Abstract
Metallothioneins (MTs) are cysteine-rich, metal-sequestering cytosolic proteins that play a key role in maintaining metal homeostasis and detoxification. We had previously characterized NmtA, a MT from the heterocystous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of Anabaena NmtA by AzuR (Alr0831) belonging to the SmtB/ArsR family of transcriptional repressors. There is currently no experimental evidence for any functional role of AzuR. It is observed that azuR is located within the znuABC operon but in the opposite orientation and remotely away from the nmtA locus. Sequence analysis of AzuR revealed a high degree of sequence identity with Synechococcus SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in Escherichia coli and purified it by chitin affinity chromatography. Far-UV circular dichroism spectroscopy indicated that the recombinant AzuR protein possessed a properly folded structure. Glutaraldehyde cross-linking and size-exclusion chromatography revealed that AzuR exists as a dimer of ∼28 kDa in solution. Analysis of its putative promoter region [100 bp upstream of nmtA open reading frame (ORF)] identified the presence of a 12–2–12 imperfect inverted repeat as the cis-acting element important for repressor binding. Electrophoretic mobility shift assays (EMSAs) showed concentration-dependent binding of recombinant dimeric AzuR with the promoter indicating that NmtA is indeed a regulatory target of AzuR. Binding of AzuR to DNA was disrupted in the presence of metal ions like Zn2+, Cd2+, Cu2+, Co2+, Ni2+, Pb2+, and Mn2+. The metal-dependent dissociation of protein–DNA complexes suggested the negative regulation of metal-inducible nmtA expression by AzuR. Overexpression of azuR in its native strain Anabaena 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of Anabaena MT by an α5 SmtB/ArsR metalloregulator AzuR.
Highlights
Trace metal ions are crucial for most aspects of metabolism in the prokaryotic cells
The ArsR-SmtB family of transcriptional metalloregulators represses the expression of genes/operons involved in maintaining metal homeostasis or toxic metal detoxification (Osman and Cavet, 2010)
We have largely focused on the role of AzuR in MT regulation, the presence of cis-regulatory elements important for repressor binding at several locations in the Anabaena 7120 genome indicates that AzuR might act as a global transcriptional regulator
Summary
Trace metal ions are crucial for most aspects of metabolism in the prokaryotic cells These are involved in various biological processes like enzymatic reactions that require metal ions as cofactors, for folding and structural stabilization of the proteins or for the maintenance of the metal-sensing regulatory factors (Rees, 2002; Bertini et al, 2007; Chandrangsu et al, 2017). Metallothioneins (MTs) are cysteine-rich, low-molecularweight, metal-sequestering proteins that are known to bind metal ions via metal–thiolate clusters and are involved in maintaining homeostasis of physiologically important metals like zinc (Zn2+) and copper (Cu2+) (Klaassen et al, 1999; Blindauer, 2011). The smtA gene expression is negatively regulated by a zinc responsive transcriptional repressor SmtB (Erbe et al, 1995; Turner et al, 1996) of the SmtB/ArsR family of transcriptional regulatory proteins. Derepression of transcription by such regulators results from direct binding of the metal to the repressor, which inhibits its binding to the operator/promoter (O/P) region of the gene under regulation (Busenlehner et al, 2003; Osman and Cavet, 2010)
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