Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing The GenBank accession numbers for the sequences reported in this paper are AJ011838, AJ250015–AJ250023 and AJ251434–AJ251437.

Abstract

Mycobacterium avium subsp. paratuberculosis is a pathogen that causes chronic inflammation of the intestine in many animals, including primates, and is implicated in Crohn's disease in humans. It differs from other members of the M. avium complex in having 14-18 copies of IS900 inserted into conserved loci in its genome. In the present study, genomic DNA flanking 14 of these insertions was characterized and homologues in the Mycobacterium tuberculosis and M. avium subsp. avium genomes were identified. These included regions encoding a sigma factor (sigJ) at locus 3, a nitrate reductase (nirA) at locus 4, a transcription regulator (tetR) and polyketide synthase at locus 6, and a 6-O-methylguanine methyltransferase at locus 9. In addition, locus numbers were assigned to 9 of 15 RFLP bands previously described. IS900 insertion at 7 of the 14 characterized loci was into the RBS of a gene substituting an RBS encoded by IS900 sited two bases closer to the initiation codon. IS900 insertion at five loci interrupted an ORF at the target site, one of which encoded a homologue of the immunodominant mycobacterial DesA1 protein. Eleven of eighty-one M. avium subsp. paratuberculosis isolates lacked the insertion site at locus 6 together with flanking genomic DNA. This region was also absent from seven reference strains of M. avium subsp. avium, from one M. avium subsp. silvaticum and from six other mycobacterial species. A multiplex PCR of IS900 loci (MPIL) typing method was developed which was able to discriminate 10 different types of M. avium subsp. paratuberculosis from the panel of 81 isolates with consistent differences between those of bovine and ovine origin. Nine MPIL types corresponded with a single PstI/BstEII RFLP type, suggesting that this method may be applicable to typing of M. avium subsp. paratuberculosis directly from a sample without the need for culture. The remaining MPIL type corresponded with seven PstI/BstEII RFLP types. Further resolution of these may come from sequencing the remaining four uncharacterized IS900 loci.

Toxin-Antitoxin Systems of Mycobacterium smegmatis Are Essential for Cell Survival

“…DNA Manipulation and Cloning of Constructs-Genomic DNA from M. smegmatis was isolated using the cetyltrimethylammonium bromide method (26). Restriction or DNA-modifying enzymes and other molecular biology reagents were obtained from Roche Diagnostics or New England Biolabs.…”

Distribution of Mycobacterium avium subsp.paratuberculosis in tissues of naturally infected cattle as affected by age

“…Methods for differentiation or subtyping of MAP, such as IS900 multiplex PCR (Bull et al, 2000), RFLP -random fragment length polymorphism (Pavlik et al, 1999), AFLP -amplified fragment length polymorphism (Motiwala et al, 2003) or MLSSR -multilocus short sequence repeat (Amonsin et al, 2004) have been previously developed. RFLP analysis of the examined tissue isolates identified RFLP type B-C1 in all cases.…”

Rapid differentiation of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis by amplification of insertion element IS901

“…No. AJ250015; Bull et al, 2000). The distance of this flanking site from the beginning of IS900 is 47 bp.…”