|Kumas T.m.a., Senthil|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/18/2008
Publication Date: 4/18/2008
Citation: Janagama, H.K., Bannantine, J.P., Kumas T.M.A., S., Rodriguez, G.M., Smith, I., Sreevatsan, S. 2008. Functional Characterization of Iron Dependent Regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis [abstract]. Johne's Disease Integrated Program. p. 31 Interpretive Summary:
Technical Abstract: In this study we investigated an iron dependent regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis (MAP). IdeR is a transcriptional factor that plays a global iron regulatory role in Mycobacterium tuberculosis (MTB) with a 19-bp recognition sequence. IdeR recognition sites within MAP genome were identified using computational methods. Analysis of the annotated sequence of MAP IdeR (MAP2827c) did not share amino acid homology to IdeR family of proteins. The complementary sequence of MAP2827c was identified to carry a second open reading frame (MAP2827) that shared >93% amino acid identity to MTB IdeR and this is speculated to be the functional protein in MAP. The consensus sequence containing regions were amplified to demonstrate that IdeR of MAP recognizes and binds to a consensus region by electro mobility shift assays (EMSA). The physical binding of IdeR to consensus sequences was functionally characterized using reporter gene assays in an IdeRnull M. smegmatis (SM3) background. EMSA results suggested that both MAP2827 and MAP2827c demonstrated a sequence specific binding to MAP mbtB promoter. We further investigated if MAP2827 and/or MAP2827c regulated gene expression of MAP mbtB (iron storage gene) using reporter gene assays. Our results demonstrated that MAP2827 up regulated mbtB transcription under low iron condition while repressing transcription under high iron condition, a classic feature of MTB IdeR. Furthermore, MAP2827 only but not MAP2827c bind the promoters of MTB mbtB (iron acquisition gene) and bfrB (iron storage gene) and regulate transcription similar to MTB IdeR. Despite a sequence specific binding to MAP mbtB promoter, MAP2827c did not regulate transcription under the current experimental conditions. It is possible that MAP2827c triggers transcription of a part of IdeR regulated genes under special circumstances. In summary, our results suggest that MAP2827 functions as an IdeR in MAP.