Structure, Organization, and Expression of the lct Gene for Lacticin 481, a Novel Lantibiotic Produced by Lactococcus lactis

The structural gene for the lactococcal lantibiotic lacticin 481 (lct) has been identified and cloned using a degenerated 20-mer DNA oligonucleotide based on the amino-terminal 7 amino acid residues of the purified protein. The transcription of the lct gene was analyzed, and its promoter was mapped. DNA sequence analysis of the lct gene revealed an open reading frame encoding a peptide of 51 amino acids. Comparison of its deduced amino acid sequence with the amino-terminal sequence and the amino acid composition of lacticin 481 indicates that the 61-residue peptide is prelacticin 481, containing a 27-residue carboxyl-terminal propeptide and a 24-residue amino-terminal leader peptide which lacks the properties of a typical signal sequence and which is significantly different from the leaders of other lantibiotics. The predicted amino acid sequence of prolacticin 481 contains 3 cysteines, 2 serines, and 2 threonines which were not detectable in amino acid analyses of mature lacticin 481. Based on these results and on characterization by two-dimensional NMR techniques, a structural model is proposed in which 2 cysteine residues are involved in lanthionine and one in β-methyllanthionine formation, and a 4th threonine residue is dehydrated. This model predicts a molecular mass for lacticin 481 of 2,901, which is in excellent agreement with that obtained from mass spectrometry.

Ability of Lactobacillus fermentum to overcome host α-galactosidase deficiency, as evidenced by reduction of hydrogen excretion in rats consuming soya α-galacto-oligosaccharides

<p>Abstract</p> <p>Background</p> <p>Soya and its derivatives represent nutritionally high quality food products whose major drawback is their high content of α-galacto-oligosaccharides. These are not digested in the small intestine due to the natural absence of tissular α-galactosidase in mammals. The passage of these carbohydrates to the large intestine makes them available for fermentation by gas-producing bacteria leading to intestinal flatulence. The aim of the work reported here was to assess the ability of α-galactosidase-producing lactobacilli to improve the digestibility of α-galacto-oligosaccharides <it>in situ</it>.</p> <p>Results</p> <p>Gnotobiotic rats were orally fed with soy milk and placed in respiratory chambers designed to monitor fermentative gas excretion. The validity of the animal model was first checked using gnotobiotic rats monoassociated with a <it>Clostridium butyricum </it>hydrogen (H₂)-producing strain. Ingestion of native soy milk by these rats caused significant H₂emission while ingestion of α-galacto-oligosaccharide-free soy milk did not, thus validating the experimental system. When native soy milk was fermented using the α-galactosidase-producing <it>Lactobacillus fermentum </it>CRL722 strain, the resulting product failed to induce H₂emission in rats thus validating the bacterial model. When <it>L. fermentum </it>CRL722 was coadministered with native soy milk, a significant reduction (50 %, <it>P </it>= 0.019) in H₂emission was observed, showing that α-galactosidase from <it>L. fermentum </it>CRL722 remained active <it>in situ</it>, in the gastrointestinal tract of rats monoassociated with <it>C. butyricum</it>. In human-microbiota associated rats, <it>L. fermentum </it>CRL722 also induced a significant reduction of H₂emission (70 %, <it>P </it>= 0.004).</p> <p>Conclusion</p> <p>These results strongly suggest that <it>L. fermentum </it>α-galactosidase is able to partially alleviate α-galactosidase deficiency in rats. This offers interesting perspectives in various applications in which lactic acid bacteria could be used as a vector for delivery of digestive enzymes in man and animals.</p

The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis:location of the thioether bridges

AbstractThe lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26

Ciblage de protéines hétérologues chez les bactéries lactiques (vers l'utilisation de Lactococcus lactis comme véhicule de molécules utiles dans le tube digestif de l'hôte)

Les bactéries lactiques (BL) sont les microorganismes les plus utilisés dans l'industrie alimentaire. Elles sont ainsi ingérées en grande quantité au travers des aliments et certaines espèces de BL font partie de la flore naturelle de l'homme ou des animaux. Du fait de ces intérêts, les BL sont très étudiées et les connaissances accumulées sur leur biologie permettent aujourd'hui d'envisager des applications nouvelles dans lesquelles ces bactéries seront utilisées comme vecteurs pour délivrer dans l'organisme des molécules biologiquement actives. Suivant l'utilisation souhaitée, ces molécules devront être produites dans des compartiments cellulaires différents d'où la nécessité de maîtriser le ciblage de protéines hétérologues chez les BL. Mes travaux de thèse ont permis la mise au point d'un système qui permet l'expression et le ciblage de protéines hétérologues dans le cytoplasme, la paroi bactérienne ou le milieu extracellulaire chez différentes espèces de BL.. Pour optimiser l'ancrage des protéines à la paroi des BL, nous avons entrepris la caractérisation de la sortase chez Lactococus lactis, la BL modèle. La sortase est une enzyme qui fixe les protéines, de façon covalente et selon un mécanisme conservé chez les bactéries à Gram positif. Nous avons identifié deux gènes de sortase sur le génome de Lactococcus lactis et démontré l'activité d'ancrage à la paroi du produit de l'un d'entre eux. Nous avons utilisé notre système de ciblage pour exprimer des antigènes du virus de la maladie de Gumboro chez Lactococcus lactis. La maladie de Gumboro est une affection immunodépressive du poulet qui cause des pertes importantes dans les élevages avicoles. Les souches de Lactococcus lactis exprimant les antigènes viraux ont été utilisées dans des tests de vaccination orale chez le poulet. Une réponse immunitaire significative a été obtenue avec une souche exprimant une forme ancrée à la paroi de l'antigène majeur du virus de Gumboro.Lactic acid bacteria (LAB) are widely used in the alimentary industry and are thus ingested in great quantities through food products. Furthermore, certain LAB species are members of the natural flora of man and animals. Because of these interests, LAB have been actively studied and the knowledge accumulated on their biology allow today to envisage new applications in which these bacteria will be used as vectors for the delivery of biologically active molecules into the body. According to the wished use, these molecules should be produced in different cellular compartments, which stress the need of controlling the targeting of heterologous proteins in LAB My thesis works allowed the settling of a system that permits the expression and the targeting of heterologous proteins in the cytoplasm, the cell wall or the extracellular medium of various LAB species. To optimize the anchoring of proteins in the cell wall, we tried to characterize the sortase of Lactococus lactis, the model LAB. Sortase is an enzyme that covalently anchors proteins, in a mechanism universal in Gram-positive bacteria. We identified two sortase genes on Lactococcus lactis genome and demonstrated the cell-wall-anchoring activity the product of the one of them. We used our protein targeting system to express antigens from the Gumboro disease virus in Lactococcus lactis. Gumboro disease is a chicken immunodepressive affection that causes important losses in poultry breeding. Lactococcus lactis strains expressing the viral antigens were used to immunize chickens and cells expressing cell-wall-anchored antigen induced significant immune response.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Vers le développement de bactéries lactiques probiotiques pour améliorer la digestibilité du soja

Le développement des produits à base de soja est freiné par leur forte teneur en a-galactosides (raffinose, stachyose). Du fait de l'absence d'a-galactosidase (a-Gal) dans l'intestin grêle, ils s'accumulent dans le côlon où ils sont métabolisés par des bactéries gazogènes, entraînant des problèmes de flatulences. L'objectif de cette étude est de dégrader les a-galactosides grâce à des bactéries lactiques a-Gal+ en fermentation de lait de soja et in vivo dans la partie supérieure du tractus digestif. Des fermentations de lait de soja ont été réalisées avec L. plantarum ATCC8014, L. fermentum CRL722, B. longum R0070 et B. breve R0175. L. plantarum ATCC8014 dégrade seulement le stachyose alors que les autres souches permettent de dégrader plus de 90% des deux a-galactosides majoritaires du soja. L. fermentum CRL722, testée dans le tractus digestif du rat monoxénique placé en chambre respiratoire, permet de réduire significativement l'émission de gaz intestinaux, indiquant sa capacité à exercer l'activité a-Gal in vivo. Un modèle d'étude a été mis au point chez L. lactis afin de définir les facteurs influençant l'activité a-Gal dans le tractus digestif. Dans ce cadre, les gènes melA et melAF, codant respectivement pour l'a-Gal de L. plantarum ATCC8014 et de L. fermentum CRL722, ont été caractérisés. Nous avons choisi de faire varier la localisation cellulaire de l'enzyme chez la bactérie (cytoplasme, surnageant, ou ancrée à la paroi). Dans les conditions expérimentales appliquées, les vecteurs construits n'ont pas permis de diminuer les flatulences chez le rat nourri avec un régime riche en a-galactosides. Les améliorations à apporter à ce système sont discutées.The development of soy products has been limited because of their high a-galactosides contents (raffinose, stachyose). Since humans do not posses a-galactosidase (a-Gal) in the small intestine, they accumulate in the large intestine where they are metabolized by gasogenic microorganisms which cause flatulence problems. The objective of this study was to degrade the a-galactosides in soy using a-Gal producing lactic acid bacteria in soymilk fermentation and in vivo in the upper gastrointestinal tract. Soymilk fermentations were performed using L. plantarum ATCC8014, L. fermentum CRL722, B. longum R0070 and B. breve R0175. L. plantarum ATCC8014 only degrades stachyose whereas the other strains degraded more than 90% of the two major a-galactosides found in soymilk. The activity of L. fermentum CRL722 was evaluated in vivo in the digestive tract of monoxenic rats placed in respiratory chambers. This strain reduced significantly the intestinal gas production, suggesting that it is able to exert a-Gal activity in vivo. Also, a study model was developed in L. lactis for evaluation of factors that affect a-Gal activity in the digestive tract. For this purpose, the melA and melAF genes, which code for the a-Gal of L. plantarum ATCC8014 and L. fermentum CRL722 respectively, have been characterized. In this study, we chose to vary the cellular localization of the enzyme in the bacterium. In the applied experimental conditions, these vectors did not reduce the flatulence of rats fed an a-galactosides rich diet. Improvements to this system are discussed.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations

Plasmid profiles of 35 Lactobacillus plantarum strains isolated from different green olive fermentors were obtained. A large number of plasmids in the CCC form (from 5 to 16) were present in all the tested strains as confirmed by a second dimension electrophoresis of DNA. These plasmids, all of which remain cryptic, ranged from 2.0 to 68 kb in size. Novobiocin, sodium dodecyl sulphate and ethidium bromide were used as plasmid-curing agents but only novobiocin induced loss of extrachromosomal DNA at a high frequency in these strains.Peer reviewe

Pseudomonas grimontii biofilm protects food contact surfaces from Escherichia coli colonization

Sous presseEscherichia coli typically colonizes food contact surfaces in the presence of other bacterial strains. The aimof this work was to evaluate the influence of a resident strain isolated from a fresh-cut salad industry (Pseudomonas grimontii 13A10) on the development of a model pathogen (E. coli) on bare stainless steel(SST) and stainless steel coated with diamond-like carbon (DLC) films, a-C:H:Si:O designated by SICON ®and a-C:H:Si designated by SICAN. The bacterial composition and spatial organization of single- anddual-species biofilms were analyzed by confocal laser scanning microscopy (CLSM). Biofilms weredeveloped for 1 and 3 days at 10degreesC and it was observed that the biovolume of E. coli biofilms in thepresence of P. grimontii was lower than in axenic conditions, suggesting that the isolate can protect foodcontact surfaces from pathogen colonization. After 3 days, the dual-species biofilms contained essentiallyP. grimontii cells and no preferential vertical distribution of bacterial strains was observed. The use of a-C:H:Si:O coated surfaces reduced the short-term colonization of the model pathogen in single- and dual-species biofilms, whereas decreased colonization by the non-pathogenic strain was only observed after 3days

Bacterias lacticas. As bacterias lacticas no centro dos novos desafios tecnologicos

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