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Bejar W.,University of Sfax | Gabriel V.,CNRS Laboratory for Applied Biology in Agro-food and the Environment | Amari M.,CNRS Laboratory for Applied Biology in Agro-food and the Environment | Morel S.,National Polytechnic Institute of Toulouse | And 8 more authors.
International Journal of Biological Macromolecules | Year: 2013

Pear-derived Weissella sp. TN610 produced extracellular glycosyltransferase activity responsible for the synthesis of soluble exopolysaccharide from sucrose. Acid and dextranase-catalyzed hydrolysis revealed that the synthesized polymer was a glucan. According to 1H and 13C NMR analysis, the glucan produced by TN610 was a linear dextran made of 96% α-(1→6) and 4% α-(1→3) linkages. Zymogram analysis confirmed the presence of a unique glucansucrase of approximately 180kDa in the cell-free supernatant from TN610. The crude enzyme, optimally active at 37°C and pH 5, has promising potential for application as a food additive since it catalyzes dextran synthesis in sucrose-supplemented milk, allowing its solidification. A 4257-bp product corresponding to the mature glucansucrase gene was amplified by PCR from TN610. It encoded a polypeptide of 1418 residues having a calculated molecular mass of 156.089kDa and exhibiting 96% and 95% identity with glucansucrases from Lactobacillus fermentum Kg3 and Weissella cibaria CMU, respectively. © 2012 Elsevier B.V.


PubMed | CNRS Laboratory for Applied Biology in Agro-food and the Environment, University Paul Sabatier and French National Institute for Agricultural Research
Type: Journal Article | Journal: The FEBS journal | Year: 2015

Leuconostoc citreum NRRL B-1299 has long been known to produce -glucans containing both -(16) and -(12) linkages, which are synthesized by -transglucosylases of the GH70 family. We sequenced the genome of Leuconostoc citreum NRRL B-1299 to identify the full inventory of GH70 enzymes in this strain. Three new genes (brsA, dsrM and dsrDP) putatively encoding GH70 enzymes were identified. The corresponding recombinant enzymes were characterized. Branching sucrase A (BRS-A) grafts linear -(16) dextran with -(12)-linked glucosyl units, and is probably involved in the -(12) branching of L. citreum NRRL B-1299 dextran. This is the first report of a naturally occurring -(12) branching sucrase. DSR-M and DSR-DP are dextransucrases that are specific for -(16) linkage synthesis and mainly produce oligomers or short dextrans with molar masses between 580 and 27 000 gmol(-1) . In addition, DSR-DP contains sequences that diverge from the consensus sequences that are typically present in enzymes that synthesize linear dextran. Comparison of the genome with five other L. citreum genomes further revealed that dsrDP is unique to L. citreum NRRL B-1299. The presence of this gene in a prophage represents the first evidence of phage-mediated horizontal transfer of genes encoding such enzymes in lactic acid bacteria. Finally, brsA and dsrM are located in a chromosomal region in which genes encoding strain-specific GH70 enzymes are consistently located. This region may be a good target on which to focus in order to rapidly evaluate the diversity of GH70 enzymes in L. citreum strains.


PubMed | CNRS Laboratory for Applied Biology in Agro-food and the Environment and French National Institute for Agricultural Research
Type: Journal Article | Journal: FEMS microbiology letters | Year: 2015

The whole set of putative glucansucrases from Leuconostoc citreum LBAE-E16 and LBAE-C11 was retrieved from the draft genome sequence of these two sourdough strains previously suggested as alternan producers. Four and five putative glycoside hydrolase family 70 (GH70) encoding genes were identified in the genome sequence of strain C11 and E16, respectively. Some putative genes have high sequence identity to known Leuconostoc dextransucrases. Molecular and biochemical data confirmed that L. citreum C11 could be considered as a new alternan-producing strain, unlike strain E16. In the latter, two new putative glucansucrases with unusual structural features were retrieved. In particular, the GSE16-5 gene encodes for a protein of 2063 amino acids with a theoretical molecular mass of 229 kDa that shares 61% identity with the alternansucrase (ASR) of L. citreum NRRL B-1355, due to the presence of seven APY repeats identified in the C-terminal peptide sequence. Cloning and expression of the corresponding coding sequence revealed synthesis of a low molecular weight (10(4) Da) linear dextran polymer with glucosyl residues only linked by -1,6 linkages. This novel GH70 enzyme may thus be viewed as a natural chimeric enzyme resulting from the addition of the ASR C-terminal region in a dextransucrase.


PubMed | CNRS Laboratory for Applied Biology in Agro-food and the Environment, University Paul Sabatier, French National Institute for Agricultural Research and Agrocampus Ouest
Type: Journal Article | Journal: Genome announcements | Year: 2016

We report here the complete genome sequence of Lactococcus lactis subsp. lactis strain A12, a strain isolated from sourdough. The circular chromosome and the four plasmids reveal genes involved in carbohydrate metabolism that are potentially required for the persistence of this strain in such a complex ecosystem.


Bounaix M.-S.,CNRS Laboratory for Applied Biology in Agro-food and the Environment | Robert H.,CNRS Laboratory for Applied Biology in Agro-food and the Environment | Gabriel V.,CNRS Laboratory for Applied Biology in Agro-food and the Environment | Morel S.,INSA Toulouse | And 5 more authors.
FEMS Microbiology Letters | Year: 2010

The study of exopolysaccharide production by heterofermentative sourdough lactic acid bacteria has shown that Weissella strains isolated from sourdoughs produce linear dextrans containing α-(1→6) glucose residues with few α-(1→3) linkages from sucrose. In this study, several dextran-producing strains, Weissella cibaria and Weissella confusa, isolated from sourdough, were characterized according to carbohydrate fermentation, repetitive element-PCR fingerprinting using (GTG)5 primers and glucansucrase activity (soluble or cell-associated). This study reports, for the first time, the characterization of dextransucrase from Weissella strains using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ polymer production (after incubation with sucrose) from enzymatic fractions harvested from both sucrose and glucose culture media. Results demonstrate that dextransucrase activity was mainly soluble and associated with a constitutive 180-kDa protein. In addition, microsequencing of the active dextransucrase from W. cibaria LBAE-K39 allowed the design of specific primers that could detect the presence of glucansucrase encoding genes similar to GTFKg3 of Lactobacillus fermentum Kg3 and to DSRWC of W. cibaria CMU. This study hence indicates that sourdough Weissella strains synthesize original dextransucrase. © 2010 Federation of European Microbiological Societies.


Randrianjatovo I.,CNRS Laboratory for Applied Biology in Agro-food and the Environment | Girbal-Neuhauser E.,CNRS Laboratory for Applied Biology in Agro-food and the Environment | Marcato-Romain C.-E.,CNRS Laboratory for Applied Biology in Agro-food and the Environment
Applied Microbiology and Biotechnology | Year: 2015

Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7 μg to as high as 50 μg per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250 μg ml−1) showed little or no sugar interference up to 2000 μg ml−1, thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1 ± 3.1, 38.3 ± 7.1 and 0.3 ± 0.1 μg equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms. © 2015, Springer-Verlag Berlin Heidelberg.


Randrianjatovo-Gbalou I.,CNRS Laboratory for Applied Biology in Agro-food and the Environment | Girbal-Neuhauser E.,CNRS Laboratory for Applied Biology in Agro-food and the Environment | Marcato-Romain C.-E.,CNRS Laboratory for Applied Biology in Agro-food and the Environment
Analytical Biochemistry | Year: 2016

A novel approach to the quantification of extracellular polysaccharides in miniaturized biofilms presenting a wide variety of extracellular matrices was developed. The assay used the periodic acid-Schiff reagent and was first calibrated on dextran and alginate solutions. Then it was implemented on 24-h and 48-h biofilms from three strains known to produce different exopolymeric substances (Pseudomonas aeruginosa, Bacillus licheniformis, Weissella confusa). The assay allowed quantification of the total exopolysaccharides, taking into account possible interferences due to cells or other main expolymers of the matrix (eDNA, proteins). © 2016 Elsevier Inc. All rights reserved.


PubMed | CNRS Laboratory for Applied Biology in Agro-food and the Environment
Type: | Journal: Analytical biochemistry | Year: 2016

A novel approach to the quantification of extracellular polysaccharides in miniaturized biofilms presenting a wide variety of extracellular matrices was developed. The assay used the periodic acid-Schiff reagent and was first calibrated on dextran and alginate solutions. Then it was implemented on 24-h and 48-h biofilms from three strains known to produce different exopolymeric substances (Pseudomonas aeruginosa, Bacillus licheniformis, Weissella confusa). The assay allowed quantification of the total exopolysaccharides, taking into account possible interferences due to cells or other main expolymers of the matrix (eDNA, proteins).


PubMed | CNRS Laboratory for Applied Biology in Agro-food and the Environment
Type: | Journal: Analytical biochemistry | Year: 2015

An amyloid fibrils investigation within biofilm samples requires distinguishing the amyloid -sheet structure of these proteins and quantifying them. In this study, the property of amyloids to incorporate the fluorescent dye Thioflavin T has been exploited to propose a method of quantification. The experimental protocol includes the preparation of amyloids from commercial -casein (CN) and their fractionation through size exclusion chromatography (SEC) to provide calibration curves from fluorescence and absorbance signals. Finally, a bacterial biofilm extract was injected into SEC, and the amyloid fibrils could be expressed as equivalent CN, representing approximately 21% of the total proteins.


PubMed | CNRS Laboratory for Applied Biology in Agro-food and the Environment
Type: Journal Article | Journal: Applied microbiology and biotechnology | Year: 2015

Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7g to as high as 50g per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250gml(-1)) showed little or no sugar interference up to 2000gml(-1), thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.13.1, 38.37.1 and 0.30.1g equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms.

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