Construction of Chimeric Glucansucrases for Analyzing Substrate-binding Regions That Affect the Structure of Glucan Products

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

Characterisation of four Leuconostoc bacteriophages isolated from dairy fermentations

Abstract Four bacteriophages (phages) growing on the same Leuconostoc strain were characterised. Electron micrographs showed these phages to be similar in morphology to the commonly isolated lactococcal phages with head diameters ranging from 49–55 nm and tail lengths of 117–131 nm. A distinctive base plate and collar were also present. From restriction enzyme analysis of purified phage DNA, the genome sizes were 23–29 kb. All four phages showed one major structural protein (of approximately 24 kDa) on SDS polyacrylamide gels. Hybridization experiments confirmed that the phages belonged to the same homology group. There was no homology between DNA from these phages and DNA from a prolate or small isometric lactococcal phage.     

Asp-196-->Ala mutant of Leuconostoc mesenteroides sucrose phosphorylase exhibits altered stereochemical course and kinetic mechanism of glucosyl transfer to and from phosphate

Mutagenesis of Asp-196 into Ala yielded an inactive variant of Leuconostoc mesenteroides sucrose phosphorylase (D196A). External azide partly complemented the catalytic defect in D196A with a second-order rate constant of 0.031 M-1 s-1 (pH 5, 30 degrees C) while formate, acetate and halides could not restore activity. The mutant utilized azide to convert alpha-D-glucose 1-phosphate into beta-D-glucose 1-azide, reflecting a change in stereochemical course of glucosyl transfer from alpha-retaining in wild-type to inverting in D196A. Phosphorolysis of beta-D-glucose 1-azide by D196A occurred through a ternary complex kinetic mechanism, in marked contrast to the wild-type whose reactions feature a common glucosyl enzyme intermediate and Ping-Pong kinetics. Therefore, Asp-196 is identified unambiguously as the catalytic nucleophile of sucrose phosphorylase, and its substitution by Ala forces the reaction to proceed via single nucleophilic displacement. D196A is not detectably active as alpha-glucosynthase.     

Identification of a replicon from pCC3, a cryptic plasmid from <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> C4 derived from kimchi,and development of a new host–vector system

Analysis of the structural properties of pCC3, a cryptic plasmid from Leuconostoc citreum C4 isolated from kimchi, determined its length as 3,338 bp and revealed three open reading frames (ORFs): ORF1–ORF3. ORF3 showed high homology with a replication initiation protein of the theta-type plasmid pTXL1. The fragment encompassing ORF3 and its upstream sequences (nt 1,299–1,634) was found to contain a functional plasmid replicon. A new shuttle vector, pUCC3E1, was constructed based on pCC3. Using Southern hybridization analysis, no single-stranded DNA intermediate was detected from Leu. citreum harboring pUCC3E1, which indicates that pCC3 replicated via the theta mechanism. The pUCC3E1 could be replicated in E. coli TG1 (5.8 × 10⁴ CFU/μg DNA) and the developed cloning hosts, Leu. citreum C16 (2.1 × 10² CFU/μg DNA) and Leu. citreum GJ7 (8.0 × 10¹ CFU/μg DNA). pUCC3E1 was stably maintained in Leu. citreum C16 (for 100 generations, ca. 94.2%) in the absence of erythromycin (5 μg/ml).     

Lactic acid bacteria associated with a heat-processed pork product and sources of variation affecting chemical indices of spoilage and sensory characteristics

Aims:  To evaluate the potential for developing a quality index for a Danish modified atmosphere packaged (MAP) heat-processed and naturally contaminated pork meat product stored at 5°C.
Methods and Results:  The composition of the predominating microflora and changes in contents of tyramine, arginine, organic acids and sensory characteristics were analysed. The microflora was predominated by Lactobacillus sakei , Leuconostoc carnosum and Carnobacterium divergens . The presence of each species varied between products and batches resulting in limited usefulness of the concentrations of these bacteria or their metabolites as indices of quality. Furthermore, the three species differed in their metabolic activities as shown by use of a model meat extract. However, when MAP storage of the processed pork product was followed by aerobic storage then acetic acid showed some potential as a chemical indicator of sensory quality.
Conclusion:  Variation in processing parameters and spoilage microbiota limited the usefulness of concentrations of micro-organisms and their metabolites as indices of spoilage for the studied processed MAP pork product.
Significance and Impact of the Study:  The present study contributes to an understanding of the difficulties experienced in developing quality indices to be used in the control of microbial spoilage of processed MAP meat products.     

Novel Leuconostoc citreum starter culture system for the fermentation of kimchi, a fermented cabbage product

To determine the dominant microorganisms involved in kimchi fermentation and to examine their effect on kimchi fermentation, we randomly isolated and characterized 120 lactic acid bacteria from kimchi during a 5-day fermentation at 15°C. Leuconostoc citreum was dominant during the early and mid-phases of kimchi fermentation whereas Lactobacillus sake/Lactobacillus curvatus or Lactobacillus brevis were found during later stages. Eighty-two out of 120 isolates (68%) were identified as Leuconostoc citreum by means of a polyphasic method, including 16S rDNA sequencing and DNA/DNA hybridization. A few Weissella confusa-like strains were also isolated during the mid-phase of the fermentation. Strain IH22, one of the Leuconostoc citreum isolates from kimchi, was used as an additive to evaluate growth and acid production in kimchi fermentation. This strain was consistently over 95% of the population in IH22-treated kimchi over a 5-day fermentation, while heterogeneous lactic acid bacteria were observed in the control kimchi. The pH in IH22-treated kimchi dropped rapidly but was stably maintained for 5 days, compared to its slow and prolonged decrease in the control kimchi. These results indicate that Leuconostoc citreum IH22 dominates over and retards the growth of other lactic acid bacteria in kimchi, suggesting it can be used as a bacterial starter culture to maintain the quality of kimchi for prolonged periods. This revised version was published online in June 2006 with corrections to the Cover Date.     

Leuconostoc mesenteroides growth kinetics with application to bacterial profile modification

Bacterial profile modification (BPM) is being developed as an oil recovery technique that uses bacteria to selectively plug oil depleted zones within a reservoir to divert displacing fluids (typically water) into oil-rich zones. Leuconostoc mesenteroides, which produces dextran when supplied with sucrose, is a bacterium that is technically feasible for use in profile modification. However, the technique requires controlled bacterial growth to produce selective plugging.A kinetic model for the production of cells and polysaccharides has been developed for L. mesenteroides bacteria. This model, based on data from batch growth experiments, predicts saccharide utilization, cell generation, and dextran production. The underlying mechanism is the extracellular breakdown of sucrose into glucose and fructose and the subsequent production of polysaccharide (dextran). The monosaccharides are then available for growth. Accompanying sucrose consumption is the utilization of yeast extract. The cell requires a complex media that is provided by yeast extract as a source of vitamins and amino acids. Varying the concentration ratio of yeast extract to sucrose in the growth media provides a means of controlling the amount of polymer produced per cell. Consequently, in situ bacteria growth can be controlled by the manipulation of nutrient media composition, thereby providing the ability to create an overall strategy for the use of L. mesenteroides bacteria for profile modification.     

Bacteriocin production ofLeuconostoc carnosum LA54A at different combinations of pH and temperature

Summary Leuconostoc carnosum LA54A produces a bacteriocin which is active againstListeria monocytogenes andListeria innocua. The ability ofLc. carnosum to produce the bacteriocin at various combinations of the growth parameters pH and temperature was analyzed. In the case of this strain bacteriocin production seems to be coupled to growth rate. This fact enables the prediction ifLc. carnosum can produce the bacteriocin at a given set of growth parameters, simply by predicting the growth rate of this organism. In addition we have analyzed the growth behavior of the target organismL. innocua WS2258 at the same set of growth parameters.     

A novel family of glucosyl 1,5-anhydro-d-fructose derivatives synthesised by transglucosylation with dextransucrase from Leuconostoc mesenteroides NRRL B-512F

1,5-Anhydro-d-fructose (AF), a metabolite of starch/glycogen degradation, is a good antioxidant. With the prospect of increasing its applications and use as a food ingredient, AF glucosylation catalysed by the dextransucrase from Leuconostoc mesenteroides NRRL B-512F was performed in the presence of sucrose. This led to AF glucosylated derivatives containing alpha-(1-->6) linkages named 1,5-anhydro-d-fructo-glucooligosaccharides (AFGOS). LC-MS analyses showed that AFGOS with a degree of polymerisation (DP) of up to 7 were synthesised. The amount of AFGOS produced and the average DP increased by using a high sucrose/AF molar ratio and high total sugar concentration. AFGOS were proved to present antioxidant properties quite similar to AF.