Comprehensive insights in the Mycobacterium avium subsp. paratuberculosis genome using new WGS data of sheep strain JIII-386 from Germany

Abstract

Mycobacterium avium (M. a.) subsp. paratuberculosis (MAP) - the etiologic agent of Johne's disease - affects cattle, sheep and other ruminants worldwide. To decipher phenotypic differences among sheep and cattle strains (belonging to MAP-S [Type-I/III] respectively MAP-C [Type-II]) comparative genome analysis needs data from diverse isolates originating from different geographic regions of the world. The current study presents the so far best assembled genome of a MAP-S-strain: sheep isolate JIII-386 from Germany. One newly sequenced cattle isolate (JII-1961, Germany), four published MAP strains of MAP-C and MAP-S from U.S. and Australia and M. a. subsp. hominissuis (MAH) strain 104 were used for assembly improvement and comparisons. All genomes were annotated by BacProt and results compared with NCBI annotation. Corresponding protein-coding sequences (CDSs) were detected, but also CDSs that were exclusively determined either by NCBI or BacProt. A new Shine-Dalgarno sequence motif (5'AGCTGG3') was extracted. Novel CDSs including PE-PGRS family protein genes and about 80 non-coding RNAs exhibiting high sequence conservation are presented. Previously found genetic differences between MAP-types are partially revised. Four out of ten assumed MAP-S-specific large sequence polymorphism regions (LSP^Ss) are still present in MAP-C strains; new LSP^Ss were identified. Independently of the regional origin of the strains, the number of individual CDSs and single nucleotide variants confirm the strong similarity of MAP-C strains and show higher diversity among MAP-S strains. This study gives ambiguous results regarding the hypothesis that MAP-S is the evolutionary intermediate between MAH and MAP-C, but it clearly shows a higher similarity of MAP to MAH than to M. intracellulare.

Keywords: Johne’s disease; MAP-S; SNV/SNP; Shine-Dalgarno sequence; evolution of MAP-types; ncRNA; new LSPSs.

Fig. 1.—

Overview of M. avium strains compared in this study and their assembly and annotation status. Strains of MAP-S (Type I/III): JIII-386, S397, CLIJ361 (red); strains of MAP-C (Type II): K-10, K-10′, MAP4, JII-1961 (green); MAH strain 104 (blue); M. tuberculosis strain H37Rv (brown, used for extended ncRNA annotation). Underlined, annotations available; scaff, scaffolds; con, contigs; full, finished genome. Pictograms describe host origin.

Fig. 2.—

Genome comparison of K-10′ (top), JIII-386 (middle), and S397 (bottom) calculated with Mauve. Colored blocks connected by lines indicate homologous regions which are internally free from genomic rearrangements. White areas within blocks indicate sequence regions of lower similarity. Blocks below the center line are aligned reverse complementary. See detailed supplementary figure S10a, Supplementary Material online.

Fig. 3.—

Shine–Dalgarno sequence motifs of investigated M. avium strains and M. tuberculosis strain H37Rv. Detailed information and the motif of MAP strain K-10 (similar to K-10′) can be found in supplementary table S11, Supplementary Material online.

Fig. 4.—

Phylogenetic reconstructions for all investigated M. avium strains based on sequence comparison of 790 corresponding CDSs on nucleotide (A) and amino acid level (B) and 70 corresponding ncRNAs (C). Mycobacterium tuberculosis strain H37Rv was used as an outgroup. Mycobacterium intracellulare (MI) strains were included as members of the MAC. Float numbers correspond to substitutions per site and integer numbers represent RAxML bootstrap values. Long branches are shrinked. Detailed figures, all multiple sequence alignments, and tree representations in Newick format can be found in the supplementary figures S22–S26, Supplementary Material online. Strains of MAP-S, Type I: CLIJ361 and Type III: JIII-386, S397 (red); Strains of MAP-C, Type II: K-10, K-10′, MAP4, JII-1961 (green); MAH strain 104 (blue); MI MOTT-64 and MI MOTT-02 (orange); and M. tuberculosis strain H37Rv (brown, used as outgroup) are shown.