Stand. Genomic Sci. 2013 8:3
doi:10.4056/sigs.4217923
Draft genome sequence of Francisella tularensis subsp. holarctica BD11-00177

Jordy P. M. Coolen1, Andreas Sjödin2, Boulos Maraha3, Gerard F. Hajer4, Mats Forsman2, Ellen Verspui5, Hendrina M.E. Frenay3, Daan W. Notermans6, Maaike C. de Vries6, Frans A.G. Reubsaet6, Armand Paauw1, Guus Roeselers1

1 TNO, The Netherlands
2 Division for CBRN Defence and Security, FOI - Swedish Defence Research Agency, Umeå, Sweden
3 Department of Medical Microbiology, Beatrix Hospital, Gorinchem and Albert Schweitzer Hospital, Dordrecht, The Netherlands.
4 Department of Surgery, Beatrix Hospital, Gorinchem, The Netherlands
5 Public Health Service Zuid Holland Zuid, Dordrecht, The Netherlands
6 Diagnostic Laboratory for Infectious Diseases and Perinatal Screening (LIS), Center for Infectious Disease Control, National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Correspondence: Guus Roeselers (guus.roeselers@tno.nl)

epub-ppub publication date: August 10, 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: tularaemia, biodefence, zoonotic infection, phylogeography, Netherlands.

Copyright © retained by original authors
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
Figure 1
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
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
Figure 3
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.

Acknowledgements

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

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Acknowledgements

We would like to gratefully acknowledge the support of many members of the Genomic Standards Consortium, the broader genomic science community, and those who have indicated their willingness to serve as editors, reviewers and contributors.

SIGS was founded with grants from the Office of the Vice President for Research and Graduate Studies at Michigan State University, the Michigan State University Foundation, and the US Department of Energy Biological and Environmental Research DE-FG02-08ER64707. The journal became self-supporting on October 1, 2011.

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