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.

Fig. 1.

Electrophoretic mobility shift assay to demonstrate specificity of MAP2827 protein with the predicted promoters. Recombinant MAP2827 was reacted with mbtB and bfrA promoters for this assay. MAP IdeR reacts with the promoters only in presence of nickel (second and third lanes from left). P, Promoter; +/− IB, presence/absence of iron box; cold, unlabelled DNA; oligo, non-specific DNA.

Fig. 2.

DNase footprint assay showing the binding of the MAP2827 protein to the iron box sequence located on the MAP mbtB promoter. Red peaks in the electropherogram represent DNA alone and black peaks represent DNA pre-incubated with MAP2827 (bottom panel). Both reactions were partially digested with DNase I and analysed by capillary electrophoresis in a genetic analyser (Applied Biosystems 3130xl). The top panel shows an electropherogram of the promoter sequence with the terminal bases identified (iron box sequence is underlined). The protected region is indicated which overlaps the iron box sequence. Note the difference in the heights of black peaks (lost completely) relative to red peaks, indicating protection from DNase digestion.

Fig. 3.

Genotypes of SM3 constructs used to transform M. smegmatis and MAP transformants used in reporter assays. The polymorphisms present in the promoter regions of MAP mbtB and bfrA promoters are highlighted. Red boxes, iron boxes; black arrow, ideR ORF; c, cattle; s, sheep.

Fig. 4.

Reporter activity of mbtB promoter fusions of MTB mbtB, cattle MAP mbtB or sheep MAP mbtB to lacZ driven by cIdeR, sIdeR or MTB IdeR. Under iron-rich conditions, cIdeR, sIdeR and MTB IdeR repressed transcription of mbtB. Open bars, transcription in iron-depleted medium; filled bars, transcription in iron-rich medium. Means of three independent experiments, each performed in triplicate, are shown.

Fig. 5.

Reporter activity of bfrA promoter fusions of MTB bfrA, cattle MAP bfrA or sheep MAP bfrA to lacZ driven by cIdeR, sIdeR or MTB IdeR. Under iron-rich conditions, cIdeR, sIdeR and MTB IdeR showed repressed transcription with the sheep bfrA promoter. Open bars, transcription in iron-depleted medium; closed bars, transcription in iron-rich medium. Means of three independent experiments, each performed in triplicate, are shown.

Fig. 6.

Reporter activity of mbtB and bfrA promoter fusions to lacZ in two MAP strains. The line indicates the background reporter activity of the empty vector (pSM128). Both mbtB and bfrA are active in MAP. The activity of MAP mbtB promoters is lower in comparison to that of MTB mbtB. The bfrA promoter from a sheep MAP strain showed consistently lower activity under high-iron conditions, as shown in the SM3 background. Open bars, transcription in iron-depleted medium; closed bars, transcription in iron-rich medium; c, cattle; s, sheep. Means of three independent experiments, each performed in triplicate, are shown.