Deletion of an mmpL gene and multiple associated genes from the genome of the S strain of Mycobacterium avium subsp. paratuberculosis identified by representational difference analysis and in silico analysis

Abstract

Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) can be divided into two major strains, sheep (S) and cattle (C), based on cultural requirements, host specificity, degree of clumping of cells in suspension and minor genomic differences including copy number of insertion elements and point mutations. Representational difference analysis (RDA) with S strain as driver and C strain as tester was used to identify unique genomic regions. Three sequences (RDA1, RDA3 and RDA4) were identified. RDA1 (229bp) contained a single base difference between S and C strains. RDA4 (163bp) was an artefact. RDA3 (206bp) was similar to several sequences in the incomplete genome sequences of M. avium subsp. paratuberculosis K10 and M. avium subsp. avium 104. In silico analysis led to the identification of a deletion that may be as large as 17kb in the sheep strain of M. a. paratuberculosis. PCR analysis of this region confirmed the deletion of 11,584bp that included 10 genes (MAP1734 to MAP1743c) of the M. a. paratuberculosis K10 genome. This included the loss of mmpL5 and mmpS5 genes and homologues of the M. tuberculosis genes: Rv2002 (fabG3), Rv2017c (lipW), Rv3132c (devS), Rv2032 (acg) and the conserved hypothetical genes Rv2005c and Rv2026c. PCR reactions designed to detect the single nucleotide polymorphism in RDA1 and the deletion in the mmpL region can be used to distinguish these strains. MmpL genes, found in M. tuberculosis and other mycobacteria are part of the resistance-nodulation-division (RND) family but contain domains unique to mycobacteria thought to play a role in cell wall biogenesis, virulence and other phenotypic characteristics. Absence of mmpL5 in the S strain of M. a. paratuberculosis is unlikely to account for the difference in clumping in suspension but may explain the difference in cultural requirements and host specificity compared to the C strain but the impact of the remainder of the deletion is yet to be ascertained.