|Mcgarvey, Jeffery - Jeff|
Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/11/2009
Publication Date: 11/1/2009
Citation: Janagama, H.K., Senthilkumar, T., Bannantine, J.P., Rodriquez, M.G., Smith, I., Paustian, M., Mcgarvey, J.A., Sreevatsan, S. 2009. Identification and Functional Characterization of the Iron-dependent Regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis. Microbiology. 155(Pt 11):3683-3690. Interpretive Summary: This manuscript describes the thorough characterization of a transcriptional regulator called ideR. The protein encoded by this gene regulates iron acquisition and storage mechanisms by binding to genes that promote these activities in the bacterium that causes Johne’s disease. We discovered that there is a very slight genetic difference in the ideR regulators in the cattle isolates when compared to the sheep isolates of Mycobacterium avium subspecies paratuberculosis (aka. MAP, the bacterium that causes Johne’s disease). The key discovery from this work is that the ideR regulator from sheep isolates of Mycobacterium avium subspecies paratuberculosis inhibits both iron storage and iron acquisition when iron is present in the environment. However, the ideR regulator from cattle isolates only inhibits iron acquisition, not iron storage. Findings from this study have important implications for the differences observed between cattle and sheep strains of Mycobacterium avium subspecies paratuberculosis.
Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP), a ruminant pathogen, has unique iron requirements based on the observation that it is mycobactin dependent for successful cultivation in vitro. Thus an elucidation of iron regulation in MAP is expected to provide an understanding of its survival in a variety of environments. Iron dependent regulator (IdeR) is very well characterized as a global regulator in maintaining iron homeostasis of Mycobacterium tuberculosis (MTB). We identified that the complementary sequence (MAP2827) of the annotated MAP IdeR (MAP2827c) shared >93% amino acid identity to MTB IdeR. Our results suggested that recombinant MAP2827 protein binds the 19 base pair consensus motif (Iron box) on the MAP genome as demonstrated by gel mobility shift assays and DNase protection assays. Re-sequencing MAP ideR of diverse MAP strains revealed a non-synonymous change (R91G) in sheep MAP strains (n=3) relative to cattle, human or bison isolates (n=9). Reporter gene assays utilizing an IdeR deletion mutant of M. smegmatis (mc2155) suggested that sheep MAP IdeR (sIdeR; with R91G) repressed transcription of both bfrA (iron storage gene) and mbtB (iron acquisition gene) whereas cattle MAP IdeR (cIdeR) repressed transcription of only mbtB but not bfrA under high iron growth condition. Under low iron growth condition both cIdeR and sIdeR de-repressed transcription of mbtB and bfrA. Taken together, the results show that the functional IdeR of MAP is MAP2827 and not the annotated coding sequence on the complementary strand designated as MAP2827c. Furthermore, sIdeR differentially regulates transcription of genes relative to cIdeR. These results underscore their differential iron regulatory mechanisms and may directly contribute to the differential growth characteristics of sheep and cattle MAP strains.