 to -400 mV has a strong effect on the Leuc . mesenteroides physiology . The constancy of the maximum carbon and energetic fluxes (qglu, qATP) under the two Eh7 conditions accompanied by the decrease of YX/S and YATP suggested the existence of an uncoupling phenomenon, namely that some catabolized glucose and hence ATP was not associated with biomass production . SIGNIFICANCE AND IMPACT OF THE STUDY: This paper demonstrates the usefulness of taking into account, the effect of the oxidoreduction potential on the growth of Leuc . mesenteroides in the fermentation process.

Appl Environ Microbiol, 2003 Jan, 69(1), 304 - 11
Plasmid-encoded diacetyl (acetoin) reductase in Leuconostoc pseudomesenteroides; Rattray FP et al.; A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned . Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin) reductases reported previously . Downstream of the butA gene of L . pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified . A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114 . FPR02 had significantly reduced diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L . pseudomesenteroides . Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L . pseudomesenteroides.

J Gen Appl Microbiol, 1998 Apr, 44(2), 153 - 159
Characterization of Leuconostoc species isolated from vacuum-packaged ham; Cai Y et al.; Thirty-six isolates of Leuconostoc spp . were isolated from yellow spots that occurred on the surface of vacuum-packaged ham . All isolates were Gram-positive, catalase-negative cocci that produced gas from glucose and formed more than 90% of their lactate as D(-) isomer . These isolates could grow at 4 degrees C but not above 30 degrees C and most strains produced yellow spots on the ham . The isolates were divided into three groups by sugar fermentation patterns . Representative strains from three groups showed intergroup DNA homology values of above 88.8%, showing that these groups were composed of a single species . This organism was positioned at a separate branch in the genus Leuconostoc on the phylogenetic tree based on 16S rRNA sequences, which was assigned to Leuconostoc gelidum on the basis of DNA-DNA relatedness.

Carbohydr Res, 2002 Nov 29, 337(24), 2427 - 35
Synthesis of acarbose analogues by transglycosylation reactions of Leuconostoc mesenteroides B-512FMC and B-742CB dextransucrases; Yoon SH et al.; Two new acarbose analogues were synthesized by the reaction of acarbose with sucrose and dextransucrases from Leuconostoc mesenteroides B-512FMC and B-742CB . The major products for each reaction were subjected to yeast fermentation, and then separated and purified by Bio-Gel P2 gel permeation chromatography and descending paper chromatography . The structures of the products were determined by one- and two-dimensional 1H and 13C NMR spectroscopy and by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) . B-512FMC-dextransucrase produced one major acarbose product, 2(I)-alpha-D-glucopyranosylacarbose and B-742CB-dextransucrase produced two major acarbose products, 2(I)-alpha-D-glucopyranosylacarbose and 3(IV)-alpha-D-glucopyranosylacarbose.

Scand J Infect Dis, 2002, 34(10), 766 - 7
Multiple liver abscesses associated with bacteremia due to Leuconostoc lactis; Vagiakou-Voudris E et al.; Leuconostoc species, which are members of the family Streptococcacae, have only recently been recognized as potential pathogens . We describe a patient with type II diabetes mellitus who had multiple liver abscesses associated with bacteremia due to Leuconostoc lactis . To our knowledge, this is the first case of this association to be reported in the literature.

Appl Environ Microbiol, 2002 Dec, 68(12), 6451 - 6
Identification of a replicon from pTXL1, a small cryptic plasmid from Leuconostoc mesenteroides subsp . mesenteroides Y110, and development of a food-grade vector; Biet F et al.; A 2,665-bp cryptic plasmid, pTXL1, isolated from Leuconostoc mesenteroides subsp . mesenteroides Y110 was identified . This plasmid harbors a replicon localized on a 1,300-bp fragment . Two observations suggested that pTXL1 does not belong to rolling-circle replication (RCR)-type plasmids and most likely replicates via a theta mechanism . These hypotheses are supported by the observation that no detectable single-stranded intermediate was found for the replicon and that, unlike in RCR-type plasmids, the pTXL1 replicon sequence lacks an open reading frame encoding a replicase . The small-sized pTXL1 plasmid is stable and, according to its origin, can be considered in the "generally recognized as safe" category . Its ability to replicate in several lactic acid bacteria was exploited to develop a vector producing mesentericin Y105, a class II anti-Listeria bacteriocin . With this new vector, a recombinant industrial Leuconostoc cremoris strain able to produce mesentericin Y105 was constructed.

Pediatr Nephrol, 2002 Nov, 17(11), 966 - 8 Epub 2002 Aug 27.
Peritonitis due to Leuconostoc species in a child receiving peritoneal dialysis; Gillespie RS et al.; Leuconostoc species are rarely pathogenic in humans, but may cause infection in patients at risk . A 7-year-old girl with p-ANCA-positive crescentic glomerulonephritis, treated with peritoneal dialysis, developed peritonitis due to Leuconostoc species . She had a history of treatment with vancomycin and a brief course of immunosuppressive therapy . The peritonitis responded well to ampicillin therapy . To date, only 47 cases of Leuconostoc infection, including our patient, have been reported in the medical literature; 25 of the cases occurred in children . Only 1 prior case has been reported in the setting of peritoneal dialysis . The risk factors for Leuconostoc infections are not clear, but commonly associated conditions include immunocompromised status and indwelling medical devices . Leuconostoc species are easily misidentified as streptococci in culture, but they possess inherent resistance to vancomycin despite sensitivity to most other antibiotics . In patients with gram-positive peritonitis, Leuconostoc should be considered as a possible etiological agent, particularly if vancomycin resistance is noted in an organism thought to be a Streptococcus species.

Biochimie, 2002 May-Jun, 84(5-6), 569 - 76
Heterologous expression of bacteriocins using the mesentericin Y105 dedicated transport system by Leuconostoc mesenteroides; Morisset D et al.; Mesentericin Y105 (MesY105) is a class IIa anti-Listeria bacteriocin, produced by Leuconostoc (Ln.) mesenteroides Y105 and with potential food grade application . This bacterium produces a second bacteriocin, mesentericin B105 (MesB105), that does not belong to the same class . To study secretion of bacteriocins by the use of the MesY105 dedicated transport system (DTS), plasmids were constructed for heterologous expression by Ln . mesenteroides . pFBYC04 (Microbiology 144 (1998) 2845) harbours two divergent operons required for MesY105 secretion, i.e . the mesYI operon, encoding pre-MesY105 and immunity, respectively, and the mesCDE operon for secretion . A pFBYC04 derivative, pDMJF01 was constructed by divergent PCR to remove the mesY gene . Ln . mesenteroides DSM20484(pDMJF01) was unable to produce MesY105 . The mesYI operon and mesB, mesH and mesF genes, encoding pre-MesB105, MesB105 immunity and a putative protein with unknown function, respectively, were cloned independently into a compatible pDMJF01 plasmid to produce, respectively, pDMJF:YI and pDMJF:BHF . DSM20484 transformed independently with these plasmids was unable to secrete any bacteriocin . MesY105 and MesB105 secretion was observed for DSM20484(pDMJF01) harbouring both pDMJF:YI and pDMJF:BHF . This indicates that the MesY105 DTS permits the transport of MesB105 . MesY105 secretion machinery was used to secrete pediocin PA-1 (PedPA-1) by DSM20484 by an in-frame gene fusion strategy where the gene portions corresponding to the MesY105 leader peptide and the mature PedPA-1 were ligated . Thus, MesY105 secretion machinery appears to be a useful tool for secretion of class II bacteriocins by Leuconostoc.

Appl Environ Microbiol, 2002 Nov, 68(11), 5452 - 8
Characterization of six Leuconostoc fallax bacteriophages isolated from an industrial sauerkraut fermentation; Barrangou R et al.; Six bacteriophages active against Leuconostoc fallax strains were isolated from industrial sauerkraut fermentation brines . These phages were characterized as to host range, morphology, structural proteins, and genome fingerprint . They were exclusively lytic against the species L . fallax and had different host ranges among the strains of this species tested . Morphologically, three of the phages were assigned to the family Siphoviridae, and the three others were assigned to the family Myovidae: Major capsid proteins detected by electrophoresis were distinct for each of the two morphotypes . Restriction fragment length polymorphism analysis and randomly amplified polymorphic DNA fingerprinting showed that all six phages were genetically distinct . These results revealed for the first time the existence of bacteriophages that are active against L . fallax and confirmed the presence and diversity of bacteriophages in a sauerkraut fermentation . Since a variety of L . fallax strains have been shown to be present in sauerkraut fermentation, bacteriophages active against L . fallax are likely to contribute to the microbial ecology of sauerkraut fermentation and could be responsible for some of the variability observed in this type of fermentation.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1477 - 84
Phylogenetic, amino acid content and indel analyses of the beta subunit of DNA-dependent RNA polymerase of gram-positive and gram-negative bacteria; Morse R et al.; 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.

Carbohydr Res, 2002 Sep 27, 337(17), 1529 - 33
Production of insoluble dextran using cell-bound dextransucrase of Leuconostoc mesenteroides NRRL B-523; Padmanabhan PA et al.; Water-insoluble, cell-free dextran biosynthesis from Leuconostoc mesenteroides NRRL B-523 has been examined . Cell-bound dextransucrase is used to produce cell-free dextran in a sucrose-rich acetate buffer medium . A comparison between the soluble and insoluble dextrans is made for various sucrose concentrations, and 15% sucrose gave the highest amount of cell-free dextran for a given time . L . mesenteroides B-523 produces more insoluble dextran than soluble dextran . The near cell-free synthesis was validated in a batch reactor, by monitoring the cell growth which is a small (10(6)-10(7) CFU/mL) and constant value throughout the synthesis.

J Bacteriol, 2002 Oct, 184(20), 5753 - 61
Molecular characterization of DSR-E, an alpha-1,2 linkage-synthesizing dextransucrase with two catalytic domains; Bozonnet S et al.; 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.

Biochem Biophys Res Commun, 2002 Jul 26, 295(4), 826 - 7
The complete amino acid sequence of the pediocin-like antimicrobial peptide leucocin C; Fimland G et al.; 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.

Lett Appl Microbiol, 2002, 35(1), 68 - 73
Identification and characterization of an oligopeptide transport system in Leuconostoc mesenteroides subsp . mesenteroides CNRZ 1463; Germain-Alpettaz V et al.; AIMS: To identify and characterize an oligopeptide transport system in Leuconostoc mesenteroides CNRZ 1473 . METHODS AND RESULTS: The uptake of a model substrate was monitored by determining intracellular concentrations of the corresponding amino acids by means of reversed-phase HPLC analysis . The oligopeptide transport system is specific for peptides containing at least four amino acid residues and operative under physiological conditions of growth . It is expressed maximally in the presence of oligopeptides, enhanced in the presence of Mg2+ or Ca2+ ions, and driven by ATP or a related energy-rich phosphorylated intermediate . CONCLUSIONS: The study showed evidence for and characterized the oligopeptide transport system of Leuc . mesenteroides for the first time . SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the growth of Leuc . mesenteroides in mixed-strain cultures for the dairy industry.

J Ind Microbiol Biotechnol, 2002 Jul, 29(1), 44 - 9
High-level production of D-mannitol with membrane cell-recycle bioreactor; von Weymarn N et al.; Ten heterofermentative lactic acid bacteria were compared in their ability to produce D-mannitol from D-fructose in a resting state . The best strain, Leuconostoc mesenteroides ATCC-9135, was examined in high cell density membrane cell-recycle cultures . High volumetric mannitol productivity (26.2 g l(-1) h(-1)) and mannitol yield (97 mol%) were achieved . Using the same initial biomass, a stable high-level production of mannitol was maintained for 14 successive bioconversion batches . Applying response surface methodology, the temperature and pH were studied with respect to specific mannitol productivity and yield . Moreover, increasing the initial fructose concentration from 100 to 120 and 140 g l(-1) resulted in decreased productivities due to both substrate and end-product inhibition of the key enzyme, mannitol dehydrogenase (MDH) . Nitrogen gas flushing of the bioconversion media was unnecessary, since it did not change the essential process parameters.

J Ind Microbiol Biotechnol, 2002 Feb, 28(2), 112 - 7
Characterization of a cell-associated inulosucrase from a novel source: a Leuconostoc citreum strain isolated from Pozol, a fermented corn beverage of Mayan origin; Olivares-Illana V et al.; A cell-associated fructosyltransferase was extracted from a novel source, a strain of Leuconostoc citreum isolated from Pozol, a Mexican traditional fermented corn beverage, where lactic microflora are partially responsible for the transformation process . The enzyme is associated with the cell wall . It was characterized both in its cell-associated insoluble form and after separation by urea treatment . The fructosyltransferase has a molecular mass of 170 kDa, the highest reported for this type of enzyme, and in its insoluble form is highly specific for polymer synthesis, with low fructose transferred to maltose and lactose added to the reaction medium (acceptor reactions) . The synthesized polymer has an inulin-like structure with beta2-1 glycosidic linkages, as demonstrated by 13C nuclear magnetic resonance (NMR) . Bacterial inulosucrases have only been reported in Streptococcus mutans.

Appl Environ Microbiol, 2002 Jun, 68(6), 2910 - 6
Variations in the membrane fatty acid composition of resistant or susceptible Leuconostoc or Weissella strains in the presence or absence of Mesenterocin 52A and Mesenterocin 52B produced by Leuconostoc mesenteroides subsp . mesenteroides FR52; Limonet M et al.; Mesenterocins 52A (Mes52A) and 52B (Mes52B) are antimicrobial peptides produced by Leuconostoc mesenteroides subsp . mesenteroides FR 52 . Mes52A is a class IIa bacteriocin of lactic acid bacteria with a broad spectrum of activity . Mes52B is an atypical class II bacteriocin with a narrow spectrum of activity . Four Leuconostoc and Weissella wild-type strains were selected for their susceptibility or insensitivity to these mesenterocins . Four strains resistant to Mes52A or Mes52B were generated from the three susceptible wild-type strains by increasing bacteriocin concentrations in culture media . These resistant strains were at least 30 times more resistant than the wild-type strains . No cross-resistance to Mes52A and Mes52B was observed in these strains . No significant differences in membrane fatty acid composition were observed among the three susceptible wild-type strains and the four resistant strains cultured in MRS broth . Thus, the mesenterocin resistance is unlikely to be due to changes in membrane fatty acid composition . When cultured with Mes52A or Mes52B, the membranes of insensitive and resistant strains contained more saturated fatty acids (1 to 10% more) and less unsaturated fatty acids (3 to 6% less), resulting in a more rigid membrane . Thus, the presence of mesenterocin in the culture media of insensitive or resistant strains induced a significant increase in saturated fatty acid contents and a decrease in unsaturated fatty acid contents . Weissella paramesenteroides DSM 20288BR, resistant to Mes52B, responded atypically, probably due to the production of an inhibitor.

Appl Environ Microbiol, 2002 Jun, 68(6), 2877 - 84
Identification and characterization of Leuconostoc fallax strains isolated from an industrial sauerkraut fermentation; Barrangou R et al.; Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages . Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides . Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains . Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another . The growth and fermentation patterns of the L . fallax isolates were highly similar to those of L . mesenteroides . The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species . However, none of the L . fallax strains exhibited the malolactic reaction, which is characteristic of most L . mesenteroides strains . These results indicated that in addition to L . mesenteroides, a variety of L . fallax strains may be present in the heterofermentative stage of sauerkraut fermentation . The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L . fallax require further investigation.

Biochemistry, 2002 Jun 4, 41(22), 6939 - 45
The catalytic mechanism of glucose 6-phosphate dehydrogenases: assignment and 1H NMR spectroscopy pH titration of the catalytic histidine residue in the 109 kDa Leuconostoc mesenteroides enzyme; Cosgrove MS et al.; The chemical shifts of the C(epsilon1) and C(delta2) protons of His-240 from the 109 kDa Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) were assigned by comparing 1H and 13C spectra of the wild-type and mutant G6PDs containing the His-240 to asparagine mutation (H240N) . Unambiguous assignment of the His-240 1H(epsilon1) resonance was obtained from comparing 13C-1H heteronuclear multiple quantum coherence NMR spectra of wild-type and H240N G6PDs that were selectively labeled with 13C(epsilon1) histidine . The results from NOESY experiments with wild-type and H240N variants were consistent with these assignments and the three-dimensional structure of G6PD . pH titrations show that His-240 has a pK(a) of 6.4 . This value is, within experimental error, identical to the value of 6.3 derived from the pH dependence of kcat {Viola, R . E . (1984) Arch . Biochem . Biophys . 228, 415-424}, suggesting that the pK(a) of His-240 is unperturbed in the apoenzyme despite being part of a His-Asp catalytic dyad . The results obtained for this 109 kDa enzyme indicate that 1H NMR spectroscopy in combination with heteronuclear methods can be a useful tool for functional analysis of large proteins.

J Ind Microbiol Biotechnol, 2002 May, 28(5), 291 - 6
Effect of process parameters on the production and drying of Leuconostoc mesenteroides cultures; Champagne CP et al.; Leuconostoc mesenteroides BLAC was grown on MRS broth or on a carrot juice medium, and the effects of sugar concentration, type of pH control, aeration and fermentor size on viable counts were examined . The effect on viability of the type of centrifuge used to concentrate the bacterial culture was also examined . When the MRS broth had the traditional 110 mM glucose, pH control did not increase the final population . However, using a zone pH control mode, increasing the glucose content of MRS both from 110 to 220 mM almost doubled the population . In MRS broth, the amount of acetic acid produced was the same for all treatments, and was proportional to the amount of citrate consumed . There was a significantly lower cell yield in the carrot juice medium when the pH was not regulated . In the carrot juice medium, pH had a more pronounced effect on the final population level than did aeration, even though the quantity of viable cells was greater when the culture was aerated . In MRS broth, glucose was completely consumed during fermentation, but this was not the case in carrot juice medium . Aeration resulted in increased acetic acid content of the fermented medium . Viable counts were not affected by scaling the volume of the fermentation from 2 to 15 l,or by the type of centrifuge used to concentrate the cells . Cells were concentrated by a factor of 10, but in both centrifuge types, viable counts showed only an eightfold average increase . However, freeze-dried powders obtained from the continuous pilot-plant-centrifuged cultures had, on the average, 33% lower populations than those obtained from the laboratory unit.

Lett Appl Microbiol, 2002, 34(2), 82 - 5
Random amplified polymorphic DNA analysis for differentiation of Leuconostoc mesenteroides subspecies isolated from Tenerife cheese; Perez G et al.; AIMS: In the present study, a RAPD-PCR fingerprinting method was developed to assign Tenerife cheese Leuconostoc mesenteroides strains to its three subspecies (mesenteroides, cremoris and dextranicum) . METHODS AND RESULTS: Arbitrarily primed-PCR gave different DNA banding patterns for each type strain of Leuc . mesenteroides subspecies consisting in three major and intense bands of: 1800, 1600 and 1150 bp for subspecies mesenteroides 1800, 1150 and approximately 350 bp for subspecies cremoris; and 1800, 1600 and 1500 bp for subspecies dextranicum . DNA fingerprints of Tenerife cheese Leuc . mesenteroides subspecies were coincident to that of their respective type strain . RAPD profiles were reproducible with DNA template obtained by two different extraction methods . CONCLUSIONS: Tenerife cheese Leuc . mesenteroides strains were rapidly and unequivocally assigned to one of the subspecies by comparing their RAPD-PCR fingerprints with those displayed by type strains used as standards . This technique can be applied to complement preliminary identification of Leuc . mesenteroides to the species level by other molecular methods such as protein fingerprinting . SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD-PCR allows reliable, reproducible and rapid molecular differentiation of Tenerife cheese Leuc . mesenteroides subspecies with no need to use time-consuming and often ambiguous biochemical tests.

J Clin Lab Anal, 2001, 15(6), 319 - 23
Homogenous enzyme immunoassay for cyclosporine in whole blood using the EMIT 2000 cyclosporine specific assay with the COBAS MIRA-plus analyzer; Kimura S et al.; We describe the evaluation of the EMIT 2000 cyclosporine specific assay kit, an enzyme-multiplied immunoassay for cyclosporine in whole blood, with a COBAS MIRA-plus analyzer . The enzyme used for the assay was glucose-6-phosphate dehydrogenase (EC 1.1.1.49 G6PDH) from Leuconostoc mesenteroides; the monoclonal antibody is fairly specific for cyclosporine, and is not reactive with most metabolites . The assay principle is based on competitive immunoassay with G6PDH-labeled cyclosporine and cyclosporine in sample to the anticyclosporine mouse monoclonal antibody binding site . The within-assay coefficient of variation (CV) of this method was 2.7-4.2% (n = 10) at the levels of 56.2-339.7 microg/L . Day-to-day CVs ranged from 4.2-8.1% at the levels of 47.2-350.2 microg/L . The within-day CVs ranged from 2.0-6.4% at the levels of 43.3-330.5 microg/L . The functional detection limit was 24.9 microg/L . Samples treated with pretreatment reagent were stable at least 5 hr . Calibration was stable at least 10 days . The analytical recovery was 81-109% . The correlation between values obtained with the EMIT 2000 cyclosporine specific assay kit (y) and fluorescence polarization immunoassay (FPIA) (TDxFLx) (x) was: y = 0.880x - 13.053 microg/L (r = 0.984, Sy/x = 15.968, n = 71) with a mean difference of 31.42 +/- 19.89 microg/L ((TDxFLx - EMIT 2000) +/- SD); for the FPIA (AxSYM) (x): y = 0.989 - 4.144 microg/L (r = 0.981, Sy/x = 17.478, n = 71) with a mean difference of 5.56 +/- 17.38 microg/L ((AxSYM - EMIT 2000) +/- SD); and for the radioimmunoassay (RIA, CYCLO-Trac SP) (x): y = 0.893 - 6.764 microg/L (r = 0.993, Sy/x = 10.582, n = 71) with a mean difference of 22.18 +/- 14.98 microg/L ((RIA - EMIT 2000) +/- SD) using the Bland-Altman technique .

Biotechnol Bioeng, 2002 Mar 5, 77(5), 577 - 88
Pore-scale investigation of biomass plug development and propagation in porous media; Stewart TL et al.; Biomass plugging of porous media finds application in enhanced oil recovery and bioremediation . An understanding of biomass plugging of porous media was sought by using a porous glass micromodel through which biomass and nutrient were passed . This study describes the pore-scale physics of biomass plug propagation of Leuconostoc mesenteroides under nutrient-rich conditions . It was found that, as the nutrient flowed through the micromodel, the initial biomass plug occurred at the nutrient-inoculum interface due to growth in the larger pore throats . As growth proceeded, biomass filled and closed these larger pore throats, until only isolated groupings of pore throats with smaller radii remained empty . As nutrient flow continued, a maximum pressure drop was reached . At the maximum pressure drop, the biomass yielded in a manner similar to a Bingham plastic to form a breakthrough channel consisting of a path of interconnected pore throats . The channel incorporated the isolated groupings of empty pore throats that had been present before breakthrough . As the nutrient flow continued, subsequent plugs developed as breakthrough channels refilled with biomass and in situ growth was stimulated in the region just downstream of the previous plug . The downstream plugs had a higher fraction of isolated groupings of empty pore throats, which can be attributed to depletion of nutrient downstream . When the next breakthrough channel formed, it incorporated these isolated groupings, causing the breakthrough channels to be branched . It was observed that the newly formed plug could be less stable with this higher fraction of empty pore throats and that the location of breakthrough channels changed in subsequent plugs . This change in breakthrough channel location could be attributed to the redistribution of nutrient flow and the changes in flowrate in the pore throats .

Microbiology, 2002 Jan, 148(Pt 1), 325 - 32
NAD(P)H regeneration is the key for heterolactic fermentation of hexoses in Oenococcus oeni; Maicas S et al.; Oenococcus oeni (formerly Leuconostoc oenos) can perform malolactic fermentation, converting L-malate to L-lactate and carbon dioxide, in wines . The energy and redox potential required to support the growth of the micro-organism are supplied mainly by the consumption of carbohydrates via the heterolactic pathway . In the first steps of hexose metabolism two molecules of NAD(P)(+) are consumed, which must be regenerated in later reactions . The aim of this work was to test if aerobic growth of O . oeni promotes higher cell yields than anaerobic conditions, as has been shown for other lactic acid bacteria . O . oeni M42 was found to grow poorly under aerobic conditions with glucose as the only carbohydrate in the medium . It was demonstrated that O(2) inactivates the enzymes of the ethanol-forming pathway, one of the two pathways which reoxidizes NAD(P)(+) cofactors in the heterolactic catabolism of glucose . These results suggest that the regeneration of cofactors is the limiting factor for the aerobic consumption of glucose . When external electron acceptors, such as fructose or pyruvate, were added to glucose-containing culture medium the growth of O . oeni was stimulated slightly; fructose was converted to mannitol, oxidizing two molecules of NAD(P)H, and pyruvate was transformed to lactate, enabling the regeneration of NAD(+) . The addition of cysteine seemed to suppress the inactivation of the ethanol-forming pathway enzymes by O(2), enabling glucose consumption in aerobic conditions to reach similar rates to those found in anaerobic conditions.

J Infect, 2001 Aug, 43(2), 155 - 7
Spontaneous bacterial peritonitis and bacteremia due to Leuconostoc species in a patient with end-stage liver disease: a case report; Templin KS et al.; This report describes a case of spontaneous bacterial peritonitis in a patient with end stage liver disease in whom Leuconostoc spp . was isolated from blood and ascitic fluid . In common with several previously described patients with cultures positive for Leuconostoc from other body sites, this patient had recently received vancomycin . The antibiotic susceptibilities and mechanism of vancomycin resistance of this Gram-positive bacteria are reviewed .

Biotechnol Appl Biochem, 2001 Oct, 34(Pt 2), 93 - 7
Production of dextran from sucrose by a newly isolated strain of Leuconostoc mesenteroides (PCSIR-3) with reference to L . mesenteroides NRRL B-512F; Ul-Qader SA et al.; A newly isolated strain of Leuconostoc mesenteroides (PCSIR-3) produced a different dextran compared with that of L . mesenteroides NRRL B-512F . Different media compositions used for dextran production showed that media containing CaCl(2) produced dextran in higher quantities compared with other media . The viscosity of the dextran produced in different media varied in nature . Dextran from media 1 and 2 was of higher molecular mass compared with that from media 3, 4 and 5 . Dextran production is also effected by the sucrose concentration in the media . The higher the initial concentration of sucrose, the higher is the yield of dextran produced per unit volume; however, the percentage conversion of sucrose into dextran decreases . A continuous drop in pH was associated with growth and dextran production . The yield of dextran increases during the growth phase and maximum yield was obtained at the end of the exponential phase . Dextran produced by L . mesenteroides PCSIR-3 is quite different from the dextran produced by NRRL B-512F . Maximum dextran production from L . mesenteroides PCSIR-3 occurs in 18 h compared with 12 h for NRRL B-512F.

Prikl Biokhim Mikrobiol, 2001 Jul-Aug, 37(4), 487 - 93
{Biological deacidification of wines using lactic-acid bacteria and yeasts}; Eliseeva GS et al.; Based on a study of 200 lactic-acid bacteria monocultures and 30 associating lactic bacteria and yeasts cultures, a stable association was created formed by Leuconostoc oenos, Pediococcus pentosaceus and Saccharomyces cerevisiae yeasts, intended for the biological deacidification of wine . Physiology of microorganisms and their effect on the wine chemical composition was studied . By means of selective association, high quality fine wines were produced from the high-acid wines.

Biotechnol Bioeng, 2001 Sep 20, 74(6), 498 - 504
Factors affecting alpha,-1,2 glucooligosaccharide synthesis by Leuconostoc mesenteroides NRRL B-1299 dextransucrase; Dols-Lafargue M et al.; The optimization of alpha-1,2 glucooligosaccharide (GOS) synthesis from maltose and sucrose by Leuconostoc mesenteroides NRRL B-1299 dextransucrase was achieved using experimental design and consecutive analysis of the key parameters . An increase of the pH of the reaction from 5.4 to 6.7 and of the temperature from 25 to 40 degrees C significantly favored alpha-1,2 GOS synthesis, thanks to a significant decrease of the side reactions, i.e., dextran and leucrose synthesis . These positive effects were not sufficient to compensate for the decrease of enzyme stability caused by the use of high pH and temperature . However, the critical parameters were the sucrose to maltose concentration ratio (S/M) and the total sugar concentration (TSC) . Alpha1,2 GOS synthesis was favored at high S/M ratios . But using these conditions also led to an increase of side reactions which could be modulated by choosing the appropriate TSC . Finally, with S/M = 4 and TSC = 45% w/v, dextran and leucrose productions were limited and the final alpha-1,2 GOS yield reached 56.7%, the total GOS yield being 88% .

Carbohydr Res, 2001 Aug 3, 334(1), 19 - 25
Water-soluble and water-insoluble glucans produced by Escherichia coli recombinant dextransucrases from Leuconostoc mesenteroides NRRL B-512F; Funane K et al.; Two dextransucrase genes, dsrS and dsrT5, from Leuconostoc mesenteroides NRRL B-512F were expressed in Escherichia coli, and recombinant dsrT5 dextransucrase was shown to produce a water-insoluble glucan . In contrast, native dextran from L . mesenteroides B-512F is water-soluble . The water-insoluble glucan was shown by 13C NMR and glycosyl-linkage composition analysis to contain about 50% 6-linked Glcp and 40% 3-linked Glcp . The 'primitive' B-512F strain is suggested to have produced water-insoluble glucan containing 3-linked Glcp . The glucans produced by dextransucrases expressed in E . coli contained 4-linked Glcp, as shown by glycosyl-linkage composition analysis . The amount of 4-linked Glcp was increased when the truncated, water-insoluble, glucan-producing dextransucrase, which does not have C-terminal repeating units, was added to the water-soluble, glucan-producing dextransucrase . Trace amounts of 4-linked Glcp were also detected in the dextran obtained from the B-512F culture supernatant, in dextran produced by dextransucrase purified from the B-512F strain culture supernatant, and in clinical dextran . The results of glycosyl-linkage composition analysis suggest that dextransucrases produce 4-linked Glcp as well as 6- and 3-linked Glcp.

Biotechnol Bioeng, 2001 Mar 20, 72(6), 603 - 10
Alteration of the growth rate and lag time of Leuconostoc mesenteroides NRRL-B523; Wolf BF et al.; Bacterial profile modification is an important enhanced oil recovery technique used to direct injected water into a reservoir's low permeability zone containing trapped crude oil . During water flooding, the use of bacteria to plug the high permeability water zone and divert flow into the oil-bearing low-permeability zone will have a significant economic impact . However, during the field implementation of bacterial profile modification, the rapid growth of bacteria near the injection well bore may hinder the subsequent injection of growth media so that profile modification of the reservoir occurs only in the immediate vicinity of the well bore . By slowing the growth rate and prolonging the lag phase, the onset of pore-space plugging may be delayed and the biologically active zone extended deep into the reservoir . High substrate loading, high pH values, and the addition of the growth inhibitors sodium dodecylsulfate and sodium benzoate have been used in combination to alter the growth characteristics of Leuconostoc mesenteroides NRRL-B523 grown in batch conditions . The highest sucrose concentration used in these studies, 500 g/L, produced lag times 12-fold greater than the slowest lag times achieved at low sucrose concentrations . When L . mesenteroides was grown in media containing 500 g/L sucrose, an alkaline pH value threshold was found above which bacteria did not grow . At this threshold pH value of 8.1, an average lag time of 200 h was observed . Increasing the concentration of sodium benzoate had no effect on lag time, but reduced the growth rate until the threshold concentration of 0.6%, above which bacteria did not grow . Last, it was found that a solution of 0.075 mM sodium dodecylsulfate in media containing 15 g/L sucrose completely inhibited bacterial growth .

Carbohydr Res, 2001 Apr 23, 331(4), 403 - 11
Novel oligosaccharides synthesized from sucrose donor and cellobiose acceptor by alternansucrase; Arguello Morales MA et al.; Cellobiose was tested as acceptor in the reaction catalyzed by alternansucrase (EC 2.4.1.140) from Leuconostoc mesenteroides NRRL B-23192 . The oligosaccharides synthesized were compared to those obtained with dextransucrase from L . mesenteroides NRRL B-512F . With alternansucrase and dextransucrase, overall oligosaccharide synthesis yield reached 30 and 14%, respectively, showing that alternansucrase is more efficient than dextransucrase for cellobiose glucosylation . Interestingly, alternansucrase produced a series of oligosaccharides from cellobiose . Their structure was determined by mass spectrometry and {13C-1H} NMR spectroscopy . Two trisaccharides are first produced: alpha-D-glucopyranosyl-(1-->2)-{beta-D-glucopyranosyl-(1-->4)}-D-glucopyranose (compound A) and alpha-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl-(1-->4)-D-glucopyranose (compound B) . Then, compound B can in turn be glucosylated leading to the synthesis of a tetrasaccharide with an additional alpha-(1-->6) linkage at the non-reducing end (compound D) . The presence of the alpha-(1-->3) linkage occurred only in the pentasaccharides (compounds C1 and C2) formed from tetrasaccharide D . Compounds B, C1, C2 and D were never described before . They were produced efficiently only by alternansucrase . Their presence emphasizes the difference existing in the acceptor reaction selectivity of the various glucansucrases.

Carbohydr Res, 2001 Jun 4, 332(3), 299 - 303
Enzymic alpha-galactosylation of a cyclic glucotetrasaccharide derived from alternan; Biely P et al.; Alternanase catalyzes the hydrolysis of alternan, an alpha-(1-->3)-alpha-(1-->6)-D-glucan produced by Leuconostoc mesenteroides, resulting in the formation of a cyclic tetramer cyclo -->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->(2) (cGlc(4)) . Two alpha-galactosidases, one from coffee bean and the other produced by a fungus, currently described as Thermomyces lanuginosus, were found to catalyze an efficient 6-O-alpha-D-galactopyranosylation of cGlc(4) . The attachment of a nonreducing alpha-D-galactopyranosyl residue to the cGlc(4) molecule opens new possibilities for future applications of the cyclic tetramer, since the D-galactopyranosyl residue can be easily modified by D-galactose oxidase to introduce a reactive aldehyde group . The results also extend our knowledge about the synthetic potential of T . lanuginosus alpha-galactosidase.

Appl Environ Microbiol, 2001 Jun, 67(6), 2867 - 70
Spontaneous formation of a mannitol-producing variant of Leuconostoc pseudomesenteroides grown in the presence of fructose; Grobben GJ et al.; We report the spontaneous formation of a stable mannitol-producing variant of Leuconostoc pseudomesenteroides . The mannitol-producing variant showed mannitol dehydrogenase activity which was absent in the parental strain . It was also able to use fructose and glucose simultaneously, whereas the parental strain showed diauxic growth with these sugars . A possible explanation of these observations is discussed.

J Clin Pathol, 2001 May, 54(5), 371 - 6
A microbiological study of Papillon-Lefévre syndrome in two patients; Robertson KL et al.; AIM: To analyse the microflora of subgingival plaque from patients with Papillon-Lefevre syndrome (PLS), which is a very rare disease characterised by palmar-plantar hyperkeratosis with precocious periodontal destruction . METHODS: Bacterial isolates were identified using a combination of commercial identification kits, traditional laboratory tests, and gas liquid chromatography . Some isolates were also subjected to partial 16S rDNA sequencing . Plaque samples were also assayed for the presence of Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in a quantitative enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies . RESULTS: The culture results showed that most isolates were capnophilic and facultatively anaerobic species-mainly Capnocytophaga spp and Streptococcus spp . The latter included S . constellatus, S . oralis, and S . sanguis . Other facultative bacteria belonged to the genera gemella, kingella, leuconostoc, and stomatococcus . The aerobic bacteria isolated were species of neisseria and bacillus . Anaerobic species included Prevotella intermedia, P . melaninogenica, and P . nigrescens, as well as Peptostreptococcus spp . ELISA detected P gingivalis in one patient in all sites sampled, whereas A . actinomycetemcomitans was detected in only one site from the other patient . Prevotella intermedia was present in low numbers . CONCLUSIONS: Patients with PLS have a very complex subgingival flora including recognised periodontal pathogens . However, no particular periodontopathogen is invariably associated with PLS.

Acta Crystallogr D Biol Crystallogr, 2001 May, 57(Pt 5), 635 - 48 Epub 2001 Apr 24.
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; Naylor CE et al.; 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.

FEMS Microbiol Lett, 2001 Apr 1, 197(1), 29 - 33
Application of gel microdroplet and flow cytometry techniques to selective enrichment of non-growing bacterial cells; Manome A et al.; We describe an application of gel microdroplet (GMD) and flow cytometry techniques to selective enrichment of non-growing Leuconostoc mesenteroides cells, which are well culturable on other media, from a mixture with Bacillus subtilis cells in nutrient broth . After encapsulating cells of the mixed population within GMDs and a brief incubation in nutrient broth, the inability of L . mesenteroides cells to form microcolonies within GMDs allowed their discrimination from B . subtilis cells . After staining the GMD mixture with 6-carboxyfluorescein diacetate, which showed no influence on cell viability, the GMDs containing single cells of L . mesenteroides were selectively collected using flow cytometry sorting based on differences in fluorescence intensity . The cells of L . mesenteroides retained viability during the process.

J Food Prot, 2001 Feb, 64(2), 189 - 94
Novel quantitative assays for estimating the antimicrobial activity of fresh garlic juice; Unal R et al.; Novel agar diffusion and broth dilution assays were developed for quantitatively estimating the antimicrobial activity of fresh garlic juice . Bacteria found to be inhibited by garlic juice in agar diffusion assay included two gram-positive and five gram-negative species . Leuconostoc mesenteroides was not inhibited . Escherichia coli B-103 (HB101, with pJH101, ampicillin resistant, 100 microg ml(-1)) was inhibited and chosen as the standard culture for quantitative assays . The agar diffusion assay was based on the slope ratio method, where the slope of dose response for garlic juice was divided by the slope of dose response for methylmethane thiosulfonate (MMTSO2) . Juice from fresh garlic varied in activity between 1.76 and 2.31 microg of MMTSO2 per mg of garlic juice . The activity of juice decreased during 11 months of storage of garlic cloves at 5 degrees C from 2.31 to less than 0.1 microg of MMTSO2 per mg of juice . The broth dilution assay also used the E . coli B-103 culture, which permitted selective enumeration of this bacterium when 100 microg ml(-1) of ampicillin was incorporated into the enumerating agar . Selective enumeration was essential since the garlic juice was not sterile and, thus, contained natural flora . Growth of E . coli was unaffected by 0.1%, delayed by 0.25%, and completely inhibited at 0.5 and 2% garlic juice in broth during 24 h of incubation at 37 micro C . The minimum inhibition concentration of garlic juice by broth dilution assay was, thus, estimated to be 0.5%, which is equivalent to 3.46 microg of MMTSO2 per mg of garlic juice by the agar diffusion assay.

Lett Appl Microbiol, 2001 Mar, 32(3), 185 - 9
Characterization of dextran-producing Leuconostoc strains; Holt SM et al.; Leuconostoc strains were characterized according to their antibiotic susceptibilities, carbohydrate fermentation profiles, sucrase activity patterns and plasmid content . All the strains tested were resistant to the antibiotics sulphathiazole, trimethoprim and vancomycin, and could be separated into two groups based on whether or not they could ferment melibiose and raffinose . Six Leuconostoc strains possessed plasmids and many produced unique sucrase activity patterns in polyacrylamide gels . These data will aid in distinguishing among physiologically similar dextran-producing leuconostocs, frequently used in research and industry.

Syst Appl Microbiol, 2000 Dec, 23(4), 461 - 8
Valine transport and biodiversity of Leuconostoc wild strains from French raw milk cheeses; Gendrot F et al.; The rate of L-valine transport in whole cells of Leuconostoc was at the maximum at 30 degrees C, pH 6.0 in the presence of an energy source . Transport was inhibited by 40-55%, in the presence of the ionophores (valinomycin, nigericin or monensin), and uncouplers (carbonyl cyanide-m-chloro-phenylhydrazone or 2,4-dinitrophenol) confirming the previously described delta p-driven branched-chain amino acid transport system described in cytoplasmic membranes (Winters et al., 1991, Appl . Environ . Microbiol., 57, 3350-3354) . Sulfhydryl group reagents (p-chloro-mercuribenzoate, iodoacetate and N-ethyl maleimide) all inhibited valine transport by 60-70%, indicating that valine is actively transported at high valine concentration . Three kinetically distinguishable transport systems were identified for each strain using whole cells, confirming results obtained with membranes . L-valine transport Kt and Vmax could be an additional tool to estimate the biodiversity of 18 Leuconostoc strains belonging to the dominant flora of French raw milk cheeses . Kt values varied from 20 to 510 nmol/l for the very high affinity system, from 26 to 427 pmol/l for the high affinity system and from 0.65 to 4.40 mmol/l for the low affinity system . No correlation existed between valine transport rates and a particular strain's ability to acidify milk or complex media, suggesting that valine transport is not a growth-limiting function in species of the genus Leuconostoc.

FEMS Microbiol Lett, 2001 Jan 15, 194(2), 245 - 9
Purification and characterisation of acetolactate decarboxylase from Leuconostoc lactis NCW1; O'Sullivan SM et al.; A two-step strategy involving DEAE-cellulose and POROS PI anion exchange chromatography has been developed for rapid purification of acetolactate decarboxylase (ALD) from Leuconostoc lactis NCW1 . This results in 5333-fold purification with a yield of 30% . Purified ALD is a dimer of 49-kDa subunits, has a pH optimum of 6.0, a pI of 4.2 and its activity is independent of metals or branched chain amino acids . At the optimum pH, the K(m) for 2-acetolactate (ALA) was found to be 1.3 mM and the turnover number was 4000 min(-1) . N-terminal sequence comparison with other ALDs showed little sequence conservation in this region . Purified ALD does not catalyse direct production of diacetyl from ALA, unlike the crude extract.

Clin Immunol, 2001 Feb, 98(2), 293 - 300
Cross-reaction of lupus anti-dsDNA antibodies with protein translation factor EF-2; Alberdi F et al.; This report elucidates a new cross-reactive intracellular target of anti-dsDNA antibodies . Previous experiments have demonstrated that some anti-dsDNA antibodies penetrate cells grown in tissue culture and all inhibit in vitro translation . Data implicate a cross-reactive antigen directly involved in protein synthesis: elongation factor-2 (EF-2) . EF-2 was identified by N-terminal sequencing of a band identified with an antibody to the ribosomal protein S1 from Leuconostoc lactis in Western blot assay . Anti-DNA antibodies bind directly to purified EF-2 from bovine liver in dot blot assays . Anti-dsDNA antibodies were shown to inhibit in vitro translation . This inhibiting effect of anti-dsDNA antibodies was partially restored by EF-2 and abrogated by dsDNA, suggesting this cross-reactive specificity . These data demonstrate a cross-reaction between anti-dsDNA antibodies and EF-2 which may lead to cellular dysfunction, as evidenced by inhibition of protein synthesis, and provide a direct pathogenic role for cell penetrating anti-dsDNA antibodies .

J Appl Microbiol, 2000 Nov, 89(5), 862 - 9
Autolysis of dairy leuconostocs and detection of peptidoglycan hydrolases by renaturing SDS-PAGE; Cibik R et al.; The autolysis of lactic acid bacteria plays a major role during cheese ripening . The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs . Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer . The ability of dairy leuconostocs to lyse is strain dependant and not related to the species . The peptidoglycan hydrolase profile of Leuc . mesenteroides subsp . mesenteroides 10L was analysed by renaturing gel electrophoresis . Two major activity bands migrating at 41 and 52 kDa were observed . According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase . The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared . Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species . Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.

Enzyme Microb Technol, 2001 Jan 2, 28(1), 35 - 41
Controlled malolactic fermentation in cider using Oenococcus oeni immobilized in alginate beads and comparison with free cell fermentation; Herrero M et al.; Cells of Oenococcus oeni (formerly Leuconostoc oenos) immobilized in alginate beads were used as starter culture to conduct malolactic fermentation in cider production . Concentrations of major organic acids and volatile compounds were monitored during the process, and results were compared to those obtained when using free cells in the same conditions . The rates of malic acid consumption were similar but lower ethanoic acid content and higher concentration of alcohols were detected with immobilized cells . These features have beneficial effects on the organoleptic properties of cider . A comparison between the kinetic behavior in immobilized and free cells, based on the data obtained for the malic acid consumption, has been developed solving the homogeneous diffusion model when it is applied to the system with immobilized cells.

FEMS Microbiol Lett, 2000 Dec 15, 193(2), 243 - 7
Multiplex PCR-based detection and identification of Leuconostoc species; Lee HJ et al.; A multiplex polymerase chain reaction (PCR) assay has been developed for rapid and reliable identification of Leuconostoc species, by using species-specific primers targeted to the genes encoding 16S rRNA . This assay can detect and differentiate Leuconostoc species from mixed populations in natural sources as well as from pure cultures, within 3 h . This assay system consists of a total of 10 primers, two primers from each target species, and comprises two multiplex PCR reactions: one reaction for Leuconostoc carnosum, Leuconostoc citreum and Leuconostoc mesenteroides, and another reaction for Leuconostoc gelidum and Leuconostoc lactis . This multiplex PCR assay was used to identify 31 Leuconostoc strains isolated from kimchi, a fermented-cabbage product, and the results showed perfect correlation with the results of a polyphasic method, including 16S rDNA sequencing and DNA-DNA hybridization . In addition, this assay enables simultaneous detection of the above-mentioned Leuconostoc species when chromosomal DNA from these Leuconostoc species was mixed . Thus, these results suggest that this multiplex PCR is a rapid and reliable method for identification of Leuconostoc species in pure cultures or in mixed populations.

Biochemistry, 2000 Dec 12, 39(49), 15012 - 21
Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase; Vought V et al.; 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.

Biochemistry, 2000 Dec 12, 39(49), 15002 - 11
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; Cosgrove MS et al.; 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.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 53 - 7
Location of repeat elements in glucansucrases of Leuconostoc and Streptococcus species; Janecek S et al.; Glucosyltransferases of oral streptococci, dextransucrases and alternansucrase of Leuconostoc mesenteroides, collectively referred to as glucansucrases, are large extracellular enzymes that synthesise glucans with a variety of structures and properties . A characteristic of all these glucansucrases is the possession of a C-terminal domain consisting of a series of tandem amino acid repeats . These repeat units are thought to interact with glucan but closely resemble the cell wall binding domain motif found in choline binding proteins in Streptococcus pneumoniae and surface-located proteins in a range of other bacteria . Analysis of dextransucrase and alternansucrase sequences has now shown that they also contain these repeat motifs in the N-terminal region, raising questions about their evolutionary origin and functional importance.

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1915 - 9
Leuconostoc kimchii sp . nov., a new species from kimchi; Kim J et al.; A Gram-positive, catalase-negative, facultatively anaerobic, coccus-shaped bacterium, designated IH25T, was isolated from kimchi, a traditional Korean vegetable product . Phylogenetic analysis based on almost complete 16S rDNA sequences placed the isolate in a monophyletic clade corresponding to the genus Leuconostoc . All validly described species in the genus Leuconostoc, with the exception of Leuconostoc fallax, showed high sequence identity of over 97% . The 16S rDNA sequence of strain IH25T showed the highest homology to those of Leuconostoc gelidum DSM 5578T (98.9%) and Leuconostoc citreum KCTC 3526T (98.3 %) . However, DNA-DNA hybridization experiments indicated that the organism represents a novel genomic species in the genus, since the previously known leuconostocs share DNA homology with strain IH25T of less than 70% . In this work, it is proposed that isolate IH25T be classified in the genus Leuconostoc as Leuconostoc kimchii sp . nov . The type strain of Leuconostoc kimchii is IH25T (= KCTC 2386T = IMSNU 11154T).

J Biol Chem, 2000 Dec 15, 275(50), 39130 - 6
Arg-425 of the citrate transporter CitP is responsible for high affinity binding of di- and tricarboxylates; Bandell M et al.; The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes exchange of divalent anionic citrate from the medium for monovalent anionic lactate, which is an end product of citrate degradation . The exchange generates a membrane potential and thus metabolic energy for the cell . The mechanism by which CitP transports both a divalent and a monovalent substrate was the subject of this investigation . Previous studies indicated that CitP is specific for substrates containing a 2-hydroxycarboxylate motif, HO-CR(2)-COO(-) . CitP has a high affinity for substrates that have a "second" carboxylate at one of the R groups, such as divalent citrate and (S)-malate (Bandell, M., and Lolkema, J . S . (1999) Biochemistry 38, 10352-10360) . Monovalent anionic substrates that lack this second carboxylate were found to bind with a low affinity . In the present study we have constructed site-directed mutants, changing Arg-425 into a lysine or a cysteine residue . By using two substrates, i.e . (S)-malate and 2-hydroxyisobutyrate, the substrate specificity of the mutants was analyzed . In both mutants the affinity for divalent (S)-malate was strongly decreased, whereas the affinity for monovalent 2-hydroxyisobutyrate was not . The largest effect was seen when the arginine was changed into the neutral cysteine, which reduced the affinity for (S)-malate over 50-fold . Chemical modification of the R425C mutant with the sulfhydryl reagent 2-aminoethyl methanethiosulfonate, which restores the positive charge at position 425, dramatically reactivated the mutant transporter . The R425C and R425K mutants revealed a substrate protectable inhibition by other sulfhydryl reagents and the lysine reagent 2,4,6-trinitrobenzene sulfonate, respectively . It is concluded that Arg-425 complexes the charged carboxylate present in divalent substrates but that is absent in monovalent substrates, and thus plays an important role in the generation of the membrane potential.

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2701 - 5
In vitro antimicrobial properties of recombinant ASABF, an antimicrobial peptide isolated from the nematode Ascaris suum; Zhang H et al.; ASABF is a CSalphabeta-type antimicrobial peptide that contains four intramolecular disulfide bridges (Y . Kato and S . Komatsu, J . Biol . Chem . 271:30493-30498, 1996) . In the present study, a recombinant ASABF was produced by using a yeast expression system, and its antimicrobial activity was characterized in detail . The recombinant ASABF was active against all gram-positive bacteria tested (7 of 7; minimum bactericidal concentration {MBC}, 0.03 to 1 microg/ml) except Leuconostoc mesenteroides, some gram-negative bacteria (8 of 14; MBC, >0.5 microg/ml), and some yeasts (3 of 9; MBC >3 microg/ml) . Slight hemolytic activity (4.2% at 100 microg/ml) against human erythrocytes was observed only under low-ionic-strength conditions . Less than 1 min of contact was enough to kill Staphylococcus aureus ATCC 6538P . The bactericidal activity against S . aureus was inhibited by salts.

J Org Chem, 2000 Jul 28, 65(15), 4498 - 508
alpha-fluorinated phosphonates as substrate mimics for glucose 6-phosphate dehydrogenase: the CHF stereochemistry matters; Berkowitz DB et al.; Reported is a systematic study of the "fitness" (in terms of kcat/Km) of a series of phosphonate mimics of glucose 6-phosphate (G6P) as unnatural substrates for G6P dehydrogenase from Leuconostoc mesenteroides . The four G6P analogues (9, 10, 15a, and 15b) differ only in the degree of fluorination at the "bridging" phosphonate carbon . All have been synthesized from benzyl 6-O-trifluoromethanesulfonyl-2,3,4-tri-O-benzyl beta-D-glucopyranoside (6) . The phosphonates with bridging CH2 (9) and CF2 (10) groups are cleanly obtained by direct displacements with the appropriate LiX2CP(O)(OEt)2 reagents (X = H, F) in 15 min at -78 degrees C . For the (alpha-monofluoro)alkylphosphonates (15a/b), homologation of 6 is achieved via lithiodithiane-mediated triflate displacement, followed by aldehyde unmasking {CaCO3, Hg(ClO4)2, H2O} . Addition of diethyl phosphite anion produces diastereomeric, (alpha-hydroxy)phosphonates 13a/b (1.4:1 ratio) which may be readily separated by chromatography . The stereochemistry of the minor diastereomer was established as 7(S) via X-ray crystallographic structure determination of its p-bromobenzoate derivative, 16b . Treatment of the major 7(R) diastereomer with DAST produces alpha-fluorinated phosphonate 14a, in modest yield, with inversion of configuration, as established, again, by X-ray crystallography . To our knowledge, this is first example of DAST-mediated fluorination of a (nonbenzylic, nonpropargylic) secondary (alpha-hydroxy)phosphonate and thus establishes the stereochemical course of this transformation . alpha-Deprotonation/kinetic quenching of 14a provides access to the 7(R)-epimer (14b) . For all four protected phosphonates (7, 8, 14a, and 14b), diethyl phosphonate ester deprotection was carried out with TMSBr, followed by global hydrogenolytic debenzylation to produce the free phosphonates, as alpha/beta anomeric mixtures . Titrations of G6P itself and the free phosphonic acids provides second pKa values of 6.5 (1, bridging-O), 5.4 (10, bridging-CF2), 6.2 (14a, bridging-CHF), and 7.6 (9, bridging-CH2) . Leuconostoc mesenteroides G6PDH-mediated oxidation and Lineweaver-Burk analysis yields normalized kcat/Km values of 0.043 (14b, bridging-7(R)-CHF), 0.11 (10, bridging-CF2), 0.23 (14b, bridging-CH2), and 0.46 (14a, bridging-7(S)-CHF) relative to G6P itself, largely reflecting differences in Km . The fact that kcat/Km increases by more than an order of magnitude in going from the 7(R)-alpha-monofluoroalkyl phosphonate (worst substrate) to the 7(S)-diastereomer (best substrate) is especially notable and is discussed in the context of the known phosphate binding pocket of this enzyme as revealed by X-ray crystallography (Adams, M . J . et al . Structure 1994, 2, 1073-1087).

Syst Appl Microbiol, 2000 Jun, 23(2), 267 - 78
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; Cibik R et al.; 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.

J Agric Food Chem, 2000 Jun, 48(6), 2281 - 5
Strains of lactic acid bacteria isolated from sour doughs degrade phytic acid and improve calcium and magnesium solubility from whole wheat flour; Lopez HW et al.; Five strains of lactic bacteria have been isolated from sour doughs and examined for their ability to degrade phytic acid . In white flour medium in which phytic acid was the only source of phosphorus, the disappearance of phytate and an elevation of inorganic phosphate were observed after only 2 h of incubation in all strains tested (-30 and +60%, respectively) . Both phenomena correspond to phytate breakdown . No difference was observed in the levels of phytic acid hydrolysis among strains, suggesting that phytase enzymes are similar among these bacteria . Using whole wheat flour medium naturally rich in phytic acid in the presence of Leuconostoc mesenteroides strain 38, a 9 h fermentation established that the degradation of PA and the production of lactic acid lead to greater Ca and Mg solubility than in control medium.

Appl Environ Microbiol, 2000 Jul, 66(7), 3098 - 101
Absence of a putative mannose-specific phosphotransferase system enzyme IIAB component in a leucocin A-resistant strain of Listeria monocytogenes, as shown by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ramnath M et al.; Leucocin A is a class IIa bacteriocin produced by Leuconostoc spp . that has previously been shown to inhibit the growth of Listeria monocytogenes . A spontaneous resistant mutant of L . monocytogenes was isolated and found to be resistant to leucocin A at levels in excess of 2 mg/ml . The mutant showed no significant cross-resistance to nontype IIa bacteriocins including nisaplin and ESF1-7GR . However, there were no inhibition zones found on a lawn of the mutant when challenged with an extract containing 51,200 AU of pediocin PA-2 per ml as determined by a simultaneous assay on the sensitive wild-type strain . DNA and protein analysis of the resistant and susceptible strains were carried out using silver-stained amplified fragment length polymorphism (ssAFLP) and one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively . Two-dimensional SDS-PAGE clearly showed a 35-kDa protein which was present in the sensitive but absent from the resistant strain . The N-terminal end of the 35-kDa protein was sequenced and found to have an 83% homology to the mannose-specific phosphotransferase system enzyme IIAB of Streptococcus salivarius.

J Bacteriol, 2000 Jul, 182(14), 3904 - 12
Transcriptional control of the citrate-inducible citMCDEFGRP operon, encoding genes involved in citrate fermentation in Leuconostoc paramesenteroides; Martin M et al.; In this study we describe the expression pattern of the Leuconostoc paramesenteroides citMCDEFGRP operon in response to the addition of citrate to the growth medium . An 8.8-kb polycistronic transcript, which includes the citMCDEFGRP genes, was identified; its synthesis was dramatically induced upon addition of citrate to the growth medium . We also found that expression of the cit operon is subjected to posttranscriptional regulation, since processing sites included in four complex secondary structures (I, II, III, and IV) were identified by Northern blot analysis and mapped by primer extension . Upstream of the citMCDEFGRP operon a divergent open reading frame, whose expression was also increased by citrate, was identified by DNA sequencing and designated citI . The start and end sites of transcription of the cit operon and citI gene were mapped . The start sites are separated by a stretch of 188 bp with a very high A+T content of 77% and are preceded by transcriptional promoters . The end sites of the transcripts are located next to the 3' end of two secondary structures characteristic of rho-independent transcriptional terminators . The effect of the citI gene on expression of the cit operon was studied in Escherichia coli . The presence of the citI gene in cis and in trans resulted in increased activity of the cit promoter . These data provide the first evidence that citrate fermentation in Leuconostoc is regulated at the transcriptional level by a transcriptional activator rather than by a repressor.

Eur J Biochem, 2000 Jul, 267(13), 4127 - 36
Involvement of Gln937 of Streptococcus downei GTF-I glucansucrase in transition-state stabilization; Monchois V et al.; Multiple alignment of deduced amino-acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well-conserved catalytic domain . A portion of this domain displays homology to members of the alpha-amylase family (glycoside hydrolase family 13), which all have a (beta/alpha)8 barrel structure . In the glucansucrases, however, the alpha-helix and beta-strand elements are circularly permuted with respect to the order in family 13 . Previous work has shown that amino-acid residues contributing to the active site of glucansucrases are situated in structural elements that align with those of family 13 . In alpha-amylase and cyclodextrin glucanotransferase, a histidine residue has been identified that acts to stabilize the transition state, and a histidine is conserved at the corresponding position in all other members of family 13 . In all the glucansucrases, however, the aligned position is occupied by glutamine . Mutants of glucosyltransferase I were constructed in which this glutamine, Gln937, was changed to histidine, glutamic acid, aspartic acid, asparagine or alanine . The effects on specific activity, ability to form glucan and ability to transfer glucose to a maltose acceptor were examined . Only histidine could substitute for glutamine and maintain Michaelis-Menten kinetics, albeit at a greatly reduced kcat, showing that Gln937 plays a functionally equivalent role to the histidine in family 13 . This provides additional evidence in support of the proposed alignment of the (beta/alpha)8 barrel structures . Mutation at position 937 altered the acceptor reaction with maltose, and resulted in the synthesis of novel gluco-oligosaccharides in which alpha1,3-linked glucosyl units are joined sequentially to maltose.

Enzyme Microb Technol, 2000 Jun 1, 26(9-10), 834 - 839
Continuous cider fermentation with co-immobilized yeast and Leuconostoc oenos cells; Nedovic VA et al.; Ca-alginate matrix was used to co-immobilize Saccharomyces bayanus and Leuconostoc oenos in one integrated biocatalytic system in order to perform simultaneously alcoholic and malo-lactic fermentation of apple juice to produce cider, in a continuous packed bed bioreactor . The continuous process permitted much faster fermentation compared with the traditional batch process . The flavor formation was also better controlled . By adjusting the flow rate of feeding substrate through the bioreactor, i.e . its residence time, it was possible to obtain either "soft" or "dry" cider . However, the profile of volatile compounds in the final product was modified comparatively to the batch process, especially for higher alcohols, isoamylacetate, and diacetyl . This modification is due to different physiology states of yeast in two processes . Nevertheless, the taste of cider was quite acceptable.

Appl Biochem Biotechnol, 2000 Apr, 87(1), 57 - 70
Cellular association of glucosyltransferases in Leuconostoc mesenteroides and effects of detergent on cell association; Zahnley JC et al.; Most glucosyltransferase (GTF) activity in sucrose-grown cultures of some strains of Leuconostoc mesenteroides is found with the cell pellet after centrifugation . GTFs are known to bind to dextrans, and it was traditionally assumed that cell-associated GTFs were bound to those dextrans that cosedimented with the cells . We used a mutant strain (LC-17), derived from strain NRRL B-1355, which produced dextransucrase in the absence of dextrans, to investigate the extent to which GTFs were bound to cells or dextrans . Much of the GTF activity in glucose-grown cultures of strain LC-17, which do not produce dextran, was located in the cell pellets . Soluble enzyme activity increased when cell suspensions from glucose- or sucrose-grown cultures were incubated with mild nonionic detergents or zwitterionic reagents . Alternansucrase produced by the parent strain B-1355 was almost entirely associated with cells under conditions in which dextrans were or were not produced . Alternansucrase, but not dextransucrase, tended to be enriched in the particulate fraction of B-1355 cells that had been broken in a French press . The distribution of alternansucrase and the effects of detergents on the distribution of GTFs suggest that soluble GTFs sequestered in the cytoplasm, and GTFs bound or adsorbed to the cell membrane are probably the major contributors to the cell-associated GTF activity.

FEBS Lett, 2000 May 19, 473(3), 363 - 9
A novel putative transcription factor protein MYT2 that preferentially binds supercoiled DNA and induces DNA synthesis in quiescent cells; Shao W et al.; Myelin transcription factor 2 (MYT2), a putative transcription factor found in the human central nervous system, was cloned from an expression cDNA library from human T-cells . MYT2 shares weak similarity to bacterial type I topoisomerases and shares 63% sequence identity to a replicase from Leuconostoc mesenteroides . MYT2 preferentially binds supercoiled DNA (scDNA) . Incubation of MYT2 and scDNA at or above equal molar ratios generated topoisomer-like patterns that were abolished by deproteination . Thus, MYT2 appears to relax scDNA via a non-enzymatic mechanism . The banding pattern of MYT2-scDNA complexes was shown to be quantisized, saturable and sequence-independent . Microinjection of MYT2 mRNA induced G(o) growth-arrested NIH 3T3 cells to enter the S phase of the cell cycle.

Structure Fold Des, 2000 May 15, 8(5), 463 - 70
Enzymes of vancomycin resistance: the structure of D-alanine-D-lactate ligase of naturally resistant Leuconostoc mesenteroides; Kuzin AP et al.; BACKGROUND: The bacterial cell wall and the enzymes that synthesize it are targets of glycopeptide antibiotics (vancomycins and teicoplanins) and beta-lactams (penicillins and cephalosporins) . Biosynthesis of cell wall peptidoglycan requires a crosslinking of peptidyl moieties on adjacent glycan strands . The D-alanine-D-alanine transpeptidase, which catalyzes this crosslinking, is the target of beta-lactam antibiotics . Glycopeptides, in contrast, do not inhibit an enzyme, but bind directly to D-alanine-D-alanine and prevent subsequent crosslinking by the transpeptidase . Clinical resistance to vancomycin in enterococcal pathogens has been traced to altered ligases producing D-alanine-D-lactate rather than D-alanine-D-alanine . RESULTS: The structure of a D-alanine-D-lactate ligase has been determined by multiple anomalous dispersion (MAD) phasing to 2.4 A resolution . Co-crystallization of the Leuconostoc mesenteroides LmDdl2 ligase with ATP and a di-D-methylphosphinate produced ADP and a phosphinophosphate analog of the reaction intermediate of cell wall peptidoglycan biosynthesis . Comparison of this D-alanine-D-lactate ligase with the known structure of DdlB D-alanine-D-alanine ligase, a wild-type enzyme that does not provide vancomycin resistance, reveals alterations in the size and hydrophobicity of the site for D-lactate binding (subsite 2) . A decrease was noted in the ability of the ligase to hydrogen bond a substrate molecule entering subsite 2 . CONCLUSIONS: Structural differences at subsite 2 of the D-alanine-D-lactate ligase help explain a substrate specificity shift (D-alanine to D-lactate) leading to remodeled cell wall peptidoglycan and vancomycin resistance in Gram-positive pathogens.

Syst Appl Microbiol, 1999 Dec, 22(4), 507 - 13
Influence of lactose-citrate co-metabolism on the differences of growth and energetics in Leuconostoc lactis, Leuconostoc mesenteroides ssp . mesenteroides and Leuconostoc mesenteroides ssp . cremoris
Hache C, Cachon R, Wache Y, Belguendouz T, Riondet C, Deraedt A, Divies C.
The biodiversity of growth and energetics in Leuconostoc sp . has been studied in MRS lactose medium with and without citrate . On lactose alone, Ln . lactis has a growth rate double that of Ln . cremoris and Ln . mesenteroides . The pH is a more critical parameter for Ln . mesenteroides than for Ln . lactis or Ln . cremoris; without pH control Ln . mesenteroides is unable to acidify the medium under pH 4.5, while with pH control and as a consequence of a high Y(ATP) its growth is greater than Ln . lactis and Ln . cremoris . In general, lactose-citrate co-metabolism increases the growth rate, the biomass synthesis, the lactose utilisation ratio, and the production of lactate and acetate from lactose catabolism . The combined effect of the pH and the co-metabolism lactose-citrate on the two components of the proton motive force (deltap = deltapsi - ZdeltapH) has been studied using resting-cell experiments . At neutral pH deltap is nearly entirely due to the deltapsi, whereas at acidic pH the deltapH is the major component . On lactose alone, strains have a different aptitude to regulate their intracellular pH value, for Ln . mesenteroides it drastically decreases at acidic pH values (pH, = 5.2 for pH 4), while for Ln . lactis and Ln . cremoris it remains above pH 6 . Lactose-citrate co-metabolism allows a better control of pH homeostasis in Ln . mesenteroides, consequently the pHi becomes homogeneous between the three strains studied, for pH 4 it is in an interval of 0.3 pH unit (from pHi = 6.4 to pHi = 6.7) . In this metabolic state, and as a consequence of the variation in deltapH, and to some extent in the deltapsi, the difference of deltap between the three strains is restricted to an interval of 20 mV.

J Appl Microbiol, 2000 May, 88(5), 756 - 63
Dose-response relationships . A model for describing interactions, and its application to the combined effect of nisin and lactic acid on Leuconostoc mesenteroides; Cabo ML et al.; As well as producing bacteriocins, many lactic bacteria produce other potentially toxic compounds or growth inhibitors, especially lactic acid, which may interfere in the assays commonly used to quantify these peptides . A systematic set of modifications is proposed which, when applied to the logistical equation, enable it to describe the combined (but not additive) effects of two or more active principles . The general model thus derived is applied to the interaction of nisin and lactic acid on Leuconostoc mesenteroides.

Appl Environ Microbiol, 2000 May, 66(5), 1923 - 7
Mutagenesis of asp-569 of glucosyltransferase I glucansucrase modulates glucan and oligosaccharide synthesis; Monchois V et al.; Glucansucrases of oral streptococci and Leuconostoc mesenteroides are enzymes of medical and biotechnological interest that synthesize alpha-glucans . They can also synthesize oligosaccharides in the presence of a sugar acceptor . Previous reports have identified an amino acid residue that may affect the structure of the glucan product; therefore, random mutagenesis of the corresponding Asp-569 of Streptococcus downei glucosyltransferase I (GTF-I) was used to further understanding of its involvement in the catalytic mechanism and to evaluate how different amino acids can modulate glucan and oligosaccharide synthesis . GTF-I variants were obtained where Asp-569 was replaced by each of the different possible classes of amino acids . These were expressed in Escherichia coli and purified by means of a His(6) tag . The results showed that the amino acid in position 569 influences the structure of the glucan and the size of the oligosaccharides produced by GTF-I . The results suggest that the amino acid occupying this position is more likely to interact with the acceptor molecules (oligosaccharides or elongating glucan chain) than to be directly involved in glucosyl transfer from sucrose . Engineering of the equivalent position in glucansucrases thus appears to be a good target to expand the range of oligosaccharides synthesized.

Structure Fold Des, 2000 Mar 15, 8(3), 293 - 303
Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency; Au SW et al.; BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress . The human enzyme is in a dimer<-->tetramer equilibrium and its stability is dependent on NADP(+) concentration . G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy . Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia . RESULTS: We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution . The tetramer is a dimer of dimers . Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment . The few dimer-dimer contacts making the tetramer are charge-charge interactions . CONCLUSIONS: The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes . The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule . They also indicate that a stable dimer is essential to retain activity in vivo . As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.

Appl Environ Microbiol, 2000 Apr, 66(4), 1744 - 8
Method for rapid purification of class IIa bacteriocins and comparison of their activities; Guyonnet D et al.; A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105 . The same procedure was successfully applied to the purification of five other anti-Listeria bacteriocins identified by mass spectrometry . Specific activities of the purified bacteriocins were compared.

Biosci Biotechnol Biochem, 2000 Jan, 64(1), 134 - 41
Enzymatic synthesis of stable, odorless, and powdered furanone glucosides by sucrose phosphorylase; Kitao S et al.; Sucrose phosphorylase from Leuconostoc mesenteroides catalyzed transglucosylation from sucrose to 4-hydroxy-3(2H)-furanone derivatives . When 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone or 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (EHMF) were used as acceptors, their transfer ratios were more than 45% . In the case of glucosylation of HDMF, the major transfer product was identified as 2,5-dimethyl-3(2H)-furanone 4-O-alpha-D-glucopyranoside (DMF-G) . In the case of glucosylation of EHMF, two major transfer products were obtained, and their structures were identified as 2-ethyl-5-methyl-3(2H)-furanone 4-O-alpha-D-glucopyranoside (2E5MF-G) and 5-ethyl-2-methyl-3(2H)-furanone 4-O-alpha-D-glucopyranoside (5E2MF-G) on the bases of spectrometric investigations . These glucosides were more stable than each aglycone . The glucosylated HDMF, DMF-G, was an odorless chemical, on the other hand, HDMF had a pineapple flavor . The glucosylated EHMF (EMF-G) were white odorless powders, though aglycone EHMF was a pale yellow syrup like a caramel with an intense sweet odor . Although DMF-G and EMF-G showed little radical-scavenging activity, hydrolyzates of these glucosides by an intestinal acetone powder from pigs had antioxidative activity as well as their aglycones . It was suggested that these glucosides improved some physical properties and may become prodrugs by glucosylation.

Biosci Biotechnol Biochem, 2000 Jan, 64(1), 29 - 38
Gene encoding a dextransucrase-like protein in Leuconostoc mesenteroides NRRL B-512F; Funane K et al.; 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.

Appl Environ Microbiol, 2000 Mar, 66(3), 976 - 81
Effects of pH and trace minerals on long-term starvation of Leuconostoc mesenteroides; Kim DS et al.; Laboratory experiments have definitively shown that exopolymer-producing bacteria have the potential to modify the flow of fluids in oil reservoirs to enhance oil production . Once injected into the reservoir, they will be subjected to a wide range of pH values and to starvation resulting from nutrient depletion . For successful field implementation it is necessary to have a fundamental understanding of these effects on the viability of bacteria . This paper addresses the effects of pH and trace minerals on cell viability of Leuconostoc mesenteroides during carbon source depletion . Two different carbon sources were used to grow cells before transferring the cells to starvation conditions: sucrose and a combination of glucose and fructose . These substrates were chosen because L . mesenteroides produces a significant amount of water-insoluble exopolymers (dextran) under sucrose-fed conditions, which may enhance cell survival under harsh conditions . The effects of dextran on the cell viability were tested at different pH values with and without trace minerals . The rate of cell death followed an exponential-decay law for different values of the solution pH . The optimal solution pH for survival was pH 5, whereas cells died rapidly at pH 3 and below and at pH 13 and above . The sucrose-fed cells showed a greater viability than cells fed glucose and fructose for all pH ranges tested . The results indicated that water-insoluble exopolymers help cells survive for longer periods of time under starvation conditions . The effects of trace minerals on cell culturability were tested at two pH values, 4.5 and 7 . For both cases, cells showed a greater culturability (smaller decay rate constant) in the presence of trace minerals than without trace minerals . It was also found that the effects of trace minerals on cell culturability were greater for glucose-fructose-fed cells than for sucrose-fed cells . The Michaelis pH function theory was used for comparing the relationships between the cell decay rate and pH.

J Appl Microbiol, 1999 Dec, 87(6), 907 - 14
A method for bacteriocin quantification; Cabo ML et al.; Different aspects of the most commonly used assay methods in the study of bacteriocins were examined . The conditions under which extraction and incubation (including exposure time) take place were analysed, and several different formal models that are usually employed to calculate ID50 were compared . As an alternative designed to overcome the problems which characterize the response of micro-organisms that are sensitive to bacteriocins, an operative procedure in a liquid medium and a modified re-parameterized logistic equation is proposed . When applied to the inhibition of Leuconostoc mesenteroides by nisin, the model allows an optimal experimental procedure to be defined.

Appl Environ Microbiol, 2000 Jan, 66(1), 422 - 4
Specific detection of Leuconostoc mesenteroides subsp . mesenteroides with DNA primers identified by randomly amplified polymorphic DNA analysis; Moschetti G et al.; Randomly amplified polymorphic DNA analysis using primer 239 (5' CTGAAGCGGA 3') was performed to characterize Leuconostoc sp . strains . All the strains of Leuconostoc mesenteroides subsp . mesenteroides (with the exception of two strains), two strains formerly identified as L . gelidum, and one strain of Leuconostoc showed a common band at about 1.1 kb . This DNA fragment was cloned and sequenced in order to verify its suitability for identifying L . mesenteroides subsp . mesenteroides strains.

FEMS Microbiol Lett, 2000 Jan 1, 182(1), 81 - 5
Sequence analysis of the gene encoding alternansucrase, a sucrose glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355; Arguello-Morales MA et al.; 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.

Appl Environ Microbiol, 1999 Dec, 65(12), 5504 - 9
Induction and transcription studies of the dextransucrase gene in Leuconostoc mesenteroides NRRL B-512F; Quirasco M et al.; 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.

Appl Microbiol Biotechnol, 1999 Nov, 52(5), 660 - 5
Isolation of key amino acid residues at the N-terminal end of the core region Streptococcus downei glucansucrase, GTF-I; Monchois V et al.; Related streptococcal and Leuconostoc mesenteroides glucansucrases are enzymes of medical and biotechnological interest . Molecular modelling has suggested that the catalytic domain contains a circularly permuted version of the (beta/alpha)8 barrel structure found in the amylase superfamily, and site-directed mutagenesis has identified critical amino acids in this region . In this study, sequential N-terminal truncations of Streptococcus downei GTF-I showed that key amino acids are also present in the first one-third of the core domain . Mutations were introduced at Trp-344, Glu-349 and His-355, residues that are conserved in all glucansucrases and lie within a region which is a target for inhibitory antibodies . W344L, E349L and H355V substitutions were assayed for their effect on mutan synthesis and also on oligosaccharide synthesis with various acceptors . It appeared that Trp-344 and His-355 are involved in the action mechanism of GTF-I; His-355 may also play a role in a binding subsite necessary for oligosaccharide and glucan elongation.

J Clin Microbiol, 1999 Dec, 37(12), 4124 - 6
Leuconostoc pseudomesenteroides as a cause of nosocomial urinary tract infections; Cappelli EA et al.; The phenotypic and genotypic characterization of five clinical isolates of Leuconostoc pseudomesenteroides associated with nosocomially acquired urinary tract infections is described . All the strains were susceptible to chloramphenicol, clindamycin, erythromycin, gentamicin, and tetracycline; all were resistant to nalidixic acid, norfloxacin, and vancomycin; and all were intermediately affected by ampicillin and penicillin . Analysis of chromosomal DNA by pulsed-field gel electrophoresis after treatment with SmaI indicated a clonal relationship of the isolates . The results provide evidence for the possibility of nosocomial transmission of this unusual opportunistic, vancomycin-resistant pathogen.

FEMS Microbiol Lett, 1999 Dec 1, 181(1), 25 - 30
Proteolytic processing of dextransucrase of Leuconostoc mesenteroides; Sanchez-Gonzalez M et al.; Various dextransucrase molecular mass forms found in enzyme preparations may sometimes be products of proteolytic activity . Extracellular protease in Leuconostoc mesenteroides strains NRRL B-512F and B-512FMC dextransucrase preparations was identified . Protease had a molecular mass of 30 kDa and was the predominant form derived from a high molecular mass precursor . The production and activity of protease in culture medium was strongly dependent on pH . When L . mesenteroides dextransucrase (173 kDa) was hydrolyzed by protease, at pH 7 and 37 degrees C, various dextransucrase forms with molecular masses as low as 120 kDa conserving dextransucrase activity were obtained.

Scand J Infect Dis, 1999, 31(4), 371 - 3
Leuconostoc species: a case-cluster hospital infection; Scano F et al.; Leuconostoc species are members of the Streptococcacae family . They are generally regarded as non-pathogenic culture contaminants and are thought to be an uncommon cause of infection . We present a study of a case-cluster nosocomial infection due to Leuconostoc spp . Three patients were hospitalized at the time of the infection with significant underlying diseases and all had a compromised skin and mucous barriers . Two had received previous antibiotic therapy . This report highlights the importance of Leuconostoc spp . as an emerging pathogen, even though the modes of transmission and reservoirs of Leuconostoc spp . are as yet unknown.

Curr Microbiol, 1999 Nov, 39(5), 265 - 9
Characterization of the mesB gene and expression of bacteriocins by Leuconostoc mesenteroides Y105; Hechard Y et al.; 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.

FEMS Microbiol Lett, 1999 Aug 15, 177(2), 243 - 8
Secondary structure of Streptococcus downei GTF-1 glucansucrase; Monchois V et al.; Multiple sequence alignment and structure prediction of glucansucrases produced by oral streptococci and Leuconostoc mesenteroides showed that all have common structural features, with three major domains . There is no conservation of primary sequence or structure in the N-terminal variable region . Sequence-based structure prediction combined with circular dichroism spectrum analysis of purified truncated forms of Streptococcus downei GTF-I revealed that the core catalytic region has a defined structure consistent with the proposed (alpha/beta)8-barrel structure . The C-terminal domain is a mixed structure with significant amounts of beta-sheet and random-coil . This information contributes to the development of our understanding of structure-function relationships in glucansucrases.

J Ind Microbiol Biotechnol, 1999 Jul, 23(1), 656 - 60
Some structural features of an insoluble alpha-D-glucan from a mutant strain of Leuconostoc mesenteroides NRRL B-1355; Cote GL et al.; Leuconostoc mesenteroides strain NRRL B-1355 produces two soluble extracellular alpha-D-glucans from sucrose: alternan and dextran . An unusual mutant strain derived from NRRL B-1355 has recently been isolated which produces practically no soluble polysaccharide, but significant amounts of an insoluble D-glucan . Methylation analysis shows it contains linear (1-->3) and (1-->6) linkages as well as (1-->2) and (1-->3) branch linkages . The insoluble glucan was partially digestible by endodextranase, giving rise to a series of oligosaccharides, a high-molecular weight soluble fraction and an insoluble residue . Treatment of the soluble dextranase-limit fraction with an alpha(1-->2) debranching enzyme led to further dextranase susceptibility . Methylation, FTIR and NMR analyses of the dextranase-treated fractions indicate a non-uniform structure with domains bearing similarities to L . mesenteroides strain NRRL B-1299 dextran and to insoluble streptococcal D-glucans.

J Bacteriol, 1999 Jul, 181(14), 4411 - 6
Genetic organization of the citCDEF locus and identification of mae and clyR genes from Leuconostoc mesenteroides; Bekal-Si Ali S et al.; In this paper, we describe two open reading frames coding for a NAD-dependent malic enzyme (mae) and a putative regulatory protein (clyR) found in the upstream region of citCDEFG of Leuconostoc mesenteroides subsp . cremoris 195 . The transcriptional analysis of the citrate lyase locus revealed one polycistronic mRNA covering the mae and citCDEF genes . This transcript was detected only on RNA prepared from cells grown in the presence of citrate . Primer extension experiments suggest that clyR and the citrate lyase operon are expressed from a bidirectional A-T-rich promoter region located between mae and clyR.

FEMS Microbiol Lett, 1999 May 15, 174(2), 231 - 8
Cloning and molecular characterization of the citrate utilization citMCDEFGRP cluster of Leuconostoc paramesenteroides; Martin M et al.; The citMCDEFGRP cluster from Leuconostoc paramesenteroides involved in citrate utilization was cloned and its nucleotide sequence determined . Homology of the inferred gene products with characterized enzymes reveals that citP encodes the citrate permease P, citC the citrate ligase and citDEF the subunits of the citrate lyase of Leuconostoc . Moreover, it suggests that citM encodes a Leuconostoc malic enzyme . Analysis of citrate consumption by and citrate lyase activity of Lc . paramesenteroides J1{pCITJ1} showed that its citrate permease and its citrate lyase are induced by the presence of citrate in the growth medium . Southern blot analysis demonstrated that the citMCDEFGRP cluster is located in a plasmid.

Biotechnol Bioeng, 1999 May 5, 63(3), 308 - 15
Kinetic modeling of oligosaccharide synthesis catalyzed by leuconostoc mesenteroides NRRL B-1299 dextransucrase
Dols M, Remaud-Simeon M, Willemot RM, Demuth B, Jordening HJ, Buchholz K, Monsan P.
The kinetic behavior of soluble and insoluble forms of dextransucrase from Leuconostoc mesenteroides NRRL B-1299 was investigated with sucrose as substrate and maltose as acceptor . To study the parameters involved, a kinetic model was applied that was previously developed for L . mesenteroides NRRL B-512F dextransucrase . There are significant correlations between the parameters of the soluble form of B-1299 dextransucrase and those calculated for the B-512F enzyme; that is, their properties are comparable and differ from those of the insoluble form of B-1299 dextransucrase . Whereas the calculated parameters for high maltose concentrations describe the kinetic behavior very well, the time curves for low maltose concentrations were not described correctly . Therefore, the parameters were calculated separately for the two ranges .

Acta Crystallogr D Biol Crystallogr, 1999 Apr, 55 ( Pt 4), 826 - 34
Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement; Au SW et al.; Recombinant human glucose 6-phosphate dehydrogenase (G6PD) has been crystallized and its structure solved by molecular replacement . Crystals of the natural mutant R459L grow under similar conditions in space groups P212121 and C2221 with eight or four 515-residue molecules in the asymmetric unit, respectively . A non-crystallographic 222 tetramer was found in the C2221 crystal form using a 4 A resolution data set and a dimer of the large beta + alpha domains of the Leuconostoc mesenteroides enzyme as a search model . This tetramer was the only successful search model for the P212121 crystal form using data to 3 A . Crystals of the deletion mutant DeltaG6PD grow in space group F222 with a monomer in the asymmetric unit; 2.5 A resolution data have been collected . Comparison of the packing of tetramers in the three space groups suggests that the N-terminal tail of the enzyme prevents crystallization with exact 222 molecular symmetry.

J Appl Microbiol, 1999 Feb, 86(2), 175 - 81
Partial characterization and cloning of leuconocin J, a bacteriocin produced by Leuconostoc sp . J2 isolated from the Korean fermented vegetable Kimchi; Choi HJ et al.; Leuconostoc sp . J2, isolated from naturally fermented Kimchi, produced a bacteriocin which was named leuconocin J . This bacteriocin exhibited an inhibitory activity against several lactic acid bacteria and some food-borne pathogens . The antimicrobial substance was secreted into the medium during the late log phase . It appears to be proteinaceous since its activity was completely inactivated by a range of proteolytic enzymes, and it was also relatively heat-stable . The bacteriocin was partially purified by ammonium sulphate precipitation, following dialysis . The apparent molecular mass of partially purified bacteriocin, as indicated by activity detection after Tricine-SDS-PAGE, was 2.5-3.5 kDa . Leuconostoc sp . J2 plasmid DNA digested by EcoRI was cloned into pUC118 and transformed into Escherichia coli DH5 alpha . Phenotypic expression of the bacteriocin production was detected in transformants harboring pULBJ5.5 . Finally, Southern blotting with the 2.3 kb insert as a probe against plasmid digests of Leuconostoc sp . J2 revealed that the cloned foreign DNA originated from Leuconostoc sp . J2.

Can J Microbiol, 1998 Sep, 44(9), 807 - 18
Studies on the large subunit rRNA genes and their flanking regions of Leuconostocs; Nour M; The 16S-23S (spacer-1) and 23S-5S (spacer-2) rRNA intergenic spacer regions of Leuconostoc lactis, Leuconostoc mesenteroides, Leuconostoc mesenteroides subsp . dextranicum, and Leuconostoc mesenteroides subsp . cremoris were amplified by polymerase chain reactions and sequenced . The 23S rRNA genes of Leuconostoc lactis, Leuconostoc mesenteroides, and Leuconostoc mesenteroides subsp . dextranicum were also sequenced . The RNase III-like and RNase E processing sites, as well as putative antitermination signals, were identified within the spacer regions . A single tRNA(Ala) gene without the 3'-terminal CCA sequence was found in spacer-1 regions . Secondary structure models are proposed showing interactions between the two spacer regions of leuconostocs . For all strains studied, spacer-1 and spacer-2 were highly conserved and therefore could not be directly used for strain typing . Sequence information on 23S rRNA genes from Leuconostoc species allowed the determination of regions that can be used as targets for diagnostic probes and amplification primers . Secondary structures of variable helical elements of leuconostocs 23S rRNA were constructed and their primary structures were compared with those of several Gram-positive bacteria with low G+C contents . Comparative analysis revealed that restriction analysis of 23S rRNA variable regions appeared to be sufficient for the search for species-specific signatures . Our experimental observations revealed that one form of the rRNA operons was present in leuconostocs . We have also demonstrated the direct linkage between the three species of rRNA genes, which are organized as follows: 5'-16S rRNA-spacer-1-tRNA(Ala)-23S rRNA-spacer-2-5S rRNA-3'.

Appl Microbiol Biotechnol, 1998 Sep, 50(3), 359 - 63
Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp . mesenteroides FR52 during batch fermentation; Krier F et al.; The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp . mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation . Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates . The optimal temperature was 20 degrees C for production of mesenterocin 52A and 25 degrees C for mesenterocin 52B . Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively . Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other . The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth.

J Med Microbiol, 1998 Oct, 47(10), 923 - 8
Speciation of presumptive viridans streptococci from early onset neonatal sepsis; West PW et al.; Twenty isolates resembling viridans streptococci, 16 from blood and four from gastric aspirates, from 17 cases of early onset neonatal sepsis were identified by the API20 Strep, Rapid ID 32 Strep and conventional tests plus hydrolysis of methylumbelliferyl glycoside substrates . Nineteen of the isolates were identified as species of viridans streptococci and one as a Leuconostoc sp . Ten of the isolates were Streptococcus oralis, three S . mitis biotype 1, two S . mitis biotype 2 and one each of S . sanguis, S . vestibularis, S . salivarius and S . intermedius . The Rapid ID 32 Strep and conventional plus methylumbelliferyl tests gave the same species identity for 17 of the isolates . S . intermedius was identified by the Rapid ID 32 Strep as S . constellatus and S . salivarius as S . equinus, with S . salivarius at lower probability . The API20 Strep failed to identify S . vestibularis and identified S . salivarius as S . defectivus . The absence of certain critical tests, including urea hydrolysis, does not allow the API20 Strep to identify all the currently recognised species of viridans steptococci . The species distribution was unexpected and the incidence of S . oralis and other viridans streptococci in vaginal swabs from prenatal patients is being investigated further.

Appl Environ Microbiol, 1998 Sep, 64(9), 3313 - 9
Identification and characterization of Leuconostoc carnosum, associated with production and spoilage of vacuum-packaged, sliced, cooked ham; Bjorkroth KJ et al.; Leuconostoc carnosum was shown to be the specific spoilage organism in vacuum-packaged, sliced, cooked ham showing spoilage during 3 weeks of shelf life . Identification of the specific spoilage organism was done by use of phenotypic data and ClaI, EcoRI, and HindIII reference strain ribopatterns . One hundred L . carnosum isolates associated with the production and spoilage of the ham were further characterized by pulsed-field gel electrophoresis (PFGE), together with some meat-associated Leuconostoc species: L . citreum, L . gelidum, L . mesenteroides subsp . dextranicum, and L . mesenteroides subsp . mesenteroides . ApaI and SmaI digests divided the industrial L . carnosum strains into 25 different PFGE types, ApaI and SmaI types being consistent . Only one specific PFGE type was associated with the spoiled packages . This type also was detected in air and raw-meat mass samples . The spoilage strain did not produce bacteriocins . Only seven isolates belonging to three different PFGE types produced bacteriocins . Similarity analysis of the industrial L . carnosum strains revealed a homogeneous cluster which could be divided into eight subclusters consisting of strains having at most three-fragment differences . The L . carnosum cluster was clearly distinguished from the other meat-associated leuconostoc clusters, with the exception of the L . carnosum type strain . Ribotyping can be very helpful in the identification of L . carnosum, but its discriminatory power is too weak for strain characterization . PFGE provides good discrimination for studies dealing with the properties of homogeneous L . carnosum strains.

J Appl Microbiol, 1998 Jul, 85(1), 42 - 50
Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4; Hyronimus B et al.; A protease-sensitive antibacterial substance produced by Bacillus coagulans I4 strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named coagulin . The inhibitory spectrum included B . coagulans and unrelated bacteria such as Enterococcus, Leuconostoc, Oenococcus, Listeria and Pediococcus . Coagulin was stable at 60 degrees C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by alpha-amylase, lipase or organic solvents (10% v/v) . Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells . The apparent molecular mass was estimated to be about 3-4 kDa by SDS-PAGE . The B . coagulans I4 strain harbours a plasmid, pI4, approximately 14 kb in size . Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance . Plasmid content of these two derivatives showed that one had lost pI4, whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for coagulin production.

Appl Environ Microbiol, 1998 Aug, 64(8), 3096 - 8
Differentiation of dextran-producing leuconostoc strains by a modified randomly amplified polymorphic DNA protocol
Holt SM, Cote GL.
Seven dextran-producing Leuconostoc strains were differentiated by using a modified randomly amplified polymorphic DNA (RAPD) protocol that incorporated specific primers designed from conserved regions of dextransucrase genes . RAPD profiles showed intraspecies differences among the Leuconostoc mesenteroides strains tested . This modified RAPD protocol will aid in the differentiation of polymer-producing leuconostocs, which are currently distinguished by time-consuming analyses of the dextrans they synthesize.

Enferm Infecc Microbiol Clin, 1998 May, 16(5), 237 - 8
{Infectious endocarditis caused by Leuconostoc mesenteroides}; Vazquez E et al.; BACKGROUND: Leuconostoc spp . are gram-positive coccobacilli, catalase and oxidase negative, vancomycin resistant, with a not clearly defined role in human infectious . Cases of infection have been reported previously but it has not been described confirmed infective endocarditis due to Leuconostoc mesenteroides . METHODS: We describe a case of prosthetic valve endocarditis in a 72-years-old woman with a long previous history of cardiac disease but without other immunological disorders . She developed a heart failure because of prosthetic aortic valve dysfunction . CONCLUSIONS: Leuconostoc mesenteroides should be considered as a potential cause of infective endocarditis and listed in the group of vancomycin-resistant microorganism.

Carbohydr Res, 1997 Dec, 305(3-4), 549 - 59
Structural characterization of the maltose acceptor-products synthesized by Leuconostoc mesenteroides NRRL B-1299 dextransucrase; Dols M et al.; The glucooligosaccharides (GOS), produced by Leuconostoc mesenteroides NRRL B-1299 dextransucrase through an acceptor reaction with maltose and sucrose, were purified by reverse phase chromatography . Logarithmic plots of retention time vs . dp of the GOS gave three parallel lines suggesting the existence of at least three families of homologous molecules . The structure (13C and 1H NMR spectroscopy) and reactivity of the purified molecules of the three families were investigated . All the products bear a maltose residue at the reducing end . The GOS in the first family (named OD) contained additional glucosyl residues all alpha-(1-->6) linked . The smallest molecule in this first series was panose or alpha-D-glucopyranosyl-(1-->6)-D-maltose (dp 3) . All the OD molecules were shown to be good acceptors for dextransucrase in the presence of sucrose . The second family, named R, was composed of linear GOS containing alpha-(1-->6)-linked glucosyl residues and a terminal alpha-(1-->2)-linked residue at the non-reducing end of the molecule; the smallest molecule in this family was alpha-D-glucopyranosyl-(1-->2)-D-panose (dp 4) . The third family, R', was formed of GOS containing additional residues linked through alpha-(1-->6) linkages that constitute the linear chain, and an alpha-(1-->2)-branched residue located on the penultimate element of the chain, near the non-reducing end . The smallest molecule in this series is alpha-D-glucopyranosyl-(1-->6)-{alpha-D-glucopyranosyl-(1-->2)}-alpha-D- glucopyranosyl-(1-->6)-D-panose, dp 6 . R and R' GOS are very poor acceptors for L . mesenteroides NRRL B-1299 dextransucrase . This study makes it possible to suggest a rather simple reaction scheme, where molecules Ri, R'i and ODi of the same dp all result from the glucosylation of the same GOS: ODi-l.

J Appl Microbiol, 1998 Feb, 84(2), 143 - 51
Histidine carboxylase of Leuconostoc oenos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene; Coton E et al.; Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc oenos 9204, a wine lactic acid bacterium . Histidine decarboxylase comprised two subunits, respectively alpha and beta . The hdc gene was cloned and sequenced . The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc . oenos 9204 HDC was synthesized as a precursor proHDC pi 6 (Mr 205,000) . A cleavage between Ser-81 and Ser-82 generated the alpha (Mr 25,380) and beta (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (alpha beta)6 . At the optimal pH of 4.8, the HDC activity exhibited a simple Michaelis-Menten kinetic (K(m) = 0.33 mmol l-1, Vmax = 17.8 mumol CO2 min-1 mg-1), while at pH 7.6 it was sigmoidal (cooperativity index of 2) . Histamine acted as a competitive inhibitor (Ki = 32 mmol l-1) . The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms . These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine . Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.

J Enzyme Inhib, 1998 Apr, 13(2), 147 - 60
Studies on the inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde: evidence for the presence of an essential lysine residue at the active site; Goyal A et al.; The kinetics of inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde showed that the reaction followed pseudo-first order reaction . The loss of enzyme activity was concomitant with an increase in fluorescence at 417 nm indicating that the inhibition involved the reaction of an epsilon-amino and a thiol group of the enzyme leading to the formation of an isoindole derivative . The stoichiometry of inactivation showed that one isoindole derivative was formed per enzyme molecule . The substrates sucrose and glucose provided protection against o-phthalaldehyde inactivation which was also corroborated by fluorescence studies . Dextransucrase was not inactivated by 5,5'-dithiobis(2-nitrobenzoic acid), showing that the cysteine present in close proximity to the lysine is not essential for enzyme activity . Denaturation of dextransucrase by urea or heat treatment prior to o-phthalaldehyde addition resulted in a decrease of fluorescence intensity indicating that the native conformation of the enzyme is essential for isoindole derivative formation . These results established that a lysine residue is present at the active site and is essential for the activity of dextransucrase.

Biochem Mol Biol Int, 1998 May, 44(6), 1167 - 74
Chemical modification of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by pyridoxal 5'-phosphate: evidence for the presence of an essential lysine residue at the active site; Goyal A et al.; The treatment of Leuconostoc mesenteroides NRRL B-512F dextransucrase with lysine specific reagent, pyridoxal 5'-phosphate (PLP) at pH 5.2 and 30 degrees C resulted in the loss of enzyme activity . The inactivation by PLP could be reversed completely by dilution or dialysis . Sucrose as well as acceptor substrates, glucose and dextran protected the enzyme against inactivation by PLP . A statistical, kinetic analysis of the inactivation by PLP showed that one lysine residue is essential for the enzyme activity . All these results showed that one lysine residue present at the active is essential for the activity of dextransucrase.

Microbiology, 1998 May, 144 ( Pt 5), 1343 - 8
Sequence and structural relationships of leucocins A-, B- and C-TA33a from Leuconostoc mesenteroides TA33a; Papathanasopoulos MA et al.; 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.

Curr Microbiol, 1998 Jun, 36(6), 365 - 9
Lysogeny of Oenococcus oeni (syn . Leuconostoc oenos) and study of their induced bacteriophages; Poblet-Icart M et al.; A large number of strains of Oenococcus oeni (formerly Leuconostoc oenos) that had been isolated from wines were checked for lysogeny with mitomycin C as inducer . As a result of this test, 45% of the strains proved to be lysogenic, suggesting that lysogeny is widespread among bacteria isolated from wines during malolactic fermentation . The sensitivity of bacteria to phages was very different, depending on the strain . All the lysogenic strains were resistant to infection by the temperate phage they released . Some phages infected none of the strains . Phages of Oenoc . oeni had a classical morphology, an isometric head, and a long striated tail . With the broadest host strain as an indicator, phages were detected in wines after malolactic fermentation.

Anal Biochem, 1998 May 15, 259(1), 62 - 7
A sensitive equilibrium-based assay for D-lactate using D-lactate dehydrogenase: application to penicillin-binding protein/DD-carboxypeptidase activity assays; Gutheil WG; An assay for D-lactate (D-Lac) is described where D-Lac in the presence of NAD+ is equilibrated to NADH and pyruvate (Pyr) by Leuconostoc mesenteroides D-lactate dehydrogenase (DLDH) . This assay was standardized using known concentrations of D-Lac and a linearized form of the equilibrium expression . The assay has a lower limit of about 20 microM D-Lac in a 1 ml assay mixture (20 nmol D-Lac) . As a demonstration of this assay method it is used to characterize the hydrolysis of the standard penicillin-binding protein/DD-carboxypeptidase substrate Ac2-L-Lys-D-Ala-D-Lac by penicillin-binding protein 5 from Escherichia coli . The approach adopted here of using an inherently nonlinear response, which however follows precisely determinable physical behavior, has the advantages of providing a wider dynamic range and increased relative precision over analogous linear response-based methods . This approach may be applicable to the development of other enzyme-based assay methods.

Appl Environ Microbiol, 1998 May, 64(5), 1644 - 9
Effect of Leuconostoc mesenteroides NRRL B-512F dextransucrase carboxy-terminal deletions on dextran and oligosaccharide synthesis; Monchois V et al.; Dextransucrase (DSR-S) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes synthesis of soluble dextran from sucrose . In the presence of efficient acceptor molecules, such as maltose, the reaction pathway is shifted toward glucooligosaccharide synthesis . Like glucosyltransferases from oral streptococci, DSR-S possesses a C-terminal glucan-binding domain composed of a series of tandem repeats . In order to determine the role of the C-terminal region of DSR-S in dextran or oligosaccharide synthesis, four DSR-S genes with deletions at the 3' end were constructed . The results showed that the C-terminal region modulated the initial velocity of dextran synthesis but that the K(m) for sucrose, the optimum pH, and the activation energy were all unaffected by the deletions . The C-terminal domain modulated the rate of oligosaccharide synthesis whatever acceptor molecule was used (a good acceptor molecule such as maltose or a poor acceptor molecule such as fructose) . The C-terminal domain seemed to play no role in the catalytic process in dextran and oligosaccharide synthesis . In fact, it seems that the role of the C-terminal domain of DSR-S may be to facilitate the translation of dextran and oligosaccharides from the catalytic site.

Biochemistry, 1998 Mar 3, 37(9), 2759 - 67
On the mechanism of the reaction catalyzed by glucose 6-phosphate dehydrogenase; Cosgrove MS et al.; 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.

FEMS Microbiol Lett, 1998 Feb 15, 159(2), 307 - 15
Cloning and sequencing of a gene coding for an extracellular dextransucrase (DSRB) from Leuconostoc mesenteroides NRRL B-1299 synthesizing only a alpha (1-6) glucan; Monchois V et al.; The coding region for a novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene (dsrB) was isolated and sequenced . Using degenerate primers homologous to a conserved region present in dextransucrases from Streptococcus (GTFs) and L . mesenteroides NRRL B-512F (DSRS) and conserved amino acid sequences located in the N-terminal catalytic region of these enzymes, about 60% of the DSRB encoding gene was isolated . Two sites, BamHI and HindIII, were identified which allowed one 0.5-kbp probe to be obtained to isolate the 5' and the 3' ends of dsrB . The nucleotide sequence of the dsrB gene was determined and found to consist of an open reading frame (ORF) of 4521 base pairs (bp) coding for a 1507-amino acid protein with an M1 of 168,511 . The amino acid sequence is very close to that of DSRS . Like DSRS, the dextran produced appeared to be composed of only alpha (1-6) glucosidic bonds, and the oligosaccharides synthesized in the presence of acceptor maltose were also composed of alpha (1-6) linked glucosyl residues in addition to the maltosyl residue . DSRB thus appears to be a novel dextransucrase from L . mesenteroides NRRL B-1299 . DSRB produces a dextran different from the typical dextran containing alpha (1-6) and alpha (1-2) linkages produced when this strain is grown in the presence of sucrose.

Biosci Biotechnol Biochem, 1998 Jan, 62(1), 123 - 7
Glucan binding regions of dextransucrase from Leuconostoc mesenteroides NRRL B-512F; Funane K et al.; 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.

Eur J Biochem, 1998 Jan 15, 251(1-2), 382 - 8
Amino acid substitutions at the dimer interface of human glucose-6-phosphate dehydrogenase that increase thermostability and reduce the stabilising effect of NADP; Scopes DA et al.; Over 100 mutations of the G6PD gene have been documented . With the construction of the molecular model of glucose-6-phosphate dehydrogenase, based on the structure of the bacterial Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, it has been possible to superimpose these amino acid changes on to the structure of the glucose-6-phosphate dehydrogenase molecule . There are a large number of severe disease causing mutations at the dimer interface which usually cause decreased thermostability . We have used this knowledge to predict amino acid changes which would effect an increase in the stability of the dimer . The aspartic acid at residue 421 was chosen as it is a negatively charged residue at the centre of the dimer interface in an area rich in negatively charged residues . This residue was changed to a neutrally charged alanine or asparagine, or a positively charged lysine or arginine . The thermostability of the enzyme was increased when residue 421 was neutral (A or N) and increased further when positive (K or R) . NADP is known to exert a concentration dependent stabilising effect on the glucose-6-phosphate dehydrogenase dimer . However the concentration-dependent stabilising effect of NADP was reduced in the residue-421 substitutions in a manner which was inversely proportional to charge change . These results suggest that changes at the dimer interface can also affect the distant (> 20 A) NADP-binding site, and vice versa; an attempt has been made to explain these interactions based on the molecular model of human glucose-6-phosphate dehydrogenase.

J Bacteriol, 1998 Feb, 180(3), 647 - 54
Purification of Leuconostoc mesenteroides citrate lyase and cloning and characterization of the citCDEFG gene cluster; Bekal S et al.; 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.

Biochemistry, 1997 Dec 9, 36(49), 15062 - 72
Three-dimensional structure of leucocin A in trifluoroethanol and dodecylphosphocholine micelles: spatial location of residues critical for biological activity in type IIa bacteriocins from lactic acid bacteria; Fregeau Gallagher NL et al.; The first three-dimensional structure of a type IIa bacteriocin from lactic acid bacteria is reported . Complete 1H resonance assignments of leucocin A, a 37 amino acid antimicrobial peptide isolated from the lactic acid bacterium Leuconostoc gelidum UAL187, were determined in 90% trifluoroethanol (TFE)-water and in aqueous dodecylphosphocholine (DPC) micelles (1:40 ratio of leucocin A:DPC) using two-dimensional NMR techniques (e.g., DQF-COSY, TOCSY, NOESY) . Circular dichroism spectra, NMR chemical shift indices, amide hydrogen exchange rates, and long-range nuclear Overhauser effects indicate that leucocin A adopts a reasonably well defined structure in both TFE and DPC micelle environments but exists as a random coil in water or aqueous DMSO . Distance geometry and simulated annealing calculations were employed to generate structures for leucocin A in both lipophilic media . While some differences were noted between the structures calculated for the two different solvent systems, in both, the region encompassing residues 17-31 assumes an essentially identical amphiphilic alpha-helix conformation . A three-strand antiparallel beta-sheet domain (residues 2-16), anchored by the disulfide bridge, is also observed in both media . In TFE, these two regions have a more defined relationship relative to each other, while, in DPC micelles, the C-terminus is folded back onto the alpha-helix . The implications of these structural features with regard to the antimicrobial mechanism of action and target recognition are discussed.

Res Microbiol, 1997 Jan, 148(1), 79 - 86
Sequence of DNA 16S/23S spacer region of Leuconostoc oenos (Oenococcus oeni): application to strain differentiation; Le Jeune C et al.; Leuconostoc oenos is involved in malolactic fermentation occurring during wine-making . An increasing number of wines are being inoculated with malolactic starters to control the process, and the identification and differentiation of selected strains are now indispensable both for quality control of production and for commercial purposes . In the present work we evaluated the potential use of the intergenic regions of three L . oenos strains for their differentiation . The three 16S/23S rRNA intergenic spacers were amplified in vitro by PCR, and sequences were compared . The spacer sequence was highly conserved in all strains . Inside this spacer, a tRNA-Ala gene containing an 18-bp sequence stretch which is conserved in all tRNA genes was discovered . This sequence, together with random primers, was used for characterization of ten L . oenos strains by PCR.

J Bacteriol, 1997 Dec, 179(23), 7591 - 4
The rpoN (sigma54) gene from Listeria monocytogenes is involved in resistance to mesentericin Y105, an antibacterial peptide from Leuconostoc mesenteroides; Robichon D et al.; To gain insight into the mode of action of mesentericin Y105, a bacteriocin bactericidal agent against Listeria monocytogenes, we undertook to identify the listerial factors mediating this susceptibility by using a genetic approach . Transposon mutants resistant to the bacteriocin were obtained . One of them corresponded to a transposon insertion in a gene (rpoN) encoding a putative protein (447 amino acids) with strong homologies to alternative transcriptional sigma54 factors, including that of Bacillus subtilis (38% identity) . Complementation experiments with the wild-type rpoN gene demonstrated that the insertion in rpoN was responsible for the resistance phenotype in L . monocytogenes . Moreover, expression of the L . monocytogenes rpoN gene in an rpoN mutant strain of B . subtilis promoted transcription of a sigma54-dependent operon in the presence of the associated regulator . These results demonstrate that the L . monocytogenes rpoN gene encodes a new sigma54 factor.

Appl Microbiol Biotechnol, 1997 Oct, 48(4), 465 - 72
Characterization of Leuconostoc mesenteroides NRRL B-512F dextransucrase (DSRS) and identification of amino-acid residues playing a key role in enzyme activity; Monchois V et al.; Dextransucrase (DSRS) from Leuconostoc mesenteroides NRRL B-512F is a glucosyltransferase that catalyzes the synthesis of soluble dextran from sucrose or oligosaccharides when acceptor molecules, like maltose, are present . The L . mesenteroides NRRL B-512F dextransucrase-encoding gene (dsrS) was amplified by the polymerase chain reaction and cloned in an overexpression plasmid . The characteristics of DSRS were found to be similar to the characteristics of the extracellular dextransucrase produced by L . mesenteroides NRRL B-512F . The enzyme also exhibited a high homology with other glucosyltransferases . In order to identify critical amino acid residues, the DSRS sequence was aligned with glucosyltransferase sequences and four amino acid residues were selected for site-directed mutagenesis experiments: aspartic acid 511, aspartic acid 513, aspartic acid 551 and histidine 661 . Asp-511, Asp-513 and Asp-551 were independently replaced with asparagine and His-661 with arginine . Mutation at Asp-511 and Asp-551 completely suppressed dextran and oligosaccharide synthesis activities, showing that at least two carboxyl groups (Asp-511 and Asp-551) are essential for the catalysis process . However, glucan-binding properties were retained, showing that DSRS has a two-domain structure like other glucosyltransferases . Mutations at Asp-513 and His-661 resulted in greatly reduced dextransucrase activity . According to amino acid sequence alignments of glucosyltransferases, alpha-amylases or cyclodextrin glucanotransferases, His-661 may have a hydrogen-bonding function.

Caries Res, 1997, 31(6), 441 - 50
Adsorption of Streptococcus sobrinus dextranase inhibitor to water-insoluble alpha-D-glucans of oral streptococci; Shaw JM et al.; A low molecular weight dextranase inhibitor from Streptococcus sobrinus has previously been identified and purified . The range of conditions under which inhibition occurs, and the situations in which dextranase activity of S . sobrinus can reappear, have been examined in the chemostat . These studies have revealed that when dextranase production exceeds that of the inhibitor, all the inhibitor is tightly bound into enzyme-inhibitor complexes, and the excess enzyme remains active . Another factor that influences the activity of dextranase inhibitor has now been identified, namely the ability of the inhibitor to bind to water-insoluble glucans . Adsorption to water-insoluble alpha-D-glucans, produced by oral streptococci that were grown in batch culture, increased with their proportion of alpha-1,3-linked sequences of glucose residues . Studies with water-insoluble dextrans of Leuconostoc mesenteroides strains showed that alpha-1,6-linked sequences were also important for binding . The inhibitor was not active when adsorbed to glucan, but active inhibitor was released by incubation with soluble dextran . The interactions of sucrose, alpha-D-glucosyltransferases, alpha-D-glucans, dextranase and dextranase inhibitor are discussed in relation to the growth rate of S . sobrinus . At low growth rate in the chemostat the predominant alpha-D-glucosyltransferase (GTF) is a GTF-S that converts sucrose into soluble dextran, and the activity of free dextranase inhibitor in the culture filtrate is high . By contrast, at high growth rate the streptococci produce GTFs capable of synthesizing water-insoluble alpha-D-glucans, and no free inhibitor is found in culture filtrate . Thus the activity of free, extracellular dextranase inhibitor is controlled by (i) the extent of binding to dextranase and (ii) the extent of adsorption to water-insoluble alpha-D-glucan.

Curr Microbiol, 1997 Dec, 35(6), 331 - 5
Multiple bacteriocin production by Leuconostoc mesenteroides TA33a and other Leuconostoc/Weissella strains; Papathanasopoulos MA et al.; Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra . Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography . Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB) . Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187 . A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread . Lc . carnosum LA54a, W . paramesenteroides LA7a, and Lc . gelidum UAL 187-22 produced all three bacteriocins, whereas W . paramesenteroides OX and Lc . carnosum TA11a produced only leucocin A- and B-type bacteriocins.

J Bacteriol, 1997 Sep, 179(17), 5347 - 54
Biochemical basis for glucose-induced inhibition of malolactic fermentation in Leuconostoc oenos; Miranda M et al.; The sugar-induced inhibition of malolactic fermentation in cell suspensions of Leuconostoc oenos, recently reclassified as Oenococcus oeni (L . M . T . Dicks, F . Dellaglio, and M . D . Collins, Int . J . Syst . Bacteriol . 45:395-397, 1995) was investigated by in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and manometric techniques . At 2 mM, glucose inhibited malolactic fermentation by 50%, and at 5 mM or higher it caused a maximum inhibitory effect of ca . 70% . Galactose, trehalose, maltose, and mannose caused inhibitory effects similar to that observed with glucose, but ribose and 2-deoxyglucose did not affect the rate of malolactic activity . The addition of fructose or citrate completely relieved the glucose-induced inhibition . Glucose was not catabolized by permeabilized cells, and inhibition of malolactic fermentation was not observed under these conditions . 31P NMR analysis of perchloric acid extracts of cells obtained during glucose-malate cometabolism showed high intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate, and glycerol-3-phosphate . Glucose-6-phosphate, 6-phosphogluconate, and NAD(P)H inhibited the malolactic activity in permeabilized cells or cell extracts, whereas NADP+ had no inhibitory effect . The purified malolactic enzyme was strongly inhibited by NADH, whereas all the other above-mentioned metabolites exerted no inhibitory effect, showing that NADH was responsible for the inhibition of malolactic activity in vivo . The concentration of NADH required to inhibit the activity of the malolactic enzyme by 50% was ca . 25 microM . The data provide a coherent biochemical basis to understand the glucose-induced inhibition of malolactic fermentation in L . oenos.

Clin Transplant, 1997 Aug, 11(4), 322 - 4
Leuconostoc bacteremia after liver transplantation: another cause of vancomycin resistant gram-positive infection; Espinoza R et al.; Leuconostoc sp . are gram-positive bacteria intrinsically resistant to vancomycin, which can be confused with streptococci based on routine microbiological-characteristics . Infections secondary to Leuconostoc are uncommon, and usually affect patients with underlying diseases, prior use of vancomycin and those with central lines . The most common clinical presentation is fever secondary to a central line infection . We report the first case of Leuconostoc infection in a solid organ transplant recipient . The patient developed Leuconostoc bacteremia secondary to peritonitis, 60 d after undergoing liver transplantation . He was treated with clindamycin, gentamicin and underwent surgical debridement, but succumbed to other complications.

Eur J Clin Microbiol Infect Dis, 1997 Jul, 16(7), 533 - 5
Bacteremia caused by Leuconostoc cremoris in a patient with severe burn injuries; Jimenez-Mejias ME et al.; A case of Leuconostoc cremoris bacteremia in a patient with burn wounds over 45% of the body surface is presented . Leuconostoc bacteremia has not previously been reported in burn patients . The predisposing factors in this case were severe burn wounds resulting in immunocompromise, presence of both intravenous and enteral feeding catheters, several surgical interventions and previous treatment with imipenem plus amikacin . The patient was successfully treated with ampicillin 2 g l.v . every 6 hours for 21 days . Infection with Leuconostoc should be suspected when vancomycin-resistant organisms resembling streptococci are isolated.

J Appl Microbiol, 1997 Jul, 83(1), 127 - 32
Pediocin PD-1, a bactericidal antimicrobial peptide from Pediococcus damnosus NCFB 1832; Green G et al.; Pediocin PD-1, produced by Pediococcus damnosus NCFB 1832, is inhibitory to several food spoilage bacteria and food-borne pathogens . However, pediocin PD-1 is not active against other Pediococcus spp . and differs in this respect to other pediocins produced by Pediococcus acidilactici and Pediococcus pentosaceus . Production of pediocin PD-1 starts during early growth and reaches-a plateau at the end of exponential growth . Pediocin PD-1 was partially purified and its size was determined by tricine-SDS-PAGE as approximately 3.5 kDa . The isoelectric point (pI) of pediocin PD-1 is approximately 3.5, as determined with the Rotofor electrofocusing cell (BioRad) . Pediocin PD-1 is heat-resistant (10 min at 121 degrees C) and remains active after 30 min of incubation at pH 2-10 . Pediocin PD-1 is resistant to treatment with pepsin, papain, alpha-chemotrypsin and trypsin, but not Proteinase K . Pediocin PD-1 is bactericidal against sensitive cells of Oenococcus oeni (previously Leuconostoc oenos).

Appl Microbiol Biotechnol, 1997 Jun, 47(6), 715 - 8
Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp . mesenteroides; Schmitt P et al.; The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp . mesenteroides results in a growth stimulation, an increase in D-lactate and acetate production and repression of ethanol production . This correlated well with the levels of key enzymes involved . A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed . High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture . In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation.

Appl Environ Microbiol, 1997 Jun, 63(6), 2159 - 65
Growth and energetics of Leuconostoc mesenteroides NRRL B-1299 during metabolism of various sugars and their consequences for dextransucrase production; Dols M et al.; The metabolic and energetic properties of Leuconostoc mesenteroides have been examined with the goal of better understanding the parameters which affect dextransucrase activity and hence allowing the development of strategies for improved dextransucrase production . Glucose and fructose support equivalent specific growth rates (0.6 h-1) under aerobic conditions, but glucose leads to a better biomass yield in anaerobiosis . Both sugars are phosphorylated by specific hexokinases and catabolized through the heterofermentative phosphoketolase pathway . During sucrose-grown cultures, a large fraction of sucrose is converted outside the cell by dextransucrase into dextran and fructose and does not support growth . The other fraction enters the cell, where it is phosphorylated by an inducible sucrose phosphorylase and converted to glucose-6-phosphate (G-6-P) by a constitutive phosphoglucomutase and to heterofermentative products (lactate, acetate, and ethanol) . Sucrose supports a higher growth rate (0.98 h-1) than the monosaccharides . When fructose is not consumed simultaneously with G-1-P, the biomass yield relative to ATP is high (16.8 mol of ATP.mol of sucrose-1), and dextransucrase production is directly proportional to growth . However, when the fructose moiety is used, a sink of energy is observed, and dextransucrase production is no longer correlated with growth . As a consequence, fructose catabolism must be avoided to improve the amount of dextransucrase synthesized.

J Appl Microbiol, 1997 May, 82(5), 578 - 88
Characterization of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA; Villani F et al.; 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.

J Biol Chem, 1997 Apr 4, 272(14), 9210 - 4
D-Alanyl-D-lactate and D-alanyl-D-alanine synthesis by D-alanyl-D-alanine ligase from vancomycin-resistant Leuconostoc mesenteroides . Effects of a phenylalanine 261 to tyrosine mutation; Park IS et al.; The Gram-positive bacterium Leuconostoc mesenteroides, ATCC 8293, is intrinsically resistant to the antibiotic vancomycin . This phenotype correlates with substitution of D-Ala-D-lactate (D-Ala-D-Lac) termini for D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than the dipeptide for vancomycin binding . Overproduction of the L . mesenteroides D-Ala-D-Ala ligase (LmDdl) 2 in E . coli and its purification to approximately 90% homogeneity allow demonstration that the LmDdl2 does have both depsipeptide and dipeptide ligase activity . Recently, we reported that mutation of an active site tyrosine (Tyr), Tyr216, to phenylalanine (Phe) in the E . coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in that enzyme . The vancomycin-resistant LmDdl2 has a Phe at the equivalent site, Phe261 . To test the prediction that a Tyr residue predicts dipeptide ligase while an Phe residue predicts both depsipeptide and dipeptide ligase activity, the F261Y mutant protein of LmDdl2 was constructed and purified to approximately 90% purity . F216Y LmDdl2 showed complete loss of the ability to couple D-Lac but retained D-Ala-D-Ala dipeptide ligase activity . The Tyr-->Phe substitution on the active site omega-loop in D-Ala-D-Ala ligases is thus a molecular indicator of both the ability to make D-Ala-D-Lac and intrinsic resistance to the vancomycin class of glycopeptide antibiotics.

FEMS Microbiol Lett, 1997 Feb 15, 147(2), 279 - 85
Identification and sequence analysis of the region encoding the site-specific integration system from Leuconostoc oenos (OEnococcus oeni) temperate bacteriophage phi 10MC; Gindreau E et al.; Malolactic fermentation conducted by Leuconostoc oenos is an essential step in winemaking . L . oenos bacteriophages are thought to be responsible for fermentation failures, yet they have received little attention . The integration system of bacteriophage phi 10MC in the LOF111 L . oenos strain chromosome was studied and a 1,456 bp phage DNA fragment was cloned and sequenced . An open reading frame (int) showing homology with several temperate bacteriophage integrases was located upstream of the phage attachment site (attP) . This organisation is comparable to other phage site-specific recombination systems . The same bacterial attachment site (attB) located in at tRNA(leu) gene was found in all 15 L . oenos strains studied and was involved in phi 10MC integration in the bacterial chromosome.

Appl Environ Microbiol, 1997 Feb, 63(2), 609 - 14
Molecular characterization of the gene encoding an 18-kilodalton small heat shock protein associated with the membrane of Leuconostoc oenos; Jobin MP et al.; In Leuconostoc oenos, different stresses such as heat, ethanol, and acid shocks dramatically induce the expression of an 18-kDa small heat shock protein called Lo 18 . The corresponding gene (hsp18) was cloned from a genomic library of L . oenos constructed in Escherichia coli . A 2.3-kb DNA fragment carrying the hsp18 gene was sequenced . The hsp18 gene encodes a polypeptide of 148 amino acids with a calculated molecular mass of 16,938 Da . The Lo18 protein has a significant identity with small heat shock proteins of the alpha-crystallin family . The transcriptional start site was determined by primer extension . This experiment allowed us to identify the promoter region exhibiting high similarity to consensus promoter sequences of gram-positive bacteria, as well as E . coli . Northern blot analysis showed that hsp18 consists of a unique transcription unit of 0.6 kb . Moreover, hsp18 expression seemed to be controlled at the transcriptional level . This small heat shock protein was found to be peripherally associated with the membrane of L . oenos.

Gene, 1997 Jan 31, 185(1), 99 - 103
A Leuconostoc lactis protein with homology to ribosomal protein S1 shares common epitopes and common DNA binding properties with a mammalian DNA binding nuclear factor; Yamit-Hezi A et al.; A mouse testis cDNA expression library (Clontech) was screened with a synthetic oligonucleotide ligand containing CT-rich motifs derived from the rat skeletal muscle actin gene promoter . These motifs bind nuclear proteins, and seem to be involved in the regulation of the gene . Analysis of isolated clones, which expressed proteins that specifically bind the oligonucleotide, indicated that they were derived from a single gene . This gene was identified as a contaminant of bacterial origin (Leuconostoc lactis) . The cloned gene from L . lactis encodes a protein with significant homology to bacterial ribosomal protein S1, which we designated LrpS1-L . Band shift analysis and competition experiments indicated that both the bacterial protein and a mouse nuclear protein specifically bind to the same CT-rich motif of the skeletal muscle actin promoter . Furthermore, antibodies against the recombinant bacterial protein interfered with the formation of complex between the CT-rich element and the mouse nuclear protein . These results indicate that the bacterial LrpS1-L protein and the mammalian protein bind the same CT-rich motif and share common antigenic epitopes.

J Basic Microbiol, 1997, 37(3), 197 - 204
Effect of certain nutrients on the production of dextransucrase from Leuconostoc mesenteroides NRRL B-512F; Goyal A et al.; The effects of certain nutrients on dextransucrase (sucrose: 1,6-alpha-d-glucan 6-alpha-d-glucosyltransferase EC 2.4.1.5) production from Leuconostoc mesenteroides NRRL B-512F were studied . An increase in concentration of sucrose to 4% in the enzyme production medium resulted in the increase of activity of dextransucrase . Higher enzyme yields were obtained at low yeast extract and high phosphate concentrations . The presence of peptone and beef extract in the medium in addition to 2% yeast extract resulted in an enhanced production of dextransucrase . The enzyme activity increased by 30% by both peptone and beef extract . Addition of Tween 80 to the medium enhanced the production of enzyme and the activity was increased by 25% . Magnesium ions stimulated the activity marginally . Sodium fluoride enhanced the activity of dextransucrase by 25%.

Scand J Infect Dis, 1997, 29(3), 311 - 2
Pleural empyema caused by Leuconostoc spp; Borer A et al.; A rare case of pleural empyema caused by Leuconostoc spp . is reported . The patient was treated successfully with clindamycin . To our knowledge this is the first reported case of pleural empyema caused by Leuconostoc spp . In a patient with characteristic predisposing factors, such as a serious underlying disease, previous vancomycin therapy and thoracic access device . Our case illustrates that Leuconostoc spp . can cause pleural infection as further evidence of its human pathogenicity.

Scand J Infect Dis, 1997, 29(3), 310 - 1
Leuconostoc spp . septicaemia in a child with short bowel syndrome; Monsen T et al.; Septicaemia caused by the vancomycin-resistant Gram-positive bacteria Leuconostoc spp . is uncommon . We report a case of Leuconostoc spp . septicaemia in a child with short bowel syndrome fed through a central venous catheter and a gastrostomy . Leuconostoc spp . were isolated from several blood cultures . Despite several courses of antibiotics the fever continued and her condition deteriorated . After removal of the thrombotized central venous catheter her condition improved . Leuconostoc spp . was isolated from the thrombotic masses.

FEMS Microbiol Lett, 1996 Dec 15, 145(3), 445 - 50
Cloning and sequencing of the gene encoding alpha-acetolactate decarboxylase from Leuconostoc oenos; Garmyn D et al.; The alsD gene encoding alpha-acetolactate decarboxylase was isolated from a genomic library of Leuconostoc oenos, using a screening procedure developed on microtiter plates . The nucleotide sequence of alsD encodes a putative protein of 239 amino acids showing significant similarity with other bacterial alpha-acetolactate decarboxylases . Upstream from alsD lies an open reading frame (alsS) which is highly similar to bacterial genes coding for catabolic alpha-acetolactate synthases . Northern (RNA) blotting analyses indicated the presence of a 2.4-kb dicistronic transcript of alsS and alsD . This suggests that the alsS and alsD genes are organized in a single operon.

Gene, 1996 Dec 5, 182(1-2), 23 - 32
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; Monchois V et al.; 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.

Mayo Clin Proc, 1996 Dec, 71(12), 1171 - 4
Leuconostoc bacteremia in an infant with short-gut syndrome: case report and literature review; Dhodapkar KM et al.; In this article, we report a case of Leuconostoc bacteremia in a 7-month-old infant who had short-gut syndrome after a gastroschisis repair and who was dependent on total parenteral nutrition through a central venous catheter . The organism was initially misidentified as viridans group streptococcus . Detection of vancomycin resistance led to the correct diagnosis of Leuconostoc species . The patient was successfully treated with ampicillin and an aminoglycoside . A review of the literature revealed prematurity, short-gut syndrome, prior vancomycin use, and central venous catheters as important predisposing factors . Leuconostoc species is an emerging pathogen that should be considered in the differential diagnosis of vancomycin-resistant gram-positive bacteremia, particularly in these clinical settings.

J Bacteriol, 1996 Nov, 178(21), 6209 - 15
The citrate metabolic pathway in Leuconostoc mesenteroides: expression, amino acid synthesis, and alpha-ketocarboxylate transport; Marty-Teysset C et al.; Citrate metabolism in Leuconostoc mesenteroides subspecies mesenteroides is associated with the generation of a proton motive force by a secondary mechanism (C . Marty-Teysset, C . Posthuma, J . S . Lolkema, P . Schmitt, C . Divies, and W . N . Konings, J . Bacteriol . 178:2178-2185, 1996) . The pathway consists of four steps: (i) uptake of citrate, (ii) splitting of citrate into acetate and oxaloacetate, (iii) pyruvate formation by decarboxylation of oxaloacetate, and (iv) reduction of pyruvate to lactate . Studies of citrate uptake and metabolism in resting cells of L . mesenteroides grown in the presence or absence of citrate show that the citrate transporter CitP and citrate lyase are constitutively expressed . On the other hand, oxaloacetate decarboxylase is under stringent control of the citrate in the medium and is not expressed in its absence, thereby blocking the pathway at the level of oxaloacetate . Under those conditions, the pathway is completely directed towards the formation of aspartate, which is formed from oxaloacetate by transaminase activity . The data indicate a role for citrate metabolism in amino acid biosynthesis . Internalized radiolabeled aspartate produced from citrate metabolism could be chased from the cells by addition of the amino acid precursors oxaloacetate, pyruvate, alpha-ketoglutarate, and alpha-ketoisocaproate to the cells, indicating a broad specificity of the transamination reaction . The alpha-ketocarboxylates are readily transported across the cytoplasmic membrane . alpha-Ketoglutarate uptake in resting cells of L . mesenteroides was dependent upon the presence of an energy source and was inhibited by inhibition of the proton motive force generating F(0)F(1) ATPase and by selective dissipation of the membrane potential and the transmembrane pH gradient . It is concluded that in L . mesenteroides alpha-ketoglutarate is transported via a secondary transporter that may be a general alpha-ketocarboxylate carrier.

Res Microbiol, 1996 Oct, 147(8), 651 - 60
Species attribution and strain typing of Oenococcus oeni (formerly Leuconostoc oenos) with restriction endonuclease fingerprints; Viti C et al.; In several wines, malolactic fermentation is required to improve the organoleptic characters and to stabilize the final product . In order to establish a controlled malolactic fermentation in wine, easy identification and sensitive typing of strains of Oenococcus oeni (new name of the malolactic bacterium Leuconostoc oenos) used as starter cultures are necessary . To accomplish these tasks, several strains of Oenococcus oeni isolated from wines of the Chianti region (Italy), along with reference strains and strains of L . mesenteroides subsp . mesenteroides, L . carnosum, L . fallax, L . pseudomesenteroides, L . lactis and Weisella paramesenteroides, were studied with RFLP of ribosomal genes and ultrasensitive total DNA restriction pattern analysis performed on polyacrylamide gel . With each of four restriction endonucleases used, identical restriction profiles of ribosomal genes were obtained for all strains of O . oeni . These ribopatterns, being strongly dissimilar to profiles of the other lactic acid bacteria tested, appear to be well suited for the attribution of wine lactic acid bacteria to the species O . oeni . Cluster analysis performed on two total DNA restriction profile data sets showed that the species O . oeni possesses a good degree of genomic homogeneity . Very sensitive typing of strains of O . oeni was obtained with total DNA restriction profiles . The potential of an integrated approach using restriction profiles for species assignment and typing of selected malolactic bacteria is demonstrated.

Int J Syst Bacteriol, 1996 Oct, 46(4), 1004 - 9
Analysis of the beta' subunit of DNA-dependent RNA polymerase does not support the hypothesis inferred from 16S rRNA analysis that Oenococcus oeni (formerly Leuconostoc oenos) is a tachytelic (fast-evolving) bacterium; Morse R et al.; rRNA sequencing has shown that leuconostocs comprise three distinct phylogenetic lineages which have been designated separate genera (viz., the genera Leuconostoc sensu stricto, Oenococcus, and Weissella) . In addition, the 16S rRNA line formed by Oenococcus oeni (formerly Leuconostoc oenos) is exceptionally long; this fact, together with variations in the compositions of conserved positions in the 16S rRNA, has led to the hypothesis (D . Yang and C . R . Woese, Syst . Appl . Microbiol . 12:145-149, 1989) that this organism is a fast-evolving bacterium . Previous evidence that the leuconostocs should be divided into three genera and that O . oeni is an example of tachytelic evolution has come solely from rRNA analyses . In this study we seqenced the rpoC gene encoding the beta' subunit of DNA-dependent RNA polymerase of leuconostocs and performed a comparative phylogenetic analysis . The subdivision of the leuconostocs into three distinct lineages was confirmed by the rpoC gene data, but no evidence that indicated that O . oeni is evolving at an extraordinary rate was found . If O . oeni is truly tachytelic, then fast-evolving phenomena would be expected to occur throughout the whole genome, including this independent molecular chronometer.

FEMS Microbiol Lett, 1996 Sep 1, 142(2-3), 161 - 6
A highly vancomycin-resistant laboratory mutant of Staphylococcus aureus; Sieradzki K et al.; A resistant mutant with vancomycin MIC of 100 micrograms/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 micrograms/ml for the antibiotic . Upon addition of vancomycin (50 micrograms/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria . Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium . Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5-1.0 micrograms/ml . There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant (vanA-positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc . The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable D-lactate terminating cell wall precursors . The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remained to be established.

J Bacteriol, 1996 Sep, 178(18), 5537 - 9
In vitro reassembly of the malolactic fermentation pathway of Leuconostoc oenos (Oenococcus oeni); Salema M et al.; The mechanism of metabolic energy generation by malolactic fermentation was studied with artificial membrane vesicles of Leuconostoc oenos (Oenococcus oeni) . (Note that although L . oenos was recently reclassified as O . oeni {L . M . T . Dicks, F . Dellaglio, and M . D . Collins, Int . J . Syst . Bacteriol . 45:395-397, 1995}, the old designation was kept in the present work.) Purified malolactic enzyme was entrapped in artificial membrane vesicles prepared from L . oenos cells able to transport L-malate . We show that the in vitro reconstituted system, including an electrogenic L-malate carrier and the decarboxylating malolactic enzyme, generated a proton motive force that was able to drive intravesicular accumulation of leucine.

Lett Appl Microbiol, 1996 Aug, 23(2), 120 - 4
Leuconostoc mesenteroides subsp . mesenteroides FR52 synthesizes two distinct bacteriocins; Revol-Junelles AM et al.; Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides subsp . mesenteroides FR52, was purified from producing cells by the adsorption-desorption method, combined with reverse-phase high-performance liquid chromatography . The elution profile revealed the presence of two inhibitory peaks of activity, each displaying different inhibitory spectra . Mesenterocin 52A possessed a broad inhibitory spectrum, including anti-Listeria activity, while Mesenterocin 52B was only active against Leuconostoc spp . The amino acid sequence and M(r) of Mesenterocin 52A appeared identical to the previously described Mesentericin Y105 . In contrast, Mesenterocin 52B possessed a M(r) of 3446 Da, corresponding to 32 amino acids and a sequence that shared no homology with known bacteriocins: NH2-KGVLGWLSMASSALTGPQQPNSPWLAKIKNHK.

Microbiology, 1996 Aug, 142 ( Pt 8), 2105 - 14
16S-23S rDNA intergenic sequences indicate that Leuconostoc oenos is phylogenetically homogeneous; Zavaleta AI et al.; The study of the intra-specific phylogenetic structure of Leuconostoc oenos is essential to understand the participation of several strains in malo-lactic fermentation (MLF) . RFLP of the PCR-amplified 16S-23S rDNA intergenic spacer region (ISR) was performed in Leuc . oenos and other related species . The RFLP patterns with seven endonucleases were identical for the 37 Leuc . oenos strains, but differed from those obtained for all other species tested . This method could provide an invaluable insight for molecular identification of the wine leuconostocs . The RFLP relationships of members of the genera Leuconostoc and Weissella were highly similar to those previously reported by 16S and 23S rRNA sequencing studies . The 16S-23S rDNA ISR was sequenced in five strains of Leuc . oenos . A single tRNA(Ala) was detected . The ISR sequence seems to be identical in the two rRNA (rrn) operons found in Leuc . oenos and no significant sequence variation was observed between strains that revealed relative differences as previously shown by PFGE . Results from the present study demonstrated that Leuc . oenos is phylogenetically a very homogeneous species (according to DNA-DNA hybridization studies) and sustain that this species is different from the genus Leuconostoc . The extremely conserved ISR of these organisms suggests that Leus . oenos strains currently isolated and characterized must have spread with the transfer of viticulture rather than coming from indigenous populations.

Curr Microbiol, 1996 Aug, 33(2), 136 - 7
Purification and N-terminal amino acid sequence of dextranicin 24, a bacteriocin of Leuconostoc sp; Revol-Junelles AM et al.; 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 . . .

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