Whole-Genome Analysis of subsp. IS -.

subsp. () is the etiological agent of Johne's disease in ruminants. The IS 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 genomes, which makes it possible to analyze the distribution of IS in this slow-growing bacterium. The objective of this study is to characterize the distribution of the IS element and how it affects genomic evolution and gene function of . A secondary goal is to develop automated restriction fragment length polymorphism (RFLP) analysis using IS. 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 . IS elements were located in these genomes using BLAST software and the relevant fragments extracted. An RFLP analysis using the EII restriction site was performed to obtain exact sizes of the DNA fragments carrying a copy of IS 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 sequences to identify their position within each genome along with the exact number of copies per genome. The number of IS 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 copies within the three genomes revealed new sequence polymorphisms that define three sequevars distinguishing the subtypes. Nine IS 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 along with genes bordering all IS insertions. Rarely did IS insert within coding sequences as only three genes were disrupted in this way. This study makes it possible to automate IS distribution in genomes to enrich knowledge on the distribution dynamics of this IS for epidemiological purposes, for understanding evolution and for studying the biological implications of IS insertions.