A Temporal -omic Study of Propionibacterium freudenreichii CIRM-BIA1T Adaptation Strategies in Conditions Mimicking Cheese Ripening in the Cold

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-BIA1T 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 (P<0.05 and |fold change|>1). At 4°C, a general slowing down was observed for genes implicated in the cell machinery. On the contrary, P. freudenreichii CIRM-BIA1T 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

The Complete Genome of Propionibacterium freudenreichii CIRM-BIA1T, a Hardy Actinobacterium with Food and Probiotic Applications

Background: Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use [1]. 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 103027T), was sequenced with an 11-fold coverage. Methodology/Principal Findings: The 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/Significance: With 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

Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations

International audienceCheese microorganisms, such as bacteria and fungi, constitute a complex ecosystem that plays a central role in cheeses ripening. The molecular study of cheese microbial diversity and activity is essential but the extraction of high quality nucleic acid may be problematic: the cheese samples are characterised by a strong buffering capacity which negatively influenced the yield of the extracted rRNA. The objective of this study is to develop an effective method for the direct and simultaneous isolation of yeast and bacterial ribosomal RNA and genomic DNA from the same cheese samples. DNA isolation was based on a protocol used for nucleic acids isolation from anaerobic digestor, without preliminary washing step with the combined use of the action of chaotropic agent (acid guanidinium thiocyanate), detergents (SDS, N-lauroylsarcosine), chelating agent (EDTA) and a mechanical method (bead beating system). The DNA purification was carried out by two washing steps of phenol–chloroform. RNA was isolated successfully after the second acid extraction step by recovering it from the phenolic phase of the first acid extraction. The novel method yielded pure preparation of undegraded RNA accessible for reverse transcription–PCR. The extraction protocol of genomic DNA and rRNA was applicable to complex ecosystem of different cheese matrices

Surface properties associated with the production of polysaccharides in the food bacteria Propionibacterium freudenreichii

International audienceThis study explores the production of polysaccharides (PS) in the strain Pf2289 of the food species Propioni-bacterium freudenreichii. Pf2289 presents characteristics atypical of the species: a molar-shaped morphotype upon plating, and cells strongly aggregative in liquid medium. When plating Pf2289, another morphotype was observed with a 4% frequency of appearance: round-shaped colonies, typical of the species. A clone was isolated, designated Pf456. No reversibility of Pf456 towards the molar-shaped morphotype was observed. Pf2289 was shown to produce a surface polysaccharide (PS) bound to the cell wall, mainly during the stationary growth phase. Meanwhile, Pf456 had lost the ability to produce the PS. AFM images of Pf2289 showed that entangled filaments spread over the whole surface of the bacteria, whereas Pf456 exhibited a smooth surface. Adhesion force maps, performed with concanavalin-A grafted probes, revealed twice as much adhesion of Pf2289 to concanavalin-A compared to Pf456. Furthermore, the length of PS molecules surrounding Pf2289 measured at least 7 μm, whereas it only reached 1 μm in Pf456. Finally, the presence of PS had a strong impact on adhesion properties: Pf2289 did not adhere to hydrophobic surfaces, whereas Pf456 showed strong adhesion

Role of the β-glucan surface polysaccharide in Propionibacterium freudenreichii: physiological and immunomodulatory properties

P. freudenreichii (PF) is a Gram + species, with the GRAS (Generally Recognized As Safe) status in the USA and a Qualified Presumption of Safety (QPS) status in Europe. It is widely consumed by humans because it is an essential ripening culture in Swiss type cheeses (Emmental, Leerdammer®…), and also as a probiotic in food supplement (Propio-Fidus®). Recently, PF was shown to produce a (1→3, 1→2)-β-D-glucan polysaccharide. A single gene (named gtf) is responsible for the biosynthesis of this surface polysaccharide, that has been immunodetected for 30% of the 100 strains of PF tested. Here we investigated the role(s) of this β-D-glucan

Immunomodulatory properties of the β-glucan surface polysaccharide of Propionibacterium freudenreichii, a dairy bacterium with probiotic potential

P. freudenreichii (PF) is a Gram + species, with the GRAS (Generally Recognized As Safe) status in the USA and a Qualified Presumption of Safety (QPS) status in Europe. It is widely consumed by humans because it is an essential ripening culture in Swiss type cheeses (Emmental, Leerdammer®…), which contain more than 109 live cells / g of cheese. It is also used as a probiotic in food supplement (Propio-Fidus®). Recently, 30% of the strains of PF were shown shown to produce a (1→3, 1→2)-β-D-glucan surface polysaccharide. A single gene (named gtfF) is responsible for the biosynthesis. Here we investigated the role(s) of this β-glucan

Cold adaptation of the cheese ripening bacterium Protectionniste freudenreichii

Propionibacterium freudenreichii is known to play a key role in the formation of cheese flavor throughout Swiss-type cheese ripening and especially during cold storage of cheese. At every stage of cheese manufacture, P. freudenreichii has to face several stresses-generating conditions and especially a cold-induced stress when Swiss cheeses are transferred from a warm (24°C) ripening room to a cold (4°C) room. The aim of this study was to investigate the adaptation and survival of P. freudenreichii at cold temperature by means of the first global gene expression profile for this species. The temporal transcriptomic response of P. freudenreichii was analyzed during its growth phase at 30°C and then during further incubation at 4°C for 9 days, always preventing any exhaustion of the main carbon source (lactate). As the cells transitioned from the warm to the cold condition, most of the down-expressed genes were involved in cell division, protein turnover, translation, transcription and DNA replication and repair. During incubation at cold temperature, P. freudenreichii adopted multiple strategies for maintaining its viability. It activated a two-component quorum-sensing system (a two-component system sensor kinase and luxR) that can induce population-wide changes in gene expression. It used polyphosphate supplies as energy sources by activating genes coding for Nudix hydrolases and pyrophosphatases. It accumulated carbon supplies by up-regulating genes of lactate, alanine and serine conversion to pyruvate, of aspartate conversion to fumarate, of gluconeogenesis and of glycogen synthesis. Thus, even if its metabolic activity is slowed down at cold temperature, P. freudenreichii remains active, which could explain its ability to continue the production of aroma compounds in cheese during their ripening at low temperature. Understanding the metabolism of P. freudenreichii during cold storage could constitute a real asset for a better optimization of Swiss cheese ripening process thus decreasing the carbon footprint of Swiss-type cheese manufactur

Immunomodulatory properties of the β-glucan surface polysaccharide of Propionibacterium freudenreichii, a dairy bacterium with probiotic potential

P. freudenreichii (PF) is a Gram + species, with the GRAS (Generally Recognized As Safe) status in the USA and a Qualified Presumption of Safety (QPS) status in Europe. It is widely consumed by humans because it is an essential ripening culture in Swiss type cheeses (Emmental, Leerdammer®…), which contain more than 109 live cells / g of cheese. It is also used as a probiotic in food supplement (Propio-Fidus®). Recently, 30% of the strains of PF were shown shown to produce a (1→3, 1→2)-β-D-glucan surface polysaccharide. A single gene (named gtfF) is responsible for the biosynthesis. Here we investigated the role(s) of this β-glucan

Cold adaptation of the cheese ripening bacterium Protectionniste freudenreichii

Propionibacterium freudenreichii is known to play a key role in the formation of cheese flavor throughout Swiss-type cheese ripening and especially during cold storage of cheese. At every stage of cheese manufacture, P. freudenreichii has to face several stresses-generating conditions and especially a cold-induced stress when Swiss cheeses are transferred from a warm (24°C) ripening room to a cold (4°C) room. The aim of this study was to investigate the adaptation and survival of P. freudenreichii at cold temperature by means of the first global gene expression profile for this species. The temporal transcriptomic response of P. freudenreichii was analyzed during its growth phase at 30°C and then during further incubation at 4°C for 9 days, always preventing any exhaustion of the main carbon source (lactate). As the cells transitioned from the warm to the cold condition, most of the down-expressed genes were involved in cell division, protein turnover, translation, transcription and DNA replication and repair. During incubation at cold temperature, P. freudenreichii adopted multiple strategies for maintaining its viability. It activated a two-component quorum-sensing system (a two-component system sensor kinase and luxR) that can induce population-wide changes in gene expression. It used polyphosphate supplies as energy sources by activating genes coding for Nudix hydrolases and pyrophosphatases. It accumulated carbon supplies by up-regulating genes of lactate, alanine and serine conversion to pyruvate, of aspartate conversion to fumarate, of gluconeogenesis and of glycogen synthesis. Thus, even if its metabolic activity is slowed down at cold temperature, P. freudenreichii remains active, which could explain its ability to continue the production of aroma compounds in cheese during their ripening at low temperature. Understanding the metabolism of P. freudenreichii during cold storage could constitute a real asset for a better optimization of Swiss cheese ripening process thus decreasing the carbon footprint of Swiss-type cheese manufactur

Role of the β-glucan surface polysaccharide in Propionibacterium freudenreichii: physiological and immunomodulatory properties

P. freudenreichii (PF) is a Gram + species, with the GRAS (Generally Recognized As Safe) status in the USA and a Qualified Presumption of Safety (QPS) status in Europe. It is widely consumed by humans because it is an essential ripening culture in Swiss type cheeses (Emmental, Leerdammer®…), and also as a probiotic in food supplement (Propio-Fidus®). Recently, PF was shown to produce a (1→3, 1→2)-β-D-glucan polysaccharide. A single gene (named gtf) is responsible for the biosynthesis of this surface polysaccharide, that has been immunodetected for 30% of the 100 strains of PF tested. Here we investigated the role(s) of this β-D-glucan