<i>Francisella tularensis</i> Subtype A.II Genomic Plasticity in Comparison with Subtype A.I

<div><p>Although <i>Francisella tularensis</i> is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the <i>tularensis</i> subspecies (type A). To evaluate genetic variation within type A strains, sequencing and assembly of a new subtype A.II genome was achieved for comparison to other completed <i>F</i>. <i>tularensis</i> type A genomes. In contrast with the <i>F</i>. <i>tularensis</i> A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced <i>F</i>. <i>tularensis</i> A.II strain (WY-00W4114) and the only other publically available A.II strain (WY96-3418). Genome differences between WY-00W4114 and WY96-3418 included three major chromosomal translocations, 1580 indels, and 286 nucleotide substitutions of which 159 were observed in predicted open reading frames and 127 were located in intergenic regions. The majority of WY-00W4114 nucleotide deletions occurred in intergenic regions, whereas most of the insertions and substitutions occurred in predicted genes. Of the nucleotide substitutions, 48 (30%) were synonymous and 111 (70%) were nonsynonymous. WY-00W4114 and WY96-3418 nucleotide polymorphisms were predominantly G/C to A/T allelic mutations, with WY-00W4114 having more A+T enrichment. In addition, the A.II genomes contained a considerably higher number of intact genes and longer repetitive sequences, including transposon remnants than the A.I genomes. Together these findings support the premise that <i>F</i>. <i>tularensis</i> A.II may have a fitness advantage compared to the A.I subtype due to the higher abundance of functional genes and repeated chromosomal sequences. A better understanding of the selective forces driving <i>F</i>. <i>tularensis</i> genetic diversity and plasticity is needed.</p></div

Nucleotide polymorphisms in genome of WY-00W4114 compared to WY96-3418 and overall base pair size increase in WY-00W4114.

<p>Nucleotide polymorphisms in genome of WY-00W4114 compared to WY96-3418 and overall base pair size increase in WY-00W4114.</p

Diagram illustrating GC skew within chromosomal topology map for <i>F</i>. <i>tularensis</i> A.I and A.II strains.

<p>The circular <i>F</i>. <i>tularensis</i> chromosome of subtype A.I strains are represented by SCHU S4 (A) and NE061598 (B), and WY96-3418 (C) and WY-00W4114 (D) represent the subtype A.II strains. The origin (<i>ori</i>) and termination (<i>ter</i>) region are denoted by a vertical black line at the top and bottom, respectively, of the corresponding chromosomal map. GC skew + (gray) and GC skew—(black) is shown in the outermost circle for each genome and the kilobase pair position is indicated in the innermost circle.</p

Description and nucleotide position of locally collinear blocks (LCBs) within A.II strains WY-00W4114 and WY96-3418.

<p>Description and nucleotide position of locally collinear blocks (LCBs) within A.II strains WY-00W4114 and WY96-3418.</p

Diagram depicting large rearrangements of locally collinear blocks (LCBs) within <i>F</i>. <i>tularensis</i> A.II strains.

<p><i>F</i>. <i>tularensis</i> A.II strains WY-00W4114 and WY96-3418 chromosomal comparison showing related LCBs (A) and potential recombination events with a two-step parsimonious molecular process (B). Each LCB is represented with a different pattern and/or shading. Directionality of the LCBs is depicted with an arrow and is based on the reference strain WY96-3418 (GenBank accession number CP000608). Nucleotide positions are denoted in kilobase pairs by the corresponding genome.</p

Whole genome mapping of <i>F</i>. <i>tularensis</i> subtype A.II strains WY-00W4114 and WY96-3418.

<p><i>Nco</i>I (A) and <i>Nhe</i>I (B) whole genome maps of <i>F</i>. <i>tularensis</i> WY-00W4114 (top linearized chromosome) compared to the corresponding theoretical <i>in silico</i> digestion of <i>F</i>. <i>tularensis</i> WY96-3418 (GenBank accession number CP000608, bottom linearized chromosome). Vertical lines within the genome maps denote the restriction endonuclease sites for <i>Nco</i>I (A) or <i>Nhe</i>I (B). Lines connecting the chromosomal restriction maps of WY-00W4114 and WY96-3418 and the adjacent unshaded genomic areas denote translocated regions.</p

Genome alignment of <i>F</i>. <i>tularensis</i> A.I and A.II strains.

<p>Chromosomal alignments of representative <i>F</i>. <i>tularensis</i> A.I strains SCHU S4, NE061598, FSC198, and TI0902 (A); pairwise genome alignment of the <i>F</i>. <i>tularensis</i> A.II strains WY-00W4114 and WY96-3418 (B); and multiple chromosomal alignment of the <i>F</i>. <i>tularensis</i> A.I and A.II strains sequenced to completion and shown in panels A and B <b>(C)</b>. The relative location of the <i>Francisella</i> pathogenicity island (FPI) in duplicated region 1 (DR1) and duplicated region 2 (DR2) is identified with a bar above the associated chromosomal region for each subtype. The progressiveMauve software tool was used to align the genomes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124906#pone.0124906.ref021" target="_blank">21</a>].</p

VNTR markers and their differences between Schu S4 and NE061598.

a<p>FtM1-FtM25 VNTR markers as previously reported by Johansson et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009007#pone.0009007-Johansson1" target="_blank">[7]</a>. New VTNR polymorphisms identified in this study are listed as VNTR1 through VNTR-5.</p>b<p>Indicates repeat size in nucleotides.</p>c<p>“G” indicates that the repeat is located within an open reading frame (genic) whereas “I” indicates that the repeat is located within an intergenic region. Distance to predicted translation start site is indicated in nucleotides. “+” or “−” indicates that the translation start site is downstream or upstream of repeat motif, respectively (as reported by Johansson et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009007#pone.0009007-Johansson1" target="_blank">[7]</a>).</p

Genome rearrangement representation for NE061598 and Schu S4 genomes.

<p>Each local collinear blocks (LCB) 1-6 is represented by a different color. Upside-down blocks (i.e. LCB2) represent the location of the reverse strand, which means an inversion has occurred. Note the rearrangements of LCB4 and LCB5.</p

Genomic characteristics of <i>F. tularensis</i> subsp. <i>tularensis</i> NE061598.

<p>Genomic characteristics of <i>F. tularensis</i> subsp. <i>tularensis</i> NE061598.</p