Open Access

Draft genome sequence of Francisella tularensis subsp. holarctica BD11-00177

  • Jordy P. M. Coolen
  • , Andreas Sjödin
  • , Boulos Maraha
  • , Gerard F. Hajer
  • , Mats Forsman
  • , Ellen Verspui
  • , Hendrina M.E. Frenay
  • , Daan W. Notermans
  • , Maaike C. de Vries
  • , Frans A.G. Reubsaet
  • , Armand Paauw
  • and Guus Roeselers

DOI: 10.4056/sigs.4217923

Received: 10 August 2013

Accepted: 10 August 2013

Published: 30 July 2013

Abstract

Francisella tularensis is a facultative intracellular bacterium in the class Gammaproteobacteria. This strain is of interest because it is the etiologic agent of tularemia and a highly virulent category A biothreat agent. Here we describe the draft genome sequence and annotation of Francisella tularensis subsp. holarctica BD11-00177, isolated from the first case of indigenous tularemia detected in The Netherlands since 1953. Whole genome DNA sequence analysis assigned this isolate to the genomic group B.FTNF002–00, which previously has been exclusively reported from Spain, France, Italy, Switzerland and Germany. Automatic annotation of the 1,813,372 bp draft genome revealed 2,103 protein-coding and 46 RNA genes.

Keywords:

tularaemiabiodefencezoonotic infectionphylogeographyNetherlands

Introduction

Francisella tularensis is a Gram negative, non-motile, non-spore forming, facultative intracellular bacterium appearing as short rods or coccoid forms [1]. F. tularensis is the etiologic agent of tularemia, a zoonotic infection also known as rabbit fever and deer-fly fever. Transmission to humans has been reported by direct contact with infected animals, arthropod bites, inhalation of contaminated dust or ingestion of contaminated food or water. This pathogen is highly infectious as it can cause infection upon inhalation of as few as 10 cells. This extremely low infectious dose makes transmission via aerosols easy, and previous attempts to weaponize this microorganism have led to its recognition as a category A biothreat agent (CDC classification) [2,3]. F. tularensis contains three subspecies that are infectious to humans; the highly virulent Francisella tularensis subsp. tularensis, which often causes a lethal multi-systemic disease with a fatality rate of up to 30%, the less virulent Francisella tularensis subsp. holartica and Francisella tularensis subsp. mediasiatica, which both seldom cause infectious in humans. Here we present a summary classification together with the description of the draft genome sequence and annotation of Francisella tularensis subsp. holarctica BD11-00177, that was isolated from a vesicle on the forehead of a 72-year-old male living in The Netherlands. As the patient had not been abroad for years, this was the first documented case of indigenous tularemia in The Netherlands since 1953.

Classification and features

Francisella is the only genus within the family Francisellaceae and is a member of the order Thiotrichales and the class Gammaproteobacteria [4] [Table 1]. Besides F. tularensis, the genus Francisella includes the species Francisella halioticida, Francisella hispaniensis, Francisella noatunensis, Francisella novicida, Francisella philomiragia, Francisella cantonensis and the misclassified Wolbachia persica [4,17, Figure 1]. Only rare human infections with F. hispaniensis and F. novicida, and F. philomiragia are described, often caused after nearly drowning [18,19]. F. tularensis is capable of infecting hundreds of different vertebrate and invertebrate hosts [20]. The most widely distributed subspecies is F. tularensis subsp. holarctica, which is found throughout much of the Northern Hemisphere and is the only subspecies naturally occurring in Europe [21].

Table 1

Classification and general features

MIGS ID

     Property

      Term

    Evidence codea

      Domain Bacteria

    TAS [5]

      Phylum Proteobacteria

    TAS [6]

      Class Gammaproteobacteria

    TAS [7,8]

     Current classification

      Order Thiotrichales

    TAS [7,9]

      Family Francisellaceae

    TAS [7-10]

      Genus Francisella

    TAS [11-14]

      Species Francisella tularensis

    TAS [11,12]

      Subspecies Francisella tularensis holarctica

    TAS [15,16]

      Strain BD11-00177

    NAS

     Gram stain

      negative

    TAS [1].

     Cell shape

      short rods or coccoid forms

    TAS [1].

     Motility

      No

    TAS [1].

     Sporulation

      No

    TAS [1].

     Temperature range

      Mesophilic

    TAS [1].

     Optimum temperature

      37

    IDA

     Carbon source

      Carbohydrates

    TAS [1].

     Energy source

      Chemoorganotrophic

    TAS [1].

     Terminal electron receptor

      Facultative anaerobe

    TAS [1].

MIGS-6

     Habitat

      Host

    TAS [1].

MIGS-15

     Biotic relationship

      Obligate host-dependent

    TAS [1].

MIGS-16

     Host name     Host taxon ID     Host gender

      Homo sapiens      9606      Male

    TAS [1].    TAS [1].    NAS

MIGS-14

     Pathogenicity     Biosafety Level

      Pathogen      3

    TAS [2].

MIGS-4

     Geographic location

      The Netherlands

    IDA

MIGS-5

     Sample collection time

      October 2011

    IDA

MIGS-4.1

     Latitude

      unknown

MIGS-4.2

     Longitude

      unknown

MIGS-4.3

     Depth

      unknown

MIGS-4.4

     Altitude

      unknown

MIGS-4.5

     Isolation site     Isolation source

      Human host      vesicle on the forehead

    IDA    IDA

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.

Figure 1

Maximum likelihood tree illustrating the phylogenetic relationships among several members of the genus Francisella and members of the order Thiotrichales based on full-length 16S rRNA gene sequences.

Genome sequencing information

Genome project history

Strain BD11-00177 was sequenced because of its relevance to biodefense. The draft genome sequence was finished in August 2012. The GenBank accession number for the project is 177784. The genome project is listed in the Genome OnLine Database (GOLD) [22] as project Gi21611. Sequencing was carried out at the Dutch Organization for Applied Scientific Research (TNO) and the Swedish Defense Research Agency (FOI). Initial automatic annotation was performed using the DOE-JGI Microbial Annotation Pipeline (DOE-JGI MAP). Table 2 shows the project information and its association with MIGS 2.0 compliance.

Table 2

Project information

MIGS ID

       Property

      Term

MIGS-31

       Finishing quality

      Standard Draft

MIGS-29

       Sequencing platforms

      Illumina MiSeq, 454 Roche GS Junior

MIGS-31.2

       Fold coverage

      713×

MIGS-30

       Assemblers

      Ray Assembler V2.1

MIGS-32

       Gene calling method

      Prodigal [23]

       GOLD ID       IMG Taxon ID       NCBI PROJECT ID

      Gi21611      1244086      177784

MIGS-38

       Project relevance

      Medical, biodefence

Growth conditions and DNA isolation

For DNA preparation, strain BD11-00177 was grown on 5% sheep blood agar plates for 72 h at 35°C in the presence of 5% CO2. DNA was extracted using the Qiamp DNA Micro Kit according manufacturers guidelines (Qiagen, Westburg b.v., Leusden, The Netherlands).

Genome sequencing and assembly

Sequencing was performed by the Microbiology and Systems Biology group at TNO and the Division for CBRN Defence and Security at FOI using 454 Roche GS Junior and the Illumina MiSeq platforms. The initial draft assembly yielded 95 large (>1,000 bp) and 86 small (<1,000 bp), non-redundant contigs of 1,813,372 bp by combing 75,245 Roche/454 reads at 23× coverage and 8,289,332 Illumina reads at 690× coverage by hybrid assembly through the Ray Assembler V2.1 [24].

Genome annotation

Open Reading Frames (ORFs) were predicted using the Prodigal gene prediction algorithm [23] as part of the DOE-JGI Microbial Annotation Pipeline (DOE-JGI MAP) using default parameters, followed by a round of manual curation. CRISPR elements were predicted using CRT and PILERCR [25]. Predictions from both methods were concatenated. Identification of tRNAs was performed using tRNAScan. Ribosomal RNA genes (5S, 16S, 23S) are predicted using the program RNAmmer [26]. With the exception of tRNA and rRNA, all models from Rfam [27] are used to search the genome sequence. For faster detection, sequences are first compared to a database containing all the ncRNA genes in the Rfam database using BLAST, with a very loose cutoff. Subsequently, sequences that have hits to any genes belonging to an Rfam model are searched using the program INFERNAL [27]. Protein coding genes were compared to protein families (e.g., COGs, Pfam, KEGG) and the proteome of selected “core” genomes, which are publicly available, and the product names were assigned based on the results of these comparisons.

Genome properties

The genome was assembled into 95 large (>1,000 bp) contigs and includes one circular chromosome with a total size of 11,813,372 bp (32.23% GC content). A total of 2,149 genes were predicted, 2,103 of which are protein-coding genes. Of the protein coding genes, 1,592 were assigned to a putative function, with the remaining being annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Tables 3 and 4.

Table 3

Nucleotide content and gene count levels of the genome

Attribute

     Value

     % of totala

Genome Size (bp)

     1,813,372

     100.00%

DNA coding region (bp)

     1,611,603

     88.87%

DNA G+C content (bp)

     584,435

     32.23%

Total genesb

     2149

     100.00%

RNA genes

     46

     2.14%

Protein-coding genes

     2103

     97.86%

Genes in paralog clusters

     1262

     58.72%

Genes assigned to COGs

     1584

     73.71%

Protein coding genes connected to KEGG pathways

     611

     28.43%

not connected to KEGG pathways

     1492

     69.43%

Genes with signal peptides

     111

     5.17%

Genes with transmembrane helices

     573

     26.66%

a) The total is based either on the size of the genome in base pairs or on the total number of protein coding genes in the annotated genome.

Table 4

Number of genes associated with the 25 general COG functional categories

Code

     Value

     %agea

      Description

J

     152

     8.79

      Translation

A

     1

     0.06

      RNA processing and modification

K

     65

     3.76

      Transcription

L

     198

     11.45

      Replication, recombination and repair

B

     -

     -

      Chromatin structure and dynamics

D

     18

     1.04

      Cell cycle control, mitosis and meiosis

Y

     -

     -

      Nuclear structure

V

     31

     1.79

      Defense mechanisms

T

     24

     1.39

      Signal transduction mechanisms

M

     112

     6.47

      Cell wall/membrane biogenesis

N

     19

     1.1

      Cell motility

Z

     1

     0.06

      Cytoskeleton

W

     -

     -

      Extracellular structures

U

     44

     2.54

      Intracellular trafficking and secretion

O

     66

     3.82

      Posttranslational modification, protein turnover, chaperones

C

     107

     6.18

      Energy production and conversion

G

     118

     6.82

      Carbohydrate transport and metabolism

E

     158

     9.13

      Amino acid transport and metabolism

F

     65

     3.76

      Nucleotide transport and metabolism

H

     96

     5.55

      Coenzyme transport and metabolism

I

     64

     3.7

      Lipid transport and metabolism

P

     71

     4.1

      Inorganic ion transport and metabolism

Q

     37

     2.14

      Secondary metabolites biosynthesis, transport and catabolism

R

     172

     9.94

      General function prediction only

S

     111

     6.42

      Function unknown

-

     565

     26.29

      Not in COGs

a) The total is based on the total number of protein coding genes in the annotated genome.

Comparisons with other fully sequenced genomes

Comparison of the assembled draft genome sequence of strain BD11-00177 with publicly available F. tularensis genome sequences revealed that it clusters in the FTNF002-00 genomic group (B.Br.FTNF002-00 and BIV.FTNF002-00) defined by the FTNF002-00 genome sequence [28-30] within the B.IV clade. The presence of the 1.59 kb RD23 deletion event [31] as well as the 464 bp size of the MLVA marker FtM24 [32], both typical for the FTNF002-00 genomic group, were confirmed in silico. Notably, isolates from this genomic group had previously been exclusively reported from Spain, France, Italy, Switzerland and Germany [28,31-35].

A BLAST Ring Image Generator (BRIG) analysis comparing the F. tularensis subsp. holarctica BD11-00177 genome against the F. tularensis subsp. holarctica genomes of F92, LVS, and FTNF002-00 revealed that the BD11-00177 draft genome shows considerable resemblance to FTNF002-00 (Figure 2).

Figure 2

BRIG diagram of the F. tularensis subsp. holarctica BD11-00177, FTNF002-00 and SCHU S4 genomes using the F. tularensis subsp. holarctica FSC200 genome as a reference backbone. White regions represent absent genetic regions.

Evolutionary history of F. tularensis subspecies holarctica strain BD11-00177 was inferred using publicly available whole genome sequences.

The trees in Figure 3 A and B are drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the number of differences method and are in the units of the number of base differences per sequence. The overview of Francisella genus involved 52 public genome sequences using Piscirickettia salmonis as outgroup (Figure 3A). The detailed analysis involved 14 F. tularensis subsp. holarctica genome sequences using F. tularensis subsp. tularensis strain SCHU S4 as outgroup (Figure 3B) [17,30,33,36-41]. All positions containing gaps and missing data were eliminated. There were a total of 1,599,589 positions in the final dataset.

Figure 3

A) Overview of the Francisella genus phylogeny based on 52 public whole genome sequences. B) The phylogeny of F. tularensis subsp. holarctica strains based on whole genome sequences. The new isolate, BD11-00177 belongs to the FTNF002-00 genomic group inside the B.IV clade.

Conclusion

Here we have presented the draft genome of the first member of FTNF002-00 genomic group of F. tularensis subspecies holarctica. As more genetic information of members from this genomic group becomes available, a better understanding of the evolution and biogeography of this pathogen will be gained. This knowledge may help us to understand the epidemiology and potential expansion of the geographical distribution of this genomic group. Despite potential biases associated with discontinuous draft genomes, we would like to focus on the added value of draft bacterial genome sequencing. Taking advantage of low cost and high-throughput sequencing platforms allows us to probe the vast microbial diversity present in nature and rapidly respond to clinical outbreaks and acute biosecurity hazards. From an evolutionary ecology perspective, increased sequencing efforts allow us to characterize the biogeography of microbial taxa and differentiate between neutral and conserved genome contents.

Declarations

Acknowledgements

This work was supported by Dutch Ministry of Defence [grant number V1036] and the Swedish Defence Research Agency [project A4952].


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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