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Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr).

ABSTRACT: The transcription factor iron response regulator (Irr) is a key regulator of iron homeostasis in the nitrogen-fixating bacterium Bradyrhizobium japonicum Irr acts by binding to target genes, including the iron control element (ICE), and is degraded in response to heme binding. Here, we examined this binding activity using fluorescence anisotropy with a 6-carboxyfluorescein-labeled ICE-like oligomer (FAM-ICE). In the presence of Mn²⁺, Irr addition increased the fluorescence anisotropy, corresponding to formation of the Irr-ICE complex. The addition of EDTA to the Irr-ICE complex reduced fluorescence anisotropy, but fluorescence was recovered after Mn²⁺ addition, indicating that Mn²⁺ binding is a prerequisite for complex formation. Binding activity toward ICE was lost upon introduction of substitutions in a His-cluster region of Irr, revealing that Mn²⁺ binds to this region. We observed that the His-cluster region is also the heme binding site; results from fluorescence anisotropy and electrophoretic mobility shift analyses disclosed that the addition of a half-equivalent of heme dissociates Irr from ICE, likely because of Mn²⁺ release due to heme binding. We hypothesized that heme binding to another heme binding site, Cys-29, would also inhibit the formation of the Irr-ICE complex because it is proximal to the ICE binding site, which was supported by the loss of ICE binding activity in a Cys-29-mutated Irr. These results indicate that Irr requires Mn²⁺ binding to form the Irr-ICE complex and that the addition of heme dissociates Irr from ICE by replacing Mn²⁺ with heme or by heme binding to Cys-29.

SUBMITTER: Nam D

PROVIDER: S-EPMC7415966 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We previously identified a five-member family of hemin-binding proteins (Hbp's) of Bartonella quintana that bind hemin on the outer surface but share no homology with known bacterial heme receptors. Subsequently, we demonstrated that expression of the hbp family is significantly influenced by oxygen, heme, and temperature conditions encountered by the pathogen in the human host and the body louse vector; e.g., we observed a dramatic (>100-fold) increase in hbpC transcript levels in response to the "louse-like" temperature of 30 degrees C. The goal of the present study was to identify a transcription factor(s) involved in the coordinated and differential regulation of the hbp family. First, we used quantitative real-time PCR (qRT-PCR) to show that the same environmental conditions generate parallels in the transcript profiles of four candidate transcriptional regulators (Irr, Fur, RirA, and BatR) described in the order Rhizobiales, with the greatest overall change in the transcription of irr (a >5-fold decrease) at a "louse-like" temperature, suggesting that Irr may function as an hbpC repressor. Second, a B. quintana strain hyperexpressing Irr was constructed; it exhibits a "bloodstream-like" hbp transcript profile in the absence of an environmental stimulus (i.e., hbpC is repressed and hbpA, hbpD, and hbpE mRNAs are relatively abundant). Furthermore, when this strain is grown at a "louse-like" temperature, an inversion of the transcript profile occurs, where derepression of hbpC and repression of hbpA, hbpD, and hbpE are readily evident, strongly suggesting that Irr and temperature influence hbp family expression. Third, electrophoretic mobility shift analyses show that recombinant Irr binds specifically to the hbpC promoter region at a sequence that is highly conserved in Bartonella hbp genes, which we designated the hbp family box, or "H-box." Fourth, we used the H-box to search the B. quintana genome and discovered a number of intriguing open reading frames, e.g., five members of a six-member family of cohemolysin autotransporters. Finally, qRT-PCR data regarding the effects of Fur and RirA overexpression on the hbp family are provided; they show that Fur's effect on the hbp family is relatively minor but RirA generates a "bloodstream-like" hbp transcript profile in the absence of an environmental stimulus, as observed for the Irr-hyperexpressing strain.

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Mechanistic insights into heme-mediated transcriptional regulation via a bacterial manganese-binding iron regulator, iron response regulator (Irr).

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