|Janagama, Harish -|
|Senthilkumar, T -|
|Rodriquez, Marcela -|
|Smith, Issar -|
|Sreevatsan, Srinand -|
Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 1, 2009
Publication Date: November 1, 2009
Citation: Janagama, H.K., Senthilkumar, T., Bannantine, J.P., Rodriquez, M.G., Smith, I., Paustian, M.L., Mcgarvey, J.A., Sreevatsan, S. 2009. Identification and Functional Characterization of the Iron Dependent Regulator (IdeR) of Mycobacterium avium subspecies paratuberculosis. Microbiology. 155:3683-3690. Interpretive Summary: The ability to uptake iron from the environment and to maintain appropriate intracellular iron concentrations is essential for almost all forms of life. In Mycobacterium tuberculosis a protein called IdeR is essential for iron uptake and homeostasis. We identified a gene (map2827) in Mycobacterium avium subsp. paratuberculosis that encodes a protein that is >93% identical to the M. tuberculosis IdeR. To demonstrate that this protein functions in the same way as IdeR, we showed that it could bind to an IdeR specific DNA sequence termed an “iron box”. We also demonstrated that MAP2827 could repress the expression of the gene mbtB under high iron concentrations and de-repress its expression under low iron conditions, just like the M. tuberculosis IdeR. Taken together these data suggest that MAP2827 is an IdeR homolog in M. avium subsp. paratuberculosis. Lastly, we identified some minor differences between MAP2827 isolated from M. avium subsp. paratuberculosis associated with cattle Vs. sheep, suggesting that this gene may be involved in the subtle differences observed between these two pathovars.
Technical Abstract: Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep, has unique iron requirements in that it is mycobactin-dependent for cultivation in vitro. The iron-dependent regulator (IdeR) is a well-characterized global regulator responsible for maintaining iron homeostasis in Mycobacterium tuberculosis (MTB). We identified an orthologous segment in the MAP genome, MAP2827, with >93 % amino acid identity to MTB IdeR. Electrophoretic mobility shift assays and DNase protection assays confirmed that MAP2827 binds the 19 bp consensus motif (iron box) on the MAP genome. Sequencing of MAP2827 from multiple isolates revealed a non-synonymous change (R91G) exclusive to sheep strains. Reporter gene assays and quantitative real-time RT-PCR assays in two diverse MAP strains and in an ideR deletion mutant of M. smegmatis (mc squared 155) suggested that both sheep MAP IdeR (sIdeR) and cattle MAP IdeR (cIdeR) repress mbtB transcription at high iron concentrations and relieve repression at low iron concentrations. On the other hand, bfrA (an iron storage gene) was upregulated by cIdeR when presented with MTB or the cattle MAP bfrA promoter, and was downregulated by sIdeR in the presence of MTB, or sheep or cattle MAP bfrA promoters, at high iron concentrations. The differential iron regulatory mechanisms between IdeR-regulated genes across strains may contribute to the differential growth or pathogenic characteristics of sheep and cattle MAP strains. Taken together, our study provides a possible reason for mycobactin dependency and suggests strong implications in the differential iron acquisition and storage mechanisms in MAP.