Isolationof Deletion Mutants and Complementation Studies

Researchers in the field have continued using shuttle phasmids to introduce specific mutations in target genes. Scandurra et al. (2010) inacti­vated ppiA (MAP_0011) encoding a peptidyl- prolyl cis-trans isomerase in a clinical MAP isolate from New Zealand cattle strain WAg915 (Scandurra et al., 2010).

The role of the alter­native sigma factor, SigH, was elucidated by the construction of a knockout mutant (Ghosh et al., 2013). Using this methodology, dele­tion mutants of leuD (MAP_3025c) essential for leucine biosynthesis; mpt64 (MAP_3290 c) encoding an immunogenic protein; and secA2 (MAP_1534), a preprotein translocase subunit, were generated (Chen et al., 2012; Faisal et al., 2013). Likewise, a functionally uncharacterized gene (mptD; MAP_3733c) was shown to en­code a factor required for metabolic adaptation and persistence of MAP in the host (Meissner et al., 2014). Both manuscripts reported mutant complementation difficulties due to inefficient complementation or the complex organization of the operon where the gene was located, re­spectively. Indeed this effect seems to be depend­ent upon which gene is complemented as it was successfully accomplished for the alternative global gene regulator SigL with the use of the integrating vector pMV306 (Ghosh et al., 2014). Thus, our recommendation would be to attempt complementation using an integrating vector. If this is unsuccessful, a multi-copy vector may be used. In both cases, it is most important to maxi­mize transformation efficiency for the particular mutant strain, as this may be different from the wild-type strain. For this approach, there are three markers that have been used successfully in MAP: kanamycin, hygromycin and apramy- cin (Table 7.1). A method to unmark the original mutation may ameliorate this problem, allowing the use of one selection marker for the comple­mentation step. This procedure has been applied to M. bovis, M. smegmatis and M. tuberculosis, but the slow growth of MAP could make its applica­tion more challenging (Bardarov et al., 2002).

7.6

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