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

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 sp. strain PCC 7120 and demonstrated its role in...

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Published in:Frontiers in microbiology Vol. 12; p. 782363
Main Authors: Divya, T V, Acharya, Celin
Format: Journal Article
Language:English
Published: Switzerland Frontiers Media S.A 12-01-2022
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Summary: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 sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of 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 is located within the operon but in the opposite orientation and remotely away from the locus. Sequence analysis of AzuR revealed a high degree of sequence identity with SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in 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 open reading frame (ORF)] identified the presence of a 12-2-12 imperfect inverted repeat as the -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 , Cd , Cu , Co , Ni , Pb , and Mn . The metal-dependent dissociation of protein-DNA complexes suggested the negative regulation of metal-inducible expression by AzuR. Overexpression of in its native strain 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of MT by an α5 SmtB/ArsR metalloregulator AzuR.
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Edited by: Rob Van Houdt, Belgian Nuclear Research Centre, Belgium
Reviewed by: Vicente Mariscal, Institute of Plant Biochemistry and Photosynthesis, Spanish National Research Council (CSIC), Spain; Andrés González, Aragón Institute for Health Research (IIS Aragón), Spain
This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology
ISSN:1664-302X
1664-302X
DOI:10.3389/fmicb.2021.782363