A Novel Cyanobacterial SmtB/ArsR Family Repressor Regulates the Expression of a CPx-ATPase and a Metallothionein in Response to Both Cu(I)/Ag(I) and Zn(II)/Cd(II)

Tong Liu,Susumu Nakashima,Kazunobu Hirose,Mineo Shibasaka,Maki Katsuhara,Bunichi Ezaki,David P Giedroc,Kunihiro Kasamo

doi:10.1074/jbc.m310560200

Abstract

A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions.

Highlights

Transition metal ions such as zinc, copper, iron, and manganese are essential trace elements that play integral catalytic functions in myriad metalloenzymes and electron transfer in all organisms [1,2,3]
Despite the progress made in understanding the structure, mechanism, and metal selectivity of diverse metal sensor proteins [17, 33,34,35,36], many questions remain as to how specific heavy metal ions are sensed in a common cytosol, when challenged with multiple metal ions [37, 38]
The derepression of this complex metal homeostasis system is strongly regulated by both monovalent (Cu(I) and Ag(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions. This is achieved by the direct binding of each of these metals to BxmR, which in turn, reduces the affinity of the regulator for the operator/ promoter region, thereby inducing transcription. These findings suggest that the homeostasis governing storage and trafficking of multiple heavy metal ions in living cells could be mediated by a system that minimally employs a metallothionein, a metal transporter, and a metalloregulatory protein, analogous to zinc homeostasis in mammalian cells [43]

Summary

Introduction

Transition metal ions such as zinc, copper, iron, and manganese are essential trace elements that play integral catalytic functions in myriad metalloenzymes and electron transfer in all organisms [1,2,3]. The intensity of the individual gel bands (Ii) was quantitated using an Image Analysis System/Molecular Analyst software (Bio-Rad) and converted to mol fractions of the ith DNA species, ⌰i, using ⌰i ϭ Ii/⌺Ii. In Vivo Analysis of Expression of bmxR, bmtA, and bxa1 Induced by Heavy Metal Ions Using Real Time Quantification RT-PCR (rQRTPCR)—For the experiments designed to examine the metal concentration dependence of expression, O. brevis cells (about 0.1 g wet weight) were suspended in 200 ml of a medium containing ZnSO4 (4, 8, 16, 32, 60, 92, or 122 ␮M), CdCl2 (2, 4, 8, 16, or 32 ␮M), CuSO4 (4, 8, 12, 16, 24, or 32 ␮M), and AgNO3 (2, 4, 7, 10, 15, or 18 ␮M) salts, respectively.

Results

Conclusion