Abstract
Zinc has an abounding occurrence in the prokaryotes and plays paramount roles including catalytic, structural, and regulatory. Zinc uptake regulator (Zur), a Fur family transcriptional regulator, is connoted in maintaining zinc homeostasis in the pathogenic bacteria by binding to zinc and regulating the genes involved in zinc uptake and mobilization. Zinc homeostasis has been marginally scrutinized in Bacillus anthracis, the top-rated bio-terror agent, with no decipherment of the role of Zur. Of the three Fur family regulators in B. anthracis, BAS4181 is annotated as a zinc-specific transcriptional regulator. This annotation was further substantiated by our stringent computational and experimental analyses. The residues critical for zinc and DNA binding were delineated by homology modeling and sequence/structure analysis. ba zur existed as a part of a three-gene operon. Purified BaZur prodigiously existed in the dimeric form, indicated by size exclusion chromatography and blue native-polyacrylamide gel electrophoresis (PAGE). Computational and manual strategies were employed to decipher the putative regulon of ba zur, comprising of 11 genes, controlled by six promoters, each harboring at least one Zur box. The DNA binding capability of the purified BaZur to the upstream regions of the ba zur operon, yciC, rpmG, znuA, and genes encoding a GTPase cobalamine synthesis protein and a permease was ascertained by electrophoretic mobility shift assays. The regulon genes, implicated in zinc uptake and mobilization, were mostly negatively regulated by BaZur. The ba zur expression was downregulated upon exposure of cells to an excess of zinc. Conversely, it exhibited a marked upregulation under N, N, N′, N′-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) mediated zinc-depleted environment, adding credence to its negative autoregulation. Moreover, an increase in the transcript levels of the regulon genes znuA, rpmG, and yciC upon exposure of cells to TPEN connoted their role in combating hypo-zincemic conditions by bringing about zinc uptake and mobilization. Thus, this study functionally characterizes Zur of B. anthracis and elucidates its role in maintaining zinc homeostasis.
Highlights
Zinc has the stature of being the second most plenteously occurring transition element in the living systems after iron
Owing to the multifaceted roles that zinc plays in the bacteria, both intracellular and extracellular zinc homeostasis is cardinal for its survival
Our study provides an insightful investigation of the previously unexplored Zinc uptake regulator (Zur) of B. anthracis and its connotation in maintaining zinc homeostasis in the pathogen
Summary
Zinc has the stature of being the second most plenteously occurring transition element in the living systems after iron. The metal acts as an electrophile or Lewis acid in most of the hydrolytic reactions, thereby catalyzing them and is incorporated into a variety of metalloenzymes, storage proteins, and transcription factors (Falchuk, 1993; Coleman, 1998; Auld, 2001; Outten and O’halloran, 2001; Cerasi et al, 2013) It can act both as an antioxidant, protecting the sulfhydryl groups of proteins from the attack by the free radicals, and as a combatant preventing the formation of free radicals, perpetuating the structural stability and functionality of several proteins (Bray and Bettger, 1990). Zinc can be toxic to the cells at a concentration higher than the normal by either blocking the thiols of the proteins or by competitive mismetallation with the binding sites of the other metal ions in them, precluding various physiological processes (Beard et al, 1995; Mills et al, 2002; Imlay, 2014; Chandrangsu et al, 2017)
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