Whole-Genome Analysis of Mycobacterium avium subsp. paratuberculosis IS900Insertions Reveals Strain Type-Specific Modalities

Abstract

<i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> (<i>Map</i>) is the etiological agent of Johne's disease in ruminants. The IS<i>900</i> insertion sequence (IS) has been used widely as an epidemiological marker and target for PCR diagnosis. Updated DNA sequencing technologies have led to a rapid increase in available <i>Map</i> genomes, which makes it possible to analyze the distribution of IS<i>900</i> in this slow-growing bacterium. The objective of this study is to characterize the distribution of the IS<i>900</i> element and how it affects genomic evolution and gene function of <i>Map</i>. A secondary goal is to develop automated <i>in silico</i> restriction fragment length polymorphism (RFLP) analysis using IS<i>900</i>. Complete genomes from the major phylogenetic lineages known as C-type and S-type (including subtypes I and III), were chosen to represent the genetic diversity of <i>Map</i>. IS<i>900</i> elements were located in these genomes using BLAST software and the relevant fragments extracted. An <i>in silico</i> RFLP analysis using the <i>Bst</i>EII restriction site was performed to obtain exact sizes of the DNA fragments carrying a copy of IS<i>900</i> and the resulting RFLP profiles were analyzed and compared by digital visualization of the separated restriction fragments. The program developed for this study allowed automated localization of IS<i>900</i> sequences to identify their position within each genome along with the exact number of copies per genome. The number of IS<i>900</i> copies ranged from 16 in the C-type isolate to 22 in the S-type subtype I isolate. A loci-by-loci sequence alignment of all IS<i>900</i> copies within the three genomes revealed new sequence polymorphisms that define three sequevars distinguishing the subtypes. Nine IS<i>900</i> insertion site locations were conserved across all genomes studied while smaller subsets were unique to a particular lineage. Preferential insertion motif sequences were identified for IS<i>900</i> along with genes bordering all IS<i>900</i> insertions. Rarely did IS<i>900</i> insert within coding sequences as only three genes were disrupted in this way. This study makes it possible to automate IS<i>900</i> distribution in <i>Map</i> genomes to enrich knowledge on the distribution dynamics of this IS for epidemiological purposes, for understanding <i>Map</i> evolution and for studying the biological implications of IS<i>900</i> insertions.