title: Identification and functional characterization of the iron-dependent regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis.
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(2)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.
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, Saint Paul, MN, USA.
authors.forename: Harish K
authors.initials: HK
authors.lastname: Janagama
authors.affiliation: Department of Veterinary Population Medicine, University of Minnesota, Saint Paul, MN, USA.
authors.forename: T M A
authors.initials: TMA
authors.lastname: Senthilkumar
authors.affiliation: National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Bannantine
authors.affiliation: PHRI, UMNDJ-New Jersey Medical School, Newark, NJ, USA.
authors.forename: G Marcela
authors.initials: GM
authors.lastname: Rodriguez
authors.affiliation: PHRI, UMNDJ-New Jersey Medical School, Newark, NJ, USA.
authors.forename: Issar
authors.initials: I
authors.lastname: Smith
authors.affiliation: National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Paustian
authors.affiliation: Foodborne Contaminants Research Unit, Agricultural Research Service, United States Department of Agriculture, Albany, CA, USA.
authors.forename: Jeffery A
authors.initials: JA
authors.lastname: McGarvey
authors.affiliation: Department of Veterinary Biomedical Sciences, University of Minnesota, Saint Paul, MN, USA.|Department of Veterinary Population Medicine, University of Minnesota, Saint Paul, MN, USA.
authors.forename: Srinand
authors.initials: S
authors.lastname: Sreevatsan
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002417:Cattle; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012756:Sheep
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; D007501:Iron
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