Both Corynebacterium diphtheriae DtxR(E177K) and Mycobacterium tuberculosis IdeR(D177K) are dominant positive repressors of IdeR-regulated genes in M-tuberculosis

The diphtheria toxin repressor (DtxR) is an important iron-dependent transcriptional regulator of known virulence genes in Corynebacterium diphtheriae. The mycobacterial iron-dependent repressor (IdeR) is phylogenetically closely related to DtxR, with high amino acid similarity in the DNA binding and metal ion binding site domains. We have previously shown that an iron-insensitive, dominant-positive dtxR(E175K) mutant allele from Corynebacterium diphtheriae can be expressed in Mycobacterium tuberculosis and results in an attenuated phenotype in mice (Y. C. Manabe, B. J. Saviola, L. Sun, J. R. Murphy, and W. R. Bishai, Proc. Natl. Acad. Sci. USA 96:12844-12848, 1999). In this paper, we report the M. tuberculosis IdeR(D177K) strain that has the cognate point mutation. We tested four known and predicted IdeR-regulated gene promoters (mbtI, Rv2123, Rv3402c, and Rv1519) using a promoterless green fluorescent protein (GFP) construct. GFP expression from these promoters was abrogated under low-iron conditions in the presence of both IdeR(D177K) and DtxR(E175K), a result confirmed by reverse transcription-PCR. The IdeR regulon can be constitutively repressed in the presence of an integrated copy of ideR containing this point mutation. These data also suggest that mutant IdeR(D177K) has a mechanism similar to that of DtxR(E175K); iron insensitivity occurs as a result of SH3-like domain binding interactions that stabilize the intermediate form of the repressor after ancillary metal ion binding. This construct can be used to elucidate further the IdeR regulon and its virulence genes and to differentiate these from genes regulated by SirR, which does not have this domain.