Open Access

Complete genome sequence of Propionibacterium freudenreichii DSM 20271T

  • Patrik Koskinen1Email author,
  • Paulina Deptula2,
  • Olli-Pekka Smolander1,
  • Fitsum Tamene1,
  • Juhana Kammonen1,
  • Kirsi Savijoki2,
  • Lars Paulin1,
  • Vieno Piironen2,
  • Petri Auvinen1 and
  • Pekka Varmanen2
Standards in Genomic Sciences201510:83

DOI: 10.1186/s40793-015-0082-1

Received: 8 January 2015

Accepted: 15 October 2015

Published: 24 October 2015

Abstract

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

Introduction

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 [1]. 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. [1]. 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 [1]. 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 [3]. 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 [4], 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.

Organism information

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 20271T in a 16S rRNA sequence based tree. Cells of DSM 20271T 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 20271T is supported by carbon sources including glucose, fructose, mannose, glycerol, adonitol, inositol, erythritol and galactose (Table 1).
Fig. 1

Phylogenetic tree showing the relationship of Propionibacterium freudenreichii ssp. freudenreichii DSM 20271T (shown in bold print) to Mycobacterium tuberculosis, Corynebacterium glutamicum, Bifidobacterium longum, Propionibacterium acnes, Propionibacterium acidipropionici and Lactococcus lactis (outgroup). Tree is based on MAFFT [21] aligned 16 s rRNA gene. The tree was built using the Maximum-Likelihood method [22] with GAMMA model. Bootstrap analysis with 500 replicates was performed to assess the support of the tree topology. Tree was visualized with iTOL [23]

Fig. 2

Optical microscope image. The cells of strain 20271 grown for 72 h, Gram stained. Image from light microspope, magnification 100x

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 20271T

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 sequencing information

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 20271T 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].

Genome properties

The circular genome of Propionibacterium freudenreichii subsp. freudenreichii DSM 20271T is 2,649,166 nucleotides with 67.34 % GC content (Table 3) and contains one finalized chromosome with no plasmids. From total number of 2370 genes 2320 (97.9 %) are protein coding and 50 (2.1 %) are RNA genes (Fig. 3). 91.80 % of all proteins were functionally annotated whilst the remaining genes were annotated as “functionally unknown putative proteins”. The distribution of genes into COGs functional categories is presented in Table 4. Three sequence motifs containing methylated bases were also detected in the genome by PacBio sequencing and SMRTAnalysis Modification and Motif detection protocol. Two of these motifs, 5′-GGANNNNNNNCTT-3′ and 5′AAGNNNNNNNTCC-3′, are partner motifs and correspond to same modifications on different strands with m6A as a modified base on 3rd and 2nd position respectively. The modified base is shown in bold. Each motif is found 664 times in the genome and the marked bases are methylated in all of the 1328 motifs. The structure of the motifs and similarity to existing methyltransferases in REBASE [20] suggests that this is a Type I restriction-modification (RM) system. The third modified motif detected in the genome is 5′-TCGWCGA-3′ which partners with itself and is found 4258 times in the genome. In 3676 of these motifs (86.3 %) the 5th nucleotide (C) is found to be modified. The type of this modification could not be reliably identified. However, 319 of the detected modifications are identified as m4C although with low confidence. This finding is supported by the similarity of the recognition site to existing methyltransferases found in REBASE [20] and suggests that there is a Type II RM system acting on this motif in the genome. Therefore also the unidentified modifications are probably m4C bases. This data together suggest that there are two active RM systems present in P. freudenreichii . Comprehensive analysis of these RM systems and corresponding methylations requires further study.
Table 3

Genome statistics

Attribute

Value

% of Total

Genome size (bp)

2,649,166

100.00

DNA coding (bp)

2,321,778

87.64

DNA G + C (bp)

1,783,838

67.34

DNA scaffolds

1

100.00

Total genes

2353

100.00

Protein coding genes

2320

97.90

RNA genes

50

2.10

Pseudo genes

NA

NA

Genes in internal clusters

NA

NA

Genes with function prediction

2160

91.80

Genes assigned to COGs

1751

74.42

Genes with Pfam domains

1958

83.21

Genes with signal peptides

113

4.80

Genes with transmembrane helices

577

24.52

CRISPR repeats

0

0

Fig. 3

Genome properties of Propionibacterium freudenreichii ssp. freudenreichii DSM 20271T . The circles are from inner to outer order: GC skew, GC%, putative m4C, m6A, tRNA, rRNA, CDS reverse, CDS forward. CDS are colored according to the COG functional categories

Table 4

Number of genes associated with general COG functional categories

Code

Value

%age

Description

J

138

5.86

Translation, ribosomal structure and biogenesis

A

0

0.00

RNA processing and modification

K

152

6.46

Transcription

L

211

8.97

Replication, recombination and repair

B

0

0.00

Chromatin structure and dynamics

D

18

0.76

Cell cycle control, Cell division, chromosome partitioning

V

32

1.36

Defense mechanisms

T

82

3.48

Signal transduction mechanisms

M

77

3.27

Cell wall/membrane biogenesis

N

1

0.04

Cell motility

U

24

1.02

Intracellular trafficking and secretion

O

73

3.10

Posttranslational modification, protein turnover, chaperones

C

138

5.86

Energy production and conversion

G

157

6.67

Carbohydrate transport and metabolism

E

205

8.71

Amino acid transport and metabolism

F

57

2.42

Nucleotide transport and metabolism

H

106

4.50

Coenzyme transport and metabolism

I

58

2.46

Lipid transport and metabolism

P

123

5.23

Inorganic ion transport and metabolism

Q

31

1.32

Secondary metabolites biosynthesis, transport and catabolism

R

219

9.31

General function prediction only

S

80

3.40

Function unknown

-

1017

43.22

Not in COGs

The total is based on the total number of protein coding genes in the genome

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 .

Abbreviations

PPA: 

Propionic medium

HGAP: 

Hierarchical genome-assembly process

RM: 

Restriction-modification

Declarations

Acknowledgements

This study was supported by the Academy of Finland (grant no 257333).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Institute of Biotechnology, University of Helsinki
(2)
Department of Food and Environmental Sciences, University of Helsinki

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Copyright

© Koskinen et al. 2015