Les fonctions essentielles à la croissance dans le lait de Streptococcus thermophilus

STREPTOCOCCUS THERMOPHILUS EST UNE BACTERIE LACTIQUE LARGEMENT UTILISEE EN FABRICATION DE FROMAGES ET DE LAITS FERMENTES. AU COURS DES PROCEDES DE FABRICATION, SA CROISSANCE RAPIDE ET REGULIERE EST REQUISE POUR PERMETTRE L'ACIDIFICATION ET LE DEVELOPPEMENT DES PROPRIETES ORGANOLEPTIQUES DES PRODUITS LAITIERS. ELLE N'EST CEPENDANT PAS TOUJOURS OBTENUE ET LES INTERACTIONS BACTERIE-SUBSTRAT DE FERMENTATION RESTENT ENCORE MAL MAITRISEES. NOTRE OBJECTIF A DONC ETE D'IDENTIFIER LES FONCTIONS ESSENTIELLES A LA CROISSANCE OPTIMALE DE CETTE BACTERIE DANS LE LAIT. UNE MUTAGENESE AU HASARD DANS UNE SOUCHE DE S. THERMOPHILUS A CROISSANCE RAPIDE DANS LE LAIT NOUS A PERMIS D'OBTENIR 14 MUTANTS QUI ONT ETE CARACTERISES. NOUS AVONS IDENTIFIE LES FONCTIONS TOUCHEES DANS 9 D'ENTRE EUX. NOUS AVONS MONTRE QUE LES VOIES DE BIOSYNTHESE DES BASES PURIQUES ET DES ACIDES AMINES A CHAINE BRANCHEE SONT FONCTIONNELLES ET INDISPENSABLES A LA CROISSANCE DANS LE LAIT DE CETTE BACTERIE. CE RESULTAT SUGGERE QUE LE LAIT NE CONTIENT PAS SUFFISAMMENT DE BASES ET D'ACIDES AMINES A CHAINE RAMIFIEE POUR PERMETTRE LA CROISSANCE DE S. THERMOPHILUS. LE SYSTEME DE TRANSPORT DES OLIGOPEPTIDES JOUE EGALEMENT UN ROLE ESSENTIEL DANS LA NUTRITION AZOTEE DE S. THERMOPHILUS ET DONC DANS SA CROISSANCE OPTIMALE. CE SYSTEME RESSEMBLE, PAR SON ORGANISATION GENETIQUE, A CEUX D'AUTRES STREPTOCOQUES ET PAR SA SPECIFICITE A CELUI DE LACTOCOCCUS LACTIS. C'EST UN ABC TRANSPORTEUR COMPRENANT 3 PROTEINES AFFINES POUR LES OLIGOPEPTIDES FONCTIONNANT AVEC LE MEME SYSTEME PERMEASE. CES 3 PROTEINES SONT FORTEMENT HOMOLOGUES ET ONT DES SPECIFICITES CHEVAUCHANTES. LE SYSTEME PARAIT TRES PERFORMANT PUISQU'IL EST CAPABLE DE TRANSPORTER DES PEPTIDES DE 3 A 23 ACIDES AMINES. CE TRAVAIL A PERMIS DE MIEUX COMPRENDRE ET DONC DE MIEUX MAITRISER LES INTERACTIONS QUI EXISTENT ENTRE S. THERMOPHILUS ET SON SUBSTRAT DE FERMENTATION. MOTS CLES : STREPTOCOCCUS THERMOPHILUS, LAIT, CROISSANCE, TRANSPORT DES OLIGOPEPTIDES, MUTAGENESES, BIOSYNTHESE, ACIDES AMINES A CHAINE RAMIFIEE, PURINE.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Casein Utilization by Streptococcus thermophilus Results in a Diauxic Growth in Milk

In milk, Streptococcus thermophilus displays two distinct exponential growth phases, separated by a nonexponential one, during which proteinase synthesis was initiated. During the second exponential phase, utilization of caseins as the source of amino acids resulted in a decrease in growth rate, presumably caused by a limiting peptide transport activity

Three oligopeptide-binding proteins are involved in the oligopeptide transport of Streptococcus thermophilus

The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues

Multilocus Sequence Typing of Lactobacillus casei Reveals a Clonal Population Structure with Low Levels of Homologous Recombination▿ †

Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137T (= ATCC 393T). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (π ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution

The potential probiotic Lactobacillus rhamnosus CNCM I-3690 strain protects the intestinal barrier by stimulating both mucus production and cytoprotective response

Abstract The gut barrier plays an important role in human health. When barrier function is impaired, altered permeability and barrier dysfunction can occur, leading to inflammatory bowel diseases, irritable bowel syndrome or obesity. Several bacteria, including pathogens and commensals, have been found to directly or indirectly modulate intestinal barrier function. The use of probiotic strains could be an important landmark in the management of gut dysfunction with a clear impact on the general population. Previously, we found that Lactobacillus rhamnosus CNCM I-3690 can protect intestinal barrier functions in mice inflammation model. Here, we investigated its mechanism of action. Our results show that CNCM I-3690 can (i) physically maintain modulated goblet cells and the mucus layer and (ii) counteract changes in local and systemic lymphocytes. Furthermore, mice colonic transcriptome analysis revealed that CNCM I-3690 enhances the expression of genes related to healthy gut permeability: motility and absorption, cell proliferation; and protective functions by inhibiting endogenous proteases. Finally, SpaFED pili are clearly important effectors since an L. rhamnosus ΔspaF mutant failed to provide the same benefits as the wild type strain. Taken together, our data suggest that CNCM I-3690 restores impaired intestinal barrier functions via anti-inflammatory and cytoprotective responses

Ecological robustness of the gut microbiota in response to ingestion of transient food-borne microbes

Resident gut microbes co-exist with transient bacteria to form the gut microbiota. Despite increasing evidence suggesting a role for transient microbes on gut microbiota function, the interplay between resident and transient members of this microbial community is poorly defined. We aimed to determine the extent to which a host's autochthonous gut microbiota influences niche permissivity to transient bacteria using a fermented milk product (FMP) as a vehicle for five food-borne bacterial strains. Using conventional and gnotobiotic rats and gut microbiome analyses (16S rRNA genes pyrosequencing and reverse transcription qPCR), we demonstrated that the clearance kinetics of one FMP bacterium, Lactococcus lactis CNCM I-1631, were dependent on the structure of the resident gut microbiota. Susceptibility of the resident gut microbiota to modulation by FMP intervention correlated with increased persistence of L. lactis. We also observed gut microbiome configurations that were associated with altered stability upon exposure to transient bacteria. Our study supports the concept that allochthonous bacteria have transient and subject-specific effects on the gut microbiome that can be leveraged to re-engineer the gut microbiome and improve dysbiosis-related diseases

Identification of Restriction-Modification Systems of <i>Bifidobacterium animalis</i> subsp. <i>lactis</i> CNCM I-2494 by SMRT Sequencing and Associated Methylome Analysis

<div><p><i>Bifidobacterium animalis</i> subsp. <i>lactis</i> CNCM I-2494 is a component of a commercialized fermented dairy product for which beneficial effects on health has been studied by clinical and preclinical trials. To date little is known about the molecular mechanisms that could explain the beneficial effects that bifidobacteria impart to the host. Restriction-modification (R-M) systems have been identified as key obstacles in the genetic accessibility of bifidobacteria, and circumventing these is a prerequisite to attaining a fundamental understanding of bifidobacterial attributes, including the genes that are responsible for health-promoting properties of this clinically and industrially important group of bacteria. The complete genome sequence of <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494 is predicted to harbour the genetic determinants for two type II R-M systems, designated BanLI and BanLII. In order to investigate the functionality and specificity of these two putative R-M systems in <i>B. animalis</i> subsp. <i>lactis</i> CNCM I-2494, we employed PacBio SMRT sequencing with associated methylome analysis. In addition, the contribution of the identified R-M systems to the genetic accessibility of this strain was assessed.</p></div

Transformation efficiency of <i>B. animalis subsp. lactis</i> CNCM I-2494.

<p>Transformation with pDM1 plasmid DNA isolated from CNCM I-2494, <i>E. coli</i> pWSK29, <i>E. coli</i> pWSK29-M.BanLI, <i>E. coli</i> pWSK29-M.BanLII or <i>E. coli</i> pWSK29-M.BanLI-M.BanLII. Data presented are averages of triplicate experiments.</p