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Mycobacterium avium subsp. paratuberculosis ( Map ) is the etiological agent of Johne's disease in ruminants. The IS 900 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 Map genomes, which makes it possible to analyze the distribution of IS 900 in this slow-growing bacterium. The objective of this study is to characterize the distribution of the IS 900 element and how it affects genomic evolution and gene function of Map . A secondary goal is to develop automated in silico restriction fragment length polymorphism (RFLP) analysis using IS 900 . 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 Map . IS 900 elements were located in these genomes using BLAST software and the relevant fragments extracted. An in silico RFLP analysis using the Bst EII restriction site was performed to obtain exact sizes of the DNA fragments carrying a copy of IS 900 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 900 sequences to identify their position within each genome along with the exact number of copies per genome. The number of IS 900 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 900 copies within the three genomes revealed new sequence polymorphisms that define three sequevars distinguishing the subtypes. Nine IS 900 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 900 along with genes bordering all IS 900 insertions. Rarely did IS 900 insert within coding sequences as only three genes were disrupted in this way. This study makes it possible to automate IS 900 distribution in Map genomes to enrich knowledge on the distribution dynamics of this IS for epidemiological purposes, for understanding Map evolution and for studying the biological implications of IS 900 insertions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Conde, Price-Carter, Cochard, Branger, Stevenson, Whittington, Bannantine and Biet.)

Mycobacterium avium comprises four subspecies that contain both human and veterinary pathogens. At the inception of this study, twenty-eight M. avium genomes had been annotated as RefSeq genomes, facilitating direct comparisons. These genomes represent strains from around the world and provided a unique opportunity to examine genome dynamics in this species. Each genome was confirmed to be classified correctly based on SNP genotyping, nucleotide identity and presence/absence of repetitive elements or other typing methods. The Mycobacterium avium subspecies paratuberculosis ( Map ) genome size and organization was remarkably consistent, averaging 4.8 Mb with a variance of only 29.6 kb among the 13 strains. Comparing recombination events along with the larger genome size and variance observed among Mycobacterium avium subspecies avium ( Maa ) and Mycobacterium avium subspecies hominissuis ( Mah ) strains (collectively termed non- Map ) suggests horizontal gene transfer occurs in non- Map , but not in Map strains. Overall, M. avium subspecies could be divided into two major sub-divisions, with the Map type II (bovine strains) clustering tightly on one end of a phylogenetic spectrum and Mah strains clustering more loosely together on the other end. The most evolutionarily distinct Map strain was an ovine strain, designated Telford, which had >1,000 SNPs and showed large rearrangements compared to the bovine type II strains. The Telford strain clustered with Maa strains as an intermediate between Map type II and Mah . SNP analysis and genome organization analyses repeatedly demonstrated the conserved nature of Map versus the mosaic nature of non- Map M. avium strains. Finally, core and pangenomes were developed for Map and non- Map strains. A total of 80% Map genes belonged to the Map core genome, while only 40% of non- Map genes belonged to the non- Map core genome. These genomes provide a more complete and detailed comparison of these subspecies strains as well as a blueprint for how genetic diversity originated., (Copyright © 2020 Bannantine, Conde, Bayles, Branger and Biet.)

In recent years the diversity of the French Mycobacterium bovis population responsible for bovine tuberculosis (bTB) outbreaks since 1970 has been described in detail. To further understand bTB evolution in France, we used single nucleotide polymorphisms (SNPs) based on whole genome sequence versus classical genotyping methods in order to identify accurate phylogenetic relationships between M. bovis strains. Whole genome sequencing was carried out on a selection of 87 strains which reflect the French M. bovis population's genetic diversity. Sequences were compared to the M. bovis reference genome AF2122/97. Comparison among the 87 genomes revealed 9,170 sites where at least one strain shows a SNP with respect to the reference genome; 1,172 are intergenic and 7,998 in coding sequences, of which 2,880 are synonymous and 5,118 non-synonymous. SNP-based phylogenetic analysis using these 9,170 SNP is congruent with the cluster defined by spoligotyping and multilocus variable number of tandem repeat analysis typing. In addition, some SNPs were identified as specific to genotypic groups. These findings suggest new SNP targets that can be used for the development of high-resolving methods for genotyping as well as for studying M. bovis evolution and transmission patterns. The detection of non-synonymous SNPs on virulence genes enabled us to distinguish different clusters. Our results seem to indicate that genetically differentiated clusters could also display distinctive phenotypic traits.