Leuconostoc

Plasmid, 1996 Jul, 36(1), 67 - 74
Nucleotide sequence of plasmid p4028, a cryptic plasmid from Leuconostoc oenos; Zuniga M et al.; The Leuconostoc oenos plasmid p4028 was cloned in pBlueScript (SK+), and its complete nucleotide sequence was determined . The analysis of the nucleotide sequence revealed five open reading frames, all of them located on the same strand and grouped in two clusters separated by a short noncoding stretch . A similarity search against the other sequences deposited in the EMBL and GenBank databases showed that p4028 has no significant similarity with any of the sequences checked . Nevertheless, a putative ATP-binding motif was found in ORF2 . A more detailed analysis of this ORF suggests that it could encode for a DNA-dependent ATPase.

Free Radic Res, 1996 Jul, 25(1), 23 - 9
Determination of site-specific modifications of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal using matrix assisted laser desorption time-of-flight mass spectrometry; Grace JM et al.; Products of the reaction of 4-hydroxy-2-nonenal (4HNE) with native and heat-denatured Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) were analyzed to determine the structure and position of the protein modifications . Matrix assisted laser desorption time-of-flight mass spectrometry was used to measure molecular weights of the modified proteins and determine mass maps of peptides formed by digestion with cyanogen bromide . The molecular weight data show that one to two 4HNE molecules add to each subunit of native enzyme while approximately nineteen 4HNE molecules add to each subunit of heat-denatured enzyme . Peptides are observed in the cyanogen bromide mass map of modified native G6PDH that are consistent with selective modification of two segments of the amino acid sequence . One modified segment contains Lysine-182 that has been found to be part of the enzyme active site . Peptides are observed in the cyanogen bromide mass map of modified heat-denatured enzyme that are consistent with extensive modification of several segments of the amino acid sequence . The magnitude of the mass differences between modified and unmodified peptides were approximately 156 Da, consistent with a 1,4-addition of 4HNE . These results support the conclusion that 4HNE inactivates G6PDH by selectively modifying only two or three sites in the protein by a 1,4-addition reaction and that some aspect of the tertiary structure of the enzyme directs those modification reactions.

J Bacteriol, 1996 Jun, 178(11), 3127 - 32
The proton motive force generated in Leuconostoc oenos by L-malate fermentation; Salema M et al.; In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative . In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate . Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane . The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation . The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation . At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi . Synthesis of ATP was observed in cells performing malolactic fermentation.

FEMS Microbiol Lett, 1996 May 1, 138(2-3), 251 - 9
The putative immunity protein of the gram-positive bacteria Leuconostoc mesenteroides is preferentially located in the cytoplasm compartment; Dayem MA et al.; Immunity proteins are though to protect bacteriocin-producing bacterial strains against the bactericidal effects of their own bacteriocin . The immunity protein which protects the lactic acid bacterium Leuconostoc mesenteroides against mesentericin Y105(37) bacteriocin was detected and localized by immunofluorescence and electron microscopy, using antibodies directed against the C-terminal end of the predicted immunity protein . The antibodies recognized the immunity proteins of various strains of Leuconostoc, including Leuconostoc mesenteroides and Leuconostoc gelidum . This study demonstrated that immunity proteins produced by Leuconostoc mesenteroides accumulated in the cytoplasmic compartment of the bacteria . This is in contrast with other known immunity proteins, such as the colicin immunity proteins, which are integral membrane proteins possessing three to four transmembrane domains.

Blood, 1996 Apr 1, 87(7), 2974 - 82
Glucose 6-phosphate dehydrogenase mutations causing enzyme deficiency in a model of the tertiary structure of the human enzyme; Naylor CE et al.; Human glucose 6-phosphate dehydrogenase (G6PD) has a particularly large number of variants resulting from point mutations; some 60 mutations have been sequenced to date . Many variants, some polymorphic, are associated with enzyme deficiency . Certain variants have severe clinical manifestations; for such variants, the mutant enzyme almost always displays a reduced thermal stability . A homology model of human G6PD has been built, based on the three-dimensional structure of the enzyme from Leuconostoc mesenteroides . The model has suggested structural reasons for the diminished enzyme stability and hence for deficiency . It has shown that a cluster of mutations in exon 10, resulting in severe clinical symptoms, occurs at or near the dimer interface of the enzyme, that the eight-residue deletion in the variant Nara is at a surface loop, and that the two mutations in the A- variant are close together in the three-dimensional structure.

Arch Biochem Biophys, 1996 Apr 1, 328(1), 158 - 64
Chemical characterization of a protein-4-hydroxy-2-nonenal cross-link: immunochemical detection in mitochondria exposed to oxidative stress; Cohn JA et al.; We have previously shown that incubation of the model protein glucose-6-phosphate dehydrogenase (Glu-6-PDH) from the bacterium Leuconostoc mesenteroides with 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, results in the formation of cross-linked protein . HNE-modified protein is resistant to proteolytic degradation and acts as an inhibitor of the multicatalytic proteinase . It was therefore important to establish the chemistry of the cross-linking reaction . The formation of cross-linked Glu-6-PDH is associated with the nearly exclusive loss of lysine residues . For this reason the reaction of N-acetyllysine with HNE has been investigated . The epsilon-amino group of lysine reacts with the double bond (C3) and the carbonyl (C1) functions of HNE via Michael addition and Schiff base formation resulting in the production of a 2:1 amino acid-HNE cross-link . Chromatographic detection of this adduct in the acid hydrolysate of HNE-treated Glu-6-PDH reveals that this chemistry is responsible for the formation of cross-linked protein . Antibody to the reduced form of the 2:1 lysine-HNE adduct was prepared . The antibody was used to demonstrate that exposure of isolated liver mitochondria to oxidative stress led to the formation of intra- and intermolecular protein-HNE cross-links . The results of the present study indicate that modifications to protein by lipid peroxidation products may be physiologically relevant and could contribute to the disease- and age-related buildup of damaged protein.

J Bacteriol, 1996 Apr, 178(8), 2178 - 85
Proton motive force generation by citrolactic fermentation in Leuconostoc mesenteroides; Marty-Teysset C et al.; In Leuconostoc mesenteroides subsp . mesenteroides 19D, citrate is transported by a secondary citrate carrier (CitP) . Previous studies of the kinetics and mechanism of CitP performed in membrane vesicles of L . mesenteroides showed that CitP catalyzes divalent citrate HCit2-/H+ symport, indicative of metabolic energy generation by citrate metabolism via a secondary mechanism (C . Marty-Teysset, J . S . Lolkema, P . Schmitt, C . Divies, and W . N . Konings, J . Biol . Chem . 270:25370-25376, 1995) . This study also revealed an efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate cometabolism, suggesting that under physiological conditions, CitP may function as a precursor/product exchanger rather than a symporter . In this paper, the energetic consequences of citrate metabolism were investigated in resting cells of L . mesenteroides . The generation of metabolic energy in the form of a pH gradient (delta pH) and a membrane potential (delta psi) by citrate metabolism was found to be largely dependent on cometabolism with glucose . Furthermore, in the presence of glucose, the rates of citrate utilization and of pyruvate and lactate production were strongly increased, indicating an enhancement of citrate metabolism by glucose metabolism . The rate of citrate metabolism under these conditions was slowed down by the presence of a membrane potential across the cytoplasmic membrane . The production of D-lactate inside the cell during cometabolism was shown to be responsible for the enhancement of the electrogenic uptake of citrate . Cells loaded with D-lactate generated a delta psi upon dilution in buffer containing citrate, and cells incubated with citrate built up a pH gradient upon addition of D-lactate . The results are consistent with an electrogenic citrate/D-lactate exchange generating in vivo metabolic energy in the form of a proton electrochemical gradient across the membrane . The generation of metabolic energy from citrate metabolism in L . mesenteroides may contribute significantly to the growth advantage observed during cometabolism of citrate and glucose.

Biosci Biotechnol Biochem, 1996 Feb, 60(2), 319 - 21
Screening for bacteria producing sucrose phosphorylase and characterization of the enzymes; Kawasaki H et al.; Two microbial strains, No . 165 and No . 168, were isolated from soil as sucrose phosphorylase producers and identified as Leuconostoc mesenteroides subsp . mesenteroides and subsp . dextranicum, respectively . The sucrose phosphorylases were purified, characterized, and compared with the enzymes of L . mesenteroides AKU1102 and ATCC12291 . As for the catalytic properties, these enzymes were close to each other, while as for the enzyme molecules, the No . 165 enzyme (Mr: 58,000) was slightly different from the other (Mr: 54,000), though their N-terminal amino acid sequences were almost the same.

Arch Biochem Biophys, 1996 Feb 1, 326(1), 145 - 51
Identification of an arginine residue in the dual coenzyme-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides that plays a key role in binding NADP+ but not NAD+; Levy HR et al.; Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides can utilize either NADP or NAD as coenzyme . The enzyme's three-dimensional structure has been solved (Rowland et al., 1994, Structure 2, 1073-1087) and shown to contain a conventional nucleotide binding domain . NADP+ was modeled into the structure by superimposing the beta alpha beta domain and that of coenzyme-bound 6-phosphogluconate dehydrogenase (Adams et al., 1994, Structure 2, 651-658), enabling us to identify Arg-46 as a potentially important residue for NADP+ binding . Using site-directed mutagenesis, we constructed mutant enzymes in which Arg-46 was replaced by glutamine (R46Q) and alanine (R46A) and examined their kinetic properties . The principal effects in these mutant enzymes were that the Km and Ki values for NADP+ increased by 2 to 3 orders of magnitude over those of the wild-type enzyme . No other kinetic constant was altered more than 6.5-fold . Changing this single amino acid leads to mutant glucose-6-phosphate dehydrogenases with coenzyme specificities that favor NAD+, whereas the wild-type enzyme prefers NADP+ as coenzyme . These results confirm that Arg-46 plays a key role in NADP+ binding by contributing a positively charged planar residue that interacts primarily with the 2'-adenosine phosphate . The Arg residue corresponding to Arg-46 in L . mesenteroides glucose-6-phosphate dehydrogenase is conserved in all glucose-6-phosphate dehydrogenases and, presumably, plays the same role in all these enzymes.

Int J Food Microbiol, 1996 Jan, 28(3), 361 - 7
Sapal: a traditional fermented taro {Colocasia esculenta (L.) Schott} corm and coconut cream mixture from Papua New Guinea; Gubag R et al.; Sapal is a traditional fermented food made by mixing cooked, grated taro {Colocasia esculenta (L.) Schott} corm with coconut cream and allowing it to ferment at ambient temperature . The fermentation was primarily due to heterofermentative lactic acid bacteria, which reached 10(10) cfu/ml . Seven out of 10 isolated bacterial strains were identified as Leuconostoc mesenteroides or Leuc . paramesenteroides . The initial microbial flora was derived from the coconut cream . Yeasts grew on the surface of the sapal in the later stages of the fermentation . Overnight storage of the grated taro corm resulted in the glucose concentration increasing from 1.1 to about 5 g/l . During the fermentation the glucose concentration decreased to undetectable levels . The pH value fell from an initial value of 6.1 to 4.1 after 24 h.

J Biol Chem, 1995 Oct 27, 270(43), 25370 - 6
Membrane potential-generating transport of citrate and malate catalyzed by CitP of Leuconostoc mesenteroides; Marty-Teysset C et al.; Citrate uptake in Leuconostoc mesenteroides subsp . mesenteroides 19D is catalyzed by a secondary citrate carrier (CitP) . The kinetics and mechanism of CitP were investigated in membrane vesicles of L . mesenteroides . The transporter is induced by the presence of citrate in the medium and transports both citrate and malate . In spite of sequence homology to the Na(+)-dependent citrate carrier of Klebsiella pneumoniae, CitP is not Na(+)-dependent, nor is CitP Mg(2+)-dependent . The pH gradient (delta pH) is a driving force for citrate and malate uptake into the membrane vesicles, whereas the membrane potential (delta psi) counteracts transport . An inverted membrane potential (inside positive) generated by thiocyanide diffusion can drive citrate and malate uptake in membrane vesicles . Analysis of the forces involved showed that a single unit of negative charge is translocated during transport . Kinetic analysis of citrate counterflow at different pH values indicated that CitP transports the dianionic form of citrate (Hcit2-) with an affinity constant of approximately 20 microns . It is concluded that CitP catalyzes Hcit2-/H+ symport . Translocation of negative charge into the cell during citrate metabolism results in the generation of a membrane potential that contributes to the protonmotive force across the cytoplasmic membrane, i.e . citrate metabolism in L . mesenteroides generates metabolic energy . Efficient exchange of citrate and D-lactate, a product of citrate/carbohydrate co-metabolism, is observed, suggesting that under physiological conditions, CitP may function as an electrogenic precursor/product exchanger rather than a symporter . The mechanism and energetic consequences of citrate uptake are similar to malate uptake in lactic acid bacteria.

FEMS Microbiol Lett, 1995 Aug 15, 131(1), 57 - 62
Expression of DnaK and GroEL homologs in Leuconostoc esenteroides in response to heat shock, cold shock or chemical stress; Salotra P et al.; The mechanism of adaptation of bacteria to survive at elevated temperature in the human host and the expression of heat-shock proteins in response to stress was examined by labelling with {35S}methionine . An increase in culture temperature from 26 degrees C to 37 degrees C induced expression of certain bacterial proteins (70 and 60 kDa) . Heat shock at 40 degrees C, cold shock (10 degrees C), ethanol treatment or arsenite treatment also led to an increased expression of heat shock proteins of 70 and 60 kDa . Actinomycin D completely blocked the induction, indicating that transcription is required for the overexpression of stress proteins in Leuconostoc mesenteroides . N-terminal sequence analysis showed that these proteins were homologous to the highly conserved chaperone proteins DnaK and GroEL of Escherichia coli, respectively.

Int J Food Microbiol, 1995 Aug, 26(3), 345 - 52
Differentiation of dextran-producing Leuconostoc strains from fermented rice cake (puto) using pulsed-field gel electrophoresis; Kelly WJ et al.; Lactic acid bacteria were isolated from puto, a fermented rice cake consumed as a breakfast and snack food in the Philippines . The microflora was dominated by dextran-producing leuconostocs, and these were differentiated into four groups using pulsed-field gel electrophoresis of restriction enzyme digested chromosomal DNA, in conjunction with taxonomic tests . The four groups corresponded to the species Leuconostoc mesenteroides subsp . mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc citreum and Leuconostoc fallax . Several strains showed an unusual clumping phenotype, and two of these were capable of inhibiting other strains of lactic acid bacteria.

Biochem Mol Biol Int, 1995 Jul, 36(3), 639 - 47
2,4,6-Trinitrobenzenesulphonic acid as a probe for lysine at the active site of dextransucrase from Leuconostoc mesenteroides NRRL B-512F; Goyal A et al.; Modification of Leuconostoc mesenteroides NRRL B-512F dextransucrase with 2,4,6-trinitrobenzenesulphonic acid (TNBS) at pH 5.2 and 30 degrees C resulted in the loss of enzyme activity . The kinetic profiles of inactivation showed that the reaction followed pseudo-first order reaction . Absorption spectra of TNBS modified enzyme gave characteristic maxima at 367 nm . The inactivation could not be reversed by dilution or dialysis . The substrate sucrose, provided protection to the enzyme against inactivation by TNBS, indicating that the essential residues are present at or near the active site . The stoichiometric results indicated that four mol of lysine are modified per mol of dextransucrase upon complete inactivation . However, more than 50% of the activity loss was accompanied by modification of 1 lysine residue . All these approaches suggested that one lysine residue present near or at the active site is essential for the enzymatic activity of dextransucrase.

Biochem Mol Biol Int, 1995 Jul, 36(3), 579 - 85
Involvement of a lysine residue in the inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde; Goyal A et al.; Leuconostoc mesenteroides NRRL B-512F dextransucrase was rapidly and irreversibly inactivated by o-phthalaldehyde . The dextransucrase-o-phthalaldehyde adduct showed a characteristic fluorescence maxima at 417 nm when excited at 337 nm . These results were consistent with the isoindole derivative formation in which the sulfhydryl group of cysteine and epsilon-amino group of lysine participate in the reaction . The stoichiometric determinations gave one isoindole derivative per enzyme molecule upon complete inactivation by o-phthalaldehyde . The enzyme showed no inhibition on treatment with thiol specific reagents . This indicated that cysteine is present in close proximity of the lysine and is involved in the isoindole derivative formation but is not participating in the catalysis . These results established for the first time that one lysine residue present at the active site is required for the activity of dextransucrase.

Lett Appl Microbiol, 1995 May, 20(5), 268 - 70
Isolation of a dextranase constitutive mutant of Lipomyces starkeyi and its use for the production of clinical size dextran; Kim D et al.; A derepressed and partially constitutive mutant for dextranase of Lipomyces starkeyi was selected after ethyl methane sulphonate mutagenesis by zone clearance on blue dextran agar plates . The mutant produced dextranase when grown on glucose, fructose and sucrose as well as on dextran, and more enzyme was produced by the mutant than by the parental strain when grown on 1% dextran . The pH and temperature optima for the mutant dextranase were 5.5 and 55 degrees C, respectively . Dextranase produced on sucrose produced more isomaltose and less glucose after dextran hydrolysis than the equivalent enzyme produced on dextran . The clinical size dextran (average mol . wt of 75,000 +/- 25,000) yield of mixed culture fermentation with the mutant and Leuconostoc mesenteroides was 94% of the total dextran produced.

Res Microbiol, 1995 May, 146(4), 291 - 302
Purification, properties and DNA sequence of the D-lactate dehydrogenase from Leuconostoc mesenteroides subsp . cremoris; Dartois V et al.; The complete sequence of the D-lactate dehydrogenase (D-ldh) gene from Leuconostoc mesenteroides subsp . cremoris, cloned in Escherichia coli, were determined . The deduced amino acid sequence showed homologies with all members of the D-specific-2-hydroxyacid dehydrogenase family . Furthermore, the essential residues detected so far as being involved in catalysis were also conserved . Purification of the enzyme revealed physico-chemical properties corresponding to those predicted from the sequence . The active enzyme was a dimer of 40-kDa subunits . The Km values for pyruvate, lactate, NADH and NAD were 0.3, 19, 0.03 and 0.16 mM, indicating that the enzyme reduced pyruvate in vivo . Besides the D-LDH activity, L . mesenteroides subsp . cremoris also displayed HicDH enzymatic activity, catalysing the reduction of pyruvate analogs . The purified D-LDH displayed low HicDH-type activity; therefore, differences in specificity profiles between the crude extract and the purified enzyme suggested the occurrence of a specific HicDH.

Biosci Biotechnol Biochem, 1995 May, 59(5), 776 - 80
Aggregated form of dextransucrases from Leuconostoc mesenteroides NRRL B-512F and its constitutive mutant; Funane K et al.; Purified dextransucrases {EC 2.4.1.5}, DSW-D and DSW-G, from Leuconostoc mesenteroides B-512F were obtained from affinity chromatography with DEAE-Sephadex A-50 by elution with clinical dextran and guanidine-HCl, respectively . DSM-G was purified from the B-512F mutant strain SH 3002, which produces dextransucrase constitutively . Although the sugar contents of the purified enzymes were different, their molecular masses by SDS-PAGE were all 170 kDa . DSW-D and DSW-G were highly aggregated and the all the activities were eluted at the void volume (V0) on Sepharose 6B, while the DSM-G was eluted at 1.2 x V0 volume . On rechromatography, DSM-G was separated into three peaks corresponding to the aggregated form, monomeric form, and partially digested form, respectively . The aggregation of Leuconostoc dextransucrase was looser than that of streptococcal glucosyltransferases, but the structures of these enzymes had high homology with each other.

J Clin Microbiol, 1995 Apr, 33(4), 885 - 7
Evaluation of three disk tests for identification of enterococci, leuconostocs, and pediococci; Facklam R et al.; Simple rapid tests for presumptive identification of catalase-negative non-beta-hemolytic cocci (i.e., enterococci, leuconostocs, and pediococci) have not previously been available . Seven hundred thirty-four strains of aerobic and facultatively anaerobic, catalase-negative, non-beta-hemolytic gram-positive cocci were tested for susceptibility to vancomycin (Vans) by a screening procedure and production of leucine aminopeptidase (LAPase) and pyrrolidonylarylamidase (PYRase) in disk tests . Three unique patterns of activity in response to the three disks (30 micrograms of vancomycin, PYRase, and LAPase) can be used to presumptively identify the vancomycin-resistant (Vanr) enterococci (Vanr and PYRase and LAPase positive), leuconostocs (Vanr and PYRase and LAPase negative), and pediococci (Vanr, PYRase negative, and LAPase positive) . The results indicate that, together with Gram stain characteristics and the catalase test, the vancomycin, LAPase, and PYRase disk tests can be used to presumptively identify Vanr strains of enterococci as well as Leuconostoc and Pediococcus strains from human infections.

Int J Syst Bacteriol, 1995 Apr, 45(2), 395 - 7
Proposal to reclassify Leuconostoc oenos as Oenococcus oeni {corrig.} gen . nov., comb . nov.; Dicks LM et al.; Wine strains belonging to the genus Leuconostoc were classified as Leuconostoc oenos by Garvie in 1967, and this name was confirmed on the Approved Lists of Bacterial Names in 1980 . L . oenos is distinguished from other Leuconostoc spp . by its growth in acidic media, by its requirement for a growth factor in tomato juice, and by a number of carbohydrate fermentation characteristics . In addition, the results of a total soluble cell protein analysis, an electrophoretic analysis of NAD-dependent D-(-)-lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and alcohol dehydrogenase, and an analysis of cross-reactivity with anti-glucose-6-phosphate dehydrogenase and anti-NAD-dependent D-(-)-lactate dehydrogenase performed with other Leuconostoc spp . clearly indicated that L . oenos should be distinguished from the other Leuconostoc species . Phylogenetic studies, in particular 16S and 23S rRNA sequencing studies, have revealed that L . oenos represents a distinct subline that is separate from other Leuconostoc spp . and lactic acid bacteria . In view of the phenotypic and phylogenetic distinctiveness of L . oenos, we propose that this species should be assigned to a new genus as Oenococcus oeni {corrig.} gen . nov., comb . nov . The type strain of O . oeni is NCDO 1674 (= ATCC 23179).

Lett Appl Microbiol, 1995 Apr, 20(4), 204 - 8
Detection of dairy Leuconostoc strains using the polymerase chain reaction; Ward LJ et al.; This paper reports the design of a Leuconostoc-specific oligonucleotide based on 16S rRNA sequence data . When this oligonucleotide was used in a polymerase chain reaction (PCR) in conjunction with an oligonucleotide to a conserved region of the 16S rRNA sequence, a Leuconostoc-specific PCR product of approximately 470 bp was produced . The use of a second oligonucleotide to a conserved region allowed the production of an approximately 350 bp product in all PCRs, acting as a positive control . The PCR procedure described was particularly useful for detecting the presence of Leuconostoc in mixed mesophilic starter cultures . The Leuconostoc-specific oligonucleotide was used also as a specific hybridization probe.

Antimicrob Agents Chemother, 1995 Feb, 39(2), 362 - 8
Heterogeneity of the vanA gene cluster in clinical isolates of enterococci from the northeastern United States; Handwerger S et al.; In several strains of Enterococcus faecium isolated in Europe, the cluster of genes encoding high-level resistance to vancomycin (VanA phenotype) resides on a 10.85-kb transposon, Tn1546, or closely related elements . To determine whether Tn1546 was conserved in recent enterococcal isolates from the northeastern United States, seven strains were compared by restriction mapping and DNA hybridization with probes from within the van cluster . Two of the seven strains contained intact Tn1546-like sequences; however, in five of the strains, the organization of the van cluster differed from that of Tn1546 . Three of the five strains with variations harbored a novel DNA segment within the van gene cluster . This 1,496-bp segment was similar to IS1165 of Leuconostoc mesenteroides and IS1181 of Staphylococcus aureus and was flanked by 24- and 23-bp imperfect inverted repeats and 8-bp direct repeats . On the basis of these findings, we propose that this element comprises a novel insertion-like sequence, IS1251 . Multiple copies of IS1251 were also present at other sites in both resistant and susceptible clinical isolates . Our findings suggest that the van cluster in recent isolates from the northeastern United States differs from that present in the early European VanA phenotype strains.

J Enzyme Inhib, 1995, 8(4), 291 - 5
Inactivation of Leuconostoc mesenteroids NRRL B-512F dextransucrase by specific modification of lysine residues with pyridoxal-5'-phosphate; Goyal A et al.; Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was inactivated by pyridoxal-5'-phosphate (PLP) . The inactivation was reversible in as much as the loss of enzyme activity was completely reversed by prolonged dialysis . PLP-modified dextransucrase after reduction with sodium borohydride showed a characteristic fluorescence emission maximum at 397 nm when excited at 325 nm . The stoichiometric results indicated that four lysine residues are modified by PLP under the experimental conditions . These results established for the first time that lysine residues are essential for the activity of dextransucrase.

Eur J Biochem, 1994 Dec 1, 226(2), 641 - 8
Enzymically produced cyclic alpha-1,3-linked and alpha-1,6-linked oligosaccharides of D-glucose; Cote GL et al.; A new type of bacterial enzyme hydrolyzed alternan (Leuconostoc mesenteroides NRRL B-1355 fraction S dextran, an alternating alpha-1,3-alpha-1,6-D-glucan) to give rise to a series of oligosaccharides . The oligosaccharide formed in the greatest proportion was a cyclic tetrasaccharide of D-glucosyl residues linked in an alternating alpha-1,3-alpha-1,6 fashion . Other saccharide products included isomaltose and alpha-D-glucopyranosyl-1,3-alpha-D-glucopyranosyl-1,6-D-glucose . Oligosaccharides of higher degrees of polymerization were also formed, and included alpha-D-glucosylated derivatives of the cyclic tetrasaccharide . This is the first report of a naturally produced cyclic tetrasaccharide.

Eur J Biochem, 1994 Dec 1, 226(2), 633 - 9
Purification and properties of alternanase, a novel endo-alpha-1,3-alpha-1,6-D-glucanase; Biely P et al.; A newly isolated soil bacterium strain NRRL B-21195, tentatively identified as a Bacillus species, was found to be a constitutive producer of a novel type of glycanase that hydrolyses in an endo-fashion the polysaccharide alternan, an alpha-1,3-alpha-1,6-D-glucan, referred to in the literature as B-1355 dextran (fraction S), synthesized from sucrose by alternansucrase of Leuconostoc mesenteroides . The glycanase, named alternanase, has been purified to homogeneity from a cell-free culture fluid of the bacillus grown in a liquid medium containing D-glucose, and has been characterized . The enzyme has a molecular mass of 110000 Da (SDS/PAGE) and an isoelectric point of approximately 4.0 . Optimum activity occurs at pH 7 and at a temperature of 40 degrees C . The enzyme is stable up to 50 degrees C but loses activity rapidly at 60 degrees C . Its action is inhibited by EDTA and stimulated by Ca2+ . The enzyme requires, for its action, D-glucan chains in which alpha-1,3-linkages alternate with alpha-1,6-linkages; i.e., it is specific for alternan . Monitoring of alternan hydrolysis by determination of liberated reducing sugars pointed to an unusually low extent of hydrolysis and a low specific activity of the enzyme . As shown in the accompanying paper {Cote, G . L . & Biely, P . (1994) Eur . J . Biochem . 226, 641-648} the reason for this finding is that the main hydrolytic products are non-reducing, novel types of cyclic oligosaccharides.

Enzyme Microb Technol, 1994 Dec, 16(12), 1010 - 5
Properties of Leuconostoc mesenteroides B-512FMC constitutive dextransucrase; Kim D et al.; Leuconostoc mesenteroides B-512FMC, a constitutive mutant for dextransucrase, was grown on glucose, fructose, or sucrose . The amount of cell-associated dextransucrase was about the same for the three sugars at different concentrations (0.6% and 3%) . Enzyme produced in glucose medium was adsorbed on Sephadex G-100 and G-200, but much less enzyme was adsorbed when it was produced in sucrose medium . Sephadex adsorption decreased when the glucose-produced enzyme was preincubated with dextrans of molecular size greater than 10 kDa . The release of dextransucrase activity from Sephadex by buffer (20 mM acetate, pH 5.2) was the highest at 28 degrees-30 degrees C . The addition of dextran to the enzyme stimulated dextran synthesis but had very little effect on the temperature or pH stability . Dextransucrase purified by ammonium sulfate precipitation, hydroxyapatite chromatography, and Sephadex G-200 adsorption did not contain any carbohydrate, and it synthesized dextran, showing that primers are not necessary to initiate dextran synthesis . The purified enzyme had a molecular size of 184 kDa on SDS-PAGE . On standing at 4 degrees C for 30 days, the native enzyme was dissociated into three inactive proteins of 65, 62, and 57 kDa . However, two protein bands of 63 and 59 kDa were obtained on SDS-PAGE after heat denaturation of the 184-kDa active enzyme at 100 degrees C . The amount of 63-kDa protein was about twice that of 59-kDa protein . The native enzyme is believed to be a trimer of two 63-kDa and one 59-kDa monomers.

Int J Food Microbiol, 1994 Dec, 24(1-2), 75 - 81
Bacteriocins of leuconostocs isolated from meat; Hastings JW et al.; Several bacteriocin-producing Leuconostoc strains have been isolated from meat and identified as Leuconostoc gelidum UAL 187, Leuconostoc paramesenteroides-La7a, Leuconostoc carnosum-Ta11a and Leuconostoc carnosum-La54a . All strains produce bacteriocins that are active against Listeria monocytogenes and other lactic acid bacteria of concern in meat spoilage . All of the bacteriocins studied are heat stable in acidic environments and are inactivated by a range of proteolytic enzymes but not by catalase or lysozyme . Most are detected early in the growth cycle and are produced at refrigeration temperatures and in a pH range of 4.0-7.0 . Leucocin A-UAL187, produced by Leuconostoc gelidium UAL 187, is a small peptide (MW 3930) translated as a 61 amino acid prepeptide consisting of a 24 amino acid leader region and 37 amino acid active bacteriocin that is secreted . A probe designed from a region of the leucocin gene has been used to locate the bacteriocin genes in the other strains (La7a, La54a and Ta11a) . Strong hybridization signals were detected from 8.9 MDa, 32 MDa and 8.9 MDa plasmids in strains La7a, La54a and Ta11a, respectively . The bacteriocin structural gene from Leuconostoc carnosum-Ta11a (leucocin B-Ta11a) has been cloned and sequenced and the bacteriocin shows 100% homology to leucocin A-UAL187; however, the prepeptide differs in six residues . The mature extracellular bacteriocin from strain UAL 187 was purified and characterized by precipitation, gel filtration, hydrophobic interaction chromatography followed by RP-HPLC and amino-terminal sequencing, whilst those of the other strains are in the process of being purified and characterized using similar techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure, 1994 Nov 15, 2(11), 1073 - 87
The three-dimensional structure of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides refined at 2.0 A resolution; Rowland P et al.; BACKGROUND: Glucose 6-phosphate dehydrogenase (G6PD) is the first enzyme of the pentose phosphate pathway . Normally the pathway is synthetic and NADP-dependent, but the Gram-positive bacterium Leuconostoc mesenteroides, which does not have a complete glycolytic pathway, also uses the oxidative enzymes of the pentose phosphate pathway for catabolic reactions, and selects either NAD or NADP depending on the demands for catabolic or anabolic metabolism . RESULTS: The structure of G6PD has been determined and refined to 2.0 A resolution . The enzyme is a dimer, each subunit consisting of two domains . The smaller domain is a classic dinucleotide-binding fold, while the larger one is a new beta+ alpha fold, not previously seen, with a predominantly antiparallel nine-stranded beta-sheet . There are significant structural differences in the coenzyme-binding domains of the two subunits, caused by Pro 149 which is cis in one subunit and trans in the other . CONCLUSIONS: The structure has allowed us to propose the location of the active site and the coenzyme-binding site, and suggests the role of many of the residues conserved between species . We propose that the conserved Arg46 would interact with both the adenine ring and the 2'-phosphate of NADP . Gln47, which is not conserved, may contribute to the change from NADP to dual coenzyme specificity . His178, in a nine-residue peptide conserved for all known sequences, binds a phosphate in the active site pocket . His240 is the most likely candidate for the base to oxidize the 1-hydroxyl group of the glucose 6-phosphate substrate.

Gene, 1994 Oct 11, 148(1), 125 - 9
Cloning, sequence and in vitro transcription/translation analysis of a 3.2-kb EcoRI-HindIII fragment of Leuconostoc oenos bacteriophage L10; Sutherland M et al.; A 3.2-kb EcoRI-HindIII DNA fragment of Leuconostoc oenos bacteriophage L10 was cloned and sequenced . Computer-assisted analysis of the sequence identified eleven possible open reading frames (ORFs) that were all on the same strand . In vitro transcription/translation analysis of the full-length DNA fragment yielded five prominent proteins that were correlated with ORFs by their sizes and expression from deleted clones . Only those ORFs containing recognizable Shine-Dalgarno sequences coded for proteins . Neither the nucleotide sequence, nor deduced amino-acid sequences showed significant homology with other known sequences.

J Appl Bacteriol, 1994 Oct, 77(4), 401 - 7
Histamine production by wine lactic acid bacteria: isolation of a histamine-producing strain of Leuconostoc oenos; Lonvaud-Funel A et al.; Populations of Leuconostoc oenos were harvested from wines containing a relatively high concentration of biogenic amines . Cultivation of the biomass in synthetic media and wine showed that it consisted of histamine-producing strains . Histamine levels after culture depended on the quantity of precursor available and on the presence of yeast lees, which certainly enriched the medium in histidine . Ethanol and pH, which control bacterial growth rate and total population, were also significant factors: pH and low ethanol concentration enhanced histamine production . Strain Leuc . oenos 9204 was isolated and studied since it retained its ability to produce histamine after several transfers . In synthetic medium this strain produced large amounts of histamine especially in the poorest nutritional conditions (no glucose, no L-malic acid) . These results clearly demonstrate that Leuc . oenos involved in wine-making might play a role in biogenic amine production . The vinification method might also influence the final amine concentration in wine.

Eur J Biochem, 1994 Oct 1, 225(1), 289 - 95
Uniport of monoanionic L-malate in membrane vesicles from Leuconostoc oenos; Salema M et al.; L-malate transport was studied in membrane vesicles from Leuconostoc oenos MLE(-) (mutant lacking malolactic enzyme) which were fused with liposomes containing beef heart cytochrome c oxidase as a proton-motive-force-generating system . In these hybrid membranes, accumulation of L-malate was observed in response to a pH gradient (delta pH), with the inside alkaline, but was strongly inhibited by a membrane potential (delta psi) of normal polarity (inside negative) . Imposition of a delta psi, with the inside positive, by means of valinomycin-mediated potassium influx, resulted in a rapid accumulation of L-malate, indicating that L-malate was taken up in an anionic form . The results are consistent with a uniport mechanism facilitating the uptake of monoanionic L-malate, the dominant species at the low pH of the experiments . Kinetic analysis of delta pH-driven L-malate uptake in the pH range 3.0-5.8, yielded apparent affinity constants that varied less than twofold when calculated on the basis of the concentrations of monoanionic L-malate, whereas the values differed 2-3 orders of magnitude for the other species . At L-malate concentrations above 1 mM, a non-saturable transport component became apparent which may reflect passive influx of L-malic acid . Substrate specificity studies indicated that citrate and L-malate (and possibly D-lactate and L-lactate) compete for a single general carboxylate transport system . The carboxylate transport system catalysed homologous L-malate and heterologous L-malate/citrate exchange with rates similar to the rate of L-malate efflux . Since metabolic energy is conserved during malolactic fermentation in L . oenos, the underlying mechanism most likely involves electrogenic monoanionic L-malate uptake, in combination with H+ consumption in the cytoplasm, followed by diffusion outwards of lactic acid plus carbon dioxide.

Curr Microbiol, 1994 Oct, 29(4), 207 - 12
Characterization of leucocin B-Ta11a: a bacteriocin from Leuconostoc carnosum Ta11a isolated from meat; Felix JV et al.; Leuconostoc (Lc.) carnosum Ta11a, isolated from vacuum-packaged processed meats, produced a bacteriocin designated leucocin B-Ta11a . The crude bacteriocin was heat stable and sensitive to proteolytic enzymes, but not to catalase, lysozyme, or chloroform . It was active against Listeria monocytogenes and several lactic acid bacteria . Leucocin B-Ta11a was optimally produced at 25 degrees C in MRS broth at an initial pH of 6.0 or 6.5 . An 8.9-MDa plasmid in Leuconostoc carnosum Ta11a hybridized to a 36-mer oligonucleotide probe (JF-1) that was homologous to leucocin A-UAL187 . A 4.9-kb Sau3A fragment from a partial digest of the 8.9-MDa plasmid was cloned into pUC118 . The 8.1-kb recombinant plasmid (pJF8.1) was used for sequencing and revealed the presence of two open reading frames (ORFs) . ORF1 codes for a protein of 61 amino acids comprising a 37-amino-acid bacteriocin that was determined to be the leucocin B-Ta11a structural gene by virtue of its homology to leucocin A-UAL 187 (Hastings et al . 1991 . J . Bacteriol 173:7491-7500) . The 24-amino-acid N-terminal extension, however, differs from that of leucocin A-UAL187 by seven residues . The predicted protein of the ORF2 has 113 amino acids and is identical with the amino acid sequence of the cognate ORF of the leucocin A-UAL 187 operon.

Enzyme Microb Technol, 1994 Oct, 16(10), 844 - 8
A new process for the production of clinical dextran by mixed-culture fermentation of Lipomyces starkeyi and Leuconostoc mesenteroides; Kim D et al.; A mixed-culture fermentation system was designed for the production of size-limited dextrans . This process was simpler and more economical than traditional methods . It required the establishment of microbial consortia of Lipomyces starkeyi ATCC 74054 and Leuconostoc mesenteroides ATCC 10830 . Controlling initial conditions, growth, and enzyme production by both organisms controlled the product size . In this process, both strains were grown separately and then mixed . Dextran fermentation was then allowed to proceed . At the desired time (and molecular size), the fermentation was harvested . The optimum pH and temperature for production of clinical dextran (75,000 MW) were 5.2 (+/- 0.1) and 28 (+/- 0.5) degrees C, respectively . Varying the ratio of L . mesenteroides to L . starkeyi in the inoculum did not significantly affect either the final cell ratios or dextran production.

J Appl Bacteriol, 1994 Sep, 77(3), 271 - 80
Inter-strain relationships among wine leuconostocs and their divergence from other Leuconostoc species, as revealed by low frequency restriction fragment analysis of genomic DNA; Tenreiro R et al.; Thirty Leuconostoc oenos strains, representing 28 different isolates, were distributed into 20 genomic groups according to PFGE patterns of restriction digests . The 8 bp-specific enzymes Sfi I, Not I and Asc I cleaved the Leuc . oenos DNA in a mean of 17, 11 and four fragments respectively and Sma I produced more than 50 fragments per genome . The strain differentiating capacity of the four enzymes was similar; only two related genomic groups failed to be distinguished by Asc I or Not I . Genomic relationships between Leuc . oenos strains were quantified by numerical analysis of Not I and Sfi I banding patterns . More than half of the strains, including the starters ML34 and PSU-1, formed a major cluster . The average size of the Leuc . oenos genome was estimated as 1.86 Mb . Although similar values were obtained for the genomes of Leuc . mesenteroides, Leuc . pseudomesenteroides, Leuc . gelidum and Leuc . citreum, a significant divergence between wine and non-wine species was inferred from comparisons of genome cleavage frequencies, determined with five different enzymes.

Kansenshogaku Zasshi, 1994 Sep, 68(9), 1084 - 92
{Microbiological and clinical studies of vancomycin resistant Leuconostoc spp . and Pediococcus spp . isolated from septicemia patients}; Kikuchi K et al.; We described three septicemia cases in which blood cultures yielded gram-positive cocci identified as Leuconostoc spp . and Pediococcus spp . Patients were three male adults aged 63 to 71 years with severe underlying diseases, pancreatic cancer, esophageal cancer and diabetes mellitus with chronic renal failure . They had fever and chills at the onsets of septicemia with acute obstructive suppurative cholangitis, acute pneumonia, and infection complicated with invasion sites of esophageal cancer contagious to bronchus and subcutaneous tissue . Blood cultures yielded catalase and oxidase negative highly vancomycin-resistant (MIC: 1024 micrograms/ml <) gram-positive cocci showing alpha or gamma hemolysis on blood agar plates . Two cases were polymicrobial infections . In one case with esophageal cancer, clinical symptoms persisted after the start of antimicrobial chemotherapy and the patient died 10 days later associated with complications of esophageal cancer . Leuconostoc lactis, Leuconostoc mesenteroides subsp . dextranicum, and Pediococcus acidilactici wee identified by physiological reactions . These strains were also highly resistant to teicoplanin and fosfomycin, and tolerant to all rested beta-lactams such as benzylpenicillin . This is the first report in Japan to our knowledge on the identification of Leuconostoc spp . and Pediococcus spp . isolated from human infectious diseases.

J Dairy Sci, 1994 Sep, 77(9), 2718 - 24
Bacteriocins produced by Leuconostoc species; Stiles ME; Leuconostoc spp . are lactic acid bacteria that are commonly associated with foods and that are used as starter bacteria in some dairy fermentations . Lactic acid bacteria are inhibitory to other bacteria because of pH, organic acids, hydrogen peroxide, and other chemicals produced during their growth, including bacteriocins . Bacteriocin production by Leuconostoc spp . was first observed in the 1950s, but only since 1984, when antagonistic activity of Leuconostoc spp . was reported, have more extensive studies of bacteriocins produced by Leuconostoc spp . been conducted, including mesentericin Y105, produced by Leuconostoc mesenteroides spp . mesenteroides; leucocin A-UAL 187, produced by Leuconostoc gelidum; carnosin 44A, produced by Leuconostoc carnosum; and leuconocin S, produced by Leuconostoc paramesenteroides . Bacteriocins produced by leuconostocs may or may not be active against other lactic acid bacteria, but all include Listeria in their activity spectra . Mesentericin Y105 is reported to be exclusively active against Listeria spp . The amino acid sequences for leucocin A and mesentericin Y105 have been determined . Despite considerable differences in antibacterial spectra, only two amino acids differ between these bacteriocins . The prevalence of leuconostocs in many adventitious fermentations of food and the use of leuconostocs as starter bacteria in controlled fermentations make the bacteriocins produced by these bacteria of interest as possible food preservatives by addition of the bacteriocin or its producer organism to foods.

Lett Appl Microbiol, 1994 Sep, 19(3), 165 - 8
Identification of Carnobacterium spp . and Leuconostoc spp . in meat by genus-specific 16S rRNA probes; Nissen H et al.; Oligonucleotide probes specific for Carnobacterium and Leuconostoc species were constructed from the variable regions of 16S rRNA obtained from the literature and sequence data bases . The probes were hybridized with crude nucleic acid extract from 32 type strains of lactic acid bacteria (LAB) commonly found on meat . Two of the probes hybridized only to the four Carnobacterium species whereas the other two hybridized only to five of the six Leuconostoc species tested . The probes were also hybridized with nucleic acids from unknown strains of LAB . The identification was consistent with the results of biochemical tests used to characterize the two genera.

J Biol Chem, 1994 Aug 26, 269(34), 21639 - 43
Modification of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal . Formation of cross-linked protein that inhibits the multicatalytic protease; Friguet B et al.; Incubation of glucose-6-phosphate dehydrogenase (Glu-6-PDH) from Leuconostoc mesenteroides with the lipid peroxidation product 4-hydroxy-2-nonenal leads to the formation of cross-linked protein . This is accompanied by the appearance of protein-associated fluorescence with excitation and emission maxima of 340 and 415 nm, respectively, and with the disappearance of histidine and lysine residues . Cross-linked protein is less susceptible than native Glu-6-PDH to proteolysis by the multicatalytic protease, a multienzymic proteolytic complex involved in the intracellular degradation of damaged proteins . In addition, 4-hydroxy-2-nonenal-modified Glu-6-PDH inhibits the multicatalytic protease and can therefore prevent the efficient degradation of oxidized protein . These findings may have important implications for the accumulation of altered protein and fluorescent material in vivo, processes that are believed to be involved in age- and disease-related impairment of cellular function.

J Bacteriol, 1994 Aug, 176(16), 4899 - 905
Uniport of anionic citrate and proton consumption in citrate metabolism generates a proton motive force in Leuconostoc oenos; Ramos A et al.; The mechanism and energetics of citrate transport in Leuconostoc oenos were investigated . Resting cells of L . oenos generate both a membrane potential (delta psi) and a pH gradient (delta pH) upon addition of citrate . After a lag time, the internal alkalinization is followed by a continuous alkalinization of the external medium, demonstrating the involvement of proton-consuming reactions in the metabolic breakdown of citrate . Membrane vesicles of L . oenos were prepared and fused to liposomes containing cytochrome c oxidase to study the mechanism of citrate transport . Citrate uptake in the hybrid membranes is inhibited by a membrane potential of physiological polarity, inside negative, and driven by an inverted membrane potential, inside positive . A pH gradient, inside alkaline, leads to the accumulation of citrate inside the membrane vesicles . Kinetic analysis of delta pH-driven citrate uptake over a range of external pHs suggests that the monovalent anionic species (H2cit-) is the transported particle . Together, the data show that the transport of citrate is an electrogenic process in which H2cit- is translocated across the membrane via a uniport mechanism . Homologous exchange (citrate/citrate) was observed, but no evidence for a heterologous antiport mechanism involving products of citrate metabolism (e.g., acetate and pyruvate) was found . It is concluded that the generation of metabolic energy by citrate utilization in L . oenos is a direct consequence of the uptake of the negatively charged citrate anion, yielding a membrane potential, and from H(+)-consuming reactions involved in subsequent citrate metabolism, yielding a pH gradient . The uptake of citrate is driven by its own concentration gradient, which is maintained by efficient metabolic breakdown (metabolic pull).

Enzyme Microb Technol, 1994 Aug, 16(8), 659 - 64
Production and selection of mutants of Leuconostoc mesenteroides constitutive for glucansucrases; Kim D et al.; After chemical mutagenesis using ethyl methane sulfonate, we isolated mutants constitutive for glucansucrases from Leuconostoc mesenteroides NRRL B-512FM, B-1142, and B-1355 . Those mutants produced glucansucrases when grown on D-glucose as well as on sucrose . They produced higher glucansucrase activities (3 to 22 times) when grown on D-glucose than the parent strains grown on sucrose . Glucansucrases from mutants B-1355C and B-1142C grown on glucose formed glucans that were highly resistant to Penicillium dextranase hydrolysis . Mutant B-512FMC dextransucrase formed the same kind of dextran as the parent strain; however, it showed higher thermal stability, even when dextran was absent.

Arch Biochem Biophys, 1994 May 15, 311(1), 168 - 73
Susceptibility of glucose-6-phosphate dehydrogenase modified by 4-hydroxy-2-nonenal and metal-catalyzed oxidation to proteolysis by the multicatalytic protease; Friguet B et al.; Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is inactivated when exposed to metal-catalyzed oxidation or when modified by the lipid peroxidation product, 4-hydroxy-2-nonenal (HNE) . Although in each case inactivation appears to be the result of the selective modification of an active site lysine residue, only the oxidized enzyme becomes more susceptible to proteolysis by purified rat liver multicatalytic protease, a multienzymatic proteolytic complex involved in the intracellular degradation of damaged proteins . The HNE-treated enzyme remains as resistant to proteolysis by the multicatalytic protease as the native enzyme . In contrast to the HNE-treated Glu-6-PDH, enzyme modified by Fe2+ and citrate is more thermolabile and exhibits increased binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid (ANSA) . Heat inactivation is characterized, in part, by dissociation of the dimer to inactive subunits . No change in the secondary structure and only small variations in the fluorescence and circular dichroism of the aromatic residues are observed for the two modified forms of the enzyme as compared with the native enzyme . The increased heat sensitivity, ANSA binding, and proteolytic susceptibility are likely related to a decrease in the structural stability of oxidatively modified Glu-6-PDH . Conversely, modification of Glu-6-PDH with HNE has no apparent effect on its structural stability or proteolytic susceptibility . This finding may have important implications for the accumulation of altered protein in vivo, a process that is believed to be involved in age- and disease-related impairment of cellular function.

Plasmid, 1994 May, 31(3), 251 - 64
Isolation and characterization of IS1181, an insertion sequence from Staphylococcus aureus; Derbise A et al.; The repeated nucleotide sequence isolated from a methicillin-resistant Staphylococcus aureus isolate displays the characteristic features of an insertion sequence and was named IS1181 . It has a size of 1512 bp and consists of a 1359-bp open reading frame that encodes a 439-amino-acid protein which is predicted to be highly basic and 23-bp terminal inverted complementary repeated sequences exhibiting six mismatches . The three copies of IS1181 isolated from distinct parts of the chromosome of S . aureus, BM3121, are flanked at their ends by 8-bp direct repeats, suggesting a duplication of the target sequence . IS1181 exhibits similarities with IS1165 from Leuconostoc mesenteroides and IS1001 from Bordetella parapertusis . IS1181 was detected in at least two to eight copies in 41 of the 52 S . aureus isolates tested, whereas none of the 26 coagulase-negative staphylococci, 24 streptococci, or 11 enterococci analyzed carried nucleotide sequences hybridizing with IS1181.

Appl Environ Microbiol, 1994 May, 60(5), 1459 - 66
Growth and energetics of Leuconostoc oenos during cometabolism of glucose with citrate or fructose; Salou P et al.; The metabolic and energetic characterization of the growth of Leuconostoc oenos on glucose-citrate or glucose-fructose mixtures enables the potential role of this bacterium in the wine-making process to be ascertained . Moreover, mixotrophic conditions remain a suitable means for improving biomass productivities of malolactic starter cultures . When the malolactic bacterium L . oenos was grown in batch cultures on complex medium at pH 5.0 with glucose-citrate or glucose-fructose mixtures, enhancement of both the specific growth rate and biomass production yields was observed . While growth was possible on fructose as the sole source of energy, citrate alone did not allow subsequent biomass production . The metabolic interactions between the catabolic pathways of the glucose cosubstrates and the heterofermentation of hexoses led to an increased acetate yield as a result of modified NADH oxidation . However, the calculated global coenzyme regeneration showed that the reducing equivalent balance was never equilibrated . The stimulatory effects of these glucose cosubstrates on growth resulted from increased ATP synthesis by substrate-level phosphorylation via acetate kinase . While the energetic efficiency remained close to 10 g of biomass produced per mol of ATP, the increase in the specific growth rate and biomass production yields was directly related to the rate and yield of ATP generation.

J Appl Bacteriol, 1994 Feb, 76(2), 135 - 41
Antimicrobial activity of shredded carrot extracts on food-borne bacteria and yeast; Babic I et al.; Purified ethanolic extracts of peeled and shredded carrots showed an antimicrobial effect against a range of food-borne micro-organisms . The minimum inhibitory concentration, expressed as mg ml-1 dried carrot material used for the extraction were: Leuconostoc mesenteroides, 27; Listeria monocytogenes, > 27 < 55; Staphylococcus aureus, > 27 < 55; Pseudomonas fluorescens, > 55 < 110; Candida lambica, > 55 < 110; Escherichia coli, > 110 < 220 . The antimicrobial activity was not linked to phenolic compounds but was presumably due to apolar components . Free saturated fatty acid (dodecanoic acid) and methyl esters of saturated fatty acids (of dodecanoic and pentadecanoic acids) were identified in purified active extracts of carrots by gas chromatography coupled to mass spectrometry and could be responsible for the antimicrobial activity . This effect did not seem to play a role in the resistance of shredded carrots to microbial spoilage, although the antimicrobial activity was present in fresh carrots at concentrations sufficient to inhibit spoilage bacteria.

Appl Biochem Biotechnol, 1994 Feb, 44(2), 101 - 17
Production and use of glucosyltransferases from Leuconostoc mesenteroides NRRL B-1299 for the synthesis of oligosaccharides containing alpha-(1-->2) linkages; Remaud-Simeon M et al.; Glucosyltransferase activities, produced by batch culture of Leuconostoc mesenteroides NRRL B-1299, were recovered both in the culture supernatant (SGT) and associated with the insoluble part of the culture (IGT) . A total glucosyltransferase activity of 3.5 U/mL was measured in batch culture . The enzymes from the supernatant were purified 313 times using aqueous two-phase partition between dextran and PEG phases, yielding a preparation with 18.8 U/mg protein . It was shown that both SGT and IGT preparations catalyze acceptor reactions and transfer the glucose unit from sucrose onto maltose to produce glucooligosaccharides . Some of the glucooligosaccharides synthesized (Ln series) contain alpha-(1-->6) osidic linkages and a maltose residue at the reducing end . They were completely hydrolyzed by glucoamylase and dextranase . The other glucooligosaccharides synthesized (Bn series) resisted the action of these enzymes . The tetrasaccharide of this series has been characterized by 13C NMR . Its structure was determined as 2-O-alpha-D-glucosylpanose . The oligosaccharides synthesized by the maltose acceptor reaction with the SGT and IGT preparations only differed in the relative amounts in which they were produced . The difference may arise from diffusional limitations appearing when the insoluble catalyst is used . Under the assay conditions, the glucanase resistant oligosaccharide yield was 35% with both glucosyltransferase preparations.

Arch Biochem Biophys, 1994 Feb 1, 308(2), 471 - 6
Determination of the number of sucrose and acceptor binding sites for Leuconostoc mesenteroides B-512FM dextransucrase, and the confirmation of the two-site mechanism for dextran synthesis; Su D et al.; In previous studies on dextransucrase using pulse and chase experiments with {14C}sucrose, Robyt et al . {Arch . Biochem . Biophys . 165 (1974) 634-640} proposed a two-site insertion mechanism to explain the data for the synthesis of dextran . To further establish the validity of the two-site mechanism, the number of sucrose binding sites at the active site have been determined by using equilibrium dialysis with 6-deoxysucrose, a strong competitive inhibitor for dextransucrase . A ligand binding plot gave a straight line that indicated there were two sucrose binding sites at the active site . A similar experiment was performed using the acceptor, maltose . The ligand binding plot for maltose also gave a straight line and indicated that there was one acceptor binding site at the active site . These results corroborate the proposed two-site mechanism for dextran synthesis . To further test the two-site mechanism, dextransucrase was partially inactivated to varying extents by reaction with diethylpyrocarbonate, which chemically modifies essential active-site histidines . The various partially inactivated enzymes were assayed for dextran synthesis and for the synthesis of maltose acceptor products . A plot of the log of the relative percentage of dextran synthesized and acceptor products synthesized against varying degrees of enzyme inactivation showed that the synthesis of dextran decreased to a greater extent than did the decrease of the synthesis of acceptor product . The proposed mechanism requires two sucrose sites for the synthesis of dextran and only one sucrose site for the synthesis of acceptor product . When one site is modified, the synthesis of dextran stops, but the synthesis of acceptor products can continue at the other site . Thus, the greater loss of dextran synthesis in comparison with the lesser loss of acceptor product synthesis by enzymes modified to varying degrees, gives further evidence for the two-site mechanism for dextran synthesis.

J Basic Microbiol, 1994, 34(3), 173 - 82
Antibacterial activity of three Leuconostoc strains isolated from vacuum-packaged processed meats; Papathanasopoulos MA et al.; One hundred and fifty lactic acid bacteria (LAB) isolated from vacuum-packaged processed meats were screened for antagonistic activity against various food spoilage microorganisms and foodborne pathogens . Nineteen strains produced bacteriocins active against closely related LAB and Listeria strains . Leuconostoc carnosum (LA54a and TA26b) and Leuconostoc mesenteroides subspecies dextranicum (TA33a) produced bacteriocins that were susceptible to proteolytic enzymes, but not to catalase, lysozyme or chloroform . They were heat stable up to 100 degrees C for thirty minutes at pH 2 to 7, and exerted a bacteriolytic effect . Bacteriocin production by all Leuconostoc strains was growth associated, occurring at incubation temperatures of 0 degrees C to 30 degrees C and initial medium pH 4.5 to 7.5 . Probing of plasmid DNA from the three Leuconostoc strains with an oligonucleotide probe homologous to the nucleotide sequence of leucocin A-UAL 187 indicated plasmid-mediated bacteriocin production . Homology of the three Leuconostoc bacteriocin-coding genes to the amino-terminal end of the leucocin A-UAL 187 gene from Leuconostoc gelidum UAL 187 is therefore suggested . This evidence implies that all three Leuconostoc strains produce type 2, Listeria active bacteriocins.

Biochemistry, 1993 Dec 14, 32(49), 13696 - 702
An active-site peptide containing the second essential carboxyl group of dextransucrase from Leuconostoc mesenteroides by chemical modifications; Funane K et al.; The treatment of Leuconostoc mesenteroides B-512F dextransucrase with 10 mM 1-ethyl-3-{3-(dimethylamino)propyl}carbodiimide (EDC) and glycine ethyl ester (GEE) inactivated the enzyme almost completely within 24 min where the modification of one carboxyl group/mol of the enzyme by EDC was attained . Though 30 mM diethyl pyrocarbonate (DEP) also inactivated the enzyme, about 35% of the activity remained during a 36-min incubation . When 10 mol of imidazole residues/mol of the enzyme was modified by DEP, 50% of the activity was still retained . The addition of the substrate sucrose greatly retarded the enzyme inactivation by EDC . However, the addition of dextran slightly protected the inactivation of the glucosyl-transferring activity and accelerated the inactivation of the sucrose-cleaving activity . In the case of DEP, the addition of sucrose or dextran gave no influence on the inactivation of the enzyme . Therefore, the carboxyl group seemed to play a more important role in the substrate binding and in the catalytic activity of the dextransucrase than the imidazolium group . Differential labeling of Leuconostoc dextransucrase by EDC was conducted in the presence of a sucrose analog, sucrose monocaprate . The fluorescent probe N-(1-naphthyl)ethylenediamine (EDAN) was used as the nucleophile instead of GEE . A fluorescent labeled peptide was isolated from a trypsin digest of the EDC-EDAN modified enzyme . The amino acid sequence of the isolated peptide was Leu-Gln-Glu-Asp-Asn-Ser-Asn-Val-Val-Val-Glu-Ala.(ABSTRACT TRUNCATED AT 250 WORDS)

J Appl Bacteriol, 1993 Dec, 75(6), 595 - 603
Taxonomic studies on some leuconostoc-like organisms from fermented sausages: description of a new genus Weissella for the Leuconostoc paramesenteroides group of species; Collins MD et al.; Taxonomic studies were performed on some unknown Leuconostoc-like organisms from fermented Greek sausage . Comparative 16S rRNA sequence analysis showed the unidentified organisms represent a new line within the Leuconostoc paramesenteroides group of species . On the basis of the results of this and earlier phylogenetic investigations, it is proposed that Leuconostoc paramesenteroides and related species be reclassified in a new genus Weissella . In addition a new species, Weissella hellenica, is proposed for the isolates from fermented sausage.

Plasmid, 1993 Nov, 30(3), 212 - 23
Sequence analysis of Leuconostoc oenos DNA: organization of pLo13, a cryptic plasmid; Fremaux C et al.; A Leuconostoc oenos plasmid, pLo13, was studied to analyze its genetic organization and to define its functions . The nucleotide sequence (3948 bp) revealed three major open reading frames . Features commonly found in plasmids that replicate via a rolling-circle mechanism were identified within pLo13: first, a sequence coding for a protein with an amino acid sequence homologous to the plasmid recombination enzymes (Pre), but for which a specific target site similar to those previously described was not found; second, a sequence probably encoding a replication protein (Rep) . The putative pLo13 Rep protein amino acid sequence is divergent from the pC194-pUB110 family Rep proteins . However, the consensus sequence of the Rep protein active site was found, as well as the Rep protein consensus target site . No sequence similar to the previously described minus-origins (SSOs) are present in pLo13; nevertheless, a 200-bp sequence rich in imperfect palindromes may be involved in the minus-strand replication . These overall differences are in agreement with the previously reported important phylogenetic distance existing between Ln . oenos and other lactic acid bacteria.

J Biol Chem, 1993 Oct 15, 268(29), 21632 - 6
Thermal switching between enhanced and arrested reactivation of bacterial glucose-6-phosphate dehydrogenase assisted by GroEL in the absence of ATP; Hansen JE et al.; Facilitation of protein folding by GroEL usually requires involvement of GroES and ATP . In their absence nascent proteins tend to be arrested on GroEL or, if released, fail to show enhancement of reactivation yield relative to that observed without the chaperonin . In contrast, the yield of reactivation of glucose-6-phosphate dehydrogenase (Glu-6-PDH) from Leuconostoc mesenteroides at 20 degrees C is increased 2-3-fold (to over 80%) by GroEL alone . ATP greatly enhances the rate of GroEL-assisted reactivation and slightly increases its yield to 90% . The efficiency of the GroEL-assisted reactivation of Glu-6-PDH is strongly dependent on temperature . A switch from enhanced to fully arrested reactivation occurs over a narrow temperature range from 25 to 30 degrees C in the presence of GroEL when ATP is absent . At physiological temperature therefore, reactivation is fully arrested by GroEL if ATP is absent and in its presence the protein is released in a form not committed to correct folding . The data shows that the committing step in Glu-6-PDH refolding occurs while the nascent protein is bound to GroEL, a step which is temperature-sensitive . The extreme temperature sensitivity of this step indicates a sharp structural transition in GroEL.

Carbohydr Res, 1993 Oct 4, 248, 339 - 48
Control of the synthesis of dextran and acceptor-products by Leuconostoc mesenteroides B-512FM dextransucrase; Su D et al.; In the maltose-acceptor reaction of Leuconostoc mesenteroides B-512FM dextransucrase, some of the D-glucose moieties of sucrose are diverted from the synthesis of dextran and are transferred to the nonreducing end of maltose to form panose . Glucose is also transferred to panose and to subsequent acceptor products to give a homologous series of isomaltosyl dextrins attached alpha-(1-->6) to maltose . Three experimental parameters were studied to obtain quantitative information about the yield and distribution of acceptor products and the yield of dextran: (a) the ratio of maltose to sucrose, (b) the concentration of maltose and sucrose, and (c) the amount of enzyme . The reactions were run with {14C}sucrose and the amount of each acceptor product and the amount of dextran synthesized were determined for (a), (b), and (c) by TLC separation and measurement of the radioactivity with a PhosphorImager . It was found that an increase in the ratio of maltose to sucrose increased the amount of acceptor products with a concomitant decrease in the synthesis of dextran . Further, as the ratio was increased, the number of acceptor-products decreased . When the concentrations of maltose and sucrose were increased and the ratio was maintained at 1:1, there also was a decrease in the amount of dextran and an increase in the amount of acceptor-products . In addition, there was a decrease in the amount of dextran and an increase in the amount and number of acceptor-products when the amount of enzyme was increased . The first acceptor-product can be exclusively obtained without the formation of any dextran, by using a specific ratio and concentration of maltose and sucrose and a specified amount of enzyme.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1993 Aug, 26(3), 116 - 31
Antimicrobial activity of sulfur dioxide to certain lactic acid bacteria isolated from wines; Fang TJ et al.; This study was conducted to investigate the relationship between pH and the toxicity of sulfur dioxide against homo- and hetero-lactic acid bacteria isolated from American acidic wines . Malolactic fermentation was found to be growth-associated in homo- and hetero-lactic strains . The pH of CBB broth had an insignificant effect on the specific death rate of these strains; the concentration of molecular sulfur dioxide had a great effect on the specific death rate and malate degradation . Laboratory or freshly isolated strains were exposed to various concentrations of free sulfur dioxide at pH 3.8 and 3.4 . At pH 3.4, sulfur dioxide was more effective against lactic acid bacteria than at pH 3.8 since more was present as molecular sulfur dioxide and causing an increase in the specific death rate . Pediococcus strain BB was more resistant to sulfur dioxide than those hetero-lactic fermentation strains such as Leuconostoc oenos PSU-1 and ML-34 . The effect of sulfur dioxide concentration on the viability of L . oenes ML-34, PSU-1, and Pediococcus strain BB appeared to be biphasic . No or little L-malic acid was degraded when the homo- and hetero-fermentative strains were killed by sulfur dioxide.

J Bacteriol, 1993 Jul, 175(13), 3941 - 8
Pathway and regulation of erythritol formation in Leuconostoc oenos; Veiga-da-Cunha M et al.; It was recently observed that Leuconostoc oenos GM, a wine lactic acid bacterium, produced erythritol anaerobically from glucose but not from fructose or ribose and that this production was almost absent in the presence of O2 . In this study, the pathway of formation of erythritol from glucose in L . oenos was shown to involve the isomerization of glucose 6-phosphate to fructose 6-phosphate by a phosphoglucose isomerase, the cleavage of fructose 6-phosphate by a phosphoketolase, the reduction of erythrose 4-phosphate by an erythritol 4-phosphate dehydrogenase and, finally, the hydrolysis of erythritol 4-phosphate to erythritol by a phosphatase . Fructose 6-phosphate phosphoketolase was copurified with xylulose 5-phosphate phosphoketolase, and the activity of the latter was competitively inhibited by fructose 6-phosphate, with a Ki of 26 mM, corresponding to the Km of fructose 6-phosphate phosphoketolase (22 mM) . These results suggest that the two phosphoketolase activities are borne by a single enzyme . Extracts of L . oenos were also found to contain NAD(P)H oxidase, which must be largely responsible for the reoxidation of NADPH and NADH in cells incubated in the presence of O2 . In cells incubated with glucose, the concentrations of glucose 6-phosphate and of fructose 6-phosphate were higher in the absence of O2 than in its presence, explaining the stimulation by anaerobiosis of erythritol production . The increase in the hexose 6-phosphate concentration is presumably the result of a functional inhibition of glucose 6-phosphate dehydrogenase because of a reduction in the availability of NADP.

Appl Microbiol Biotechnol, 1993 Jul, 39(4-5), 547 - 52
Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles; Lamoureux M et al.; The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains . Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines . The genomic DNA of 11 strains was analysed initially with NotI and only four different restriction profiles were observed . The genome size ranged from 1.8 to 2.1 megabase pairs (Mbp) . Constant field electrophoresis applied to DNA treatment with 19 different restriction enzymes showed that the size of the fragments obtained increased proportionally to the percentage G + C present at the site of restriction . EcoRI and HindIII profiles revealed that the zone between 9 and 23 kbp allowed differentiation of the strains tested . Thus, the 41 strains fell into 30 restriction groups using only two enzymes . Hybridization with a non-radioactive DNA probe coding for 16S rRNA revealed that there were two 16S genes on the chromosome.

J Clin Microbiol, 1993 May, 31(5), 1030 - 3
Identification of Leuconostoc spp . by analysis of soluble whole-cell protein patterns; Elliott JA et al.; Leuconostoc spp . share several physiologic characteristics, which sometimes makes it difficult to identify these organisms to the species level . We developed a system, based on the patterns of soluble whole-cell proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that was able to discriminate between the six Leuconostoc spp . that are capable of growth at 37 degrees C . Nine unidentified Leuconostoc-like bacterial isolates that were included in the study did not fit any of the protein profiles of the type strains and may represent new Leuconostoc spp.

J Bacteriol, 1993 May, 175(10), 3232 - 5
Membrane permeabilization of Listeria monocytogenes and mitochondria by the bacteriocin mesentericin Y105; Maftah A et al.; Mesentericin Y105, a bacteriocin produced by a Leuconostoc mesenteroides strain, dissipates the plasma membrane potential of Listeria monocytogenes and inhibits the transport of leucine and glutamic acid . It also induces an efflux of preaccumulated amino acids from cells . In addition, the bacteriocin uncouples mitochondria by increasing state 4 respiration and decreasing state 3 respiration . The bacteriocin inhibits ATP synthase and adenine nucleotide translocase of the organelle while the affinity of ADP for its carrier is not modified . The results suggest that mesentericin Y105 acts by inducing, directly or indirectly, pore formation in the energy-transducing membranes, especially those of its natural target.

Biochim Biophys Acta, 1993 Apr 21, 1163(1), 89 - 96
Renaturation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides after denaturation in 4 M guanidine hydrochloride: kinetics of aggregation and reactivation; Plomer JJ et al.; In 4 M guanidine hydrochloride (GdnHCl), the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (G6PD) dissociated to subunits and was extensively unfolded . Rapid dilution of this high GdnHCl concentration allowed G6PD to partially renature, as measured by enzyme reactivation, to a level which depended on the conditions employed . The fraction of the enzyme which did not renature aggregated and precipitated out of solution, a process which could not be substantially prevented by stabilizing additives . Based on the enzyme concentration dependence of the reactivation yield and on a comparison of the aggregation and reactivation rates, it was determined that aggregation and reactivation compete kinetically for a partially-folded intermediate only very early in the process, during the rapid GdnHCl-dilution step . The kinetics of G6PD reactivation were sigmoidal, indicating that this process involves more than one rate-limiting reaction . The kinetics depended on enzyme concentration in a higher than first-order manner, indicating that association of subunits is one of the rate-limiting reactions . A renaturation mechanism compatible with these observations is described, which involves a bi-unimolecular (subunit association-folding) reaction sequence, with rate constants equal to 2.19 microM-1 min-1 and 0.140 min-1, respectively . This mechanism involves an inactive, dimeric, G6PD-folding intermediate, a species whose existence has recently been established by equilibrium denaturation experiments (Plomer, J.J . and Gafni, A . (1992) Biochim . Biophys . Acta 1122, 234-242).

Arch Biochem Biophys, 1993 Mar, 301(2), 391 - 5
Oxidative modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by an iron(II)-citrate complex; Szweda LI et al.; Incubation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with Fe2+ and citrate results in rapid O2-dependent inactivation of the enzyme . Maximal rate of inactivation occurred at equimolar concentrations of Fe2+ and citrate . Loss of enzyme activity appears to be the result of selective oxidative modification, as evidenced by a corresponding increase in protein carbonyl content . Partially inactivated enzyme remained predominantly in the dimeric form with no change in the apparent affinity of the remaining active subunits for substrate . Modified Glu-6-PDH was, however, more susceptible to heat denaturation . Our results suggest that the Fe(2+)-citrate complex binds to the glucose 6-phosphate binding site and then undergoes reaction with H2O2 formed in solution leading to the oxidative modification of amino acids essential for enzyme activity.

J Biol Chem, 1993 Feb 15, 268(5), 3342 - 7
Inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal . Selective modification of an active-site lysine; Szweda LI et al.; Incubation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with 4-hydroxy-2-nonenal (HNE) results in a pseudo first-order loss of enzyme activity . The pH dependence of the inactivation rate exhibits an inflection around pH 10, and the enzyme is protected from inactivation by glucose 6-phosphate . Loss of enzyme activity corresponds with the formation of one carbonyl function per enzyme subunit and the appearance of a lysine-HNE adduct . The data presented in this paper are consistent with the view that the epsilon-amino group of a lysine residue in the glucose 6-phosphate-binding site reacts with the double bond (C3) of HNE, resulting in the formation of a stable secondary amine derivative and loss of enzyme activity . We have described a mechanism by which HNE may, in part, mediate free radical damage . In addition, a method for the detection of the lysine-HNE adduct is introduced.

Appl Environ Microbiol, 1993 Feb, 59(2), 607 - 9
Development and use of a selective medium for isolation of Leuconostoc spp . from vegetables and dairy products; Benkerroum N et al.; A selective medium (LUSM medium) for the isolation of Leuconostoc spp . was developed . This medium contained 1.0% glucose, 1.0% Bacto Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25% sodium acetate, 0.1% (vol/vol) Tween 80, 15% tomato juice, 30 micrograms of vancomycin (Sigma) per ml, 0.20 microgram of tetracycline (Serva) per ml, 0.5 mg of cysteine hydrochloride per ml, and 1.5% agar (Difco) . LUSM medium was used successfully for isolation and enumeration of Leuconostoc spp . in dairy products and vegetables . Of 116 colony isolates obtained from fresh raw milk, curdled milk, or various vegetables, 115 were identified as members of the genus Leuconostoc . A total of 89 of these isolates were identified to species; 13.5% of the isolates were Leuconostoc cremoris, 7.9% were Leuconostoc mesenteroides subsp . mesenteroides, 11.2% were Leuconostoc mesenteroides subsp . dextranicum, 16.9% were Leuconostoc mesenteroides subsp . paramesenteroides, 10.1% were leuconostoc lactis, and 40.4% were Leuconostoc oenos . When we compared the counts obtained for two Leuconostoc strains, Leuconostoc dextranicum 181 and L . cremoris JLL8, on MRS agar and LUSM medium, we found no significant difference between the values obtained on the two media.

Z Lebensm Unters Forsch, 1993 Jan, 196(1), 45 - 8
Determination of optimum pH and temperature for pasteurization of citrus juices by response surface methodology; Ulgen N et al.; Optimization of microbial death, enzyme inactivation and vitamin C retention during pasteurization of pH-adjusted orange juice is discussed free of equipment-dependent parameters such as the heating lag . The pH-temperature optimum was determined by response surface methodology in the range of 65 degrees C-75 degrees C and pH 2.5-4.0 . The results implied that there was no pectinesterase activity below pH 3.5 . Leuconostoc mesenteroides had its maximum and minimum thermal resistance at pH 3.5 and pH 2.7, respectively . For an ideal theoretical process requiring four log cycles of microbial reduction the optimum pasteurization conditions were 12 min at 75 degrees C and pH 2.7.

J Gen Microbiol, 1992 Dec, 138 ( Pt 12), 2725 - 31
Characterization and purification of mesentericin Y105, an anti-Listeria bacteriocin from Leuconostoc mesenteroides; Hechard Y et al.; A Leuconostoc mesenteroides ssp . mesenteroides was isolated from goat's milk on the basis of its ability to inhibit the growth of Listeria monocytogenes . The antimicrobial effect was due to the presence in the culture medium of a compound, named mesentericin Y105, excreted by the Leuconostoc mesenteroides Y105 . The compound displayed known features of bacteriocins from lactic acid bacteria . It appeared as a proteinaceous molecule exhibiting a narrow inhibitory spectrum limited to genus Listeria . The apparent relative molecular mass, as indicated by activity detection after SDS-PAGE, was 2.5-3.0 kDa . The bacteriocin was purified to homogeneity by a simple three-step procedure: a crude supernatant obtained from an early-stationary-phase culture in a defined medium was subjected to affinity chromatography on a blue agarose column, followed by ultrafiltration through a 5 kDa cut-off membrane, and finally by reverse-phase HPLC on a C4 column . Microsequencing of the pure bacteriocin and of tryptic fragments showed that mesentericin Y105 is a 36 amino acid polypeptide whose primary structure is close to that of leucocin A-UAL 187, which contains an extra residue at the C-terminus and displays only two differences in the overlapping sequence . However, unlike leucocin A-UAL 187, mesentericin Y105 displayed a bactericidal mode of action.

Clin Biochem, 1992 Dec, 25(6), 457 - 62
An enzymatic method for measuring serum mannitol and its use in hemodialysis patients; Diamandis EP et al.; We evaluated two chemical methods for quantifying mannitol in serum, based on the oxidation of mannitol by periodate, and measurement of the formaldehyde formed with chromotropic acid (colorimetry) or acetylacetone (fluorometry) . We found interference in these methods by serum glycerol . Additionally, a high-performance liquid chromatography (HPLC) method was evaluated and found to be specific but impractical for routine use . We therefore, developed an enzymatic fluorometric procedure, based on the oxidation of mannitol by beta-NAD to fructose and NADH, in the presence of the enzyme mannitol dehydrogenase (MD) . MD is not commercially available and was partially purified from cultures of Leuconostoc mesenteroides . This new method is specific, sensitive, simple, and accurate and is proposed as the method of choice for measuring mannitol in the serum of patients who received this sugar alcohol during routine hemodialysis treatment.

Appl Environ Microbiol, 1992 Nov, 58(11), 3784 - 6
On-line visualization of the competitive behavior of antagonistic bacteria; Hechard Y et al.; To study the interaction between cocultured Listeria monocytogenes and an antagonistic Leuconostoc strain producing an anti-Listeria bacteriocin, flow cytometry, a technique allowing on-line and real-time analysis, was used along with classical microbiological methods . Culture methods and flow cytometric measurements of the mixed culture over time point to a bactericidal action of the lactic acid-producing bacterial strain against L . monocytogenes cells.

Diagn Microbiol Infect Dis, 1992 Sep-Oct, 15(7), 641 - 4
In vitro activity of mersacidin (M87-1551), an investigational peptide antibiotic tested against gram-positive bloodstream isolates; Barrett MS et al.; We measured the in vitro activity of mersacidin (formerly M87-1551) against 183 clinical isolates (vancomycin susceptible) and 12 additional vancomycin-resistant strains of Gram-positive bacteria . The activity for mersacidin increased an average twofold (range, 1.7- to 7.6-fold) in a calcium-enriched medium . The minimum inhibitory concentration (MIC)90 for mersacidin was 8-32 times higher than vancomycin for staphylococci, 4-64 times higher for enterococci, and up to 32 times higher for other organisms tested . The MIC90 for MDL 62873, a comparison compound, was less than or equal to 0.5 micrograms/ml for all species except Staphylococcus haemolyticus (MIC90, 4 micrograms/ml), and it was greater than or equal to 4-fold more active than vancomycin . Against selected vancomycin-resistant strains, mersacidin had MICs greater than or equal to 16 micrograms/ml for enterococci, 4-32 micrograms/ml for Pediococcus, and less than or equal to 2 micrograms/ml for Leuconostoc species . Mersacidin may have some clinical utility in documented infections caused by staphylococci, nonenteric streptococci, Pediococcus, and Leuconostoc.

Biochim Biophys Acta, 1992 Aug 21, 1122(3), 234 - 42
Denaturation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride; identification of inactive, partially unfolded, dimeric intermediates; Plomer JJ et al.; The denaturation of the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride has been studied using enzymatic activity, intrinsic fluorescence, circular dichroism, and light scattering measurements . Equilibrium experiments at 25 degrees C revealed that between 0.9 and 1.2 M denaturant the enzyme underwent a conformational change, exposing tryptophan residues to solvent, with some loss of secondary structure and a complete loss of enzymatic activity but without dimer dissociation to subunits . This inactive, partially unfolded, dimeric intermediate was susceptible to slow aggregation, perhaps due to exposure of 'sticky' hydrophobic stretches of the polypeptide chain . A second equilibrium transition, reflecting extensive unfolding and dimer dissociation, occurred only at denaturant concentrations above 1.4 M . Kinetics experiments demonstrated that in the denaturant concentration range of 1.7-1.9 M the fluorescence change occurred in two distinct steps . The first step involved a large, very rapid drop in fluorescence whose rate was strongly dependent on the denaturant concentration . This was followed by a small, relatively slow rise in the emission intensity, the rate of which was independent of denaturant concentration . Enzymatic activity was lost with a denaturant-concentration-dependent rate, which was approx . 3-times slower than the rate of the first step in fluorescence change . A denaturation mechanism incorporating several unfolding intermediates and which accounts for all the above results is presented and discussed . While the fully unfolded enzyme regained up to 55% of its original activity upon dilution of denaturant to a concentration that would be expected to support native enzyme, denaturation intermediates were able to reactivate only minimally and in fact were found to aggregate and precipitate out of solution.

J Bacteriol, 1992 Aug, 174(16), 5302 - 8
Electrogenic malate uptake and improved growth energetics of the malolactic bacterium Leuconostoc oenos grown on glucose-malate mixtures; Loubiere P et al.; Growth of the malolactic bacterium Leuconostoc oenos was improved with respect to both the specific growth rate and the biomass yield during the fermentation of glucose-malate mixtures as compared with those in media lacking malate . Such a finding indicates that the malolactic reaction contributed to the energy budget of the bacterium, suggesting that growth is energy limited in the absence of malate . An energetic yield (YATP) of 9.5 g of biomass.mol ATP-1 was found during growth on glucose with an ATP production by substrate-level phosphorylation of 1.2 mol of ATP.mol of glucose-1 . During the period of mixed-substrate catabolism, an apparent YATP of 17.7 was observed, indicating a mixotrophy-associated ATP production of 2.2 mol of ATP.mol of glucose-1, or more correctly an energy gain of 0.28 mol of ATP.mol of malate-1, representing proton translocation flux from the cytoplasm to the exterior of 0.56 or 0.84 H+.mol of malate-1(depending on the H+/ATP stoichiometry) . The growth-stimulating effect of malate was attributed to chemiosmotic transport mechanisms rather than proton consumption by the malolactic enzyme . Lactate efflux was by electroneutral lactate -/H+ symport having a constant stoichiometry, while malate uptake was predominantly by a malate -/H+ symport, though a low-affinity malate- uniport was also implicated . The measured electrical component (delta psi) of the proton motive force was altered, passing from -30 to -60 mV because of this translocation of dissociated organic acids when malolactic fermentation occurred.

J Dairy Res, 1992 Aug, 59(3), 359 - 67
Intracellular pH and the role of D-lactate dehydrogenase in the production of metabolic end products by Leuconostoc lactis; FitzGerald RJ et al.; The kinetics of lactate dehydrogenase from Leuconostoc lactis NCW1 were studied . The pH optimum for the enzyme depended on the concentration of pyruvate used in the assay and the enzyme displayed an ordered mechanism with respect to substrate binding . The Km for pyruvate and NADH and the Vmax of the enzyme decreased 20-, 30- and 6-fold respectively as the pH decreased from 8.0 to 5.0 . No activators were found and none of the intermediates of the phosphoketolase pathway tested inhibited the enzyme . ATP, ADP, GTP and NAD+ were inhibitory . The intracellular volume (Vol(in)) and intracellular pH (pH(in)) decreased as the extracellular pH (pH(ex)) decreased . Co-metabolism of citrate and glucose affected the Vol(in) but did not affect the pH(in), which decreased by 0.6 units per unit change in pH(ex); at pH 7.0, the pH(in) and pH(ex) were equal . The results suggest that pH(in) may play a role in determining the production of diacetyl and acetoin at low pH by Leuconostoc.

J Bacteriol, 1992 Jul, 174(13), 4475 - 81
Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes; David S et al.; A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase . Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment . Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively . The L . lactis beta-galactosidase was overproduced in E . coli by using a lambda pL expression system . Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL . The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences . Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E . coli . The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E . coli . DNA and protein sequence alignments suggest that the L . lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.

Singapore Med J, 1992 Jun, 33(3), 241 - 3
Leuconostoc bacteraemia; Ling ML; Leuconostoc species, a gram-positive coccal bacterium that is classically moderately susceptible to ampicillin and penicillin and resistant to vancomycin is a potential pathogen in the immunocompromised host . Twenty-eight isolates were collected from patients' blood culture specimens in the year 1989-1990 . The clinical history and course of nineteen of these patients were studied . Five of them recovered without any antimicrobial therapy and in eight patients Leuconostoc species was isolated with other organisms in the blood culture specimens . The question arises as to when Leuconostoc species is of clinical significance when isolated in the blood culture specimens of patients.

Protein Sci, 1992 Mar, 1(3), 329 - 34
Lysine-21 of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase participates in substrate binding through charge-charge interaction; Lee WT et al.; Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase (G6PD) was isolated in high yield and purified to homogeneity from a newly constructed strain of Escherichia coli which lacks its own glucose 6-phosphate dehydrogenase gene . Lys-21 is one of two lysyl residues in the enzyme previously modified by the affinity labels pyridoxal 5'-phosphate and pyridoxal 5'-diphosphate-5'-adenosine, which are competitive inhibitors of the enzyme with respect to glucose 6-phosphate (LaDine, J.R., Carlow, D., Lee, W.T., Cross, R.L., Flynn, T.G., & Levy, H.R., 1991, J . Biol . Chem . 266, 5558-5562) . K21R and K21Q mutants of the enzyme were purified to homogeneity and characterized kinetically to determine the function of Lys-21 . Both mutant enzymes showed increased Km-values for glucose 6-phosphate compared to wild-type enzyme: 1.4-fold (NAD-linked reaction) and 2.1-fold (NADP-linked reaction) for the K21R enzyme, and 36-fold (NAD-linked reaction) and 53-fold (NADP-linked reaction) for the K21Q enzyme . The Km for NADP+ was unchanged in both mutant enzymes . The Km for NAD+ was increased 1.5- and 3.2-fold, compared to the wild-type enzyme, in the K21R and K21Q enzymes, respectively . For the K21R enzyme the kcat for the NAD- and NADP-linked reactions was unchanged . The kcat for the K21Q enzyme was increased in the NAD-linked reaction by 26% and decreased by 30% in the NADP-linked reaction from the values for the wild-type enzyme . The data are consistent with Lys-21 participating in the binding of the phosphate group of the substrate to the enzyme via charge-charge interaction.

J Biol Chem, 1992 Feb 15, 267(5), 3096 - 100
Iron-catalyzed oxidative modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides . Structural and functional changes; Szweda LI et al.; As a variety of eukaryotic cells age, the specific activity of glucose-6-phosphate dehydrogenase (Glu-6-PDH) declines as much as 50% . Because of the central role of this enzyme in metabolism, it is important to define factors responsible for this loss in enzyme activity . We report that Glu-6-PDH from Leuconostoc mesenteroides is rapidly inactivated by micromolar concentrations of Fe2+ and H2O2 . Inactivation correlated with the formation of one carbonyl functionality/enzyme subunit, indicating that inactivation is the result of site-specific oxidative modification . Our results suggest that Fe2+ binds to the glucose 6-phosphate binding site and that interaction of the enzyme-bound Fe2+ with H2O2 leads to the oxidative modification of amino acids essential for enzyme activity . Partially inactivated enzyme remained predominantly in the dimeric form, and no change in the apparent affinity of the remaining active subunits for substrate was observed . Partial inactivation did, however, lead to a decrease in the thermal stability of the remaining activity . This decrease in thermal stability could be largely overcome by the addition of glucose 6-phosphate . Thus, although exposure to H2O2 and Fe2+ results in the irreversible inactivation of Glu-6-PDH, the resulting modification is selective, leads to the formation of heterodimers of both active and inactive subunits, and does not appear to cause large scale structural changes . Our results demonstrate the inherent susceptibility of Glu-6-PDH from L . mesenteroides to modification by an oxidation system known to exist in vivo . An assessment of the physiological significance of Fe(2+)-catalyzed oxidation of Glu-6-PDH awaits extension of these studies to mammalian sources known to accumulate less active or inactive forms of the enzyme as a function of age.

Appl Biochem Biotechnol, 1991 Dec, 31(3), 237 - 46
Inhibition of dextransucrase by alpha-D-glucose derivatives; Michiels AG et al.; alpha-D-Glucopyranosyl fluoride was modified at positions 2, 3, or 5 and these analogs were tested as substrates and inhibitors of dextransucrase from Leuconostoc mesenteroides B-512F . The analogs studied were 2-deoxy-2-fluoro-alpha-D-glucopyranosyl fluoride, 3-deoxy-3-fluoro-alpha-D-glucopyranosyl fluoride, 3-deoxy-3-thio-alpha-D-glucopyranosyl fluoride, and 5-thio-alpha-D-glucopyranosyl fluoride . Kinetic constants for alpha-D-glucopyranosyl fluoride were also determined . None of the alpha-D-glucopyranosyl fluorides were accepted as substrates for dextransucrase . 2-Deoxy-2-fluoro-alpha-D-glucopyranosyl fluoride, 3-deoxy-3-fgluoro-alpha-D-glucopyranosyl fluoride, and 3-deoxy-3-thio-alpha-D-glucopyranosyl fluoride were competitive inhibitors with Ki values of 63, 93, and 53 mM, respectively . The Km for alpha-D-glucopyranosyl fluoride was found to be 26 mM . The data indicate that the hydroxyl groups at C2 and C3 are important for proper binding of alpha-D-glucopyranosyl fluoride for the active site of dextransucrase and that the C3-hydroxyl probably acts as a hydrogen-bond donor.

East Afr Med J, 1991 Dec, 68(12), 969 - 74
Occurrence of Leuconostoc mesenteroides and leuconostoc-like organisms in Lagos, Nigeria; Idika N et al.; A total of 91 catalase--negative Gram-positive coccal isolates obtained from 245 clinical specimens in Lagos were characterized . Ten (11.0%) of the isolates were vancomycin resistant, they fermented glucose, sucrose, fructose, lactose, mannose, mannitol, ribose, salicin, sorbitol, arabinose and xylose with acid production . One of the isolates produced in addition gas inclusive and ethanol, thus identified as Leuconostoc mesenteroides . The ten vancomycin-resistant Gram-positive coccal organisms (VRGPC) showed variable sensitivity patterns to penicillin, tetracycline, erythromycin, ampicillin, streptomycin, chloramphenicol, cloxacillin and co-trimoxazole . The possible role of Leuconostoc spp . and VRGPC in clinical infections in hospital setting is still to be defined.

Appl Environ Microbiol, 1991 Dec, 57(12), 3450 - 5
Detection and activity of a bacteriocin produced by Leuconostoc mesenteroides; Daba H et al.; Leuconostoc mesenteroides UL5 was found to produce a bacteriocin, referred as mesenterocin 5, active against Listeria monocytogenes strains but with no effect on several useful lactic acid bacteria . The antimicrobial substance is a protein, since its activity was completely destroyed following protease (pronase) treatment . However, it was relatively heat stable (100 degrees C for 30 min) and partially denaturated by chloroform . The inhibitory effect of the bacteriocin on sensitive bacterial strains was determined by a critical-dilution micromethod . Mutants of L . mesenteroides UL5 which had lost the capacity to produce the bacteriocin were obtained . The mutant strain was stable and phenotypically identical to parental cells and remained resistant to the bacteriocin . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect bacteriocin activity corresponding to an apparent molecular mass of about 4.5 kDa.

Carbohydr Res, 1991 Sep 18, 217, 201 - 11
Maltodextrin acceptor reactions of Streptococcus mutans 6715 glucosyltransferases; Fu DT et al.; The maltodextrin (maltose through maltoheptaose) acceptor reactions of two Streptococcus mutans 6715 glucosyltransferases (GTF-I and GTF-S) were studied . The acceptor product structures were determined by comparing them with the known structures of the acceptor products of Leuconostoc mesenteroides B-512FM dextransucrase (EC 2.4.1.5) and L . mesenteroides B-1355 alternansucrase (EC 2.4.1.140) . When reacted with maltose (G2), both GTF-I and GTF-S transferred a D-glucopyranose from sucrose to the nonreducing glucosyl residue to give panose (6(2)-alpha-D-glucopyranosyl maltose) . Panose then served as an acceptor to give two further acceptor products, 6(2)-alpha-isomaltosyl maltose and 6(2)-alpha-nigerosyl maltose . 6(2)-alpha-Isomaltosyl maltose then went on to serve as an acceptor to give a series of homologous acceptor products with isomaltodextrin chains attached to C-6 of the nonreducing-end residue of maltose, while 6(2)-alpha-nigerosyl maltose did not further react . When reacted with other maltodextrins (G3-G7), both GTF-I and GTF-S transferred a D-glucopyranose to C-6 of either the nonreducing-end or the reducing-end residues of the maltodextrins, forming alpha(1----6) linkages . When D-glucopyranose was transferred to the nonreducing-end residue by GTF-I or GTF-S, the first product was also an acceptor to give the second product, which then served as an acceptor to give the third product, etc., to give a homologous series of products . When D-glucopyranose was transferred to the reducing-end residue, the acceptor product that formed did not readily serve as an acceptor, or served only as a very poor acceptor, to give a small amount of the next homologue, as was the case for G7 with GTF-S . In addition, GTF-I also transferred D-glucopyranose to the reducing-end or to the nonreducing-end residue of maltotriose, forming alpha(1----3) linkages, to give 3(3)-alpha-D-glucopyranosyl maltotriose and 3(1)-alpha-D-glucopyranosyl maltotriose . Neither of these acceptor products further served as acceptors to give a homologous series . Under equivalent conditions of equimolar amounts of acceptor and sucrose, maltose and maltotriose are much better acceptors with GTF-I than they are with GTF-S, which is better than L . mesenteroides B-512FM dextransucrase . The three enzymes display significantly different efficiencies for the different maltodextrin acceptor reactions, GTF-I and GTF-S having much higher efficiencies than L . mesenteroides B-512FM dextransucrase.

J Gen Microbiol, 1991 Sep, 137 ( Pt 9), 2135 - 9
Lysogeny in Leuconostoc oenos; Arendt EK et al.; Thirty strains of Leuconostoc oenos were exposed to mitomycin C to induce lysogenic bacteriophages . Lysis curves typical for lysogenic strains were obtained with 19 strains . Indicator strans were found for 17 of these phages . Five were characterized by electron microscopy, lytic spectrum, molecular masses of the proteins, sequencing of five N-terminal amino acids of the two major proteins and DNA analysis (restriction patterns, cross hybridization) . The results revealed a very close relationship between the phages . Hybridization experiments between the DNAs of the temperate phages and the appropriate lysogenic strains revealed phage-related sequences in the DNA of the lysogenic strain.

J Biol Chem, 1991 Jul 15, 266(20), 13028 - 34
Cloning of the gene and amino acid sequence for glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides; Lee WT et al.; Amino acid sequencing of glucose 6-phosphate dehydrogenase (Glc6PD) from Leuconostoc mesenteroides yielded sequence for over 75% of the protein . Two oligonucleotides based on the amino acid sequence were used to isolate a partial Glc6PD gene clone (pLmz delta N65), from a pUC9 library, containing 85% of the coding sequence and the 3'-untranslated DNA, but lacking the 5'-noncoding DNA sequence and the portion of the gene encoding the 65 N-terminal amino acids . Attempts to obtain a full-length clone from lambda libraries were unsuccessful, possibly due to restriction of L . mesenteroides DNA by Escherichia coli host cells . The 5'-untranslated DNA was amplified by the polymerase chain reaction and partially sequenced . To obtain unmodified DNA for the gene, oligonucleotides corresponding to the 5'- and 3'-noncoding sequences were used to amplify the gene by the polymerase chain reaction, and a 1.8-kilobase pair fragment was isolated and cloned into pUC19 . The recombinant plasmid, pLmz, contains the entire Glc6PD gene and expresses the gene in E . coli . pLmz was sequenced showing that the enzyme consists of 485 amino acids . L . mesenteroides Glc6PD is 31% identical to the human enzyme.

FEMS Microbiol Lett, 1991 Jul 15, 66(1), 55 - 9
A phylogenetic analysis of an atypical leuconostoc: description of Leuconostoc fallax sp . nov; Martinez-Murcia AJ et al.; The 16S rRNA sequence of an unknown leuconostoc originally isolated from sauerkraut was investigated by reverse transcription . A comparison of the sequence with those from other lactic acid bacteria revealed the unknown organism represents a new albeit peripheral line within the genus Leuconostoc sensu stricto . A new species, Leuconostoc fallax, is proposed for this organism . The type strain is DSM 20189.

Diagn Microbiol Infect Dis, 1991 Jul-Aug, 14(4), 337 - 45
In vitro activity of 43 antimicrobial agents tested against ampicillin-resistant enterococci and gram-positive species resistant to vancomycin; Yamane N et al.; A total of 57 strains of ampicillin-resistant and -susceptible enterococci representing 10 species and 23 strains of vancomycin-resistant Gram-positive bacteria (Leuconostoc and Pediococcus) were tested to determine their susceptibility to 43 antimicrobial agents by the reference broth microdilution method . The drug MICs for the ampicillin-resistant enterococci were generally similar to those of ampicillin-susceptible strains, that is, highly resistant to cephalosporins, moderately susceptible or resistant to quinolones, and susceptible to "glycopeptides." Some investigational quinolones (PD127391, sparfloxacin, WIN57273), minocycline, and rifampin were highly active . Vancomycin-resistant strains were usually resistant to other "glycopeptides," for which correlation coefficients of MICs ranged from 0.881 to 0.978, except ramoplanin (MICs, 0.008-0.5 micrograms/ml) . Most isolates resistant to vancomycin were susceptible to the newer quinolones, penicillins, aminoglycosides, clindamycin, and erythromycin, but highly resistant to cephalosporins . Discrepancies between the MICs and MBCs for glycopeptides were noted (greater than or equal to 8-fold, MBC50/MIC50), but not for ramoplanin . The vancomycin disk test was in 96.1% absolute agreement by identifying resistant strains without contributing false-susceptible, very major error.

Agric Biol Chem, 1991 Jul, 55(7), 1805 - 10
Purification and some properties of sucrose phosphorylase from Leuconostoc mesenteroides; Koga T et al.; Sucrose phosphorylase (EC 2.4.1.7) was purified to homogeneity from Leuconostoc mesenteroides cells with a specific activity of 173.8 units per mg protein by ammonium sulfate fractionation, anion exchange HPLC on TSKgel DEAE-5PW, and hydrophobic HPLC on TSKgel Ether-5PW . The purified enzyme was an acidic protein having an isoelectric point of pH 4.6 and s0(20),W of 4.34S . The molecular weight of this enzyme was estimated to be 56,400 by sedimentation equilibrium, 55,000 by SDS-polyacrylamide gel electrophoresis, and HPLC gel filtration on TSKgel G3000SW, suggesting that the enzyme is a monomeric protein . With regard to molecular weight, amino acid composition, and N-terminal amino acid sequence of 30 residues, this enzyme is close to the glucosyltransferase A of Streptococcus mutans.

Eur J Clin Microbiol Infect Dis, 1991 Jun, 10(6), 505 - 9
Leuconostoc species as a cause of bacteremia: two case reports and a literature review; Bernaldo de Quiros JC et al.; Two new cases of significant bacteremia caused by Leuconostoc spp . are reported and five others described in the literature are reviewed . Four of the seven patients were under one year old and presented with prolonged diarrhea related to gastrointestinal disorders . The remaining three patients were over 50 years of age and being treated in intensive care units . Six patients had nosocomially acquired catheter-related bacteremia . Leuconostoc spp . are naturally resistant to vancomycin, and five patients had received this antibiotic for prior bacteremia caused by methicillin-resistant staphylococci . The majority of patients presented with fever without severe complications . Penicillin is the treatment of choice and there is no report of any death directly attributable to infection by these microorganisms . Infection with Leuconostoc spp . should be suspected if "vancomycin-resistant streptococci" are isolated from the blood, and recorded as a potential cause of bacteremia in patients with indwelling intravenous catheters.

J Biol Chem, 1991 Mar 25, 266(9), 5558 - 62
Interaction of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase with pyridoxal 5'-diphospho-5'-adenosine . Affinity labeling of Lys-21 and Lys-343; LaDine JR et al.; Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides competitively with respect to glucose 6-phosphate and noncompetitively with respect to NAD+ or NADP+, with Ki = 40 microM in the NADP-linked and 34 microM in the NAD-linked reaction . Incubation of glucose-6-phosphate dehydrogenase with {3H}PLP-AMP followed by borohydride reduction shows that incorporation of 0.85 mol of PLP-AMP per mol of enzyme subunit is required for complete inactivation . Both glucose 6-phosphate and NAD+ protect against this covalent modification . The proteolysis of the modified enzyme and isolation and sequencing of the labeled peptides revealed that Lys-21 and Lys-343 are the sites of PLP-AMP interaction and that glucose 6-phosphate and NAD+ protect both lysyl residues against modification . Pyridoxal 5'-phosphate (PLP) also modifies Lys-21 and probably Lys-343 . Lys-21 is part of a highly conserved region that is present in all glucose-6-phosphate dehydrogenases that have been sequenced . Lys-343 corresponds to an arginyl residue in other glucose-6-phosphate dehydrogenases and is in a region that is less homologous with those enzymes . PLP-AMP and PLP are believed to interact with L . mesenteroides glucose-6-phosphate dehydrogenase at the glucose 6-phosphate binding site . Simultaneous binding of NAD+ induces conformational changes (Kurlandsky, S . B., Hilburger, A . C., and Levy, H . R . (1988) Arch . Biochem . Biophys . 264, 93-102) that are postulated to interfere with Schiff's-base formation with PLP or PLP-AMP . One or both of the lysyl residues covalently modified by PLP or PLP-AMP may be located in regions of the enzyme undergoing the NAD(+)-induced conformational changes.

Arch Biochem Biophys, 1990 Dec, 283(2), 379 - 87
Acceptor reactions of maltodextrins with Leuconostoc mesenteroides B-512FM dextransucrase; Fu DT et al.; The acceptor products of maltose with Leuconostoc mesenteroides B-512FM dextransucrase are panose (6(2)-alpha-D-glucopyranosyl maltose) and a homologous series of 6(2)-isomaltodextrinosyl maltoses . The structures of the acceptor products of dextransucrase with other maltodextrins, maltotriose to maltooctaose (G3-G8), were determined by using the known specificities of alpha-glucosidase and porcine pancreatic alpha-amylase, and by methylation analysis . It has been found that dextransucrase transfers a D-glucopyranosyl residue to C-6 of either the nonreducing end or the reducing end residues of the maltodextrins, G3-G8, forming an alpha(1----6) linkage . When a D-glucose was transferred to the nonreducing residue, the first product was also an acceptor to give the second product, which served as an acceptor to give the third product, etc . to give a homologous series . When D-glucose was transferred to the reducing residue, the first product did not readily serve as an acceptor to give products or it served only as a very poor acceptor to give a small amount of the next homologue . The effectiveness of maltodextrins as acceptors decreased as the size of the maltodextrin chain increased . Maltotriose was 40% as effective as maltose and maltooctaose was only 6% as effective.

Immunobiology, 1990 Dec, 182(1), 100 - 15
T cells regulate the IgM antibody response of BALB/c mice to dextran B1355; Haslov KR et al.; The IgM antibody response of BALB/c mice to bacterial (Leuconostoc) dextran B1355 is influenced in a positive and negative manner by regulatory CD4+ and CD8+ T cells, respectively . Treatment with concanavalin A (ConA) at the time of immunization or 2 days later caused suppression and enhancement of the antibody response, respectively . Priming of mice with a sub-immunogenic dose of dextran resulted in profound suppression upon subsequent immunization 3 days later . None of these effects were demonstrable in athymic mice . Transfer of T cells from mice primed 18 h previously with a subimmunogenic dose of dextran suppressed the antibody response in immunized recipients; such suppression was abolished by the treatment of transferred cells with anti Thy 1.2 or anti Lyt 2.2 (CD8) antibody in the presence of complement . By contrast, the transfer of T cells from mice, which had been given an immunogenic dose of dextran 4 days previously, increased the antibody response in immunized recipients; such enhancement was abolished by treating transferred cells with anti Thy 1.2 or anti L3T4 (CD4) antibody in the presence of complement . These findings indicate that the immune response to dextran B1355 is regulated by CD4+ T-amplifier cells (Ta cells) and by CD8+ T-suppressor cells (Ts cells) which are activated during the course of a normal antibody response.

J Clin Microbiol, 1990 Sep, 28(9), 2125 - 6
Meningitis in a neonate caused by Leuconostoc sp; Friedland IR et al.; A case of meningitis in a neonate caused by vancomycin-resistant Leuconostoc mesenteroides is presented . This case was complicated by severe ventriculitis and was ultimately fatal . Infection with Leuconostoc spp . is rare but should be suspected when vancomycin-resistant organisms resembling streptococci are isolated . Previous reports of this infection are reviewed.

J Clin Microbiol, 1990 Aug, 28(8), 1728 - 33
Characterization of Leuconostoc lactis strains from human sources; Barreau C et al.; An examination of 23 vancomycin-resistant, streptococcuslike isolates of clinical origin revealed a group of 14 to be related to Leuconostoc lactis (type strain, CIP 102422) on the basis of chemotaxonomic studies . These isolates were initially shown to be atypical by classical biochemical tests . However, they were characterized in particular by their polar lipid patterns by thin-layer chromatography and, additionally, by whole-cell protein patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . It is feasible, therefore, that common biochemical tests may continue to serve the purpose of routine identification, even though such isolates were formerly thought to be only of dairy origin.

Biochemistry, 1990 Jul 17, 29(28), 6698 - 706
Vanadate dimer and tetramer both inhibit glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; Crans DC et al.; Vanadate dimer and tetramer inhibit glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides . The inhibition by a vanadate mixture containing vanadate monomer, dimer, tetramer, and pentamer was determined by measuring the rates of glucose 6-phosphate oxidation and reduction of NAD (or NADP) catalyzed by glucose-6-phosphate dehydrogenase . The inhibition by vanadate is competitive with respect to NAD or NADP and noncompetitive (a mixed type) with respect to glucose 6-phosphate (G6P) when NAD or NADP are cofactors . This inhibition pattern varies from that observed with phosphate and thus suggests vanadate interacts differently than a phosphate analogue with the enzyme . 51V NMR spectroscopy was used to directly correlate the inhibition of vanadate solutions to the vanadate dimer and/or tetramer, respectively . The activity of the vanadate oligomer varied depending on the cofactor and which substrate was being varied . The vanadate dimer was the major inhibiting species with respect to NADP . This is in contrast to the vanadate tetramer, which was the major inhibiting species with respect to G6P and with respect to NAD . The inhibition by vanadate when G6P was varied was weak . The competitive inhibition pattern with respect to NAD and NADP suggests the possibility that vanadate oligomers may also inhibit catalysis of other NAD- or NADP-requiring dehydrogenases . Significant concentrations of vanadate dimer and tetramer are only found at fairly high vanadate concentrations, so these species are not likely to represent vanadium species present under normal physiological conditions . It is however possible the vanadate dimer and/or tetramer represent toxic vanadate species.

Rev Infect Dis, 1990 Jul-Aug, 12(4), 602 - 10
Infection due to Leuconostoc species: six cases and review; Handwerger S et al.; Leuconostocs, members of the family Streptococcacae, have only recently been recognized as potential pathogens . We describe six cases of leuconostoc bacteremia and review 11 additional cases of infection reported in the literature . Fifteen patients with bacteremia ranged from neonates to persons aged 78 years . Almost all were hospitalized with significant underlying diseases, had received previous antibiotic therapy, and had undergone procedures that interrupted the normal integumentary defense . Leuconostoc bacteremia was associated with fever, leukocytosis, and gastrointestinal complaints . Eight of 15 patients had polymicrobial bacteremia, seven of these eight with staphylococcal species . Clinical isolates of Leuconostoc were frequently misidentified, usually as viridans streptococci . All clinical isolates identified to date--and most agricultural isolates--demonstrate a high level of resistance to vancomycin . Successful regimens for treatment of Leuconostoc include high-dose penicillin, clindamycin, and where appropriate, removal of infected intravascular catheters . Susceptibility testing of all gram-positive bacteria isolated from normally sterile body sites is recommended.

Arch Biochem Biophys, 1990 Feb 1, 276(2), 460 - 5
Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen; Fu DT et al.; The specificity of acceptor binding to the active site of dextransucrase was studied by using alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen . 2-Deoxy-alpha-methyl-D-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-alpha-methyl-D-glucopyranosides were synthesized from alpha-methyl-D-glucopyranoside; and alpha-methyl-D-allopyranoside was synthesized from D-glucose . The analogs were incubated with {14C}sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry . Structures of the acceptor products were determined by methylation analyses and optical rotation . The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy . The enzyme transfers D-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of alpha-methyl-D-glucopyranoside . The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4 . The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the alpha-methyl-D-glucopyranoside ring.

Arch Oral Biol, 1990, 35(1), 55 - 62
Immunochemical studies on levans from several strains of Actinomyces viscosus; Allen PZ et al.; High molecular-weight levans elaborated by 8 separate strains of Actinomyces viscosus were purified: the inteactions of these levans with concanavalin A and anti-fructan myeloma immunoglobulins UPC-10 and J606 were examined by the quantitative precipitin method . Oligosaccharides released from the levans by partial acid hydrolysis were separated by partition chromatography on paper and characterized in situ by selective spray reagents . The liberated oligomers were compared with oligomers of known structure released from levans of Aerobacter levanicum and Leuconostoc mesenteriodes B512 as well as with plant inulin . The fragmentation analysis indicated a structure for Actinomyces levans comprising chains of beta (2----6)-linked fructofuranosyl units joined through multiple (1,2,6)-linked fructosyl branch units.

Prep Biochem, 1990, 20(2), 93 - 106
A facile purification of Leuconostoc mesenteroides B-512FM dextransucrase; Fu DT et al.; Leuconostoc mesenteroides NRRL B-512F has been mutated by treatment with N-nitrosoguanidine . The resulting mutant (designated as B-512FM) produces 300 times as much enzyme as the parent strain . B-512FM dextransucrase was treated extensively with Sigma crude dextranase, followed by column chromatography on Bio-Gel A-5m . The purified dextransucrase had a specific activity of 84 IU/mg, a 100-fold purification with 42% yield, and was shown by SDS-PAGE to have a single protein of molecular weight of 158,000 with dextransucrase activity . The procedure has been used to produce purified enzyme for sequencing . The molecular weight of 158,000 agrees with that calculated from its amino acid sequence.

J Basic Microbiol, 1990, 30(8), 577 - 85
Purification and properties of a malolactic enzyme from Leuconostoc oenos ATCC 23278; Naouri P et al.; The malolactic enzyme of Leuconostoc oenos ATCC 23278 was purified 136fold . The molecular weight was estimated at 132,000 when determined by gel filtration . The enzyme contained two identical subunits (Mw = 66,000 using sodium dodecyl sulfate gel electrophoresis) . The malolactic enzyme catalyzes the NAD(+)- and Mn(+)-dependent reaction L-malate----L-lactate + CO2 . The apparent Km values for malic acid, NAD+, and Mn2+ were 17 mM, 0.044 mM, and 0.017 mM, respectively . The optimal pH and the optimal temperature for activity were 5.0, and 37 degrees C, respectively and the isoelectric point was pH 4.30 . L-lactate and ethanol were non-competitive inhibitors, whereas succinate, citrate, and D-tartrate showed competitive type inhibitions.

Anal Biochem, 1989 Nov 1, 182(2), 399 - 404
Assay of creatine kinase in microtiter plates using thio-NAD to allow monitoring at 405 nM; Florini JR; An assay system for creatine kinase using microtiter plates and a plate reader that records absorbancies at 405 nM has been devised . The system is an adaptation of well-established assays that couple creatine kinase with the reactions catalyzed by hexokinase and glucose-6-phosphate dehydrogenase (G6PDH), to give a measurable increase in reduced pyridine nucleotide quantitated by absorbance at 340 nM . Two features of this system are modified for reading at 405 nM: (i) The thioamido derivative of NAD is used because its reduced form exhibits a substantial increase in absorbance at 405 nM, the most commonly available wavelength on microplate readers; and (ii) glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is used because it can reduce either NAD or NADP (unlike most other G6PDH enzymes, which require NADP), thus making it unnecessary to use the more expensive thio-NADP . The rate of thio-NAD reduction is linear with enzyme concentration and time over a 20-fold range of concentrations of purified creatine kinase, and the assay also works well with myogenic cells allowed to grow and differentiate in the 96-well plate in which the assay is performed . This system offers considerable savings in cells, time, and material in studies of muscle cell differentiation, for which creatine kinase levels are frequently measured . It also provides a potential method for the convenient and economical measurement of activities of many other enzymes that can be coupled to reduction of thio-NAD.

J Bacteriol, 1989 Oct, 171(10), 5750 - 2
Chemiosmotic energy from malolactic fermentation; Cox DJ et al.; By using the luciferase-luciferin ATP assay and whole cells of Leuconostoc oenos, we have demonstrated that malolactic fermentation does yield ATP . This energy-yielding mechanism did not occur in a cell extract and was inhibited in the presence of dicyclohexylcarbodiimide or an ionophore such as monensin . A lactate:proton efflux mechanism for this proposed pathway is presented.

Carbohydr Res, 1989 Feb 15, 186(1), 87 - 94
Synthesis of 4,6-dideoxysucrose, and inhibition studies of Leuconostoc and Streptococcus D-glucansucrases with deoxy and chloro derivatives of sucrose modified at carbon atoms 3, 4, and 6; Tanriseven A et al.; Starting from sucrose, 2,3,1',3',4',6'-hexa-O-benzoyl-6-deoxy-6-iodosucrose (1) was synthesized . Reaction of 1 with sulfuryl chloride in pyridine gave 2,3,1',3',4',6'-hexa-O-benzoyl-4-chloro-4,6-dideoxy-6-iodogalactosucr ose (2) . Compound 2 was treated with tributyltin hydride in toluene in the presence of a radical initiator, alpha, alpha-azobis(isobutanonitrile) (AIBN), to remove iodine and chlorine groups and give hexa-O-benzoyl-4,6-dideoxysucrose . Benzoyl groups were removed by sodium methoxide in methanol to give 4,6-dideoxysucrose . Sucrose was modified at carbon atom 3, carbon atom 4, or carbon atoms 4 and 6, and these analogs were tested as inhibitors of the D-glucansucrases (D-glucosyltransferases) of Streptococcus mutans 6715 and Leuconostoc mesenteroides B-512F . Sucrose analogs used in this study are 4-deoxysucrose and 4-chloro-4-deoxygalactosucrose with S . mutans 6715 D-glucansucrases (GTF-S and GTF-I), and 3-deoxysucrose, 4-deoxysucrose, 4-chloro-4-deoxygalactosucrose, 6-deoxysucrose, and 4,6-dideoxysucrose with L . mesenteroides B-512F D-glucansucrase . The data indicate that 3-deoxysucrose, 4-deoxysucrose, and 4-chloro-4-deoxygalactosucrose are weak noncompetitive inhibitors for B-512F dextransucrase, with Ki values of 530, 201, and 202mM respectively . For the same enzyme, 6-deoxysucrose was a strong competitive inhibitor, with Ki of 1.60mM, and 4,6-dideoxysucrose was a good competitive inhibitor, with Ki of 20.3mM . 4-Deoxysucrose was a weak noncompetitive inhibitor for both GTF-I and GTF-S, with Ki values of 672 and 608mM, respectively . 4-Chloro-4-deoxygalactosucrose was also a weak noncompetitive inhibitor for GTF-I and GTF-S with Ki values of 391 and 308mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydr Res, 1989 Feb 1, 185(2), 239 - 48
Use of 13C-n.m.r . spectroscopy for the quantitative estimation of 3-O- and 3,6-di-O-substituted D-glucopyranosyl residues in alpha-D-glucans formed by the D-glucosyltransferases of Streptococcus sobrinus; Shimamura A; The 13C-n.m.r . spectra of the three alpha-D-glucans from Streptococcus sobrinus and the dextran from Leuconostoc mesenteroides, which differ widely in the ratios of omega (terminal, nonreducing) D-glucopyranosyl groups: 3-:6-:3,6-linked D-glucopyranosyl (Glc) residues, were measured in 0.5M NaOH at 22 degrees . The C-1 signals of 3-O-substituted Glc in a linear sequence, 6-O-substituted Glc in a linear sequence, 3,6-di-O-substituted Glc in a (1----6)-linked sequence, and Glc attached to O-3 of 3,6-di-O-substituted Glc were distinguished from each other . The C-3 signal of 3,6-linked Glc appeared downfield by 0.6 to 1.0 p.p.m . compared to the C-3 signal of 3-linked Glc in a linear sequence . The C-6 signals of omega-terminal, 3-linked, 6-linked, and 3,6-linked Glc were also assigned . The C-2 signal of 3-linked Glc in a linear sequence appeared separately, at 73.76 p.p.m . Based on these assignments, the various D-glucopyranosyl residues of the S . sobrinus alpha-D-glucans were quantitatively estimated from the signal areas of the C-2 atom of 3-linked Glc, the C-3 atom of 3-linked and 3,6-linked Glc, the C-6 atom of 6-linked and 3,6-linked Glc, and the C-6 atom of the omega-Glc groups and 3-linked Glc residues . The figures thus derived for the linkage ratios were close to those obtained by methylation analysis.

Carbohydr Res, 1988 Nov 15, 183(1), 97 - 109
Essential histidine residues in dextransucrase: chemical modification by diethyl pyrocarbonate and dye photo-oxidation; Fu D et al.; Treatment of Leuconostoc mesenteroides B-512F dextransucrase with diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees or photo-oxidation in the presence of Rose Bengal or Methylene Blue at pH 6.0 and 25 degrees, caused a rapid decrease of enzyme activity . Both types of inactivation followed pseudo-first-order kinetics . Enzyme partially inactivated by DEP could be completely reactivated by treatment with 100 mM hydroxylamine at pH 7 and 4 degrees . The presence of dextran partially protected the enzyme from inactivation . At pH 7 or below, DEP is relatively specific for the modification of histidine . DEP-modified enzyme showed an increased absorbance at 240 nm, indicating the presence of (ethoxyformyl)ated histidine residues . DEP modification of the sulfhydryl group of cysteine and of the phenolic group of tyrosine was ruled out by showing that native and DEP-modified enzyme had the same number of sulfhydryl and phenolic groups . DEP modification of the epsilon-amino group of lysine was ruled out by reaction at pH 6 and reactivation with hydroxylamine, which has no effect on DEP-modified epsilon-amino groups . The photo-oxidized enzyme showed a characteristic increase in absorbance at 250 nm, also indicating that histidine had been oxidized, and no decrease in the absorbance at 280 nm, indicating that tyrosine and tryptophan were not oxidized . A statistical, kinetic analysis of the data on inactivation by DEP showed that two histidine residues are essential for the enzyme activity . Previously, it was proposed that two nucleophiles at the active site attack bound sucrose, to give two covalent D-glucosyl-enzyme intermediates . We now propose that in addition, two imidazolium groups of histidine at the active site donate protons to the leaving, D-fructosyl moieties . The resulting imidazole groups then facilitate the formation of the alpha-(1----6)-glycosidic linkage by abstracting protons from the C-6-OH groups, and become reprotonated for the next series of reactions.

J Bacteriol, 1988 Nov, 170(11), 5006 - 11
Dextransucrase secretion in Leuconostoc mesenteroides depends on the presence of a transmembrane proton gradient; Otts DR et al.; The relationship between proton motive force and the secretion of dextransucrase in Leuconostoc mesenteroides was investigated . L . mesenteroides was able to maintain a constant proton motive force of -130 mV when grown in batch fermentors at pH values 5.8 to 7.0 . The contribution of the membrane potential and the transmembrane pH gradient varied depending on the pH of the growth medium . The differential rate of dextransucrase secretion was relatively constant at 1,040 delta mU/delta mg (dry weight) when cells were grown at pH 6.0 to 6.7 . Over this pH range, the internal pH was alkaline with respect to the external pH . When cells were grown at alkaline pH values, dextransucrase secretion was severely inhibited . This inhibition was accompanied by an inversion of the pH gradient as the internal pH became more acidic than the external pH . Addition of nigericin to cells at alkaline pH partially dissipated the inverted pH gradient and produced a fourfold stimulation of dextransucrase secretion . Treatment of cells with the lipophilic cation methyltriphenylphosphonium had no effect on the rate of dextransucrase secretion at pH 5.5 but inhibited secretion by 95% at pH 7.0 . The reduced rate of secretion correlated with the dissipation of the proton motive force by this compound . Values of proton motive force greater than -90 mV were required for maximal rates of dextransucrase secretion . The results of this study indicate that dextransucrase secretion in L . mesenteroides is dependent on the presence of a proton gradient across the cytoplasmic membrane that is directed into the cell.

Cell Immunol, 1988 Oct 15, 116(2), 482 - 8
A comparison of the IgG isotype expression in the so-called thymus-independent immune responses to alpha(1----3) and alpha(1----6) dextran; Boss K et al.; This paper presents data on the IgG antibody response against two "thymus-independent" dextran (Dex) antigens from Leuconostoc mesenteroides, alpha(1----3) Dex B 1355S and alpha(1----6) Dex B 512F in BALB/c and C57BL/6 mice, which are considered to be responders or low responders to the respective antigen . The data point toward three common rules governing the two anti-Dex responses despite immunogenetic and antigenic disparities: (1) age dependency of the IgG isotype regulation of the response; (2) down-regulation of IgG isotype expression by T cells; and (3) individually determined preposition for IgG isotype formation in a given animal.

J Clin Microbiol, 1988 Sep, 26(9), 1893 - 4
Odontogenic infection secondary to Leuconostoc species; Wenocur HS et al.; Leuconostoc species are gaining importance as pathogenic organisms . We present the first case of odontogenic infection caused by Leuconostoc spp . Isolates initially identified as streptococci were found to be vancomycin resistant . Rigorous bacteriologic investigation subsequently classified these organisms as Leuconostoc mesenteroides.

Arch Biochem Biophys, 1988 Jul, 264(1), 93 - 102
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: ligand-induced conformational changes; Kurlandsky SB et al.; Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is inactivated by trypsin, chymotrypsin, pronase E, thermolysin, 4.0 M urea, and by heating to 49 degrees C . It is protected, to varying degrees, against all these forms of inactivation by glucose 6-phosphate, NAD+, and NADP+ . When these ligands are present at 10 times their respective KD concentrations, protection by NAD+ or glucose 6-phosphate is substantially greater than protection by NADP+ . A detailed analysis was undertaken of the protective effects of these ligands, at varying concentrations, on proteolysis of glucose-6-phosphate dehydrogenase by thermolysin . This study confirmed the above conclusion and permitted calculation of KD values for NAD+, NADP+, and glucose 6-phosphate that agree with such values determined by independent means . For NADP+, two KD values, 6.1 microM and 8.0 mM, can be derived, associated with protection against thermolysin by low and high NADP+ concentrations, respectively . The former value is in agreement with other determinations of KD and the latter value appears to represent binding of NADP+ to a second site which causes inhibition of catalysis . A Ki value of 10.5 mM for NADP+ was derived from inhibition studies . The principal conclusion from these studies is that NAD+ binding to L . mesenteroides glucose-6-phosphate dehydrogenase results in a larger global conformational change of the enzyme than does NADP+ binding . Presumably, a substantially larger proportion of the free energy of binding of NAD+, compared to NADP+, is used to alter the enzyme's conformation, as reflected in a much higher KD value . This may play an important role in enabling this dual nucleotide-specific dehydrogenase to accommodate either NAD+ or NADP+ at the same binding site.

Eur J Biochem, 1988 Feb 1, 171(3), 485 - 9
Human erythrocyte glucose-6-phosphate dehydrogenase . Identification of a reactive lysyl residue labelled with pyridoxal 5'-phosphate; Camardella L et al.; Human erythrocyte glucose-6-phosphate dehydrogenase contains a reactive lysyl residue, which can be labelled with pyridoxal 5'-phosphate . The binding of one mole of pyridoxal 5'-phosphate per mole of enzyme subunit produces substantial inactivation . The substrate glucose-6-phosphate prevents the loss of activity, suggesting that the reaction site is close to the substrate-binding site . A tryptic peptide containing the pyridoxal-5'-phosphate-binding lysyl residue has been isolated and characterised . The reactive lysyl residue has been identified in the glucose-6-phosphate dehydrogenase amino acid sequence . Comparison with glucose-6-phosphate dehydrogenase from other sources shows a high homology with a peptide containing a reactive lysyl residue, isolated from the enzyme from Saccharomyces cerevisiae; glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides also contains a region highly homologous with the sequence around the reactive lysyl residue in the human enzyme . The results of this communication provide the first direct evidence for the association of an essential catalytic function with a specific region of the molecule of human erythrocyte glucose-6-phosphate dehydrogenase.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Jan, 267(3), 379 - 82
Vancomycin-resistant Streptococcaceae from clinical material; Lutticken R et al.; Three strains of vancomycin-resistant Gram-positive cocci, belonging to the family Streptococcaceae, were isolated from patient samples . Two were identified as Leuconostoc species, the other one as Enterococcus (Streptococcus) faecium . The clinical significance of vancomycin-resistant Gram-positive bacteria is discussed.

Allerg Immunol (Leipz), 1988, 34(4), 269 - 73
Anti-dextran antibodies of carp detected by the enzyme-linked immunosorbent assay (ELISA); Richter RF et al.; Carp produce anti-alpha (1-6) dextran antibodies after immunization with a vaccine of Leuconostoc mesenteroides B512 . These antibodies can be determined by passive haemagglutination, quantitative precipitation or by ELISA . In our hands the ELISA has proved to be 100 times more sensitive than the passive haemagglutination test . We could not found any correlation between the anti-dextran activity estimated with the passive haemagglutination on the one and the ELISA on the other hand.

J Clin Microbiol, 1987 Sep, 25(9), 1784 - 5
Meningitis caused by vancomycin-resistant Leuconostoc sp; Coovadia YM et al.; A rare case of purulent meningitis caused by vancomycin-resistant Leuconostoc sp . is reported . The patient was treated successfully with penicillin G, and there were no neurological sequelae . We recommend that all gram-positive cocci isolated from cerebrospinal fluid and blood be fully identified so as not to confuse Leuconostoc spp . with more commonly isolated pathogens such as pneumococci and other alpha-hemolytic streptococci.

Plasmid, 1987 Mar, 17(2), 173 - 5
Plasmids in Leuconostoc oenos; Janse BJ et al.; A new procedure was used to isolate 11 plasmids from eight Leuconostoc oenos strains . Plasmid DNA was not detected in 34 other strains of this species . Plasmid sizes ranged from 2.47 to 4.61 kilobase pairs . This is the first report of extrachromosomal elements in L . oenos.

Arch Biochem Biophys, 1987 Feb 15, 253(1), 156 - 67
Factors underlying significant underestimations of glucokinase activity in crude liver extracts: physiological implications of higher cellular activity; Davidson AL et al.; M . Kuwajima, C . B . Newgard, D . W . Foster, and J . D . McGarry (1986, J . Biol . Chem . 261, 8849-8853) have concluded that the reason postprandial hepatic glycogenesis occurs primarily from gluconeogenic precursors rather than glucose is because glucokinase activity is insufficient to support the observed rates of glycogen synthesis . F . L . Alvares and R . C . Nordlie (1977, J . Biol . Chem . 252, 8404-8414) have concluded that the combined activities of glucokinase and hexokinase are less than the apparent rates of hepatic glucose uptake . We have identified several factors in the assays used in these studies which lead to substantial underestimations of glucokinase activity . Glucokinase was assayed either by allowing glucose 6-phosphate to accumulate over 10 min (discontinuous assay) or by coupling the formation of glucose 6-phosphate with its oxidation by Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase and NAD (continuous assay) . Accurate determinations of glucokinase at 37 degrees C with subsaturating glucose require both 100 mM KCl and 2.5 mM dithioerythritol in the assay medium; 2-mercaptoethanol will not substitute for dithioerythritol . When both KCl and dithioerythritol are absent (Kuwajima et al.) glucokinase activity is underestimated by 3- to 5-fold . The discontinuous assay as used previously (Alvares and Nordlie) underestimates glucokinase activity in crude extracts by 2- to 2.5-fold, due in part to the hydrolysis of glucose 6-phosphate and its transformation to other hexose monophosphates . Under optimized conditions at 37 degrees C both assays yield similar results in extracts from fed rats, i.e., 2-3 and 4-5 units/g liver at 10 and 100 mM glucose, respectively . Some implications of the finding that total hepatic glucose phosphorylating capacity at physiological concentrations significantly exceeds the observed rates of postprandial glycogen synthesis are discussed.

Appl Environ Microbiol, 1987 Feb, 53(2), 352 - 7
Conjugal transfer of nisin plasmid genes from Streptococcus lactis 7962 to Leuconostoc dextranicum 181; Tsai HJ et al.; Acriflavine-generated mutants of Streptococcus lactis 7962 with various combinations of plasmid molecular masses were screened for nisin production . Nisin was produced by both the wild type and mutants that contained a 17.5-megadalton plasmid, which was obscured by chromosomal fragments . No nisin was produced by plasmid-free mutants . Sucrose fermentation and nisin production were simultaneously expressed . A transconjugant obtained from nisin-producing donor S . lactis 7962 and recipient Leuconostoc dextranicum 181 was a "supernisin" producer . The L . dextranicum Nis+ transconjugant was resistant to S . lactis 7962 phage and vancomycin (greater than 1,000 micrograms/ml), and it contained an extra 17.5-megadalton plasmid.

FEBS Lett, 1987 Jan 26, 211(2), 243 - 6
Sequence identity between a lysine-containing peptide from Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and an active site peptide from human erythrocyte glucose-6-phosphate dehydrogenase; Bhadbhade MM et al.; Peptides recently isolated and sequenced from a bacterial (Leuconostoc mesenteroides) glucose-6-phosphate dehydrogenase are remarkably homologous to an active site region of the human erythrocyte enzyme, although the enzymes differ in their overall amino acid composition and kinetic properties . The computer program ALIGN, used to determine the best alignment between the two enzyme sequences, gives match-scores which are statistically highly significant.

J Biol Chem, 1987 Jan 25, 262(3), 1223 - 9
Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+); White BJ et al.; Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate . The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM) . NADP+ and NAD+ protect the enzyme from inactivation by oNADP+ . The pK of inactivation is 8.1 . oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM) . Kinetic evidence and binding studies with {14C} oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated . The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.

J Bacteriol, 1987 Jan, 169(1), 334 - 9
Expression of the gene for NAD-dependent glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides cloned in Escherichia coli K-12; Murphy NB et al.; The structural gene (zwf) for Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase has been cloned into Escherichia coli on pBR322 by selecting for complementation of a zwf mutation of E . coli which eliminates glucose-6-phosphate dehydrogenase . The gene has been subcloned on a 3.4-kilobase DNA segment . The encoded glucose-6-phosphate dehydrogenase displays the dual nucleotide specificity (reacting with NAD and NADP) of the L . mesenteroides enzyme and has a subunit Mr of 55,000 . A second gene encoding protein of subunit Mr 24,000 is also encoded on the 3.4-kilobase DNA segment . The genes have been located by the analysis of deletions and insertions generated in vitro and by transcriptional mapping with a promoterless chloramphenicol acetyl transferase cartridge inserted at different sites in the 3.4-kilobase fragment.

Dev Comp Immunol, 1987 Winter, 11(1), 147 - 53
Immune response of carps (Cyprinus carpio L.) against dextran; Richter RF et al.; In carps no antibodies could be evoked by immunization with soluble high molecular weight dextran using doses of 1-5000 mg dextran per kg body weight . On the other hand, an immune response against dextran could be elicited by immunization with a vaccine of Leuconostoc mesenteroides B512 as well as a dextran-BSA-conjugate . These carp anti-dextran antibodies showed precipitating and agglutinating activities with specificity against alpha linked dextran determined by the almost complete inhibition of the dextran-anti-dextran reaction by isomaltodecaose.

J Biochem (Tokyo), 1986 Sep, 100(3), 615 - 21
Electrophoretic analysis of the multiple forms of dextransucrase from Leuconostoc mesenteroides; Kobayashi M et al.; Multiple forms of the extracellular dextransucrase {EC 2.4.1.5} from Leuconostoc mesenteroides NRRL B-512F strain were characterized by polyacrylamide gel electrophoresis . Based on the Rm (Relative mobility) values, a newly devised simple plot of log (Rm X 10/(1-Rm)) vs . degree of association of the enzyme showed a good correlation with the results obtained by the Hedrick-Smith method . Both results indicated that the B-512F dextransucrase aggregates were a mixture of two types of forms, i.e., oligomers of a 65 kDa protomer and their charge isomers . Boiling and treatment of the enzyme at pH 10.5 suggested that enzyme aggregates contained dextran or its fragments bound to the enzyme and the enzyme-dextran complex showed the charge isomerism . Since the highly aggregated forms showed higher activity for dextran synthesis than the dissociated forms, the endogenous dextran may serve as a source of primer and may stabilize the enzyme molecule . Besides allosteric regulation of the activity, the occurrence of oligomeric forms of the enzyme may play an important role in the control of dextran synthesis in vivo.

Mol Immunol, 1986 Sep, 23(9), 999 - 1028
Immunochemical and related interactions with dextrans reviewed in terms of improved structural information; Jeanes A; Structurally diverse dextrans from Leuconostoc mesenteroides and related bacteria have been used extensively in fundamental immunochemical studies such as induction and characterization of anti-dextran antibodies, as well as in studies of their interaction with pneumococcal antisera, normal bovine serum, concanavalin A, dextran-binding myeloma immunoglobulins and hybridoma antibodies . The inherent lack of specificity of structural data obtained by POSA and general lack of insight into other limitations of these analyses has often led to inaccurate and superficial interpretations . Proper interpretation of past and future studies necessitates pointing out previous inadequacies of dextran structural data and detailing more recently acquired structural information on the dextrans . Unambiguous terminology has been achieved by a new system of linkage symbols that includes the designation of structural positions, such as (1----3; l)- and (1----3; b) as linear-chain and branch-point positions, respectively . Results of immunological studies are reviewed . Improved interpretations and correlations are made on the basis of structural data from MSA and several other techniques which have become available on the dextrans.

Biochim Biophys Acta, 1986 Mar 28, 870(2), 198 - 203
Functional molecular size and structure of dextransucrase by radiation inactivation and gel electrophoresis; Miller AW et al.; Robyt et al . have proposed a mechanism for dextransucrase in which dextran is synthesized by the cooperative action of two equivalent nucleophiles (Robyt, J.F., Kimble, B.K . and Walseth, T.F . (1974) Arch . Biochem . Biophys . 165, 634-640) . To distinguish between the possibilities that the enzyme is a monomer bearing both nucleophiles, or a dimer with each subunit bearing one nucleophile, the molecular weight of the enzyme was determined by SDS-polyacrylamide gel electrophoresis and by radiation inactivation . Two major forms of dextransucrase from Leuconostoc mesenteroides NRRL B-512F were found on SDS-polyacrylamide gel electrophoresis, with Mr 177 000 and 158 000, and sometimes a minor form with Mr 168 000 . No form of dextransucrase smaller than Mr 158 000 was found, either in the presence or absence of dextran T10, although levansucrase was detected at Mr 92 000 and 116 000 . On irradiation with 60Co, dextransucrase behaved as a single species with a maximum size of Mr 201 000 . Because Mr 201 000 is much smaller than the minimum dimer size of Mr 316 000 (= 2 X 158 000), it is concluded that both nucleophiles are probably located on the same peptide, rather than one on each subunit of a dimer, and that peptide association is probably not required for dextran synthesis.

Carbohydr Res, 1986 Mar 1, 147(1), 119 - 33
Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F; Miller AW et al.; A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield . Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography . The method could be scaled up to produce gram quantities of purified enzyme . The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification . The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL . The enzyme had two major forms, of molecular weight 177,000 and 158,000 . The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.

Biochim Biophys Acta, 1986 Jan 15, 880(1), 32 - 9
Activation and inhibition of dextransucrase by calcium; Miller AW et al.; Initial rate kinetics of dextran synthesis by dextransucrase (sucrose:1,6-alpha-D-glucan-6-alpha-D-glucosyltransferase, EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F showed that below 1 mM, Ca2+ activated the enzyme by increasing Vmax and decreasing the Km for sucrose . Above 1 mM, Ca2+ was a weak competitive inhibitor (Ki = 59 mM) . Although it was an activator at low concentration, Ca2+ was not required for dextran synthesis, either of main chain or branch linkages . Neither was it required for sucrose hydrolysis, acceptor reactions, or enzyme renaturation after SDS-polyacrylamide gel electrophoresis . A model for dextran synthesis is proposed in which dextransucrase has two Ca2+ sites, one activating and one inhibitory . Ca2+ at the inhibitory site prevents the binding of sucrose.

Microbiol Immunol, 1986, 30(3), 261 - 8
Immunochemical similarity of synthetic alpha-(1----3)-branched D-glucans to dextran B1375; Torii M et al.; Anti-dextran B1375 antibodies were raised in rabbits by injecting formalin-killed Leuconostoc mesenteroides strain NRRL B1375, and the anti-dextran serum was used to examine native dextran B1375, and synthetic linear and four alpha-(1----3)-branched alpha-(1----6)-D-glucopyranans for similarities . The antiserum reacted with the homologous dextran B1375 and also with all the synthetic linear and branched glucans . Precipitation and precipitation-inhibition studies indicated that the antiserum contained at least three groups of antibodies with different specificities, the first specific for linear alpha-(1----6)-D-glucopyranan structure, the second specific for alpha-D-glycopyranosyl-(1----3)-branching and the last specific for another, unknown structure present in the dextran B1375 molecule . Two samples of the synthetic branched glucans were shown to be immunochemically the most similar to natural dextran B1375 by inhibition experiments.

Appl Environ Microbiol, 1985 Oct, 50(4), 872 - 6
Occurrence and properties of bacteriophages of Leuconostoc oenos in Australian wines; Davis C et al.; Bacteriophages specific for Leuconostoc oenos were isolated from four red wines undergoing malolactic fermentation in one winery . Bacteriophages were not found in samples of 16 other wines . The morphology of the phages was examined by electron microscopy . The phages did not lyse all strains of L . oenos, and susceptibility correlated to some extent with the colony morphology of the strain . Phage survived in wines at pH values greater than 3.5 but was inactivated in wines of lower pH and by the addition of sulfur dioxide or bentonite . Phage did not affect the growth of a sensitive strain of L . oenos in filter-sterilized wine.

Antimicrob Agents Chemother, 1985 Sep, 28(3), 458 - 60
Vancomycin-resistant streptococci or Leuconostoc sp; Buu-Hoi A et al.; Two strains of gram-positive cocci highly resistant to vancomycin (MICs of 512 and 1,024 micrograms/ml) were isolated from blood cultures in two compromised patients . These organisms were identified as Leuconostoc spp . Leuconostoc spp . are gram-positive cocci found in vegetables and dairy products; they had not been isolated previously from clinical specimens . The susceptibility of eight Leuconostoc spp . strains, including the two clinical isolates, to 23 antimicrobial agents was determined.

J Appl Bacteriol, 1985 Sep, 59(3), 239 - 42
A note on the occurrence of Leuconostoc oenos as a spoilage organism in canned mango juice; Ethiraj S et al.; A strain of Leuconostoc oenos was isolated from a blown can of mango juice . Various tests to identify and characterize the bacterium suggested that it could be a strain of L . oenos . This is the first report of L . oenos as a spoilage organism in fruit products other than wine.

Anal Biochem, 1985 Jun, 147(2), 468 - 77
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is a reliable internal standard for radiation-inactivation studies of membranes in the frozen state; McIntyre JO et al.; The target size of four soluble enzymes (beta-galactosidase, pyruvate kinase, alcohol dehydrogenase, and glucose-6-phosphate dehydrogenase) in the presence or absence of subcellular membrane fractions has been determined by the radiation-inactivation method using samples in the frozen state . For each of the four enzymes, full activity was recovered after freezing and thawing in the absence of radiation . We found minimal (less than 20%) binding of the enzymes to either submitochondrial vesicles or sarcoplasmic reticulum vesicles . Under the conditions tested, beta-galactosidase, pyruvate kinase, and alcohol dehydrogenase exhibited target sizes which varied according to the experimental conditions, i.e., the buffer selected and also the presence or absence of membrane preparations . For these tetrameric enzymes, the target sizes were generally comparable to either a monomer or a dimer . By contrast, the target size of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be essentially invariant when frozen in a variety of buffers and in the presence or absence of either cryoprotectant (sucrose or glycerol) or different membrane preparations . The target size from 19 separate determinations gave an average value of 104 +/- 16 kDa, which is comparable to the molecular weight of the enzyme (104 kDa) . We conclude that glucose-6-phosphate dehydrogenase from L . mesenteroides is a reliable internal standard for radiation-inactivation studies of membrane preparations in the frozen state.

J Biochem (Tokyo), 1985 May, 97(5), 1279 - 88
Purification and characterization of NADH oxidase from a strain of Leuconostoc mesenteroides; Koike K et al.; An NADH oxidase was purified to homogeneity from Leuconostoc mesenteroides with a specific activity 100-fold higher than that of the crude extract . The purified NADH oxidase was an acidic protein having an S0 20,W of 5.49S and a molecular weight of 104,000, consisting of a dimer with 53,000 subunit size . The enzyme could use O2, dichlorophenolindophenol and methylene blue as oxidants, but not H2O2, cytochrome c, or ferricyanide . The physiological substrate was beta-NADH (Km = 0.12 mM) with O2 as the oxidant, probably forming H2O, rather than H2O2 . Activity toward alpha-NADH was observed (Km = 0.14 mM), but the maximum velocity was 3 orders of magnitude lower than that with beta-NADH . alpha-NADPH and beta-NADPH were inert for the reaction . The enzyme showed a flavoprotein absorption spectrum with maxima at 273, 379, and 450 nm with a shoulder at 465 nm: the absorption at 450-465 nm disappeared on adding excess NADH or hydrosulfite . One mol of the holoenzyme contained approximately 2 mol of FAD . The apoenzyme was obtained by treatment with EDTA-KBr solution and could be reconstituted partially by adding FAD, but not riboflavin or FMN . The maximum activity of the reaction was observed at pH 6.5 in a temperature range of 35-45 degrees C . The activation energy was estimated to be 3.77 kcal/mol . The enzyme was inhibited by SH reagents, quinacrine, quinine, and Cu2+, but not by EDTA . Adenine and its nucleoside 5'-di- and triphosphates showed competitive inhibitions, while various metabolites, such as H2O2, FDP, acetyl phosphate, lactate, ethanol, and acetate, did not affect the reaction.

Eur J Biochem, 1985 Mar 1, 147(2), 249 - 53
Coenzymic activity of NADP derivatives alkylated at 2'-phosphate and 6-amino groups; Okuda K et al.; Coenzymic activities of the following NADP derivatives were investigated: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III), 2'-O-{N-(2-aminoethyl)carbamoylethyl}phosphono-NAD (IV), N6-{N-(2-aminoethyl)carbamoylethyl}-NADP (Va), 2',3'-cyclic NADP, and 3'-NADP . Derivatives I and IV show the effects of modification at the 2'-phosphate group, and derivatives II and Va show those at the 6-amino group of NADP . As for enzymes, alcohol, isocitrate, 6-phosphogluconate, glucose, glucose-6-phosphate, and glutamate dehydrogenases were used . These enzymes were grouped on the basis of the ratio of the activities for NAD and NADP into NADP-specific enzymes (ratio less than 0.01), NAD(P)-specific enzymes (0.01 less than ratio less than 100), and NAD-specific enzymes (ratio greater than 100) . For NADP-specific enzymes, modifications at the 2'-phosphate group of NADP caused great loss of cofactor activity . The relative cofactor activities (NADP = 100%) of derivatives I and IV for these enzymes were 0.5-20 and 0.01-0.5%, respectively . On the other hand, NAD(P)-specific enzymes showed several types of responses to the NADP derivatives . The relative cofactor activities of I and IV for Leuconostoc mesenteroides and Bacillus stearothermophilus glucose-6-phosphate dehydrogenases and beef liver glutamate dehydrogenase were 60-200%; whereas, for B . megaterium glucose dehydrogenase and L . mesenteroides alcohol dehydrogenase, the values were 0.8-8% . For NAD-specific enzymes, these values were 20-50% . The relative cofactor activities of 2',3'-cyclic NADP and 3'-NADP were very low (less than 0.2%) except for beef liver glutamate dehydrogenase, B . stearothermophilus glucose-6-phosphate dehydrogenase, and horse liver alcohol dehydrogenase . Kinetic studies showed that the losses of the cofactor activity of NADP by these modifications were mainly due to the increase of the Km value . The mechanisms of coenzyme specificity of dehydrogenases are discussed . Unlike the 2'-phosphate group, the 6-amino group is common to NAD and NADP, and the effects of modification at the 6-amino group were independent of the coenzyme specificity of enzymes used for the assay . Derivatives II and Va had high relative cofactor activities (65-130%) for most of the enzymes except for isocitrate and glucose dehydrogenases (less than 1%) and L . mesenteroides alcohol dehydrogenase (20-60%) . The cofactor activity of derivative III was generally lower than those of I and II.

Biochemistry, 1985 Jan 29, 24(3), 666 - 71
Glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae: characterization of a reactive lysine residue labeled with acetylsalicylic acid; Jeffery J et al.; Glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae (bakers' yeast) reacts with acetylsalicylic acid, and this is accompanied by inactivation and modification of essentially one lysine residue per subunit . The amino acid sequence of an 11-residue tryptic peptide containing the reactive lysine residue of the yeast enzyme is given and establishes the existence of different subgroups of glucose-6-phosphate dehydrogenases . Thus, the labeled yeast structure has few similarities to the known structure around the reactive lysine residue of the enzyme from Leuconostoc mesenteroides, although it has extensive similarities with a structure in the human enzyme . It is further shown that amino acid sequences around reactive lysine residues of dehydrogenases in general vary, even though similarities occur around reactive lysine residues in 6-phosphogluconate, glutamate, and glyceraldehyde-3-phosphate dehydrogenases.

Immunogenetics, 1985, 22(3), 269 - 76
Genes on different chromosomes influence the antibody response to bacterial antigens; Baker PJ et al.; B6.C congenic strains of mice, possessing histocompatibility (H) alleles from high responding BALB/cBy (C) mice on the genetic background of low responding C57BL/6By (B6) mice, were assayed for their ability to make an antibody response to Type III pneumococcal polysaccharide (SSS-III) and the alpha(1----3) epitope of bacterial (Leuconostoc) dextran B-1355 . The results affirmed that the antibody response to SSS-III is multigenic and that genes making a positive contribution to responsiveness are located on different chromosomes, i.e., chromosomes 1, 3, 4, 5, and 9 . At least one other gene also influences responsiveness to SSS-III; it is linked to the H-17 locus, which has not yet been assigned to a specific chromosome . Genes on chromosomes 1, 4, and 5 influence the magnitude of the antibody response to dextran B-1355 . Some of these genes may be antigen-specific in their mode of action; however, others may not since they appear to exert a positive influence on the antibody response to both SSS-III and dextran B-1355.

Appl Environ Microbiol, 1984 Dec, 48(6), 1129 - 33
Common occurrence of plasmid DNA and vancomycin resistance in Leuconostoc spp; Orberg PK et al.; Resistance to vancomycin permitted detection, in a culture of Streptococcus cremoris 290PC, of a contaminant gram-positive coccus . Morphological and physiological characteristics indicated that this bacterium was a strain of Leuconostoc sp., designated PO184 . This strain contained four plasmid species, which were distinct from those harbored by S . cremoris 290PC . Antibiotic disk susceptibility tests indicated that Leuconostoc sp . strain PO184 was also resistant to sulfathiazole and trimethoprim and susceptible to 17 other antimicrobials . The MIC of vancomycin for this strain was greater than 2,000 micrograms/ml, and resistance did not depend on drug inactivation . Leuconostoc sp . strain PO184 produced a substance which was inhibitory to S . cremoris U134, but not to S . lactis ATCC 11454 . Five other leuconostocs produced substances with antibacterial activity . Of 18 strains of Leuconostoc sp., 14 were resistant to at least 500 micrograms of vancomycin per ml, including four L . oenos strains which harbored no plasmid DNA in the 1- to 76-megadalton range . Twelve Leuconostoc sp . strains contained at least one plasmid species in this mass range . These findings are discussed from the physiological, taxonomical, and ecological standpoints and with regard to their potential applications.

Anal Biochem, 1984 Nov 1, 142(2), 556 - 61
A one-step enzymatic assay for sucrose with sucrose phosphorylase; Birnberg PR et al.; A one-step, enzymatic assay for sucrose using sucrose phosphorylase is described . Sucrose phosphorylase, which is now commercially available, was isolated from Leuconostoc mesenteroides strain B-1200 and partially purified by ammonium sulfate precipitation . Samples containing 5 to 80 nmol of sucrose are mixed with potassium phosphate, NAD, sucrose phosphorylase, and two commercial enzymes, phosphoglucomutase and NAD-accepting glucose-6-phosphate dehydrogenase . After 30 min incubation at room temperature, absorbance at 340 nm is proportional to initial sucrose content . A 20-fold molar excess of glucose or a twofold excess of fructose have no effect on the assay, while a fourfold excess of fructose interferes with the assay by decreasing absorbance ca . 20% . This assay was designed to provide a rapid method for determining sucrose in studies of sugar transport by plants . To test the assay, corn pedicel extracts were assayed enzymatically and by high-pressure liquid chromatography . Estimates of sucrose content made by the two methods were equivalent, and exogenous addition of sucrose to these samples resulted in the expected increase in apparent sucrose content.

J Appl Bacteriol, 1984 Aug, 57(1), 23 - 9
Psychrotrophic bacterial flora of raw ewes' milk, with particular reference to gram negative rods; Nunez JA et al.; The microbial flora of 141 samples of raw ewes' milk was determined, before and after storage for 72 h at 4 degrees and 7 degrees C . Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples . Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated (ca 86%) in the Gram negative psychrotrophic microflora . Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.

J Periodontol, 1984 Jul, 55(7), 424 - 30
Dextran penetration through nonkeratinized and keratinized epithelia in monkeys; Nasjleti CE et al.; The purpose of this study was to determine if polysaccharide dextrans would pass through intact-nonkeratinized and induced-keratinized sulcular epithelia in monkeys . Dextran penetration through normally keratinized oral gingival epithelium also was evaluated in the same gingival specimens . Each of three Rhesus monkeys received a thorough prophylaxis 1 week prior to the experiment . During this week, the monkeys also received daily IV injections of Achromycin . After the antibiotic treatment, the teeth were polished and cleaned with a rubber cup using prophylactic paste for 2 consecutive months, as follows: (1) the right maxillary and mandibular teeth received daily prophylaxes on weekdays and (2) the left maxillary and mandibular teeth received one prophylaxis weekly . These frequencies of plaque removal on one-half of the mouth maintained clinically healthy gingiva and produced keratinization of the sulcular epithelium . At the end of the 2-month prophylaxes, a 5% solution of dextrans derived from Leuconostoc mesenteroides was applied topically to the gingiva once daily for 3 consecutive weeks . During this time, the monkeys continued having dental prophylaxes following the previous time schedule . The study showed that induced-keratinized sulcular epithelium as well as normally keratinized oral gingival epithelium resisted penetration of dextrans, whereas intact-nonkeratinized sulcular epithelium apparently lacked a surface layer resistant to penetration.

Carbohydr Res, 1984 Apr 2, 127(1), 95 - 107
The formation of alpha-D-(1----3) branch linkages by a D-glucansucrase from Streptococcus mutans 6715 producing a soluble D-glucan; Cote GL et al.; An exocellular D- glucansucrase that synthesizes a water-soluble, alpha-D-(1----6)-linked D-glucan having a high proportion of alpha-D-(1----3) branches was purified from the culture broth of Streptococcus mutans 6715 . The rate of incorporation of D-{14C}glucose from {14C}sucrose into D-glucan of high molecular weight by this enzyme was increased (stimulated) by the presence of exogenous Leuconostoc mesenteroides B- 512F dextran, and it was found that this dextran could act as an acceptor . A highly branched dextran, containing 45-50% of alpha-D-(1----3) branch linkages, did not stimulate the enzyme nearly so much as B- 512F dextran, which has a low degree (5%) of alpha-D-(1----3) branches . We interpret this as evidence that the stimulating effects of dextran are not due to priming . If they were, the more highly branched dextran should have produced the greatest stimulation per unit weight, because a much greater number of nonreducing-end, priming sites would be available . We show that the D- glucansucrase was capable of transferring D-glucosyl groups from sucrose to B- 512F dextran to form alpha-D-(1----3) branches, thereby rendering the dextran more resistant to hydrolysis by endodextranase . The presence of 1.6M ammonium sulfate caused the enzyme to synthesize a D-glucan having a much higher percentage of alpha-D-(1----3) linkages.

Biochim Biophys Acta, 1984 Mar 29, 785(3), 89 - 96
Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by nonionic detergents, poly(ethylene glycol) and high-molecular-weight dextran; Miller AW et al.; Dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5) (3 IU/ml culture supernatant) was obtained by a modification of the method of Robyt and Walseth (Robyt, J.F . and Walseth, T.F . (1979) Carbohydr . Res . 68, 95-111) from a nitrosoguanidine mutant of Leuconostoc mesenteroides NRRL B-512F selected for high dextransucrase production . Dialyzed, concentrated culture supernatant (crude enzyme) was treated with immobilized dextranase (EC 3.2.1.11) and chromatographed on a column of Bio-Gel A-5m . The resulting, purified enzyme lost activity rapidly at 25 degrees C or on manipulation, as did the crude enzyme when diluted below 1 U/ml . Both enzyme preparations could be stabilized by low levels of high-molecular-weight dextran (2 micrograms/ml), poly(ethylene glycol) (e.g., 10 micrograms/ml PEG 20 000), or nonionic detergents (e.g., 10 micrograms/ml Tween 80) . The stabilizing capacity of poly(ethylene glycol) and of dextran increased with molecular weight . Calcium had no stabilizing action in the absence of other additions, but reduced the inactivation that occurred in the presence of 0.5% bovine serum albumin or high concentrations (greater than 0.1%) of Triton X-100 . In summary, dextransucrase could be stabilized against activity losses caused by heating or by dilution through the addition of low concentrations of nonionic polymers (dextran, PEG 20000, methyl cellulose) or of nonionic detergents at or slightly below their critical micelle concentrations.

Arch Biochem Biophys, 1984 Feb 1, 228(2), 415 - 24
Kinetic studies of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: pH variation of kinetic parameters; Viola RE; The specificity and kinetic parameters of the reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has been examined under a range of conditions in order to elucidate details about the mechanism of action of this enzyme . The rate of oxidation of glucose 6-phosphate is inhibited by the addition of various organic solvents . However, the low, inherent glucose dehydrogenase activity of this enzyme was stimulated under these conditions, and was further activated by divalent anions that were observed to be inhibitors of the glucose 6-phosphate dehydrogenation . From an examination of the pH variation of the enzyme kinetic parameters two groups on the enzyme that appear to be involved in the binding of the phosphate group of the sugar substrate have been detected . An enzyme catalytic group, probably a carboxylic acid, has been identified that accepts the proton from the hydroxyl group at carbon-1 of the sugar substrate during its oxidation to a lactone . The ionization of a group on the enzyme with a pK of 8.7 resulted in an increase in the maximum velocity of the glucose-6-phosphate dehydrogenase activity of the enzyme as a consequence of a pH-dependent product release step that is no longer rate limiting at high pH . Stabilization of gluconic acid-delta-lactone against nonenzymatic hydrolysis by organic solvents has allowed the kinetic parameters of the reverse reaction to be reliably measured for the first time in a narrow pH range.

Appl Biochem Biotechnol, 1984, 10, 61 - 71
Selection and microencapsulation of an "NADH-oxidizing" bacterium and its use for NAD regeneration; Ergan F et al.; An alternative approach to the regeneration of coenzymes is described here using immobilized microorganisms possessing "NADH-oxidase" function . Bacteria containing NADH-oxidase activity are immobilized by microencapsulation within artificial cells . In this form, the microencapsulated bacteria can recycle NADH back to NAD in the presence of molecular oxygen as an electron acceptor . The only byproduct of the recycling reaction is water . In order to perform the biological regeneration of NAD, the activity of NADH-oxidase was investigated in 13 strains of aerobic bacteria and yeast . The NADH-oxidizing bacteria Leuconostoc mesenteroides exhibited the highest activity among the microorganisms tested . The permeabilized bacteria showed 10% of their initial activity after microencapsulation . Light and electron microscopy studies of bacteria loaded microcapsules have been done . Enzymatic properties of microcapsule-immobilized bacteria were investigated in comparison with those of the free enzyme complex . Leuconostoc mesenteroides, containing NADH-oxidase, has been microencapsulated together with 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH) for stereospecific steroid oxidation . In a batch reactor, 2 mg of NAD, with recycling, allowed the same substrate consumption as 4.4 mg of NAD without recycling . The microencapsulated system can be used repeatedly . The system is functional for 10 h, during which time each molecule of NAD has been used 7.6 times.

Intervirology, 1984, 22(4), 181 - 90
New species definitions in phages of gram-positive cocci; Ackermann HW et al.; About 290 phages of the bacterial genera Leuconostoc, Micrococcus, Staphylococcus, and Streptococcus were surveyed . The phages belong to the Myoviridae, Siphoviridae, and Podoviridae families and comprise seven basic morphotypes . Fourteen new species are proposed, bringing the number of phage species for gram-positive cocci to 27 . The distribution of particular phage types may indicate phylogenetic relationships between host bacteria.

Microbios, 1984, 39(155), 29 - 39
Cloning of malic acid assimilating activity from Leuconostoc oenos in E . coli; Lautensach A et al.; High molecular weight DNA was extracted from a malo-lactic fermenting strain of Leuconostoc oenos by a specifically designed lysis procedure, restricted, and ligated into Escherichia coli cloning vector pTR 262, which allows for positive selection for inserts . Malic acid assimilating activity was directly selected for using a host blocked in malic acid utilization . Transformants grew on malate minimal medium but were genetically unstable and contained plasmid DNA that was altered through recA independent events . Analogous results were obtained from a test system using prototrophic transformants of a proline auxotrophic host.

Carbohydr Res, 1983 Dec 23, 124(2), 287 - 99
p-Nitrophenyl alpha-D-glucopyranoside, a new substrate for glucansucrases; Binder TP et al.; p-Nitrophenyl alpha-D-glucopyranoside has been shown to be a substrate for the glucansucrases of various strains of Leuconostoc mesenteroides and Streptococcus mutans . The products from a digest of p-nitrophenyl alpha-D-glucopyranoside with L . mesenteroides B-512F dextransucrase were found to include dextran, a series of p-nitrophenyl isomaltodextrin glycosides, and p-nitrophenyl nigeroside . The kinetics of the reaction were non-Michaelis-Menten, possibly because p-nitrophenyl alpha-D-glucopyranoside has a dual role in the reaction as both a D-glucosyl donor and acceptor.

Carbohydr Res, 1983 Dec 23, 124(2), 275 - 86
Disproportionation reactions catalyzed by Leuconostoc and Streptococcus glucansucrases; Binder TP et al.; Glucansucrases from Leuconostoc mesenteroides NRRL B-512F and Streptococcus mutans 6715 were found to utilize a number of D-gluco-oligosaccharides as D-glucosyl donors and as acceptors . These donors included isomaltotriose and its homologs, panose, maltotriose, and dextran . In each case, D-glucosyl groups were transferred from the donor to an acceptor sugar . When the donor sugar also acted as an acceptor, disproportionation reactions occurred . Isomaltotriose, for example, gave rise to isomaltose and isomaltotetraose initially, and to a series of isomalto-oligosaccharides eventually . In addition to forming alpha-D-(1----6) linkages in the reactions, dextransucrase from S . mutans 6715 was capable of forming alpha-D-(1----3)-linked products.

Carbohydr Res, 1983 Sep 16, 121, 279 - 86
Relative, quantitative effects of acceptors in the reaction of Leuconostoc mesenteroides B-512F dextransucrase; Robyt JF et al.; The acceptor reaction of dextransucrase consists of the transfer of D-glucosyl groups from sucrose to other carbohydrates, and occurs at the expense of dextran synthesis . In the present study, solutions of {14C}sucrose and of each of seventeen acceptor sugars were digested with highly purified Leuconostoc mesenteroides B-512F dextransucrase . The products were separated by paper chromatography, and quantitated by liquid scintillation counting . Maltose was the most effective acceptor; its products, members of an isomaltodextrinyl-maltose series (d.p . 3 to 6), accounted for greater than 75% of the D-glucosyl groups of sucrose . Other acceptors giving rise to a similar series of oligosaccharide products were (in order of decreasing effectiveness): isomaltose, nigerose, methyl alpha-D-glucoside, 1,5-anhydro-D-glucitol, D-glucose, turanose, methyl beta-D-glucoside, cellobiose, and L-sorbose . Lactose, raffinose, melibiose, D-galactose, and D-xylose each gave a single, mono-D-glucosylated product; D-fructose and D-mannose each gave a pair of mono-D-glucosylated (disaccharide) products . Another series of digests contained sucrose and various proportions of maltose . As the level of maltose increased, the size of the largest oligosaccharide acceptor-product decreased, and less dextran was produced . The virtual absence of high-d.p . (8 to 13) oligosaccharide products in all acceptor digests is interpreted as evidence against a role for acceptors as primers of dextran synthesis.

Antonie Van Leeuwenhoek, 1983 Sep, 49(3), 297 - 312
The use of mesophilic cultures in the dairy industry; Daly C; The use of mesophilic starter cultures, containing group N Streptococcus and Leuconostoc species, in the dairy industry is examined . Bacteriophage attack is identified as the main cause of culture inhibition and criteria used to select stable mixed-strain starter cultures and phage-insensitive defined strains are established . The key aspects of culture propagation and storage are highlighted, as well as bulk-culture protection based on physical exclusion of phage and the use of phage-inhibitory media . Developments in the application of starter culture systems in different countries are examined and show that significant process control and elimination of phage problems can be achieved.

Comput Programs Biomed, 1983 Jun, 16(3), 189 - 94
MIR: a versatile program for the statistical analysis of enzyme kinetic data; Bianchi R et al.; We have developed a package program for the estimation of Michaelis-Menten parameters for enzymes that conform to different kinetic mechanisms . Data from different experimental schemes can be fitted with appropriate weighing factors to any of 6 mathematical models, corresponding to 5 kinetic mechanisms: ordered bi-bi, Theorell-Chance, rapid equilibrium random bi-bi, rapid equilibrium ordered bi-bi and ping pong bi-bi . The program also performs a significance test to discriminate between different candidate models . To illustrate the performance of the program, real data from kinetic experiments with glucose 6-phosphate from Leuconostoc mesenteroides have been fitted to different mathematical models, and the results are discussed . The program can be easily implemented for the fitting of kinetic data to any other model.

J Biol Chem, 1983 May 10, 258(9), 5867 - 8
Crystallization and preliminary x-ray data for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; Adams MJ et al.; The enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has been crystallized from phosphate buffer in a form suitable for x-ray crystallographic studies . The crystals diffract to better than 2.4 A . The spacegroup is P3121 (P3221) a = 105.8 A, c = 225.1 A, V = 2.18 X 10(6) A3 . The asymmetric unit probably contains a single dimer.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1983 May, 254(3), 343 - 51
Characterization of a mutant of serotype g Streptococcus mutans strain 6715 lacking dextran-induced agglutination; Hamada S et al.; A spontaneous mutant of Streptococcus mutans 6715 (serotype g) defective in dextran-induced agglutination ability was isolated . The wild type strain and its mutant were termed as 6715-DP and 6715-DN, respectively . Both strains possessed serotype g antigen, and exerted similar sugar fermentation patterns . Strain 6715-DP was rapidly and strongly agglutinated upon addition of high molecular weight dextran, whereas the mutant strain 6715-DN was not . {14C}Dextran prepared from Leuconostoc mesenteroides dextransucrase and {14C}sucrose bound to fresh or lyophilized 6715-DP cells, but not to the mutant 6715-DN cells . However, both strains adhered to a glass surface in the presence of sucrose . Furthermore, heat-treated (100 degrees C, 10 min) cells of both strains bound cell-free glucosyltransferase, although dextran agglutination ability of strain 6715-DP was destroyed by this treatment, indicating that receptors for dextran and glucosyltransferase were different entities . Furthermore, serotype c, e, and f strains S . mutans did not agglutinate upon addition of dextran, nor did they bind {14C}dextran . However, all these strains and both 6715-DP and 6715-DN strains induced marked dental caries in SPF rats . It is concluded that dextran-induced agglutination ability is not a necessary condition for S . mutans to induce dental caries.

Arch Biochem Biophys, 1983 Apr 15, 222(2), 473 - 88
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: revised kinetic mechanism and kinetics of ATP inhibition; Levy HR et al.; The kinetic mechanisms of the NAD- and NADP-linked reactions catalyzed by glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides were examined using product inhibition, dead-end inhibition and alternate substrate experiments . The results are consistent with a steady-state random mechanism for the NAD-linked and an ordered, sequential mechanism with NADP+ binding first for the NADP-linked reaction . Thus, the enzyme can bind NADP+, NAD+, and glucose 6-phosphate, but the enzyme-glucose 6-phosphate complex can react only with NAD+, not with NADP+ . This affects the rate equation for the NADP-linked reaction by introducing a term for a dead-end enzyme-glucose 6-phosphate complex . The kinetic mechanisms represent revisions of those proposed previously (C . Olive, M.E . Geroch, and H.R . Levy, 1971, J . Biol . Chem . 246, 2047-2057) and provide a kinetic basis for the regulation of coenzyme utilization of the enzyme by glucose 6-phosphate concentration (H.R . Levy, and G.H . Daouk, 1979, J . Biol . Chem . 254, 4843-4847) and NADPH/NADP+ concentration ratios (H.R . Levy, G.H . Daouk, and M.A . Katopes, 1979, Arch, Biochem . Biophys . 198, 406-413) . The kinetic mechanisms were found to be the same at pH 6.2 and pH 7.8 . The kinetics of ATP inhibition of the NAD- and NADP-linked reactions were examined at pH 6.2 and pH 7.8 . The results are interpreted in terms of ATP addition to binary enzyme-coenzyme and enzyme-glucose 6-phosphate complexes.

Biochem J, 1983 Feb 1, 209(2), 363 - 71
Tandem dye-ligand chromatography and biospecific elution applied to the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; Hey Y et al.; 1 . A total of 65 immobilized triazine dyes were screened for their ability to purify the dual-nucleotide-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides . From this screen a 'negative' (Matrex Gel Purple A) and a 'positive' (Matrex Gel Orange B) adsorbent were found to be the best in terms of overall purification and yield and were therefore combined to give the best purification . 2 . Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified approx . 56-fold in a two-step tandem chromatographic system using Matrex Gel Purple A followed by Matrex Gel Orange B chromatography to a specific activity of 228 units/mg of protein in a final yield of 73% . 3 . A study of the elution characteristics of glucose-6-phosphate dehydrogenase bound to Matrex Gel Orange B by KCl (pulse and gradient) and biospecific eluents (pulse) was carried out . NADP+, NADPH and adenosine 2',5'-bisphosphate were found to be the only effective biospecific eluents . A pulse of 50 microM-NADP+ (1/2 column vol.) was found to give a better purification than a 0-1 M-KCl gradient and therefore was the preferred method of elution . 4 . Presaturation of the enzyme with various nucleotides was carried out to determine the effect on the subsequent binding of glucose-6-phosphate dehydrogenase to Matrex Gel Orange B . The results of these and biospecific-elution studies lead us to propose two possible schemes to explain the mechanism of the dye-protein interaction . 5 . Reusability, capacity of the adsorbent and effect of varying the ligand concentration were also studied in the purification of glucose-6-phosphate dehydrogenase on Matrex Gel Orange B.

Anal Biochem, 1983 Jan, 128(1), 186 - 90
Correcting a potential defect in an enzymatic cycle for NADP; Hintz CS et al.; An enzymatic cycle for NADP which uses as one of its enzymes glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has occasionally caused trouble due to failure to completely heat-kill this enzyme before the indicator step . It was found that a very small increase in pH was the cause of this . It was also found that the other two proteins present in the reagent greatly increase heat inactivation of the enzyme . The inactivation problem is completely overcome by keeping the pH below 7.2.

Biochemistry, 1982 Dec 7, 21(25), 6429 - 34
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides . Kinetics of reassociation and reactivation from inactive subunits; Haghighi B et al.; Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is denatured in 8 M urea and dissociated into its two inactive subunits . Denaturation leads to an approximately 80% decrease in protein fluorescence and a 20-nm red shift in the emission maximum . Upon dilution, the urea-treated enzyme regains catalytic activity (approximately 70%) . The reactivated enzyme is indistinguishable from the native enzyme based on a number of physicochemical and enzymological criteria . The kinetics of renaturation and reactivation were monitored by measuring the rates of regain of native fluorescence and appearance of activity and the accessibility of histidine residues toward diethyl pyrocarbonate modification . Regain of native fluorescence was too rapid to measure at 25 degrees C; at 5 degrees C the initial phase was also too rapid, but a slower phase was monitored and shown to obey first-order kinetics with k = (5.9 +/- 1.3) x 10(-3) s-1 . Reappearance of activity was measured at several protein concentrations; reactivation followed second-order kinetics with k = (4.85 +/- 0.47) x 10(-3) M-1 min-1 . Reactivation was stimulated to different degrees by either the initial or delayed addition of NAD+, NADP+, or glucose 6-phosphate . During the initial, rapid phase of renaturation, approximately 3 of the enzyme's 12 histidine residues become unreactive toward diethyl pyrocarbonate; concomitant with the subsequent reactivation, approximately 7 more histidines become inaccessible to diethyl pyrocarbonate . The data are consistent with a model for enzyme renaturation and reactivation in which the unfolded subunits rapidly refold to an inactive structure that can dimerize slowly to generate native enzyme . Specific ligands stimulate reactivation by binding to refolded subunits or incompletely folded dimers.

Biochemistry, 1982 Dec 7, 21(25), 6415 - 20
Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides . Isolation and sequence of a peptide containing an essential lysine; Haghighi B et al.; Interaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with pyridoxal 5'-phosphate and sodium borohydride leads to inactivation and modification of two lysine residues per enzyme dimer that are thought to bind glucose 6-phosphate {Milhausen, M., & Levy, H.R . (1975) Eur . J . Biochem . 50, 453-461} . The amino acid sequence surrounding this lysine residue is reported . Following tryptic hydrolysis of the modified enzyme, two peptides, each containing one pyridoxyllysine residue, were purified to homogeneity and subjected to automated Edman degradation . The sequences revealed that one of these, a heptapeptide, was derived from the other, containing 11 amino acids . Supporting evidence for the role of the modified lysine is provided in the following paper {Haghighi, B., & Levy, H.R . (1982) Biochemistry (second paper of three in this issue)} . End-group analysis of the native enzyme revealed that valine is the N-terminal and glycine the C-terminal amino acid and provides support for the identity of the enzyme's two subunits.

Fortschr Med, 1982 Nov 4, 100(41), 1917 - 21
{The problem of dextran intolerance and its specific prevention with hapten inhibition . A part of the history of applied immunology}; Ring J; Dextrans are used in clinical medicine since more than 35 years . One of the main problems of dextran application are severe incompatibility reactions of the anaphylactoid type . Lethal cases have been reported . In the early fifties antibodies against high molecular weight dextrans have been described in patients with incompatibility reactions as well as in normals . By the introduction of new dextrans (produced by B . Leuconostoc mesenteroides B 512) with lower molecular weight and less branching newer solutions were developed without immunogenicity . When, in the early seventies, cases of severe incompatibility reactions to those dextrans were reported, nonimmunological mechanisms were discussed as e.g . direct mediator release--as in the model of rat dextran hypersensitivity--or direct complement activation via the alternative pathway.

Proc Natl Acad Sci U S A, 1982 Jun, 79(11), 3462 - 5
Geometric specificity of alcohol dehydrogenases and its potential for separation of trans and cis isomers of unsaturated aldehydes; Klibanov AM et al.; The geometric specificity of three different alcohol dehydrogenases (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) (from yeast, from horse liver, and from Leuconostoc mesenteroides) in the reduction of trans- and cis-cinnamaldehydes has been investigated . All three enzymes display a remarkable trans specificity: they react with the trans isomer 7 to 647 times faster than with its cis counterpart . Experiments with the enzymatic reduction of 3-phenylpropionaldehyde, a saturated analog of cinnamaldehyde, have revealed that whereas trans-cinnamaldehyde possesses the "right" configuration for the active centers of the alcohol dehydrogenases, the cis isomer apparently does not fit the active centers well . All three alcohol dehydrogenases studied also exhibit a marked trans specificity in the reaction with alpha-methylcinnamaldehyde . The geometric specificity of alcohol dehydrogenases can be used for the production of otherwise hard to synthesize cis isomers of unsaturated aldehydes from their readily available trans counterparts: trans-cinnamaldehyde was irradiated with ultraviolet light (which converted it to a mixture of trans and cis isomers) then treated with NADH and yeast alcohol dehydrogenase (which selectively reduces only trans aldehyde into the alcohol), and finally the mixture of cis-cinnamaldehyde and trans-cinnamyl alcohol was separated easily by preparative column chromatography.

Carbohydr Res, 1982 Feb 16, 101(1), 57 - 74
Isolation and partial characterization of an extracellular glucansucrase from Leuconostoc mesenteroides NRRL B-1355 that synthesizes an alternating (1 goes to 6), (1 goes to 3)-alpha-D-glucan; Cote GL et al.; Leuconostoc mesenteroides NRRL B-1355 grows on sucrose to produce two extracellular alpha-D-glucans . Although both are termed dextrans, they are chemically and physically distinct, and can be separated by fractional ethanol precipitation into fractions designated L and S . Fraction L is similar to B-512F dextran, having 95% alpha-(1 goes to 6) linkages and 5% alpha-(1 goes to 3) branch linkages, but fraction S has an alternating sequence of alpha-(1 goes to 6) and alpha-(1 goes to 3) linkages . Because of its structural differences from dextran, its different physical characteristics, and its resistance to hydrolysis by endodextranase, we have named glucan S, alternan, and the enzyme that synthesizes it from sucrose, alternansucrase . Alternansucrase has been isolated by two different methods . The first involves removal of the fraction L glucan from the culture fluid via hydrolysis by an endodextranase, followed by chromatography on Bio-Gel A5m . The void-volume fraction synthesizes only alternan, whereas the slower-migrating, second fraction synthesizes mainly dextran, together with some alternan . The second method utilized hydrophobic chromatography on O-(phenoxyacetyl) cellulose; a portion of the alternansucrase did not bind, whereas the bound portion, removed by eluting with detergent, contained both alternansucrase and dextransucrase . The glucans were identified by physical appearance, the concentration of ethanol required for precipitation, periodate-oxidation behavior, and susceptibility to hydrolysis by endodextranase . Also studied was the inhibition of the enzymes by 3-deoxy-3-fluoro-alpha-D-glucopyranosyl fluoride, tris(hydroxymethyl)aminomethane, 2-aminoethanol, and octyl beta-D-glucopyranoside.

Microbiol Immunol, 1982, 26(5), 403 - 9
Gelatin of Limulus amoebocyte lysate by simple polysaccharides; Mikami T et al.; The Limulus lysate gelatin activity of several simple polysaccharides including yeast mannans and bacterial dextrans was investigated . The mannans from Saccharomyces cerevisiae wild and mutant strains possessing dense branches showed positive gelatin activity at concentrations of 1 microgram/ml or more regardless of differences in their chemical structure . However, two synthetic mannans possessing linear structures with alpha 1 leads to 2 and alpha 1 leads to 6 linkages also gave positive reactions at concentrations of 10 microgram/ml or more and 500 microgram/ml or more, respectively . The dextran from Leuconostoc mesenteroides IAM 1046 consisting of a dense branching moiety displayed reactivity at concentrations of 100 microgram/ml or more, while the dextrans devoid of such branches were negative in this reaction . The optimal concentration for Limulus lysate gelatin could not be determined for any of the polysaccharides and lipopolysaccharides (LPS) tested in this study . The gelation activity of the polysaccharides was stable to treatment with 100 mM NaOH at 30 C for 72 hr . The minimum concentration for the gelation activity of LPS treated with 100 mM NaOH under the same conditions was reduced from 10(-6) approximately(-9) microgram/ml to 1-10 microgram/ml . The above findings demonstrate that the major part of Limulus lysate gelation activity of LPS depends on the alkali-degradable lipid A moiety, and that such simple polysaccharides are also able to participate in this activity even though the extent of participation is very low.

Steroids, 1981 Aug, 38(2), 211 - 20
Quantitative requirements for NADPH in the support of aromatization by human placental microsomes; Sheean LA et al.; Suitable incubation conditions were developed for reduced pyridine nucleotide protection and regeneration to permit quantitative assessment of the NADPH requirement for steroid aromatization by human placental microsomes . 10 mM dithiothreitol was found to protect NADP(H) from microsomal nucleotide pyrophosphatase and 2 mM nicotinamide mononucleotide was utilized to control nucleotide glycohydrolase activity . Under these assay conditions, the initial rates of aromatization obtained with restricted NADPH levels were critically dependent upon both the amount and the source of exogenous NADPH-regenerating dehydrogenase system . With excess Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, an apparent Km for NADPH of 0.20 microM was observed for aromatization which is significantly below all previous estimates of the NADPH requirement and which is at greatest only one-tenth the Km value for NADPH utilization by NADPH-cytochrome c reductase . These findings suggest a potential regulatory role for both NADPH-generating and NADPH-accepting enzymes in the support of estrogen biosynthesis.

Biochim Biophys Acta, 1981 Feb 13, 657(2), 468 - 81
Two hexokinases of Homarus americanus (lobster), one having great affinity for mannose and fructose and low affinity for glucose; Stetten MR et al.; Two major hexokinases (ATP: D-hexose 6-phosphotransferases, EC 2.7.1.1) have been identified in tissues of Homarus americanus (lobster) and separated from each other by DEAE-cellulose ion-exchange chromatography and by polyacrylamide gel electrophoresis . The molecular weight of each, determined by gel filtration, is about 50 000 . Hexokinase II, named for its column elution order, resembles hexokinase isozymes I and II of vertebrates . Km values for glucose, mannose and fructose are 0.08, 0.13 and 6.7 mM, respectively . It is strongly inhibited by the reaction products, ADP and glucose-6-P (Ki = 0.8 mM) . Hexokinase I appears to be different from any animal hexokinase previously described . It has a high affinity for mannose and fructose and low affinity for glucose . Km values are 6, 0.07 and 1.2 mM and relative maximum rates 100, 520 and 1070 for glucose, mannose and fructose, respectively . Hexokinase I is not inhibited by physiological concentrations of ATP nor by glucose-6-P , mannose-6-P or fructose-6-P even at high concentrations . Both enzymes occur in muscle at about 10% of the concentration found in the hepatopancreas . The use of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49), with NAD as cofactor, is recommended for measuring hexokinases in crude tissue preparations to avoid the variable further reduction of nucleotide caused by the action of 6-phosphogluconate dehydrogenase when NADP is used with yeast glucose-6-phosphate dehydrogenase.

Folia Microbiol (Praha), 1981, 26(3), 232 - 42
Inherent limitations to the problem of reducing the lysine microbiological assay time; Tanner RD et al.; A kinetic approach is proposed to shorten the microbiological assay time for the determination of unbound L-lysine . The present lysine bacterial assay takes from 16 to 24 h using Pediococcus cerevisiae P-60 ATCC 8042 (formerly Leuconostoc mesenteroides P-60 ATCC 8042) and uses a medium in which lysine is the limiting substance . Measurements of the final cell concentration are linearly correlated with the initial concentration of lysine, S, to provide an indirect estimate of S . We propose to understand the limitations inherent to the reduction of the assay time to 4 h by focusing in our analysis on the bacterial late lag or early growth transient phases, rather than the stationary phase of growth . Generally, the Monod equation is expected to describe a hyperbolically increasing correlation between the bacterial specific growth rate at about 2-4 h and the initial lysine concentration . A hyperbolic correlation is obtained by 3 h, but the lysine region of interest falls in the saturated portion of the curve . Discriminations between different initial lysine levels are therefore difficult with this nearly flat curve . On the other hand, when the initial inoculum level is lowered, so that substrate inhibition becomes effective, a correlation with a large negative slope is obtained by 4 h . Limitations to using absorbance measurements for the rapid assay turn up in a lack of reproducibility and, hence, a large variance associated with the measurements . Alternative microbial measuring techniques, such as impedance methods, need to be examined in order to reduce that large variance.

J Biochem (Tokyo), 1980 Dec, 88(6), 1855 - 9
Immunochemical studies on dextrans . Two distinct alpha 1 linked to 3 specificities of rabbit anti-dextran B1355; Torii M et al.; Rabbit anti-dextran B1355 sera prepared by injecting rabbits with Leuconostoc mesenteroides NRRL B1355 were separated on a Sephadex G75 column into two fractions, one binding and the other not binding to the column . Oligosaccharide inhibition of precipitation of the two fractions with dextran B1355 indicated that both fractions had alpha 1 linked to 3 specificity . However, antibodies in the non-binding fraction were shown to be directed against O-alpha-D-glucopyranosyl-(1 linked to 3)-O-alpha-D-glucopyranosyl-(1 linked to 6)-D-glucose, while those in the binding fraction were directed against O-alpha-D-glucopyranosyl-(1 linked to 6)-O-alpha-D-glucopyranosyl-(1 linked to 3)-D-glucose . These results are consistent with the proposal of Bhoopalam et al . (Proc . Soc . Exp . Biol . Med . (1979(=) 161, 430-434) that there are different epitopic groups on this dextran.

J Immunol, 1980 Oct, 125(4), 1454 - 8
Effect of dextran-S (alpha, 1-3 dextran) on the growth of plasmacytomas MOPC-104E and J558; Bhoopalam N et al.; The murine plasmacytomas MOPC-104E and J558 secrete IgM-lambda and IgA-lambda, respectively, which are antibodies to alpha, 1-3 dextran, a constituent of dextran-S (DEX-S) extracted from Leuconostoc mesenteroides . A single i.p . injection of 10 microgram DEX-S into BALB/c mice from 7 days before and up to 3 days after the implantation of 5 x 10(3) plasmacytoma cells protected the BALB/c mice from developing the tumors . Immunization with other antigens, such as Escherichia coli lipopolysaccharide, inulin (alpha, 1-6 linkage), and dextran T-10 (no known alpha, 1-3 linkages) did not protect the mice from developing the tumors . Growth of LPC-1 was not affected by DEX-S . The mechanism of this growth inhibition is unknown . It does not appear to depend solely on binding of antigen to tumor cells, and the limits of the effective time intervals between antigen and tumor injection suggest dependence on the presence of antibody to alpha-1,3 dextran.

Biochim Biophys Acta, 1980 Jul 10, 614(1), 46 - 62
Characterization of the multiple forms and main component of dextransucrase from Leuconostoc mesenteroides NRRL B-512F; Kobayashi M et al.; Multiple forms of dextransucrase (sucrose:1.6-alpha-D-glucan 6-alpha-D-glucosyltransferae EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F strain were shown by gel filtraton and electrophoretic analyses . Two components of enzyme, having different affinities for dextran gel, were separated by a column of Sephadex G-100 . The major component voided from the Sephadex column was treated with dextranase and purified to an electrophoretically homogeneous state . The }urified enzyme had a molecular weight of 64 000-65 000, pI value of 4.1, and 17% of carbohydrate in a molecule . EDTA showed a characteristic inhibition on the enzyme while stimulative effects were observed by the addition of exogenous dextran to the incubation mixture . The enzyme activity was stimulated by various dextrans and its Km value was decreased with increasing concentration of dextran . The purified enzyme showed no affinity for a Sephadex G-100 gel, and readily aggregated after the preservation at 4 degrees C in a concentrated solution.

Int Arch Allergy Appl Immunol, 1980, 63(2), 195 - 200
Clinical dextrans lack mitogenic activity for normal human lymphocytes in vitro; Cunnington PG et al.; In vitro studies on mitogenic stimulation of lymphocytes from a panel of normal volunteers revealed no transformation in response to the presence of dextran 40, 70, 110 or 150 at concentrations ranging from 0.8 to 8,000 microgram/ml . High molecular weight native dextran B512 was mitogenic in 1 individual only . In addition, neither Leuconostoc-derived nor fermentation medium-derived moieties, sometimes present in clinical dextrans, were implicated as lymphocyte mitogens . It is concluded on the basis of these findings that clinical dextrans of average molecular weight 40,000--150,000 are not B- or T-cell mitogens.

Int Arch Allergy Appl Immunol, 1980, 61(4), 457 - 66
A new immunochemical purity test for clinical dextran . Methodology and studies on clinical dextran preparations; Richter AW; A simple immunochemical procedure, based on reversed single radial immunodiffusion (RSRI) was developed to detect commonly occurring traces of antigenic contaminants in clinical dextran . High-titred antisera against non-dextran components of Leuconostoc mesenteroides NRRL B512 were produced and served as analytical reagents . Dextran samples were incorporated into a gel layer . Presence of contaminants is revealed by precipitate formation following application of antiserum to a well cut into the gel . Antigenic contaminants can be detected in concentrations exceeding 10 ppm by the Leuconostoc-RSRI test . Screen results on 119 clinical dextran samples from manufactures in different countries disclosed presence of antigen traces in 82% of preparations . Introduction of the new test and improved purification measures at Pharmacia AB resulted in purer clinical dextran with negative RSRI test scores in 99% of batches produced . Only batches passing this test are released for clinical use . Although no direct correlation was found between contaminant levels and incidence of dextran reactions, antigenic contaminants may play a contributory role in elicitation of mild reactions.

J Clin Chem Clin Biochem, 1979 Nov, 17(11), 689 - 91
Influence of indicating enzyme reaction on apparent creatine kinase activity creatine kinase in serum, VII; Szasz G et al.; Comparison of the indicative systems yeast glucose-6-phosphate dehydrogenase/NADP+, leuconostoc glucose-6-phosphate dehydrogenase/NADP+ and leuconostoc glucose-6-phosphate dehydrogenase/NAD+ showed excellent correlation and no differences in apparent creatine kinase activity with the two methods using NADP+ . By using NAD+ with the leuconostoc enzyme enzyme relative recovery of apparent creatine kinase activity is lower due to interference of other serum constituents . The mean value of the relative differences versus methods using NADP+ was 5.8% in our experiments.

Biotechnol Bioeng, 1979 Jul, 21(7), 1121 - 31
Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides; Lawford GR et al.; Leuconostoc mesenteroides NRRL B-512(F) was grown in continuous culture under conditions of energy-limited growth . The extracellular enzyme dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5), was not detected in glucose- or maltose-limited cultures . Under conditions of sucrose-limited growth, the enzyme activity of the cell-free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved . The appearance of fructose in the effluent of the sucrose-limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis . The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production . Dextransucrase was isolated from the cell-free culture supernatant by ammonium sulfate precipitation and DEAE-cellulose chromatography . The enzyme preparation exhibited both dextran biosynthetic activity and an invertase-like activity . The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10 degrees C . The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.

J Biol Chem, 1979 Jun 10, 254(11), 4843 - 7
Simultaneous analysis of NAD- and NADP-linked activities of dual nucleotide-specific dehydrogenases . Application to Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase; Levy HR et al.; A method is described which enables one to assay simultaneously the NAD- and NADP-linked reactions of dehydrogenases which can utilize both coenzymes . The method is based on the fact that the thionicotinamide analogs of NADH and NADPH absorb light maximally at 400 nm, a wavelength sufficiently far removed from the absorbance maximum of NADH and NADPH to permit measurements of the simultaneous reduction of NAD+ (or NADP+) and the thionicotinamide analog of NADP+ (or NAD+) . Application of the method to glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reveals differential effects of glucose 6-phosphate concentration on the NAD- and NADP-linked reactions catalyzed by this enzyme which can not be detected by conventional assay procedures and which may have regulatory significance.

J Assoc Off Anal Chem, 1979 Jan, 62(1), 160 - 7
Rapid automated turbidimetric assay for chlortetracycline hydrochloride, using Leuconostoc mesenteroides as the test organism; Mueller DL et al.; A rapid turbidimetric assay has been developed for chlortetracycline hydrochloride (CTC-HCl) in finished animal feeds and feed supplements, using Leuconostoc mesenteroides as the test organism . Two modifications are presented: The incubation period of modification 1 is 2.5 hr and the sensitivity of the assay is 0.03 microgram CTC-HCl/assay tube . Modification 2 has a sensitivty of 0.01 microgram CTC-HCl/assay tube and requires an incubation period of 3.5 hr . For 21 feed formulations, the turbidimetric method recovered 95.7% of label claim . Recoveries of CTC-HCl standards from the same feeds ranged from 93.4 to 134% with a mean of 103% . The relative standard deviation among day-to-day duplicates is 3.50% for the faster modification and 1.63% for the more sensitive modification.

Carbohydr Res, 1979 Jan, 68(1), 95 - 111
Production, purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F; Robyt JF et al.; The production of dextransucrase from Leuconostoc mesenteroides NRRL B-512F was stimulated 2-fold by the addition of 0.005% of calcium chloride to the medium; levansucrase levels were unaffected . Dextransucrase was purified by concentration and dialysis of the culture supernatant with a Bio-Fiber 80 miniplant, and by treatment with dextranase followed by chromatography on Bio-Gel A-Fm . A 240-fold purification, with a specific activity of 53 U/mg, was obtained . Contaminating enzyme activities of levansucrase, invertase, dextranase, glucosidase, and sucrose phosphorylase were decreased to non-detectable levels . Poly(acrylamide)-gel electrophoresis of the purified enzyme showed only two protein bands, both of which had dextransucrase activity . These bands also gave a carbohydrate stain, indicating that the dextransucrase could be a glycoprotein . Acid hydrolysis, followed by paper chromatography, of the purified enzyme showed that the major carbohydrate was mannose . Concanavalin A completely removed dextransucrase activity from solution, confirming the mannoglycoprotein character of the enzyme . Dextransucrase activity was not altered by the addition of 0.008-4 mg/ml of dextran, but its storage stability was increased by the addition of 4 mg/ml of dextran . As previously shown by others, the activity of dextransucrase was decreased by EDTA, and was restored by the addition of calcium ions . Zinc, cadmium, lead, mercury, and copper ions were inhibitory to various degrees.

J Immunol, 1978 Sep, 121(3), 962 - 5
Estimation of antibodies specific for dextran; Matsuuchi L et al.; Methods are described for the isolation and characterization of picogram quantities of anti-dextran antibodies . 14C-dextrans produced by using the dextransucrases of Leuconostoc mesenteroides strains B1355 and B512 were used in a radioimmunoassay . The specificity of this assay was verified by using cell cytoplasmic lysates from mouse plasmacytomas, J558 (anti-alpha 1 leads to 3 dextran) and W3129 (anti-alpha 1 leads to 6 dextran) . Dextran produced by strain B1355 and insolubilized with epichlorohydrin was used as an immunoabsorbent.

Appl Environ Microbiol, 1978 May, 35(5), 920 - 4
Growth inhibition of Streptococcus mutans and Leuconostoc mesenteroides by sodium fluoride and ionic tin; Yost KG et al.; Sodium fluoride caused inhibition of growth rate and growth levels of Streptococcus mutans with glucose as the primary energy and carbon source . Stannous fluoride increased growth lag nad caused a much greater inhibition of growth rate than did sodium fluoride . Neither compound was found to be bactericidal when culture viability was measured after 6 days of incubation . Leuconostoc mesenteroides, which lacks a phosphotransferase system for sugar transport, showed less inhibition of growth rate with both inhibitors than did S . mutans, which possesses a phosphotransferase system . Metabolism of glucose or lactose which requires enolase activity shoed sodium fluoride inhibition, whereas metabolism of arginine or pyruvate does not involve enolase activity and showed no inhibition of growth.

Carbohydr Res, 1978 Mar, 61, 433 - 45
The mechanism of acceptor reactions of Leuconostoc mesenteroides B-512F dextransucrase; Robyt JF et al.; Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides . The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using D-{14C}glucose, D-{14C}fructose, and 14C-reducing-end labeled maltose as acceptors . A purified dextransucrase was preincubated with sucrose, and the resulting D-fructose and unreacted sucrose were removed from the enzyme by chromatography of columns of Bio-Gel P-6 . The enzyme, which migrated at the void volume was collected and referred to as "charged enzyme" . The charged enzyme was incubated with 14C-acceptor in the absence of sucrose . Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide . It was found that all three of the acceptors were incorporated into the products at the reducing end . Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when D-glucose or maltose was the acceptor . We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the D-glucosyl and dextranosyl groups and the acceptor . We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.

J Bacteriol, 1977 Jul, 131(1), 288 - 92
Ultrastructural surface changes associated with dextran synthesis by Leuconostoc mesenteroides; Brooker BE; When Leuconostoc mesenteroides NCDO 1875 was grown in MRS broth and fixed for electron microscopy in the presence of ruthenium red, the cell wall appeared as a triple-layered structure similar to other, gram-positive bacteria . When such logarithmic-phase cultures were exposed to sucrose, the appearance and growth of a uniform layer of electron-dense material was evident on the surface of the cell wall . After 2 h in the presence of sucrose, the formation of this surface coat (110 to 130 nm thick) was complete . For 85 to 90% of the cells, continued exposure to sucrose did not produce any further change in their appearance, but the rest of the population began to accumulate insoluble capsular dextran at the surface of their coat material . Within 18 h, these cells had produced a large capsule (maximum diameter, 6 micrometer) composed mainly of an extensive reticulum of fine filaments . Periodate-reactive carbohydrate was localized cytochemically in the capsular dextran and in the surface coat of all cells . It is suggested that the surface coat of sucrose-grown cells represents a cell-bound dextran-dextransucrase complex and that the acapsulate cells produce the relatively soluble S dextran reported by previous workers.

Eur Surg Res, 1977, 9(5), 338 - 46
Immunological properties of a high molecular weight component from yeast cell autolysate in dogs and evaluation of its potential role in human dextran reactions; Ring J et al.; A high molecular weight component (HMC) of autolysate from Saccharomyces cerevisiae yeast cells was prepared . HMC was found to be immunogenic in dogs, inducing hemagglutinating antibody formation . Upon HMC challenge of immunized dogs, systemic anaphylactoid reactions were observed in 4/5 animals . The most prominent symptom was decreased cardiac output . Decrease in mean arterial pressure and increase in pulmonary arterial pressure were also observed . Consumption of total serum complement activity amounted to 22% of initial values . HMC also exhibited mitogenic activity in lymphocyte cultures from nonimmunized and immunized dogs . Since yeast autolysate is used as nitrogen source for Leuconostoc mesenteroides in the production of clinical B 512 dextran it is a theoretically possible trace contaminant of such solutions . Therefore, dogs hyperimmunized with HMC were also challenged with clinical dextran . No anaphylactoid signs were observed . These data suggest a negligible causal role of macromolecular contaminants derived from yeast cell autolysate in rare human anaphylactoid reactions following infusion of clinical dextran.

Arch Microbiol, 1976 Dec 1, 111(1-2), 99 - 104
Surface coat transformation and capsule formation by Leuconostoc mesenteroides NCDO 523 in the presence of sucrose; Brooker BE; When Leuconostoc mesenteroides NCDO 523 was grown in MRS browth, electron microscopy of cells fixed in the presence of ruthenium red showed that the cell wall was covered with a thin layer of filamentous material . When MRS-grown cells were resuspended in the same medium supplemented with 3.6% sucrose, this surface coat doubled in thickness and a number of radial thickenings appeared within it . After 3 h, the filamentous component of the surface coat had disappeared leaving only the radial projections . The progressive accumulation of polymer to produce a capsule visible by light microscopy was observed in only about 20% of the population . In this minority of cells, a dense globular dextran composed of fibrillar and particulate elements was always produced in the initial stages of synthesis . After 18 h, the dextran capsule was generally composed of an inner globular and outer fibrillar layer . It appeared that the outer layer was derived from the globular dextran of the capsule by a process of dispersion.

Biochemistry, 1976 Nov 2, 15(22), 4844 - 9
Determination of the hydride transfer stereospecificity of nicotinamide adenine dinucleotide linked oxidoreductases by proton magnetic resonance; Arnold LJ Jr et al.; A facile proton magnetic resonance technique is described for the determination of the coenzyme stereospecificity during hydride transfer reactions catalyzed by pyridine nucleotide dependent oxidoreductases . The reliability of this technique was demonstrated by examining the coenzyme stereospecificity of lactate, malate, and 3-phosphoglycerate dehydrogenases, which are known to be A-stereospecific enzymes, as well as triosephosphate and octopine dehydrogenases, which are known to be B-stereospecific enzymes . Furthermore, by applying this technique, it was shown that the previously unstudied enzymes D-beta-hydroxybutyrate and 4-aminobutanal dehydrogenases are B- and A-stereospecific enzymes, respectively . In addition, the nicotinamide adenine dinucleotide linked reaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be B stereospecific, like the reaction of the nicotinamide adenine dinucleotide phosphate linked yeast enzyme.

Hoppe Seylers Z Physiol Chem, 1976 Sep, 357(9), 1209 - 14
On the mechanism and stereochemistry of the malate-lactate fermentation of Leuconostoc mesenteroides; Kraus A et al.; During the transformation of (2S, 3R) {3-3H}malate to (S) lactate no tritium exchange takes place . The stereochemical course of the decarboxylation studied with (2S, 3R) {3-2H}-malate in 3HOH/H2O and (2S, 3R) {3-3H}malate in 2H2O occurs with retention and is therefore the same as that determined by other authors for malic enzyme from vertebrates and from Escherichia coli . The malate-lactate fermentation is a useful procedure to prepare chiral methyl groups on a preparative scale starting from (2S, 3R) {3-H}malate.

J Biochem (Tokyo), 1976 Jun, 79(6), 1301 - 8
Purification and properties of the extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299; Kobayashi M et al.; Dextransucrase {EC 2.4.1.5} activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75 . The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I . Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively . On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000 . Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl . The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees . The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively . Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme . Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.

Infect Immun, 1976 Apr, 13(4), 1228 - 34
Dextran-mediated interbacterial aggregation between dextran-synthesizing streptococci and Actinomyces viscosus; Bourgeau G et al.; Streptococcus sanguis and Streptococcus mutans bind to the surface of Actinomyces viscosus, producing large microbial aggregates . Aggregates form rapidly and are not easily dissociated by vigorous mixing . The binding is mediated by dextran . Glucose-grown streptococci will not aggregate unless they are first mixed with high-molecular-weight dextran . Aggregation is induced with dextrans isolated from Leuconostoc, S . sanguis, or S . mutans . Sucrose-grown streptococci will adhere to A . viscosus without the addition of an exogenous source of dextran . A . viscosus will bind dextran and then bind glucose-grown streptococci . Aggregation occurs over a wide pH range and is dependent on cations . The aggregating activity of A . viscosus is both protease and heat sensitive . The aggregating activity of S . sanguis is heat stable but sensitive to dextranase.

J Periodontol, 1976 Mar, 47(3), 171 - 9
The effect of topical application of dextran on the gingiva of the beagle dog; Neuman SM et al.; Dextrans derived from Leuconostoc mesenteroides were placed on clinically healthy gingiva of Beagle dogs once a day for 21 days . Control gingival tissues received saline . Both healthy controls and dextran-treated tissues were brushed daily . Inflamed control tissues were obtained by allowing plaque to accumulate for 21 days . Tissues receiving daily application of dextran solutions developed chronic gingival inflammation but displayed no clinical signs of gingivitis . Healthy control gingival tissues showed no clinical signs of gingivitis and minor histologic inflammatory changes . Tissues exposed to dental plaque showed the typical clinical and histological inflammatory changes of gingivitis . Thus dextran, a substance similar to the extracellular polysaccharide found in dental plaque, was able to penetrate the sulcular epithelium, enter healthy gingival connective tissue and cause chronic inflammation . This connective tissue inflammation occurred without inducing any of the clinical signs of gingivitis . Therefore, it is concluded that dextran produced one component of the gingivitis response, chronic histologic inflammation, independent of another major component of the disease, clinical inflammation.

J Dairy Res, 1976 Feb, 43(1), 63 - 73
Acetaldehyde: an intermediate in the formation of ethanol from glucose by lactic acid bacteria; Lees GJ; Group N streptococci formed acetaldehyde and ethanol from glucose . As the enzymes aldehyde dehydrogenase, phosphotransacetylase and acetate kinase were present this would enable these organisms to reduce acetyl-CoA to acetaldehyde and convert acetyl-CoA to acetyl phosphate and acetate . A pentose phosphate pathway which converted ribose-5-phosphate to glyceraldehyde-3-phosphate was also present . Acetaldehyde could not be formed via the hexose monophosphate shunt or by direct decarboxylation of pyruvate, as the enzymes phosphoketolase and alpha-carboxylase were absent . Phosphoketolase activity was induced in Streptococcus lactis subsp . diacetylactis after growth on D-xylose . Group N streptococci also contained an NAD-dependent alcohol dehydrogenase which reduced acetaldehyde to ethanol while both NAD- and NADP-dependent alcohol dehydrogenase activities were found in Leuconostoc cremoris.

Arch Microbiol, 1975 Dec 31, 106(3), 267 - 9
{Fermentative degradation of 2-14C-mannose with Leuconostoc mesenteroides (author's transl)}; Ziegler E et al.; Mannose-2-14C has been fermented by Leuconostoc mesenteroides, CO2 ethanol and D-lactic acid were formed in a molar ratio of 1:1:1 . A small amount of acetic acid was found as by-product . It could easily be isolated from the main products of the fermentation and it did not disturb further degradation procedures . The methyl-C-atom of ethanol, which was derived from C-2 of the mannose, had nearly the same specific radioactivity as mannose-2-14C . All other C-atoms of the degradation products were only very slightly labeled . Their content of radioactivity was in any case lower than 3% of the specific radioactivity of the degraded mannose . This procedure is applicable for the degradation of 14C-labeled mannose.

Biochim Biophys Acta, 1975 Jul 27, 397(1), 69 - 79
Purification and characterization of two activities of the intracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299; Kobayashi M et al.; Dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5) activity of Leuconostoc mesenteroides NRRL B-1299 cells has been purified by adsorption on hydroxyapatite, followed by chromatographies on DEAE-cellulose and DEAE-Sephadex . The enzyme activity was readily separated into two principal forms of the enzyme, I and II, by DEAE-cellulose chromatography . Both enzymes I and II were purified to an electrophoretically homogeneous state in which the relative enzyme activities reached 32-and 14-fold of the original specimen, respectively . Molecular weights were 69 000 for the enzyme I and 79 000 for the enzyme II as determined by electrophoresis in sodium dodecyl sulphate-polyacrylamide, and no subunit structure was observed . Enzyme I had an optimum pH at 6.3-6.5 and exhibited a maximal activity at 45 degrees C, while the optimum pH and temperature of enzyme II were pH 5.5-5.9 and 35-40 degrees C . The Km values of enzymes I and II were 10.7 and 250 mM, respectively . The effects of several metal ions, chemical reagents, and addition of various dextrans were also examined . Beside linear alpha-1,6-linkages the polymer synthesized by the enzyme II contained lesser amount of alpha-1,2-and alpha-1,3-linkages, which seems to be a primary characteristic of the B-1299 dextran.

J Dairy Res, 1975 Feb, 42(1), 139 - 46
Citrate utilization in milk by Leuconostoc cremoris and Streptococcus diacetilactis; Cogan TM; Citrate utilization and diacetyl, acetoin and acetaldehyde production by 2 strains each of Leuconostoc cremoris and Streptococcus diacetilactis in milk were studied . With the leuconostoc bacteria no growth and little citrate utilization occurred unless a stimulant (yeast extract) was present, when complete utilization of citrate without concomitant production of diacetyl or acetoin was obtained . The additon of Mn2+ stimulated growth resulted in diacetyl and acetoin production . Destruction of diacetyl and acetoin occurred when the citric acid level fell to c.1000 and 600 mug/g in the case of Leuc . cremoris FR8-1 and CAF1, respectively . Only strain FR8-1 produced acetaldehyde . In contrast, Str . diacetilactis produced diacetyl, acetoin and acetaldehyde concomitant with citrate utilization.

Eur J Biochem, 1975 Jan 2, 50(2), 453 - 61
Evidence for an essential lysine in glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides; Milhausen M et al.; 1 . Pyridoxal 5'-phosphate inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides reversibly which Ki equals 0.04-0.06 mM . 2 . This inhibition is competitive with respect to glucose 6-phosphate and non-competitive with respect to NADP+ or NAD+ . Interaction between enzyme and excess pyridoxal 5'-phosphate follows pseudo-first-order kinetics and indicates that one molecule of inhibitor reacts with each active unit of enzyme . 3 . Substrate and coenzyme protect the enzyme from inhibition by pyridoxal 5'-phosphate . Dissociation constants for NADP+ and glucose 6-phosphate were determined from their effects on the kinetics of enzyme--inhibitor interaction . 4 . Reaction of the enzyme with pyridoxal 5'-phosphate produces a typical Schiff-base absorbance peak at 430 nm . Subsequent reduction with sodium borohydride leads to spectral changes characteristic for the formation of a secondary amine . 5 . The irreversibly inactivated enzyme thus produced contains two moles of inhibitor per mole of enzyme (two subunits per mole) . After protein hydrolysis, N-6-pyridoxyllysine can be identified by paper chromatography . 6 . The enzyme is inhibited irreversibly by 1-fluoro-2,4-dinitrobenzene, even in the presence of excess 2-mercaptoethanol . At least one dinitrophenyl group is bound per active unit of enzyme; 4 to 5 moles of dinitrophenyl group are bound per mole of enzyme . NADP+ AND GLUCOSE 6-PHOSPHATE PROTECT AGAINST INHIBITION BY 1-FLUORO-2,4-DINITROBENZENE . The absorption spectrum of dinitrophenyl-enzyme corresponds to that for dinitrophenylated amino groups . 7 . These studies indicate that there is an essential lysine at the active site of the enzyme . It is suggested that the function of this lysine is to bind glucose 6-phosphate . 8 . It is proposed that a group of "active lysine" proteins may exist (in analogy with the "active serine" enzymes), which share a common structural feature at their substrate-binding site and to which pyridoxal 5'-phosphate binds specifically.

Prikl Biokhim Mikrobiol, 1975 Jan-Feb, 11(1), 141 - 4
{Use of a freeze-drief culture of Leuconostoc mesenteroides for the synthesis of dextran}; Bolotnikova FI et al.; The influence of a prolonged storage of the freeze-dried culture Leuconostoc mesenteroides strain SF-4 on its capacity for the dextrane synthesis has been studied . The culture has maintained its normal viability for 12 years (the observation time) without changes in the cell morphology and capacity for the dextrane synthesis . The structure of dextrane synthesized by the revived culture L . mesenteroides has been similar to that of dextrane synthesized by the culture L . mesenteroides preserved via passages.

Arch Microbiol, 1975, 102(2), 111 - 6
Citrate lyase from Streptococcus diacetilactis . Association with its acetylating enzyme; Kummel A et al.; Citrate lyase (EC 4.1.3.6) was purified 38-fold from cell-free extracts of Streptococcus diacetilactis . The enzyme was homogeneous in analytical ultracentrifugation and polyacrylamide gel electrophoresis . The final enzyme preparation contained acetate: HS-citrate lyase ligase--an acetylating enzyme which converts inactive HS-citrate lyase into enzymatically active acetyl-S-citrate lyase . This enzyme activity was purified 25-fold over the crude extract and seemed to be associated with citrate lyase . Partially purified citrate lyase from Leuconostoc citrovorum contained also its acetylating enzyme . Purified citrate lyases from Klebsiella aerogenes and Rhodopseudomonas gelatinosa were devoid of acetylating enzyme activity . The HS-form of citrate lyase from S . diacetilactis was completely acetylated and hence activated by incubation with ATP and acetate for 25 min at 25 degrees C . The enzyme did not acetylate the HS-lyases from R . gelatinosa and K . aerogenes . In contrast to the citrate lyases from R . gelatinosa and K . aerogenes the enzymes from S . diacetilactis and L . citrovorum showed only a very weak reaction inactivation . It is assumed that this is due to the association of the acetylating enzymes with these lyases.

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