Complete genome sequence of Propionibacterium freudenreichii DSM 20271T
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Propionibacterium freudenreichii subsp. freudenreichii DSM 20271T is the type strain of species Propionibacterium freudenreichii that has a long history of safe use in the production dairy products and B12 vitamin. P. freudenreichii is the type species of the genus Propionibacterium which contains Gram-positive, non-motile and non-sporeforming bacteria with a high G + C content. We describe the genome of P. freudenreichii subsp. freudenreichii DSM 20271T consisting of a 2,649,166 bp chromosome containing 2320 protein-coding genes and 50 RNA-only encoding genes.
Keywords+Propionibacterium Type strain Dairy starter B12 vitamin
Hierarchical genome-assembly process
Strain DSM 20271T (= van Niel 1928T = ATCC 6207) is the type strain of species Propionibacterium freudenreichii, which is the type species of its genus Propionibacterium . There are traditionally two groups described in Propionibacterium genus; the “classical” or “dairy” and the “cutaneous” propionibacteria. P. freudenreichii belongs to the dairy group and is divided into two subspecies on the basis of lactose fermentation and nitrate reductase activity. The DSM 20271T strain represents the P. freudenreichii subsp. freudenreichii distinguished from subsp. shermanii by nitrate reduction and by a lack of lactose fermentation. . Dairy propionibacteria do not belong to human microbiota but can be isolated from various habitats including raw milk, dairy products, soil and fermenting food and plant materials such as silage and fermenting olives . Strains of P. freudenreichii have a long history of safe use in human diet and for instance in the production of Swiss-type cheeses, in which they play central role as ripening starters [1, 2]. Industrial applications of P. freudenreichii include production of vitamin B12 (cobalamin), as well as several other biomolecules like propionic acid, trehalose and conjugated linoleic acid . Recently, there has been growing interest to study P. freudenreichii for its probiotic properties. Complete genome sequence of the type strain P. freudenreichii subsp. shermanii CIRM-BIA1 has been reported , but lack of other complete genome sequences has prevented the genomic level comparisons between the two subspecies. Thus, the genomic analysis of DSM 20271T strain should help us in P. freudenrichii subspecies definition that has been under debate [4, 5].
Here we present a summary classification and a set of features for P. freudenreichii DSM 20271T, together with the description of the complete genomic sequence and annotation.
Classification and features
Classification and general features of Propionibacterium freudenreichii subspecies freudenreichii DSM20271 T according to the MIGS recommendations 
Species Propionibacterium freudenreichii subspecies freudenreichii
(Type) strain: van Niel 1928 T, (DSM 20271 T = ATCC 6207)
pH range; Optimum
Glucose, fructose, mannose, glycerol, adonitol, inositol, erythritol, galactose
Genome sequencing project information for Propionibacterium freudenreichii DSM 20271T
One PacBio 10 kb standard library
PacBio RS II
SMRTAnalysis (2.1.0), HGAP2
Gene calling method
GenBank Date of Release
February 1st 2015
Source Material Identifier
Type strain, dairy starter, B12 vitamin
Genome sequencing information
This organism was selected for sequencing on the basis of its importance in food fermentations and in metabolite production.
Growth conditions and genomic DNA preparation
The strain was grown to early stationary growth phase in propionic medium (PPA), composed of 5.0 g. tryptone (Sigma-Aldrich), 10.0 g. yeast extract (Becton, Dickinson), 14.0 ml 60 % w/w DL-sodium lactate (Sigma-Aldrich) per liter and pH adjusted to 6.7. The cells were harvested by centrifugation for 5 min at 21,000 g at 4 °C and washed once with 0.1 M Tris–HCl pH 8.0. The DNA extraction was performed with ILLUSTRA™ bacteria genomicPrep Mini Spin Kit (GE Healthcare) according to the manufacturer’s instruction for Gram-positive bacteria using 100 mg/ml of lysozyme (Sigma-Aldrich) and 30 min incubation time in the lysis step.
Genome sequencing and assembly
The complete finished genome sequence of P. freudenreichii strain DSM 20271T was generated at the Institute of Biotechnology, University of Helsinki, using Pacific Biosciences RS II sequencing platform (Table 2) . One standard PacBio 10 kb library was constructed and sequenced using two SMRTCells with 180 min runtime on the RS II instrument, which generated 145,463 reads totaling up to 608.98 Mbp. For the assembly, the data was filtered using default HGAP parameters. The resulting 130,046 reads totaling up to 557.87 Mbp were used to generate the initial genome sequence. 498.74 Mbp of the filtered data mapped to the assembled genome afterwards. The assembled genome sequence was generated using SMRTAnalysis (2.1.0) HGAP2 pipeline  with default parameters, excluding the expected genome size and seed cutoff which were set to 2,700,000 and 7000 respectively. The assembly contains one contig which represents the whole genome. The resulting assembly was further improved by two consecutive rounds of mapping the full data on the reference and obtaining a new improved consensus sequence on each run. This was done using the standard SMRTAnalysis resequencing protocol with Quiver algorithm . The circular nature of the final consensus sequence was then confirmed and the start of the sequence manually set to dnaA using Gap4 tool from Staden package .
Genes were identified using Prodigal v2.50 tool  with manual curation in ARGO Genome Browser . The predicted genes were translated and functionally annotated with description lines, Gene Ontology (GO) classes and Enzyme Commission (EC) numbers with PANNZER program  using UniProtKB, Enzyme and GOA databases. PfamA domains were identified using InterProScan 48.0 , transmembrane helices and signal peptides were found with TMHMM  and SignalP , respectively. Clusters of Orthologous Groups (COG) assignments were done by using CD-Search . The tRNAscanSE tool  was used to identify tRNA genes and ribosomal RNA were predicted with RNAmmer v1.2 .
% of Total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
General function prediction only
Not in COGs
Prior to this report only a single genome sequence was available for Propionibacterium freudenreichii, from the type strain of P. freudenreichii subsp. shermanii CIRM-BIA1 . In the present study the first genome sequence of a P. freudenreichii subsp. freudenreichii strain was described. P. freudenreichii is an industrially important species and a rare producer of biologically active form of vitamin B12. Probably the characteristics of P. freudenreichii DNA such as high G + C content and regions of repeated sequences have hampered the unraveling the complete genomes of this species. The results presented here indicate that PacBio RS II sequencing platform is well-suited to overcome these potential obstacles. In this study three DNA sequence motifs containing methylated bases were detected. Our future investigations include using this platform for sequencing of several additional strains for establishing core and pan-genomes as well as methylomes to gain understanding of genome structure and evolution of P. freudenreichii.
This study was supported by the Academy of Finland (grant no 257333).
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