Francisella tularensis Insertion Sequence Elements Contribute to Differential Gene Expression

This abstract is under embargo until April 26, 2020

Application of chromosomal DNA and protein targeting for the identification of \u3ci\u3eYersinia pestis\u3c/i\u3e

Purpose: A comprehensive strategy was developed and validated for the identification of pathogens from closely related near neighbors using both chromosomal and protein biomarkers, with emphasis on distinguishing Yersinia pestis from the ancestral bacterium Yersinia pseudotuberculosis. Experimental design: Computational analysis was used to discover chromosomal targets unique to Y. pestis. Locus identifier YPO1670 was selected for further validation and PCR was used to confirm that this biomarker was exclusively present in Y. pestis strains, while absent in other Yersinia species. RT-PCR and Western blot analyses were utilized to evaluate YPO1670 expression and MRM MS was performed to identify the YPO1670 protein within cell lysates. Results: The described study validated that YPO1670 was exclusive to Y. pestis. PCR confirmed the locus to be unique to Y. pestis. The associated transcript and protein were produced throughout growth with the highest abundance occurring in stationary phase and MRM MS conclusively identified the YPO1670 protein in cell extracts. Conclusions and clinical relevance: These findings validated YPO1670 as a reliable candidate biomarker for Y. pestis and that a dual DNA and protein targeting approach is feasible for the development of next-generation assays to accurately differentiate pathogens from near neighbors

\u3ci\u3eFrancisella tularensis\u3c/i\u3e Subtype A.II Genomic Plasticity in Comparison with Subtype A.I

Although Francisella tularensis is considered a monomorphic intracellular pathogen, molecular genotyping and virulence studies have demonstrated important differences within the tularensis 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 F. tularensis type A genomes. In contrast with the F. tularensis A.I strains (SCHU S4, FSC198, NE061598, and TI0902), substantial genomic variation was observed between the newly sequenced F. tularensis 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 F. tularensis 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 F. tularensis genetic diversity and plasticity is needed

<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 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

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 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

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