Complete genome sequence of Propionibacterium freudenreichii DSM 20271^T

Classification and features

P. freudenreichii subsp. freudenreichii DSM 20271 T is a Gram-positive, non-motile, non-sporulating, anaerobic to aerotolerant, mesophilic Actinobacteria belonging to the order Propionibacterium. The strain was originally isolated as one of the three propionic acid-producing strains from Emmental cheese by von Freudenreich and Orla-Jensen as Bacterium acidi propionici a [6] during their work in Bern, Switzerland [7]. The strain was further studied by van Niel and renamed to Propionibacterium freudenreichii [6]. Figure 1 shows the phylogenetic neighborhood of DSM 20271^T in a 16S rRNA sequence based tree. Cells of DSM 20271^T are short rods with length of approximately 1,5 μm (Fig. 2). According to API 50 CH (Biomerieux, France) carbohydrate fermentation test the growth of DSM 20271^T is supported by carbon sources including glucose, fructose, mannose, glycerol, adonitol, inositol, erythritol and galactose (Table 1).

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Table 1

Classification and general features of Propionibacterium freudenreichii subspecies freudenreichii DSM20271 T according to the MIGS recommendations [24]

MIGS ID	Property	Term	Evidence codea
	Classification	Domain Bacteria	TAS [25]
		Phylum Actinobacteria	TAS [26, 27]
		Class Actinobacteria	TAS [26, 27]
		Order Propionbacteriales	TAS [28]
		Family Propionibacteriaceae	TAS [1]
		Genus Propionibacterium	TAS [1, 29]
		Species Propionibacterium freudenreichii subspecies freudenreichii	TAS [1, 29, 30]
		(Type) strain: van Niel 1928 T, (DSM 20271 T = ATCC 6207)
	Gram stain	Positive	TAS [1]
	Cell shape	Rod	TAS [1]
	Motility	Non-motile	TAS [1]
	Sporulation	Not reported	NAS
	Temperature range	Mesophile	TAS [1]
	Optimum temperature	30 °C	TAS [1]
	pH range; Optimum	~4.5–8; ~7	NAS
	Carbon source	Glucose, fructose, mannose, glycerol, adonitol, inositol, erythritol, galactose	IDA
MIGS-6	Habitat	Swiss cheese	TAS []
MIGS-6.3	Salinity	Unknown	TAS []
MIGS-22	Oxygen requirement	Anaerobic	TAS [1]
MIGS-15	Biotic relationship	Free-living	NAS
MIGS-14	Pathogenicity	Non-pathogen	NAS
MIGS-4	Geographic location	Unknown	NAS
MIGS-5	Sample collection	Unknown	NAS
MIGS-4.1	Latitude	Unknown	NAS
MIGS-4.2	Longitude	Unknown	NAS
MIGS-4.4	Altitude	Unknown	NAS

^aEvidence codes - IDA inferred from direct assay, TAS traceable author statement (i.e., a direct report exists in the literature), NAS non-traceable author statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [31]

Table 2

Genome sequencing project information for Propionibacterium freudenreichii DSM 20271^T

MIGS ID	Property	Term
MIGS 31	Finishing quality	Finished
MIGS-28	Libraries used	One PacBio 10 kb standard library
MIGS 29	Sequencing platforms	PacBio RS II
MIGS 31.2	Fold coverage	198x
MIGS 30	Assemblers	SMRTAnalysis (2.1.0), HGAP2
MIGS 32	Gene calling method	Prodigal v2.50
	Locus Tag	RM25
	Genbank ID	CP010341
	GenBank Date of Release	February 1st 2015
	GOLD ID	Gs0113908
	BIOPROJECT	PRJNA269789
MIGS 13	Source Material Identifier	DSM 20271T
	Project relevance	Type strain, dairy starter, B12 vitamin

Genome project

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 20271^T was generated at the Institute of Biotechnology, University of Helsinki, using Pacific Biosciences RS II sequencing platform [8](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 [9] 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 [9]. 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 [10].

Genome annotation

Genes were identified using Prodigal v2.50 tool [11] with manual curation in ARGO Genome Browser [12]. The predicted genes were translated and functionally annotated with description lines, Gene Ontology (GO) classes and Enzyme Commission (EC) numbers with PANNZER program [13] using UniProtKB, Enzyme and GOA databases. PfamA domains were identified using InterProScan 48.0 [14], transmembrane helices and signal peptides were found with TMHMM [15] and SignalP [16], respectively. Clusters of Orthologous Groups (COG) assignments were done by using CD-Search [17]. The tRNAscanSE tool [18] was used to identify tRNA genes and ribosomal RNA were predicted with RNAmmer v1.2 [19].

Conclusions

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 [4]. 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.