Accumulation of intracellular glycogen and trehalose by Propionibacterium freudenreichii under conditions mimicking cheese ripening in the cold.

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.

Functional genomics provides insights into the role of Propionibacterium freudenreichii ssp. shermanii JS in cheese ripening.

Propionibacterium freudenreichii is a commercially important bacterium that is essential for the development of the characteristic eyes and flavor of Swiss-type cheeses. These bacteria grow actively and produce large quantities of flavor compounds during cheese ripening at warm temperatures but also appear to contribute to the aroma development during the subsequent cold storage of cheese. Here, we advance our understanding of the role of P. freudenreichii in cheese ripening by presenting the 2.68-Mbp annotated genome sequence of P. freudenreichii ssp. shermanii JS and determining its global transcriptional profiles during industrial cheese-making using transcriptome sequencing. The annotation of the genome identified a total of 2377 protein-coding genes and revealed the presence of enzymes and pathways for formation of several flavor compounds. Based on transcriptome profiling, the expression of 348 protein-coding genes was altered between the warm and cold room ripening of cheese. Several propionate, acetate, and diacetyl/acetoin production related genes had higher expression levels in the warm room, whereas a general slowing down of the metabolism and an activation of mobile genetic elements was seen in the cold room. A few ripening-related and amino acid catabolism involved genes were induced or remained active in cold room, indicating that strain JS contributes to the aroma development also during cold room ripening. In addition, we performed a comparative genomic analysis of strain JS and 29 other Propionibacterium strains of 10 different species, including an isolate of both P. freudenreichii subspecies freudenreichii and shermanii. Ortholog grouping of the predicted protein sequences revealed that close to 86% of the ortholog groups of strain JS, including a variety of ripening-related ortholog groups, were conserved across the P. freudenreichii isolates. Taken together, this study contributes to the understanding of the genomic basis of P. freudenreichii and sheds light on its activities during cheese ripening.

The structural basis of receptor-binding by Escherichia coli associated with diarrhea and septicemia.

GafD in Escherichia coli G (F17) fimbriae is associated with diarrheal disease, and the structure of the ligand-binding domain, GafD1-178, has been determined at 1.7A resolution in the presence of the receptor sugar N-acetyl-D-glucosamine. The overall fold is a beta-barrel jelly-roll fold. The ligand-binding site was identified and localized to the side of the molecule. Receptor binding is mediated by side-chain as well main-chain interactions. Ala43-Asn44, Ser116-Thr117 form the sugar acetamide specificity pocket, while Asp88 confers tight binding and Trp109 appears to position the ligand. There is a disulfide bond that rigidifies the acetamide specificity pocket. The three fimbrial lectins, GafD, FimH and PapG share similar beta-barrel folds but display different ligand-binding regions and disulfide-bond patterns. We suggest an evolutionary path for the evolution of the very diverse fimbrial lectins from a common ancestral fold.

A temporal-omic study of Propionibacterium freudenreichii CIRM-BIA1 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-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 (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-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.

Multilocus sequence typing of Propionibacterium freudenreichii.

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 not reflect 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.