Comparative genomic analysis of Mycobacterium avium subspecies obtained from multiple host species

Abstract

Background: Mycobacterium avium (M. avium) subspecies vary widely in both pathogenicity and host specificity, but the genetic features contributing to this diversity remain unclear.

Results: A comparative genomic approach was used to identify large sequence polymorphisms among M. avium subspecies obtained from a variety of host animals. DNA microarrays were used as a platform for comparing mycobacterial isolates with the sequenced bovine isolate M. avium subsp. paratuberculosis (MAP) K-10. Open reading frames (ORFs) were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to MAP K-10 DNA. Multiple large polymorphic regions were found in the genomes of MAP isolates obtained from sheep. One of these clusters encodes glycopeptidolipid biosynthesis enzymes which have not previously been identified in MAP. M. avium subsp. silvaticum isolates were observed to have a hybridization profile very similar to yet distinguishable from M. avium subsp. avium. Isolates obtained from cattle (n = 5), birds (n = 4), goats (n = 3), bison (n = 3), and humans (n = 9) were indistinguishable from cattle isolate MAP K-10.

Conclusion: Genome diversity in M. avium subspecies appears to be mediated by large sequence polymorphisms that are commonly associated with mobile genetic elements. Subspecies and host adapted isolates of M. avium were distinguishable by the presence or absence of specific polymorphisms.

Figure 1

Distribution of log transformed microarray fluorescence intensity ratios for competitive genomic hybridizations of MAH 104 and MAP K-10 (MAH 104/MAP K-10). The results presented (n = number of results) are the average values of multiple hybridizations and replicate spots as described in Material and Methods. Log ratio distributions are displayed for (A) all probes present on the microarray; (B) MAP K-10 specific probes (as determined by BLAST E-value < 1 × 10^-5); and (C) MAH 104 specific probes.

Figure 2

Most variable ORFs amongst mycobacterial isolates examined. 474 ORFs with 3-fold or greater differences in hybridization between at least two isolates and MAP K-10 are displayed in rows. Columns represent individual mycobacterial isolates that were grouped by hierarchical clustering (Pearson correlation, average linkage). Grey colored data points represent no data. Isolate designations are listed in the right margin next to a colored bar indicating the location of the isolates on the graph. Selected gene clusters are labeled by the first and last ORFs. A complete listing of the ORFs and log ratios has also been provided [see Additional file 1].

Figure 3

Glycopeptidolipid biosynthesis gene cluster in MAP sheep isolate 397. Solid black lines represent homology (approximately 99% identity) with the MAA TMC724 (ATCC 25291) glycopeptidolipid biosynthesis cluster (accession number AF125999), while dashed green lines represent sequences absent in isolate 397. Solid or dashed red and blue lines represent regions of homology with MAH 104 and MAP K-10, respectively. Vertical lines are spaced at 1600 base intervals for reference and represent the number of nucleotides from the start of the Genbank entry.

Figure 4

Location of polymorphic regions mapped to the MAP genome. The outermost ring is the nucleotide position from the origin of replication, while the green ring is the percentage of GC nucleotides. The innermost rings represent the ORFs that were identified as variably present (log ratio < -1.2) in at least one of the mycobacterial genomes (blue) as well as ORFs conserved in all of the isolates examined (black). ORFs that are annotated as mobile genetic elements are colored red. The numbers adjacent to polymorphic sequences correspond to the LSP numbers listed in Table 2. The circular genome MAP was generated with the software program Microbial Genome Viewer [50].