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The dramatic increase in the amount of microbe descriptions in databases, reports and papers presents a two-fold challenge for accessing the information: integration of heterogeneous data in a standard ontology-based representation and normalization of the textual descriptions by semantic analysis. Recent text mining methods offer powerful ways to extract textual information and generate ontology-based representation.This paper describes the design of the Omnicrobe application that gathers comprehensive information on habitats, phenotypes and usages of microbes from scientific sources of high interest to the microbiology community. The Omnicrobe database contains around 1 million descriptions of microbe properties that are created by analyzing and combining six information sources of various kinds, i.e. biological resource catalogues, sequence database and scientific literature. The microbe properties are indexed by the Ontobiotope ontology and their taxa are indexed by an extended version of the taxonomy maintained by the National Center for Biotechnology Information.The Omnicrobe application covers all domains of microbiology. It provides an easy-to-use support in the resolution of scientific questions related to the habitats, phenotypes and uses of microbes through simple and complex ontology-based queries. We illustrate the potential of Omnicrobe with a use case from the food innovation domain.

International audience; Genomic evidence has led to the division of genus Propionibacterium into Propionibacterium and Acidipropionibacterium. They are pleomorphic rods, anaerobic to aerotolerant, mesophilic, Gram-positive and of high G+C. They produce propionic acid as a major end product from many substrates. P. freudenreichii is used as ripening culture in Swiss-type cheeses, where it produces propionic and acetic acid, CO2 and aroma compounds leading to the characteristic flavor and eyes. They also produce vitamins and other compounds of interest and may be used as probiotics. Genetic tools and genome sequences are now available for both Propionibacterium and Acidipropionibacterium genera, which will improve their applications.

Propionibacterium freudenreichii is crucial in Swiss-type cheese manufacture. Classic propionic acid fermentation yields the nutty flavor and the typical eyes. Co-metabolism of aspartate pronounces the flavor of the cheese; however, it also increases the size of the eyes, which can induce splitting and reduce the cheese quality. Aspartase (EC 4.3.1.1) catalyzes the deamination of aspartate, yielding fumarate and ammonia. The aspartase activity varies considerably among P. freudenreichii strains. Here, the correlation between aspartase activity and the locus of aspartase-encoding genes (aspA ) and dcuA encoding the C4-dicarboxylate transporter was investigated in 46 strains to facilitate strain selection for cheese culture. Low aspartase activity was correlated with a particular genomic rearrangement: low in vitro aspartase activity always occurred in strains with gene clusters aspA - dcuA where the dcuA was frameshifted, producing a stop codon or was disrupted by an ISL3-like element. The low aspartase activity could be due to the protein sequence of the aspartase or a dysfunctional DcuA. The highest values of aspartase activity were detected in strains with aspA1 - aspA2-dcuA with a DcuA sequence sharing 99.07 - 100% identity with the DcuA sequence of strain DSM 20271 T and an additional C4-dicarboxylate transporter belonging to the DcuAB family.

Objectifs Le microbiote intestinal humain est un écosystème complexe et dynamique. Il exerce de nombreuses fonctions biologiques et métaboliques. Le maintien de son équilibre (composition, activité) est important pour la santé et le bien-être de l’hôte et peut être influencé par de nombreux facteurs, comme l’alimentation. Les produits laitiers fermentés, qui contiennent une grande quantité de bactéries lactiques vivantes, peuvent contribuer à cet équilibre mais la compréhension de leur impact reste encore limitée. Ce travail a pour objectif de mieux comprendre, à partir de l’utilisation d’outils métaomiques, l’impact de la consommation de lait fermenté et de bactéries lactiques vivantes sur la composition et l’activité du microbiote intestinal. De plus, il vise à évaluer l’état physiologique et métabolique des bactéries lactiques en transit dans le tractus digestif. Matériels et Méthodes Trois groupes de 8 souris à flore humanisée (ie transplantation d’un microbiote fécal d’un individu sain à des souris axéniques) ont été alimentées avec (i) un régime contrôle sans lait (alimentation traditionnelle des souris + tampon PBS), (ii) un régime avec lait fermenté (alimentation traditionnelle des souris + un lait fermenté par les 2 bactéries du yaourt, Streptococcus thermophilus et Lactobacillus bulgaricus), ou (iii) un régime avec lait fermenté thermisé (alimentation traditionnelle des souris + un lait fermenté par les 2 bactéries du yaourt puis thermisé à 95°C pendant 25 min). La composition et l’activité du microbiote humain transplanté chez les souris ont été évaluées respectivement par métagénomique shotgun et métatranscriptomique à partir d’échantillons fécaux. Résultats principaux L’analyse quantitative et le suivi de l’activité du microbiote intestinal de souris à flore humanisée est réalisable grâce à des approches métaomiques. La consommation de lait fermenté (thermisé ou pas) ne modifie pas la composition du microbiote intestinal humain chez la souris mais a un impact significatif sur son activité. L’expression des gènes est modifiée pour la plupart des genres bactériens dominants, et modulant par conséquent leurs fonctions. Cet impact est d’autant plus important que la consommation de lait fermenté est régulière et prolongée dans le temps. En revanche, il n’a pas été possible de suivre l’activité des bactéries lactiques en transit qui sont minoritaires. Conclusion Ce travail met en évidence l’intérêt des méthodes métaomiques pour suivre l’impact de l’alimentation et ouvre des perspectives sur l’exploration des effets bénéfiques potentiels des laits fermentés sur le microbiote intestinal humain.

From strain potential to bacterial community design for targeted functions. STLOpendays

1- Introduction: Propionibacterium freudenreichii is an Actinobacterium widely used in the dairy industry as a ripening culture for Swiss-type cheeses, for vitamin B12 production and some strains display probiotic properties. It is reportedly a hardy bacterium, able to survive the cheese-making process and digestive stresses. 2- Objectives : The aim of the study was to decipher metabolic adaptations of P. freudenrechii responsible for long-term survival under gradual nutritional shortage. 3- Materials & methods: For 11 days, in a non-nutrient supplemented culture medium, eight strains were monitored by measuring their optical density, counting colony-forming units (CFU) and using LIVE/DEAD staining and microscopy observation. The strain with the highest survival rate was incubated for 11 days (9 days after entry into stationary phase) in Yeast Extract Lactate medium at 30 °C under microaerophilic conditions, without any adjunct during the incubation. Its physiological adaptation was investigated by RNA-seq analysis. The carbon and free amino acids sources available in the medium, and the organic acids produced by the strain, were monitored throughout growth and survival. 4- Results: Under these conditions, the eight studied strains displayed high survival rates, their culturability reaching more than 9 log 10 CFU/ml after 2 days. After 11 days, this value ranged from 7.8 to 8.2 log 10 CFU/ml depending on the strain, and at least 50% of the P. freudenreichii population displayed an intact envelope. P. freudenreichii with the highest survival rate was CIRM-BIA 138 (alias ITG P9). Although lactate (the preferred carbon source for P. freudenreichii) was exhausted three days after inoculation, the CIRM-BIA 138 strain sustained a high population level of 9.3 log 10 CFU/mL and revealed a complete disruption of metabolism at the entry into stationary phase as compared to exponential phase. 5- Conclusions: P. freudenreichii adapts its metabolism during entry into stationary phase by down-regulating oxidative phosphorylation, glycolysis, and the Wood-Werkman cycle, by exploiting new nitrogen (glutamate, glycine, alanine) sources, by down-regulating the transcription, translation and secretion of protein. Utilization of polyphosphates was suggested.

Propionibacterium freudenreichii, a dairy starter, can reach a population of almost 109 propionibacteria per gram in Swiss-type cheese at the time of consumption. Also consumed as a probiotic, it displays strain-dependent anti-inflammatory properties mediated by surface proteins that induce IL-10 in leukocytes. We selected 23 strains with varied anti-inflammatory potentials in order to identify the protein(s) involved. After comparative genomic analysis, 12 of these strains were further analysed by surface proteomics, eight of them being further submitted to transcriptomics. The omics data were then correlated to the anti-inflammatory potential evaluated by IL-10 induction. This comparative omics strategy highlighted candidate genes that were further subjected to gene-inactivation validation. This validation confirmed the contribution of surface proteins, including SlpB and SlpE, two proteins with SLH domains known to mediate non-covalent anchorage to the cell-wall. Interestingly, HsdM3, predicted as cytoplasmic and involved in DNA modification, was shown to contribute to anti-inflammatory activity. Finally, we demonstrated that a single protein cannot explain the anti-inflammatory properties of a strain. These properties therefore result from different combinations of surface and cytoplasmic proteins, depending on the strain. Our enhanced understanding of the molecular bases for immunomodulation will enable the relevant screening for bacterial resources with anti-inflammatory properties.

International audience; Mastitis is a mammary gland inflammatory disease often due to bacterial infections. Like many other infections, it used to be considered as a host-pathogen interaction driven by host and bacterial determinants. Until now, the involvement of the bovine mammary gland microbiota in the host-pathogen interaction has been poorly investigated, and mainly during the infectious episode. In this study, the bovine teat microbiome was investigated in 31 quarters corresponding to 27 animals, which were all free of inflammation at sampling time but which had different histories regarding mastitis: From no episode of mastitis on all the previous lactations (Healthy quarter, Hq) to one or several clinical mastitis events (Mastitic quarter, Mq). Several quarters whose status was unclear (possible history of subclinical mastitis) were classified as NDq. Total bacterial DNA was extracted from foremilk samples and swab samples of the teat canal. Taxonomic profiles were determined by pyrosequencing on 16s amplicons of the V3-4 region. Hq quarters showed a higher diversity compared to Mq ones (Shannon index: ~8 and 6, respectively). Clustering of the quarters based on their bacterial composition made it possible to separate Mq and Hq quarters into two separate clusters (C1 and C2, respectively). Discriminant analysis of taxonomic profiles between these clusters revealed several differences and allowed the identification of taxonomic markers in relation to mastitis history. C2 quarters were associated with a higher proportion of the Clostridia class (including genera such as Ruminococcus, Oscillospira, Roseburia, Dorea, etc.), the Bacteroidetes phylum (Prevotella, Bacteroides, Paludibacter, etc.), and the Bifidobacteriales order (Bifidobacterium), whereas C1 quarters showed a higher proportion of the Bacilli class (Staphylococcus) and Chlamydiia class. These results indicate that microbiota is altered in udders which have already developed mastitis, even far from the infectious episode. Microbiome alteration may have resulted from the infection itself and or the associated antibiotic treatment. Alternatively, differences in microbiome composition in udders with a history of mastitis may have occurred prior to the infection and even contributed to infection development. Further investigations on the dynamics of mammary gland microbiota will help to elucidate the contribution of this endogenous microbiota to the mammary gland health.

International audience; Propionibacterium freudenreichii belongs to the class Actinobacteria (Gram positive with a high GC content). This "Generally Recognized As Safe" (GRAS) species is traditionally used as (i) a starter for Swiss-type cheeses where it is responsible for holes and aroma production, (ii) a vitamin B12 and propionic acid producer in white biotechnologies, and (iii) a probiotic for use in humans and animals because of its bifidogenic and anti-inflammatory properties. Until now, only strain CIRM-BIA1T had been sequenced, annotated and become publicly available. Strain CIRM-BIA129 (commercially available as ITG P20) has considerable anti-inflammatory potential. Its gene content was compared to that of CIRM-BIA1 T. This strain contains 2384 genes including 1 ribosomal operon, 45 tRNA and 30 pseudogenes.

Colloque organisé par : Pr. Catherine Béal - UFR Technologie et Procédés Alimentaires - AgroParisTech Dr. Luis Bermudez - MICALIS - Institut National de la Recherche Agronomique Dr. Marie-Christine Champomier-Verges - MICALIS - Institut National de la Recherche Agronomique, Jouy en Josas Dr. Céline Delbès - Unité de Recherches Fromagères - Institut National de la Recherche Agronomique, Aurillac Dr. Stéphanie-Marie Deutsch - STLO - Institut National de la Recherche Agronomique, Rennes Dr. Véronique Monnet - Institut National de la Recherche Agronomique, Jouy en Josas Dr. Monique Zagorec - Unité SECALIM - Institut National de la Recherche Agronomique, ONIRIS - NantesColloque organisé par :Pr. Catherine Béal - UFR Technologie et Procédés Alimentaires - AgroParisTechDr. Luis Bermudez - MICALIS - Institut National de la Recherche AgronomiqueDr. Marie-Christine Champomier-Verges - MICALIS - Institut National de la Recherche Agronomique, Jouy en JosasDr. Céline Delbès - Unité de Recherches Fromagères - Institut National de la Recherche Agronomique, AurillacDr. Stéphanie-Marie Deutsch - STLO - Institut National de la Recherche Agronomique, RennesDr. Véronique Monnet - Institut National de la Recherche Agronomique, Jouy en JosasDr. Monique Zagorec - Unité SECALIM - Institut National de la Recherche Agronomique, ONIRIS - Nantes; Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF can grow on a variety of carbon and nitrogen sources. The aim of this work was to discover the genetic basis for strain-dependent traits related to its ability to use specific carbon sources. Twenty-one strains were sequenced. Each gene was attributed to either the core genome or an accessory genome. The ability of the 21 strains to degrade 50 different sugars was evaluated. Thirty-three sugars were degraded by none of the sequenced strains whereas eight sugars were degraded by all of them. The corresponding genes were present in the core genome. Lactose, melibiose were only used by some strains. The presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes and was in line the metabolic pathways described previously in other species. We also considered the genetic origin (transduction, rearrangement) of the corresponding genomic islands. Ribose and gluconate were to a greater or lesser extent (quantitative phenotype) degraded by some strains. For these sugars, the phenotypes correlated with the premature appearance of a stop codon interrupting protein synthesis, preventing the catabolism of corresponding sugar. These results illustrate (i) the power of correlation studies to discover the genetic basis of binary strain-dependent traits, and (ii) the plasticity of PF chromosomes, probably resulting from horizontal transfers, duplications, transpositions and an accumulation of mutations.

Aims Propionibacterium freudenreichii is an actinobacterium widely used in dairy industry during the ripening process of Swiss-type cheeses and which presents probiotic properties. P. freudenreichii is reportedly a hardy bacterium, able to survive during the cheese-making process and when subjected to digestive stresses. During this study the long-term survival (LTS) of P. freudenreichii was investigated for 11 days by means of phenotypic characterization in a culture medium without the addition of any nutrients. Methods and Results For 11 days, in a non-nutrient supplemented culture medium, eight strains were monitored by measuring their optical density, counting colony-forming units (CFU) and using LIVE/DEAD staining and microscopy observation. Under these conditions, all strains displayed high survival rates in the culture medium, their culturability reaching more than 9 log10 CFU ml−1 after 2 days. After 11 days, this value ranged from 7·8 to 8·2 log10 CFU ml−1 depending on the strain, and at least 50% of the P. freudenreichii population displayed an intact envelope. As lysis of part of a bacterial population may be a microbial strategy to recover nutrients, in CIRM-BIA 138 (the strain with the highest population at day 11), cell lysis was assessed by quantifying intact bacterial cells using qPCR targeting the housekeeping gene tuf. No lysis was observed. Conclusion Taken together, our results suggest that P. freudenreichii strains use a viable but nonculturable state to adapt to the LTS phase. Significance and Impact of the Study Assessing the viability of P. freudenreichii and understanding their mechanisms for survival should be of great interest regarding their potential probiotic applications.

Propionibacterium freudenreichii is a beneficial bacterium used in the food industry as a vitamin producer, as a bio-preservative, as a cheese ripening starter and as a probiotic. It is known to adhere to intestinal epithelial cells and mucus and to modulate important functions of the gut mucosa, including cell proliferation and immune response. Adhesion of probiotics and cross-talk with the host rely on the presence of key surface proteins, still poorly identified. Identification of the determinants of adhesion and of immunomodulation by P. freudenreichii remains a bottleneck in the elucidation of its probiotic properties. In this report, three complementary proteomic methods are used to identify surface-exposed proteins in a strain, previously selected for its probiotic properties. The role of these proteins in the reported immunomodulatory properties of P. freudenreichii is evidenced. This work constitutes a basis for further studies aimed at the elucidation of mechanisms responsible for its probiotic effects, in a post-genomic context. Biological significance Dairy propionibacteria, mainly the species Propionibacterium freudenreichii , are consumed in high amounts within Swiss type cheeses. These peculiar bacteria are considered both as dairy starters and as probiotics. Their consumption modulates the gut microbiota, which makes them both probiotic and prebiotic. Promising immunomodulatory properties have been identified in these bacteria, in vitro , in animals and in humans. However, the mechanisms responsible for such anti-inflammatory properties are still unknown. In this work, we identify surface proteins involved in adhesion and immunostimulation by P. freudenreichii . This opens new perspectives for its utilization in new functional fermented food products, in clinical trials, and in understanding modulation of gut inflammation by products containing propionibacteria.

Lactic acid bacteria (LAB) and propionic acid bacteria (PAB) are widely used in the manufacture of cheeses and other fermented dairy products. Bacterial species used as starters are mainly chosen according to their intrinsic properties: the milk acidifying capacity for LAB starters and the aromatizing properties of PAB, for example. Beyond the general characteristics of a bacterial species, many key phenotypic traits determining their interest for dairy applications depend on the strain within a given species. Through some examples, this review illustrates how the choice of a bacterial strain with specific technological characteristics, within a given species of LAB or PAB, can determine the final properties in the end product. This concerns the technological properties of cheeses, such as flavour, texture, and opening formation, and their functional properties, such as inhibition of undesirable microorganisms and health properties. When known, the genetic determinants of the diversity are presented. This review emphasizes the importance of preserving and exploring microbial resources at the intraspecific level, as an unending source of diversity for innovation in food fermentation.

Propionibacterium freudenreichii is widely used in Swiss-type cheese manufacture, where it contributes to flavour and eye development. It is currently divided into two subspecies, according to the phenotype for lactose fermentation and nitrate reduction (lac+/nit− and lac−/nit+ for P. freudenreichii subsp. shermanii and subsp. freudenreichii, respectively). However, the existence of unclassifiable strains (lac+/nit+ and lac−/nit−) has also been reported. The aim of this study was to revisit the relevance of the subdivision of P. freudenreichii into subspecies, by confirming the existence of unclassifiable strains. Relevant conditions to test the ability of P. freudenreichii for lactose fermentation and nitrate reduction were first determined, by using 10 sequenced strains, in which the presence or absence of the lactose and nitrate genomic islands were known. We also determined whether the subdivision based on lac/nit phenotype was related to other phenotypic properties of interest in cheese manufacture, in this case, the production of aroma compounds, analysed by gas chromatography-mass spectrometry, for a total of 28 strains. The results showed that a too short incubation time can lead to false negative for lactose fermentation and nitrate reduction. They confirmed the existence of four lac/nit phenotypes instead of the two expected, thus leading to 13 unclassifiable strains out of the 28 characterized (7 lac+/nit+ and 6 lac−/nit−). The production of the 15 aroma compounds detected in all cultures varied more within a lac/nit phenotype (up to 20 times) than between them. Taken together, these results demonstrate that the division of P. freudenreichii into two subspecies does not appear to be relevant.

S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity. Electronic supplementary material The online version of this article (doi:10.1186/s13567-014-0106-7) contains supplementary material, which is available to authorized users.

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis. Fil: Abeijon Mukdsi, Maria Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Falentin, Hélène . INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia Fil: Maillard, Marie-Bernadette . INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia Fil: Chuat, Victoria . INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia Fil: Medina, Roxana Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina Fil: Parayre, Sandrine . INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia Fil: Thierry, Anne. INRA. Science et Technologie du Lait et de l’OEuf; Francia. Agrocampus Ouest. Science et Technologie du Lait et de l’OEuf; Francia

Lactococcus lactis is widely used in the dairy industry. We report the draft genome sequence of L. lactis subsp. lactis bv. diacetylactis LD61, an industrial and extensively studied strain. In contrast to the closely related and plasmidless strain IL1403, LD61 contains 6 plasmids, and the genome sequence provides additional information related to adaptation to the dairy environment.

Background Propionibacterium freudenreichii is a food grade bacterium consumed both in cheeses and in probiotic preparations. Its promising probiotic potential, relying largely on the active release of beneficial metabolites within the gut as well as the expression of key surface proteins involved in immunomodulation, deserves to be explored more deeply. Adaptation to the colon environment is requisite for the active release of propionibacterial beneficial metabolites and constitutes a bottleneck for metabolic activity in vivo. Mechanisms allowing P. freudenreichii to adapt to digestive stresses have been only studied in vitro so far. Our aim was therefore to study P. freudenreichii metabolic adaptation to intra-colonic conditions in situ. Results We maintained a pure culture of the type strain P. freudenreichii CIRM BIA 1, contained in a dialysis bag, within the colon of vigilant piglets during 24 hours. A transcriptomic analysis compared gene expression to identify the metabolic pathways induced by this environment, versus control cultures maintained in spent culture medium. We observed drastic changes in the catabolism of sugars and amino-acids. Glycolysis, the Wood-Werkman cycle and the oxidative phosphorylation pathways were down-regulated but induction of specific carbohydrate catabolisms and alternative pathways were induced to produce NADH, NADPH, ATP and precursors (utilizing of propanediol, gluconate, lactate, purine and pyrimidine and amino-acids). Genes involved in stress response were down-regulated and genes specifically expressed during cell division were induced, suggesting that P. freudenreichii adapted its metabolism to the conditions encountered in the colon. Conclusions This study constitutes the first molecular demonstration of P. freudenreichii activity and physiological adaptation in vivo within the colon. Our data are likely specific to our pig microbiota composition but opens an avenue towards understanding probiotic action within the gut in further studies comparing bacterial adaptation to different microbiota.

Lactobacillus acidophilus is a natural inhabitant of mammalian gastrointestinal systems and is used in dairy and pharmaceutical products. Five draft genome sequences, covering 1,995,790 nucleotides (nt) on average, are divided into 19 to 34 scaffolds covering 1,995 to 2,053 genes. The draft genome sequences were compared to the sequence of the L. acidophilus NCFM dairy strain.

International audience

Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. It grows when cheeses are ripened in a warm room (about 24°C). Cheeses with an acceptable eye formation level are transferred to a cold room (about 4°C), inducing a marked slowdown of propionic fermentation, but P. freudenreichii remains active in the cold. To investigate the P. freudenreichii strategies of adaptation and survival in the cold, we performed the first global gene expression profile for this species. The time-course transcriptomic response of P. freudenreichii CIRM-BIA1(T) strain was analyzed at five times of incubation, during growth at 30°C then for 9 days at 4°C, under conditions preventing nutrient starvation. Gene expression was also confirmed by RT-qPCR for 28 genes. In addition, proteomic experiments were carried out and the main metabolites were quantified. Microarray analysis revealed that 565 genes (25% of the protein-coding sequences of P. freudenreichii genome) were differentially expressed during transition from 30°C to 4°C (P1). At 4°C, a general slowing down was observed for genes implicated in the cell machinery. On the contrary, P. freudenreichii CIRM-BIA1(T) strain over-expressed genes involved in lactate, alanine and serine conversion to pyruvate, in gluconeogenesis, and in glycogen synthesis. Interestingly, the expression of different genes involved in the formation of important cheese flavor compounds, remained unchanged at 4°C. This could explain the contribution of P. freudenreichii to cheese ripening even in the cold. In conclusion, P. freudenreichii remains metabolically active at 4°C and induces pathways to maintain its long-term survival.

Seven Propionibacterium freudenreichii strains exhibited similar responses when placed at 4°C. They slowed down cell machinery, displayed cold stress responses, and rerouted their carbon metabolism toward trehalose and glycogen synthesis, both accumulated in cells. These results highlight the molecular basis of long-term survival of P. freudenreichii in the cold.

Propionibacterium freudenreichii is a bacterial species found in Swiss-type cheeses and is also considered for its health properties. The main claimed effect is the bifidogenic property. Some strains were shown recently to display other interesting probiotic potentialities such as anti-inflammatory properties. About 30% of strains were shown to produce a surface exopolysaccharide (EPS) composed of (1→3,1→2)-β -d- glucan due to a single gene named gtfF . We hypothesized that functional properties of P. freudenreichii strains, including their anti-inflammatory properties, could be linked to the presence of β-glucan. To evaluate this hypothesis, gtfF genes of three β-glucan-producing strains were disrupted. These knockout (KO) mutants were complemented with a plasmid harboring gtfF (KO-C mutants). The absence of β-glucan in KO mutants was verified by immunological detection and transmission electron microscopy. We observed by atomic force microscopy that the absence of β-glucan in the KO mutant dramatically changed the cell's topography. The capacity to adhere to polystyrene surface was increased for the KO mutants compared to wild-type (WT) strains. Anti-inflammatory properties of WT strains and mutants were analyzed by stimulation of human peripheral blood mononuclear cells (PBMCs). A significant increase of the anti-inflammatory interleukin-10 cytokine production by PBMCs was measured in the KO mutants compared to WT strains. For one strain, the role of β-glucan in mice gut persistence was assessed, and no significant difference was observed between the WT strain and its KO mutant. Thus, β-glucan appears to partly hide the anti-inflammatory properties of P. freudenreichii ; which is an important result for the selection of probiotic strains.

Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. This study investigates the molecular diversity and the population structure of this bacterium via multilocus sequence typing (MLST). Internal fragments of seven genes sequenced for 113 strains of different subspecies and origins allowed the resolution of 46 sequence types (STs) with occurrence frequencies ranging from 1 to 11. The core genome of the species harbours a low level of nucleotide polymorphism. In our data, single nucleotide polymorphisms account for only 2.28% of the concatenated sequences, and the average polymorphism rate in pairwise comparisons is 0.46%. The analyses reveal quantitatively comparable contributions of recombination and mutation in nucleotide changes at core genome loci along cell lineages. Remarkably, the STs exhibit little if any dairy biotope specialization. Phenotypic characterisation of the strains, based on their aptitude to use lactose and nitrate, shows that the two previously identified subspecies (freudenreichii and shermani) do notreflect the ancestral relationships in the P. freudenreichii population. The considerable phenotypic heterogeneity, found even at the ST level, suggests instead a history of recurrent switches between phenotypes.

Lipolysis plays an important role in the formation of cheese flavor. In Emmental cheese, the main part of lipolysis has been associated with the presence of Propionibacterium freudenreichii , a species used as a ripening culture. Our aim was to identify the most probable lipolytic esterase(s) involved in cheese lipolysis by P. freudenreichii . Since cheese lipolysis mainly occurs during P. freudenreichii growth, we hypothesized that P. freudenreichii possesses secreted lipolytic esterase(s). For 12 putative esterase genes previously identified from the genome of P. freudenreichii CIRM1, the level of expression was quantified by real-time reverse transcriptase (RT)-PCR, and the subcellular localization of esterases was predicted in silico . The esterase activity in extracellular and intracellular extracts of P. freudenreichii was characterized by zymography, and the extracellular esterases were identified by mass spectrometry. Finally, the best candidate was overexpressed in the same strain. All of the 12 genes encoding putative esterases were expressed. Esterase PF#279 was predicted to be secreted in the medium, PF#774 to be surface exposed, and the 10 remaining putative esterases to be intracellular. Zymography revealed that esterase activities in culture supernatant differed from the ones detected in intracellular extracts. PF#279 was identified as the sole esterase present in culture supernatant. Transformed P. freudenreichii CIRM1 clones overexpressing PF#279 showed 5 to 8 times more lipolytic activity on milk fat than the wild-type strain. Combining in silico , biochemical, and genetic approaches, we showed that PF#279 is the sole secreted esterase in P. freudenreichii and is active on milk fat. Therefore, it is likely a key component in cheese lipolysis by P. freudenreichii .

BackgroundPropionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027(T)), was sequenced with an 11-fold coverage.Methodology/principal findingsThe circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters.Conclusions/significanceWith the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity.

La présente invention concerne un vecteur recombinant pour l'expression et la sécrétion, par une bactérie propionique, d'une ou plusieurs séquences d'acides aminés d'intérêt, ledit vecteur comprenant au moins : - sous le contrôle d'au moins un promoteur approprié, - au moins une séquence nucléotidique codant un peptide signal de bactérie propionique et, en fusion traductionnelle avec celle-ci, - une ou plusieurs séquences nucléotidiques codant la ou lesdites séquences d'acides aminés d'intérêt. L'invention s'intéresse également aux utilisations d'un tel vecteur dans le domaine pharmaceutique ou pour la production à grande échelle de peptides ou protéines d'intérêt.

Background Lipolysis is an important process of cheese ripening that contributes to the formation of flavour. Propionibacterium freudenreichii is the main agent of lipolysis in Emmental cheese; however, the enzymes involved produced by this species have not yet been identified. Lipolysis is performed by esterases (carboxylic ester hydrolases, EC 3.1.1.-) which are able to hydrolyse acylglycerols bearing short, medium and long chain fatty acids. The genome sequence of P. freudenreichii type strain CIP103027T was recently obtained in our laboratory. The aim of this study was to identify as exhaustively as possible the potential esterases in P. freudenreichii that could be involved in the hydrolysis of acylglycerols in Emmental cheese. The proteins identified were produced in a soluble and active form by heterologous expression in Escherichia coli for further study of their activity and specificity of hydrolysed substrates. Results The approach chosen was a genomic search approach that combined and compared four methods based on automatic and manual searches of homology and motifs among P. freudenreichii CIP103027T predicted proteins. Twenty-three putative esterases were identified in this step. Then a selection step permitted to focus the study on the 12 most probable esterases, according to the presence of the GXSXG motif of the α/β hydrolase fold family. The 12 corresponding coding sequences were cloned in expression vectors, containing soluble N-terminal fusion proteins. The best conditions to express each protein in a soluble form were found thanks to an expression screening, using an incomplete factorial experimental design. Eleven out of the 12 proteins were expressed in a soluble form in E. coli and six showed esterase activity on 1-naphthyl acetate and/or propionate, as demonstrated by a zymographic method. Conclusion We were able to demonstrate that our genomic search approach was efficient to identify esterases from the genome of a P. freudenreichii strain, more exhaustively than classical approaches. This study highlights the interest in using the automatic search of motifs, with the manual search of homology to previously characterised enzymes as a complementary method. Only further characterisations would permit the identification of the esterases of P. freudenreichii involved in the lipolysis in Emmental cheese.

Many food-grade bacteria produce exopolysaccharides (EPS) that affect the texture of fermented food products and that may be involved in probiotic properties. Propionibacterium freudenreichii is a Gram-positive food-grade bacterium with reported probiotic capabilities that is widely used as starter in Swiss-type cheese. In this study, 68 strains of P. freudenreichii were screened for the β-glucan capsular phenotype by immunoagglutination with a specific antibody and for the presence of the gtf gene coding for polysaccharide synthase. All strains were positive for PCR amplification with gtf gene-specific primers, but the presence of β-glucan capsular EPS was detected for only 35% of the strains studied. Disruption of gtf in P. freudenreichii revealed that gtf is a unique gene involved in β-glucan capsular EPS production in P. freudenreichii . The gtf gene was transferred into and expressed in Lactococcus lactis , in which it conferred an agglutination-positive phenotype. Expression of the gtf gene was measured by performing quantitative reverse transcription-PCR assays with RNA from four capsular and three noncapsular strains. A positive correlation was found between the β-glucan capsular phenotype and gtf gene expression. Sequencing of the region upstream of the gtf open reading frame revealed the presence of an insertion element (IS element) in this upstream region in the four strains with the β-glucan capsular phenotype. The role of the IS element in the expression of neighboring genes and its impact on interstrain variability of the P. freudenreichii capsule phenotype remain to be elucidated.

Lactofermented soy juices represent an interesting alternative to milk yogurt to increase the part of plant-based proteins in our diet. The choice of lactic acid bacteria (LAB) used for soy juice fermentation is crucial as it determines the organoleptic and nutritional properties of the product. LAB metabolisms are however not well known in soy juice fermentation. In this thesis, 276 strains of 25 species were screened in synthetic media on their ability to ferment stachyose, raffinose and sucrose as unique source of carbon and soy juice. We showed that i) LAB capacity to catabolize sucrose is important to ferment soy juice ii) S. thermophilus strains are the most efficient to ferment soy juice iii) Lactobacillus LAB produce diverse aromatic notes according to the specie and the strain. LAB association can help to improve fermentation, the organoleptic and the nutritional properties.We associated then LAB in duo according to their metabolic complementarities. One coculture out of 32 improved the organoleptic properties of fermented soy juices compared to the monocultures. One coculture out of 12 reduced of 30% the SJ fermentation time (L. plantarum CIRM-BIA777 (Lp777) and L. delbrueckii CIRM-BIA865 (Ld865)).Genomic, metabolomics and transcriptomics approaches were set up to identify the molecular bases of these two LAB cooperation. These approaches revealed three factors of stress of LAB in soy juice fermentation: the limitation of sucrose, the limitation of free amino acids and the oxidative stress. The level of these three stresses were lowered with Lp777 and Ld865., Le jus de soja lactofermenté représente une alternative intéressante au yaourt laitier pour augmenter la part de protéines végétales dans l’alimentation. Le choix des bactéries lactiques (LAB) utilisées pour la fermentation du jus de soja est crucial car il détermine les qualités organoleptiques et nutritionnelles des produits. Les métabolismes des LAB sont cependant mal connus en jus de soja. Dans cette thèse 276 souches de 25 espèces de LAB ont été évaluées sur leur aptitude à fermenter du jus de soja ou des milieux de culture contenant du stachyose, du raffinose ou du saccharose comme unique source de carbone. Nous avons montré que i) la capacité à cataboliser le saccharose est un critère déterminant pour identifier des souches capables de fermenter le jus de soja, ii) les souches de S. thermophilus sont les plus efficaces pour fermenter le jus de soja, iii) les Lactobacillus apportent des notes aromatiques variées selon l’espèce et la souche. L’association de LAB pour améliorer lesfermentaires, organoleptiques et nutritionnelles a été évaluée. Nous avons associé des LAB en duos selon leurs complémentarités métaboliques. Une coculture sur 32 a permis d’améliorer les qualités organoleptiques du produit par rapport aux monocultures. Une coculture sur 12 a permis de réduire de 30% le temps de fermentation du jus de soja par rapport aux monocultures (L. plantarum CIRM-BIA777 (Lp777) et L. delbrueckii CIRM-BIA865 (Ld865)).