Molecular Genetic Analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium smegmatis

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants. Moreover, the potential linkage with Crohn’s disease makes MAP a concern as a zoonotic pathogen. The generation of comprehensive random transposon mutant libraries is a fundamental genetic technology to determine the role of genes in physiology and pathogenesis. To identify the genomic regions associated with virulence a comprehensive MAP Tn5367 transposon mutant bank was constructed. Assuming random transposition of Tn5367 it was calculated that collection of approximately 14,000 mutants will generate a comprehensive MAP mutant library sufficient to analyze essentiality of all the genes. However, further studies showed that transposition of Tn 5367 is not random due to transposition biases leading to miscalculation of number of mutants required to generate a comprehensive library. To identify the sources of transposition biases and the ideal transposon for generation of comprehensive library, a whole MAP genome analysis comparing the transposon recognition sites for Tn5367 and MycoMarT7 transposons was performed. Results indicate that only MycoMarT7 provides a random representation of insertions in 99% of all MAP genes. Our novel finding is that both transposons have loci-dependent biases with Tn5367 having more bias. These loci-dependent transposition biases lead to an underestimation of the number of independent mutants required to generate a comprehensive mutant library. MycoMarT7 was used to construct a comprehensive transposon mutant library. Analysis of the library indicated that 93.1% of the MAP genes have at least one transposon insertion. Applying a 4-state hidden Markov model, we preliminarily identified 328 essential; 1,103 growth-defect; 2,603 non-essential and 258 growth-advantage genes. This study also analyzed glutamate racemase (MurI) as potential drug target. MurI catalyzes D-glutamate biosynthesis in most eubacteria and some microorganisms also synthesize D-glutamate using D-amino-acid transaminase (Dat). However, in mycobacteria the presence of Dat has not been investigated. In this study, genetic and biochemical evidence is presented for the presence of both pathways in Mycobacterium smegmatis. Thus, the presence of a redundant Dat/MurI pathway negates that specific MurI inhibitors would be effective in the treatment of mycobacterial infections, suggesting that mixed inhibitors or combination of single inhibitors may be necessary