Whole-Genome Analysis of Mycobacterium avium subsp. paratuberculosis IS 900 Insertions Reveals Strain Type- Specific Modalities

Abstract

Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of Johne's disease in ruminants. The IS900 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 IS900 in this slow-growing bacterium. The objective of this study is to characterize the distribution of the IS900 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 IS900. 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. IS900 elements were located in these genomes using BLAST software and the relevant fragments extracted. An in silico RFLP analysis using the BstEII restriction site was performed to obtain exact sizes of the DNA fragments carrying a copy of IS900 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 IS900 sequences to identify their position within each genome along with the exact number of copies per genome. The number of IS900 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 IS900 copies within the three genomes revealed new sequence polymorphisms that define three sequevars distinguishing the subtypes. Nine IS900 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 IS900 along with genes bordering all IS900 insertions. Rarely did IS900 insert within coding sequences as only three genes were disrupted in this way. This study makes it possible to automate IS900 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 IS900 insertions.

Keywords: Mycobacterium avium subsp. paratuberculosis; RFLP; complete genome; evolution; insertion sequence IS900.

FIGURE 1

Bioinformatic analysis pipeline. Details of steps performed by the IS900 RFLP in silico pipeline. From complete genome sequences, BstEII restriction site and IS900 sequence positions were identified. Both data sets are merged together to extract the BstEII fragment sizes from the genome sequence. Previous positions IS900 and sequence orientation are stored in a.rflp file for further analysis. BstEII fragment sizes are converted into migration distance based on calculation from an in vitro gel migration (see section “Materials and Methods”) and saved in the .tsv file for further visualization of the RFLP profile.

FIGURE 2

Distribution of IS900 copies on the genome of Mycobacterium avium subsp. paratuberculosis strain K-10. (A) Shown, using Circos version 0.69–8, from the outer circle to the inner circle are the megabase (Mb) positions on the chromosome, the BstEII restriction sites, the position of each IS900 element, plus strand ORFs, minus strand ORFs and a plot of the percent GC. (B) Mauve alignment of K-10 (top) with Telford (middle) and S397 (bottom) showing genomic reorganization of the genomes. The colored boxes represent homologous regions present in each genome. Blocks below the centerline indicate regions with inverse orientation. Regions outside the blocks lack homology between the genomes. Within each block there is a similarity profile of the DNA sequences and the white areas indicate sequences specific to a genome. The scale is in base pairs. Orthologous insertions are indicated, using the slider of Mauve, by a green arrow, specific insertions are indicated by a orange arrow and conserved loci only in two genomes by a blue arrow.

FIGURE 3

IS900 restriction fragment length polymorphism (RFLP) “in vitro” versus “in silico” (A) An in silico RFLP analysis was developed using complete genomes. This automated procedure identified the BstEII restriction sites to obtain the exact size of all DNA fragments carrying a copy of the IS900 sequence. IS900 RFLP profiles were compared using fragment sizes (A) or (B) by digital visualization of the restriction fragments separated according to their size by mimicking their migration in agarose gel. (C) The IS900 RFLP profiles obtained by classical Southern blot method and hybridization to IS900 were used to find the approximate fragment sizes by band analysis software (D).

FIGURE 4

IS900 RFLP Phylogeny. Phylogeny RFLP with profiles established in silico and integrated into the database are indicated by blue boxes for the three reference genomes of C-type and S-type subtypes I and III and in green boxes for the other complete genomes available for Map.

FIGURE 5

Venn diagram, showing IS900 orthologous sites and orthologous loci between the three Map genomes. (A) The diagram indicates the number of IS900 sites unique or orthologous between 2 and 3 genomes. (B) The diagram indicates the number of orthologous or unique loci upstream and downstream of the IS900 between genomes. Refer to Supplementary Table 2 for additional details.

FIGURE 6

Mah gene disrupted by IS900 insertion in Map. Shown are orthologous regions of the genomes of M. avium subspecies. The 576 bp Mah gene encoding a putative membrane protein is disrupted by insertion of IS900 into that gene sometime after the subspecies delineation. The corresponding region in the Telford strain appears to have a second genome modifying event, which changed this region upstream of the insertion.

FIGURE 7

IS900 sites associated with functional genes and pathways. The diagram indicates Gene Ontology (GO) enrichment analysis of predicted coding sequences near IS900 sites.