Transcriptional Control of the Bradyrhizobium japonicum irr Gene Requires Repression by Fur and Antirepression by Irr

Thomas H Hohle,Mark R O'Brian

doi:10.1074/jbc.m110.145979

Abstract

Bradyrhizobium japonicum Fur mediates manganese-responsive transcriptional control of the mntH gene independently of iron, but it also has been implicated in iron-dependent regulation of the irr gene. Thus, we sought to address the apparent discrepancy in Fur responsiveness to metals. Irr is a transcriptional regulator found in iron-limited cells. Here, we show that irr gene mRNA was regulated by both iron and manganese, and repression occurred only in the presence of both metals. Under these conditions, Fur occupied the irr promoter in vivo in the parent strain, and irr mRNA expression was derepressed in a fur mutant. Under low iron conditions, the irr promoter was occupied by Irr, but not by Fur, and control by manganese was lost. Fur occupancy of the irr promoter was dependent on manganese, but not iron, in an irr mutant, suggesting that Irr normally interferes with Fur binding. Correspondingly, regulation of irr mRNA was dependent only on manganese in the irr strain. The Irr binding site within the irr promoter partially overlaps the Fur binding site. DNase I footprinting analysis showed that Irr interfered with Fur binding in vitro. In addition, Fur repression of transcription from the irr promoter in vitro was relieved by Irr. We conclude that Fur mediates manganese-dependent repression of irr transcription and that Irr acts as an antirepressor under iron limitation by preventing Fur binding to the promoter.

Highlights

The Irr protein is the primary global regulator of iron homeostasis in Bradyrhizobium japonicum [4] and has been described in other ␣-proteobacteria as well [5,6,7]
The irr gene is modestly regulated by iron at the mRNA level, which is lost in a fur mutant, whereas Fur mediates manganese-dependent con
We examined irr and mntH mRNA levels by quantitative real-time PCR in cells grown in media containing different combinations of high and low iron and manganese concentrations (Fig. 2, A and B)

Summary

Introduction

The Irr protein is the primary global regulator of iron homeostasis in Bradyrhizobium japonicum [4] and has been described in other ␣-proteobacteria as well [5,6,7]. Irr recognizes and binds to an iron control element within the promoter of target genes [8]. Binding of Irr to the iron control element of a negatively regulated gene is sufficient to repress transcription in vitro [9], but the molecular basis of the positive control is unknown. Irr and mntH are the only genes known to be direct targets of B. japonicum Fur [23,24] Both gene promoters contain a conserved motif of three imperfect direct repeat hexamers necessary for Fur binding and transcriptional repression activity. The irr gene is modestly regulated by iron at the mRNA level, which is lost in a fur mutant, whereas Fur mediates manganese-dependent con-. We show that Fur mediates manganese-dependent repression of both the irr and mntH genes at the mRNA level. An explanation for the apparent discrepancy in Fur function is provided, and a novel function for Irr has been identified

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