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.

BACKGROUND:Propionibacterium freudenreichii (PF) is an actinobacterium used in cheese technology and for its probiotic properties. PF is also extremely adaptable to several ecological niches and 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. High-throughput sequencing technologies were ideal for this purpose as they have the potential to decipher genomic diversity at a moderate cost.RESULTS:21 strains of PF were sequenced and the genomes were assembled de novo. Scaffolds were ordered by comparison with the complete reference genome CIRM-BIA1, obtained previously using traditional Sanger sequencing. Automatic functional annotation and manual curation were performed. 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 and xylitol were only used by some strains. In this case, the presence/absence of genes responsible for carbon uptake and degradation correlated well with the phenotypes, with the exception of xylitol. Furthermore, the simultaneous presence of these genes 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 degraded to a greater or lesser extent (quantitative phenotype) by some strains. For these sugars, the phenotypes could not be explained by the presence/absence of a gene but correlated with the premature appearance of a stop codon interrupting protein synthesis and preventing the catabolism of corresponding carbon sources.CONCLUSION: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. Knowledge of the genetic basis of nitrogen and sugar degradation opens up new strategies for the screening of PF strain collections to enable optimum cheese starter, probiotic and white biotechnology applications.

ABSTRACT Lactobacillus delbrueckii subsp. lactis CNRZ327 is a dairy bacterium with anti-inflammatory properties both in vitro and in vivo . Here, we report the genome sequence of this bacterium, which appears to contain no less than 215 insertion sequence (IS) elements, an exceptionally high number regarding the small genome size of the strain.

Recent works have revealed highly strain-dependent immunomodulatory properties in Propionibacterium freudenreichii, a cheese starter bacterium. The underlying mechanisms are unknown but preliminary attempts have shown that surface components are involved in these immunomodulatory effects. The most promising strains/components exert promising anti-inflammatory effects through induction of regulatory cytokines.The aim of this work is to identify key surface components of Propionibacterium freudenreichii responsible for the immunomodulatory properties. 12 strains of Propionibacterium freudenreichii were sorted according to their high or low regulatory properties, measured by induction of IL-10 cytokine in peripheral blood mononuclear cells stimulated by equal amounts of the different P. freudenreichii strains. These strains were sequenced by Illumina paired-end sequencing and de novo assembled using Velvet software. Genome sequences were automatically and manually annotated on the INRA-AGMIAL platform. Correlations are studied both (i) between quantitative traits (high or low induction of IL-10) and genotypic properties, namely presence/lack of proteins predicted in silico and (ii) between quantitative traits (high or low induction of IL-10) and in vitro data (surface exposed proteins). In both cases, we account for the phylogenetic inertia induced by the shared evolutionary history of the strains. For in vitro data, three methods (shedding using guanidium chloride, surface labeling using CyDye cyanine and shaving using trypsine combined with mass spectrometry) are used to identify P. freudenreichii surface proteins. Statistical analysis is used to highlight which of these proteins can potentially be involved in immunomodulation, comparing the proteins identified and IL-10 induction. Statistical analyses of both in vitro and in silico approaches based on genome sequencing reveal targets potentially involved in immunomodulation. Both strategies are complementary. Proteomic approach followed by statistical analysis allow the identification of surface exposed proteins associated with immunomodulation, whereas in silico approach point out the mechanisms explaining the presence/ absence of the candidates on bacterial surface.Genome sequences, either directly used (in silico approach) or used as database (in vitro approach) determine key genes involved in the studied phenotypes. Candidate genes identified in the present study will be further characterized and their role in immunomodulation confirmed by knock out or overexpression of selected genes.

High-throughput sequencing technologies have the potential to decipher genomic diversity at a moderate cost. Propionibacterium freudenreichii is an Actinobacterium used in cheese technology (responsible for aroma and holes in Swiss cheese) and that has probiotic properties (bifidogenic and with anti-inflammatory properties in human and animal). 23 strains of Propionibacterium freudenreichii covering 18 different sequence types out of 46 (Dalmasso et al. AEM 2010) were (i) sequenced by Solexa-Illumina paired-end sequencing and (ii) de novo assembled using Velvet software. Depending on strain, 63 to 166 scaffolds were obtained. These scaffolds were arranged along the reference genome CIRM-BIA1, previously obtained from traditional Sanger sequencing (Falentin et al. PlosOne 2010). For each strain, single nucleotide polymorphism and insertion deletion events were plotted against reference genome. Sequences were automatically annotated (by blastp and pattern matching) on the INRA AGMIAL platform. By bidirectional best-hit, each gene was attributed either to core genome or accessory genome. In some strains, some genes belonging to the accessory genome corresponded with genomic islands and confered peculiar phenotypes (ability to lactose degradation for exemple). In the future, screening of P. freudenreichii (~500 strains) collection on genetic basis at CIRM-BIA biological resource center (Rennes) will help in strain choice for cheese starter and probiotic market.