Interplay between the Zur Regulon Components and Metal Resistance in Cupriavidus metallidurans

Abstract

The Zur regulon is central to zinc homeostasis in the zinc-resistant bacterium Cupriavidus metallidurans. It comprises the transcription regulator Zur, the zinc importer ZupT, and three members of the COG0523 family of metal-chaperoning G3Etype GTPases, annotated as CobW1, CobW2, and CobW3. The operon structures of the zur and cobW1 loci were determined. To analyze the interplay between the Zur regulon components and metal resistance, deletion mutants were constructed from the wild-type strain CH34 and various other strains. The Zur regulon components interacted with the plasmid-encoded and chromosomally encoded metal resistance factors to acquire metals from complexes of EDTA and for homeostasis of and resistance to zinc, nickel, cobalt, and cadmium. The three G3E-type GTPases were characterized in more detail. CobW1 bound only 1 Zn atom per mol of protein with a stability constant slightly above that of 2-carboxy-2=-hydroxy-5=-sulfoformazylbenzene (Zincon) and an additional 0.5 Zn with low affinity. The CobW1 system was necessary to obtain metals from EDTA complexes. The GTPase CobW2 is a zinc storage compound and bound 0.5 to 1.5 Zn atoms tightly and up to 6 more with lower affinity. The presence of MgGTP unfolded the protein partially. CobW3 had no GTPase activity and equilibrated metal import by ZupT with that of the other metal transport systems. It sequestered 8 Zn atoms per mol with decreasing affinity. The three CobWs bound to the metal-dependent protein FolE IB2 , which is encoded directly downstream of cobW1. This demonstrated an important contribution of the Zur regulon components to metal homeostasis in C. metallidurans. IMPORTANCE Zinc is an important transition metal cation and is present as an essential component in many enzymes, such as RNA polymerase. As with other transition metals, zinc is also toxic at higher concentrations so that living cells have to maintain strict control of their zinc homeostasis. Members of the COG0523 family of metal-chaperoning GE3-type GTPases exist in archaea, bacteria, and eucaryotes, including humans, and they may be involved in delivery of zinc to thousands of different proteins. We used a combination of molecular, physiological, and biochemical methods to demonstrate the important but diverse functions of COG0523 proteins in C. metallidurans, which are produced as part of the Zur-controlled zinc starvation response in this bacterium.

Behind the Shield of Czc: ZntR Controls Expression of the Gene for the Zinc-Exporting P-Type ATPase ZntA in Cupriavidus metallidurans

“…Even though at least 9 additional transport systems exist that may be able to import zinc ions into the cell, these systems could not completely replace ZupT under "normal" (no added zinc) or zinc starvation conditions (14,15). Interference of the zinc chaperone CobW 3 with some of these other uptake systems is responsible for this effect (62). Adding zinc to DzupT cells with the zupTp::gfp fusion resulted in a downregulation of the reporter activity but not to the level observed for the same fusion in the AE104 parent (Fig.…”

“…S9A). Since deletion of zur results in a constitutively high-level expression of the genes in the Zur regulon (13,62), the increased expression of zupTp::gfp in the DzupT mutant compared to the Dzur mutant indicated the presence of another, currently unknown regulatory circuit for zupT expression in the Dzur mutant. The zupTp::gfp activity in the De4 quadruple efflux mutant, which is not known to have a zinc efflux system, was similar to that of the AE104 parent strain under conditions of zinc deprivation, but zupTp::gfp activity was also elevated in the De4 DzupTp mutant at low zinc concentrations (Fig.…”

Zinc, an unexpected integrator of metabolism?

“…Zinc is a critical cofactor in a great many enzymes – as much as 9% of the entire proteome in eukaryotes and from 5% to 6% in prokaryotes (Andreini et al , 2009). Yet, its singularity has been virtually overlooked [see however studies such as (Bütof et al , 2019)]. Unexpectedly, it was recently observed that the Aspergillus fumigatus mycotoxin, gliotoxin, had a function specifically involving zinc (Seo et al , 2019), and, because this metabolite could act as a specific zinc chelator, this opens up approaches to investigate in depth the role of zinc.…”

The ancient alarmone ZTP and zinc homeostasis in Bacillus subtilis

“…The zinc‐resistant betaproteobacterium Cupriavidus metallidurans contains three COG0523 members annotated CobW1, CobW2, CobW3, plus one FolE IA and two FolE IB ‐type proteins. As discussed elsewhere in detail (Bütof et al , ), CobW1 is similar to ZagA and may be the ZagA ortholog in C. metallidurans , CobW2 may serve as zinc‐storage depot that releases zinc upon an unknown signal, and although CobW3 has no GTPase activity, it may nevertheless have a regulatory function (Bütof et al ., ). All three CobWs interact with the metal‐cambialistic FolE IB2 protein in the presence of zinc while GTP is not needed for this interaction (Bütof et al ., ).…”

“…As discussed elsewhere in detail (Bütof et al, 2019), CobW1 is similar to ZagA and may be the ZagA ortholog in C. metallidurans, CobW2 may serve as zinc-storage depot that releases zinc upon an unknown signal, and although CobW3 has no GTPase activity, it may nevertheless have a regulatory function (Bütof et al, 2019). All three CobWs interact with the metal-cambialistic FolE IB2 protein in the presence of zinc while GTP is not needed for this interaction (Bütof et al, 2019). In contrast, ZagA of B. subtilis does not bind to the FolE IB ortholog, even in the presence of ZTP (Chandrangsu et al, 2019).…”