The Components of the Unique Zur Regulon of Cupriavidus metallidurans Mediate Cytoplasmic Zinc Handling

Abstract

Zinc is an essential trace element and at the same time it is toxic at high concentrations. In the beta-proteobacterium the highly efficient removal of surplus zinc from the periplasm is responsible for its outstanding metal resistance. Rather than having a typical Zur-dependent, high-affinity ATP-binding cassette transporter of the ABC protein superfamily for zinc uptake at low concentrations, instead has the secondary zinc importer ZupT of the ZRT/IRT (ZIP) family. It is important to understand, therefore, how this zinc-resistant bacterium copes when it is exposed to low zinc concentrations. Members of the Zur regulon in were identified by comparing the transcriptomes of a Δ mutant and its parent strain. The consensus sequence of the Zur-binding box was derived for the promoter-regulatory region using a truncation assay. The motif was used to predict possible Zur-boxes upstream of Zur regulon members. Binding of Zur to these boxes was confirmed. Two Zur-boxes upstream of the gene, encoding a putative zinc chaperone, proved to be required for complete repression of and its downstream genes in cells cultivated in mineral salts medium. A Zur box upstream of each of and permitted low-expression level of these genes plus their up-regulation under zinc starvation conditions. This demonstrates a compartmentalization of zinc homeostasis in with the periplasm being responsible for removal of surplus zinc and cytoplasmic components for management of zinc as an essential co-factor, with both compartments connected by ZupT. Elucidating zinc homeostasis is necessary to understand both host-pathogen interactions and performance of free-living bacteria in their natural environment. acquires zinc under low zinc concentrations by the Zur-controlled ZnuABC importer of the ABC superfamily, and this was also the paradigm for other bacteria. In contrast, the heavy metal-resistant bacterium achieves high tolerance to zinc due to sophisticated zinc handling and efflux systems operating on periplasmic zinc ions, so that removal of surplus zinc is a periplasmic feature in this bacterium. It is shown here that this process is augmented by management of zinc through cytoplasmic zinc chaperones, whose syntheses are controlled by the Zur regulator. This demonstrates a new mechanism to organize zinc homeostasis through compartmentalization.

Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis

“…Among those, we found 12 motifs located in the promoter-operator region of Zur-regulated genes ( Table 6, indicated by boldface, and Tables 3 and 4, indicated by asterisks), i.e., the predicted ZnuABC transporter (MSMEG_6045-6047) and a second ABC transporter (MSMEG_6052), four CobW-like proteins (MSMEG_1122, MSMEG_1123, MSMEG_6048, MSMEG_6069), a methyltransferase (MSMEG_6055), and the set of genes encoding ARPs (MSMEG_6065-6068, MSMEG_6070). Some promoter-operators harbored more than one predicted motif (MSMEG_6047, MSMEG_6048, MSMEG_6055), which suggests a complex regulation of these genes, as already described for Streptomyces coelicolor and Cupriavidus metallidurans (41,43). Notably, smtB-zur was also preceded by a putative Zur binding site.…”

“…Homologous genes in MAP and MTB have been shown to be regulated zinc dependently by Zur (28,29). In Cupriavidus metallidurans, CobW proteins substitute for missing zinc importers (41). During zinc starvation, expression of genes rpmB, rpmG, rpsN, rpsR, and rpmE2 encoding ARPs was highly induced and regulated by Zur.…”

Comparative Insights Into the Complete Genome Sequence of Highly Metal Resistant Cupriavidus metallidurans Strain BS1 Isolated From a Gold–Copper Mine

“…The strain BS1 shows a high degree of conservation on gene and amino acid sequence level as well as the gene loci synteny of the metal uptake systems encoding genome regions in comparison to strain CH34. In contrast to the transportome, the knowledge about the direct interaction with the metal handling proteins of the cytoplasm, cytoplasmic membrane and the periplasm, which form the metal repositories as the second pillar of bacterial metal homeostasis (Herzberg et al, 2014b), is underrepresented but further investigation is in progress (Herzberg et al, 2014a(Herzberg et al, , 2016Bütof et al, 2017;Chandrangsu et al, 2019). In order to adapt the cellular metal homeostasis to the specific environmental conditions, its regulation is of central importance as a third pillar.…”

Bacterial zinc uptake regulator proteins and their regulons

“…The identification of a true member of a Zur regulon therefore also requires: (i) the presence of Zur boxes, (ii) proof that Zur binds to the promoter region of the selected gene, and/or (iii) in vivo reporter assays. A recent study illustrates these requirements dramatically: Of the hundreds of genes that were differentially expressed in a Δ zur mutant of Cupriavidus metallidurans , only 11 had computationally identified Zur boxes, and of those, only four were experimentally verified to be true Zur boxes [ 92 ].…”