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 . RESULT