Early Developments
Progress made during the period of 1993-2008 provided key molecular tools for development of genetic systems based on transformation and transfection, and the use of reporter genes (e.g.
β-galactosidase, firefly and bacterial luciferases, green fluorescent protein). One important use of these expression systems was the determination of consensus promoter sequence. It was found that this consensus for many MAP genes involved the hexanucleotide sequences 5,-TGMCGT-3, and 5,-CGGCCS-3, centred approximately 35 and 10 base pairs (bp) upstream from the transcription startpoints. These sequences do not correspond to the consensus hexanucleotides of Escherichia coli promoters (Bannantine et al., 1997). In addition, a novel promoter upstream sequence was identified for genes up- or downregulated by the iron repressor IdeR corresponding to the consensus 5,-TWAGGTWAGSCTWACCTWA-3,, where W = A/T and S = G/C (Janagama et al., 2009). It was shown that IdeR binds to the -10 box adjacent to the transcriptional start site repressing the expression of iron storage genes (e.g. bfrA) (Goldetal., 2001). Upon iron starvation, IdeR is released from the promoter region allowing for the expression of iron acquisition genes (e.g. bfd).
The systems described above, along with the completion of the MAP sequencing project (Li et al., 2005), led to the development of the functional genomics through generation and characterization of mutants via transposon mutagenesis, gene replacement and complementation strategies. These pioneering studies described in the previous edition of this book (Chacon and Barletta, 2010) are summarized in Table 7.1, while in the rest of the chapter we will focus on further developments in this line of research from 2009 to the present. These advances have significantly increased the understanding of MAP molecular pathogenesis to the level of progress made for more tractable mycobacteria.
7.3
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