AUTHOR=Divya T. V. , Acharya Celin 

TITLE=AzuR From the SmtB/ArsR Family of Transcriptional Repressors Regulates Metallothionein in Anabaena sp. Strain PCC 7120

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.782363

DOI=10.3389/fmicb.2021.782363

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Anabaena</italic> sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of <italic>Anabaena</italic> 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 <italic>azuR</italic> is located within the <italic>znuABC</italic> operon but in the opposite orientation and remotely away from the <italic>nmtA</italic> locus. Sequence analysis of AzuR revealed a high degree of sequence identity with <italic>Synechococcus</italic> SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in <italic>Escherichia coli</italic> 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 <italic>nmtA</italic> open reading frame (ORF)] identified the presence of a 12–2–12 imperfect inverted repeat as the <italic>cis</italic>-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 Zn<sup>2+</sup>, Cd<sup>2+</sup>, Cu<sup>2+</sup>, Co<sup>2+</sup>, Ni<sup>2+</sup>, Pb<sup>2+</sup>, and Mn<sup>2+</sup>. The metal-dependent dissociation of protein–DNA complexes suggested the negative regulation of metal-inducible <italic>nmtA</italic> expression by AzuR. Overexpression of <italic>azuR</italic> in its native strain <italic>Anabaena</italic> 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of <italic>Anabaena</italic> MT by an α5 SmtB/ArsR metalloregulator AzuR.</p>