Revisiting Francisella tularensis subsp. holarctica, Causative Agent of Tularemia in Germany With Bioinformatics: New Insights in Genome Structure, DNA Methylation and Comparative Phylogenetic Analysis

Abstract

Francisella (F.) tularensis is a highly virulent, Gram-negative bacterial pathogen and the causative agent of the zoonotic disease tularemia. Here, we generated, analyzed and characterized a high quality circular genome sequence of the F. tularensis subsp. holarctica strain 12T0050 that caused fatal tularemia in a hare. Besides the genomic structure, we focused on the analysis of oriC, unique to the Francisella genus and regulating replication in and outside hosts and the first report on genomic DNA methylation of a Francisella strain. The high quality genome was used to establish and evaluate a diagnostic whole genome sequencing pipeline. A genotyping strategy for F. tularensis was developed using various bioinformatics tools for genotyping. Additionally, whole genome sequences of F. tularensis subsp. holarctica isolates isolated in the years 2008-2015 in Germany were generated. A phylogenetic analysis allowed to determine the genetic relatedness of these isolates and confirmed the highly conserved nature of F. tularensis subsp. holarctica.

Keywords: Francisella tularensis subsp. holarctica; genome analysis; high quality genome; phylogeny; tularemia.

FIGURE 1

Plot of the complete Francisella tularensis subsp. holarctica 12T0050. The genome consists of 1890738 base pairs and 2114 predicted coding sequences. The circles represent from the inside: 1, GC skew (red above and black below zero, 10 kb window); 2, GC content (blue above and black below genome average of 32.20%, 10 kb window); 3, strand-specific genome-wide methylation analyzed in 10 kb windows; 4, strand-specific methylation per gene; 5, scale in million base pairs (Mb). Methylation color spectrum goes from blue (minimum, 5%) over yellow (mean, 12.5%) to red (maximum, 23%).

FIGURE 2

Phylogeny based on 16S rRNA performed with MOLE-BLAST.

FIGURE 3

PhyloPhlAn analysis of all Francisella isolates using amino acid sequences of more than 400 proteins. The graphic scale equals 2.0 amino acid differences in the overview (A) and in the detailed view with F. tularensis subsp. holarctica, the graphic scale equals 0.001 amino acid difference (B).

FIGURE 4

MLST⁺ of the strains showing the distance based on 1147 core genome MLST loci generated with SeqSphere. The graphic scale equals 0.1 difference of a MLST locus. Red colored isolate numbers indicate assignment to clade B.6, blue to clade B.12. The arrow marks the position of 12T0050.

FIGURE 5

Neighbor-Joining Phylogenetic Tree of the ParSNP core genome SNP analysis (A). The trees were statistically evaluated with RaXML with a bootstrap analysis with 500 bootstraps. (B and detailed view C, compared to canSNPer analysis)