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Dietrich H. Nies - One of the best experts on this subject based on the ideXlab platform.
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interplay between the zur regulon components and metal resistance in Cupriavidus metallidurans
Journal of Bacteriology, 2019Co-Authors: Lucy Bütof, Martin Herzberg, Cornelia Grose, Hauke Lilie, Dietrich H. NiesAbstract: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 G3E-type 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.
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the third pillar of metal homeostasis in Cupriavidus metallidurans ch34 preferences are controlled by extracytoplasmic function sigma factors
Metallomics, 2019Co-Authors: Cornelia Grose, Martin Herzberg, Grit Schleuder, Claudia Schwarzenberger, Anja Poehlein, Kathrin Blank, Dietrich H. NiesAbstract:The role of extracytoplasmic function (ECF) sigma factors in multiple metal homeostasis of the metallophilic bacterium Cupriavidus metallidurans was studied. RNA sequencing was used to predict 3084 operons in the genome of this bacterium, including 11 for ECF sigma factors, and to measure transcript abundances. Mutants carrying multiple deletions in genes for ECF sigma factors were constructed and characterized. Mutants and parent were challenged with a metal mix, changes in the global gene expression profile and the overall metal content determined. All 11 ECF sigma factors were involved in metal homeostasis. The three ECF sigma factors RpoI, RpoJ and RpoK synchronized iron homeostasis with that of other divalent metal cations, RpoO, RpoL and RpoM magnesium and phosphorous homeostasis with that of zinc and with cadmium resistance. Factors RpoE, CnrH and RpoP controlled the response to nickel and cobalt, RpoQ and RpoR may be assigned to the thiol and sulfide metabolism. All 11 ECF sigma factors overlap in their function and control gene expression involved in metal homeostasis, however, except CnrH, no other ECF sigma factor was needed for up-regulation of 63 predicted operons responding to metal shock, 48 of these encoding metal efflux pumps. Moreover, disturbance of the cellular metal content resulting from missing sigma factors also affected silencing and un-silencing of genomic islands. Together, these data demonstrate on a global and systemic level how a robust network of ECF sigma factors and other regulators allow C. metallidurans to handle a mixture of toxic transition metal cations, which are conditions the bacterium faces in its natural environment. Iron homeostasis is to be maintained at any cost, followed by the necessity for magnesium, phosphorous and zinc homeostasis on the second level, and cobalt plus nickel coming last.
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synergistic gold copper detoxification at the core of gold biomineralisation in Cupriavidus metallidurans
Metallomics, 2018Co-Authors: Lucy Bütof, Nicole Wiesemann, Martin Herzberg, Frank Reith, M Altzschner, Alexander W Holleitner, Dietrich H. NiesAbstract:The bacterium Cupriavidus metallidurans is capable of reducing toxic Au(I/III)-complexes into metallic gold (Au) nano-particles, thereby mediating the (trans)formation of Au nuggets in Earth surface environments. In this study we describe a novel detoxification pathway, which prevents synergistic copper (Cu)/Au-toxicity. Gold-complexes and Cu-ions exert cooperative toxicity, because cellular uptake of Au(I/III)-complexes blocks Cu(I) export from the cytoplasm by the Cu-efflux pump CupA. Using a combination of micro-analytical and biochemical methods we show that inducible resistance to these Cu/Au mixtures is mediated by the periplasmic Cu(I)-oxidase CopA, which functions as an oxygen-consuming Au(I)-oxidase. With high Au-complex loads the enzymatic activity of CopA detoxifies the reduction pathway of Au(III)-complexes via Au(I)-intermediates to Au(0) nanoparticles in the periplasm. Thereby the concentration of highly toxic Au(I) in the cytoplasm is diminished, while allowing direct reduction of Au(III) to Au nanoparticles in the periplasm. This permits C. metallidurans to thrive in Au-rich environments and biomineralise metallic Au.
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the components of the unique zur regulon of Cupriavidus metallidurans mediate cytoplasmic zinc handling
Journal of Bacteriology, 2017Co-Authors: Lucy Bütof, Martin Herzberg, Cornelia Grose, Christopher Schmidtvogler, Dietrich H. NiesAbstract:Zinc is an essential trace element, yet it is toxic at high concentrations. In the betaproteobacterium Cupriavidus metallidurans, the highly efficient removal of surplus zinc from the periplasm is responsible for the outstanding metal resistance of the organism. Rather than having a typical Zur-dependent, high-affinity ATP-binding cassette transporter of the ABC protein superfamily for zinc uptake at low concentrations, C. metallidurans has the secondary zinc importer ZupT of the zinc-regulated transporter, iron-regulated transporter (ZRT/IRT)-like protein (ZIP) family. It is important to understand, therefore, how this zinc-resistant bacterium copes with exposure to low zinc concentrations. Members of the Zur regulon in C. metallidurans were identified by comparing the transcriptomes of a Δzur mutant and its parent strain. The consensus sequence of the Zur-binding box was derived for the zupTp promoter-regulatory region by use of a truncation assay. The motif was used to predict possible Zur boxes upstream of Zur regulon members. The binding of Zur to these boxes was confirmed. Two Zur boxes upstream of the cobW1 gene, encoding a putative zinc chaperone, proved to be required for complete repression of cobW1 and its downstream genes in cells cultivated in mineral salts medium. A Zur box upstream of each of zur-cobW2, cobW3, and zupT permitted both low expression levels of these genes and their upregulation under conditions of zinc starvation. This demonstrates a compartmentalization of zinc homeostasis in C. metallidurans, where the periplasm is responsible for the removal of surplus zinc, cytoplasmic components are responsible for the management of zinc as an essential cofactor, and the two compartments are connected by ZupT.IMPORTANCE Elucidating zinc homeostasis is necessary for understanding both host-pathogen interactions and the performance of free-living bacteria in their natural environments. Escherichia coli acquires zinc under conditions of low zinc concentrations via 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 C. metallidurans achieves high tolerance to zinc through 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 the management of zinc by cytoplasmic zinc chaperones, whose synthesis is controlled by the Zur regulator. This demonstrates a new mechanism, involving compartmentalization, for organizing zinc homeostasis.
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proteomic responses to gold iii toxicity in the bacterium Cupriavidus metallidurans ch34
Metallomics, 2016Co-Authors: Carla M Zammit, Joël Brugger, Frank Reith, Dietrich H. Nies, Gordon Southam, Florian Weiland, Benjamin P Wade, Lyron J Winderbaum, Peter HoffmannAbstract:The metal-resistant β-proteobacterium Cupriavidus metallidurans drives gold (Au) biomineralisation and the (trans)formation of Au nuggets largely via unknown biochemical processes, ultimately leading to the reductive precipitation of mobile, toxic Au(I/III)-complexes. In this study proteomic responses of C. metallidurans CH34 to mobile, toxic Au(III)-chloride are investigated. Cells were grown in the presence of 10 and 50 μM Au(III)-chloride, 50 μM Cu(II)-chloride and without additional metals. Differentially expressed proteins were detected by difference gel electrophoresis and identified by liquid chromatography coupled mass spectrometry. Proteins that were more abundant in the presence of Au(III)-chloride are involved in a range of important cellular functions, e.g., metabolic activities, transcriptional regulation, efflux and metal transport. To identify Au-binding proteins, protein extracts were separated by native 2D gel electrophoresis and Au in protein spots was detected by laser absorption inductively coupled plasma mass spectrometry. A chaperon protein commonly understood to bind copper (Cu), CupC, was identified and shown to bind Au. This indicates that it forms part of a multi-metal detoxification system and suggests that similar/shared detoxification pathways for Au and Cu exist. Overall, this means that C. metallidurans CH34 is able to mollify the toxic effects of cytoplasmic Au(III) by sequestering this Au-species. This effect may in the future be used to develop CupC-based biosensing capabilities for the in-field detection of Au in exploration samples.
Rob Van Houdt - One of the best experts on this subject based on the ideXlab platform.
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phenotypic and genetic characterization of temperature induced mutagenesis and mortality in Cupriavidus metallidurans
Frontiers in Microbiology, 2021Co-Authors: Rob Van Houdt, Pieter Monsieurs, A. Provoost, Natalie Leys, Joachim Vandecraen, Wietse Heylen, Abram AertsenAbstract:Cupriavidus metallidurans strains display a decreased viability when incubated in rich medium at a temperature of 37°C compared to their normal growth temperature of 30°C. A phenomenon coined “temperature-induced mortality and mutagenesis” (TIMM). To scrutinize this aberrant phenotype further, the contribution of specific inducers and protective agents were determined. Different growth media, including Lysogeny Broth (LB) and Schatz, and components, including casamino acids, particular amino acids (proline, cysteine, glycine, glutamine, leucine, histidine and phenylalanine) and ammonium, were found to induce TIMM at 37°C. Sorbitol was found to counteract TIMM. Furthermore, although TIMM is well-conserved within the C. metallidurans species, multiple and strain-specific TIMM-inducers exist. Twenty nine percent of the TIMM survivors inherited resistance to TIMM. Whole-genome sequencing of two resistant derivatives revealed an important role of an uncharacterized oxidoreductase, indicating putative metabolic poisoning when grown in high concentration nitrogen-containing media at 37°C.
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adaptation of Cupriavidus metallidurans ch34 to toxic zinc concentrations involves an uncharacterized abc type transporter
Microorganisms, 2021Co-Authors: Rob Van Houdt, Pieter Monsieurs, Natalie Leys, Joachim Vandecraen, Abram AertsenAbstract:Cupriavidus metallidurans CH34 is a well-studied metal-resistant β-proteobacterium and contains a battery of genes participating in metal metabolism and resistance. Here, we generated a mutant (CH34ZnR) adapted to high zinc concentrations in order to study how CH34 could adaptively further increase its resistance against this metal. Characterization of CH34ZnR revealed that it was also more resistant to cadmium, and that it incurred seven insertion sequence-mediated mutations. Among these, an IS1088 disruption of the glpR gene (encoding a DeoR-type transcriptional repressor) resulted in the constitutive expression of the neighboring ATP-binding cassette (ABC)-type transporter. GlpR and the adjacent ABC transporter are highly similar to the glycerol operon regulator and ATP-driven glycerol importer of Rhizobium leguminosarum bv. viciae VF39, respectively. Deletion of glpR or the ABC transporter and complementation of CH34ZnR with the parental glpR gene further demonstrated that loss of GlpR function and concomitant derepression of the adjacent ABC transporter is pivotal for the observed resistance phenotype. Importantly, addition of glycerol, presumably by glycerol-mediated attenuation of GlpR activity, also promoted increased zinc and cadmium resistance in the parental CH34 strain. Upregulation of this ABC-type transporter is therefore proposed as a new adaptation route towards metal resistance.
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Cupriavidus metallidurans ch34 a historical perspective on its discovery characterization and metal resistance
FEMS Microbiology Ecology, 2021Co-Authors: Max Mergeay, Rob Van HoudtAbstract:Cupriavidus metallidurans, and in particular type strain CH34, became a model bacterium to study bacterial resistance to metals. Although nowadays the routine use of a wide variety of omics and molecular techniques allow refining, deepening and expanding our knowledge on adaptation and resistance to metals, these were not available at the onset of C. metallidurans research starting from its isolation in 1976. This minireview describes the early research and legacy tools used to study its metal resistance determinants, characteristic megaplasmids, ecological niches and environmental applications.
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the transcriptomic landscape of Cupriavidus metallidurans ch34 acutely exposed to copper
Genes, 2020Co-Authors: Laurens Maertens, Natalie Leys, Jeanyves Matroule, Rob Van HoudtAbstract:Bacteria are increasingly used for biotechnological applications such as bioremediation, biorecovery, bioproduction, and biosensing. The development of strains suited for such applications requires a thorough understanding of their behavior, with a key role for their transcriptomic landscape. We present a thorough analysis of the transcriptome of Cupriavidus metallidurans CH34 cells acutely exposed to copper by tagRNA-sequencing. C. metallidurans CH34 is a model organism for metal resistance, and its potential as a biosensor and candidate for metal bioremediation has been demonstrated in multiple studies. Several metabolic pathways were impacted by Cu exposure, and a broad spectrum of metal resistance mechanisms, not limited to copper-specific clusters, was overexpressed. In addition, several gene clusters involved in the oxidative stress response and the cysteine-sulfur metabolism were induced. In total, 7500 transcription start sites (TSSs) were annotated and classified with respect to their location relative to coding sequences (CDSs). Predicted TSSs were used to re-annotate 182 CDSs. The TSSs of 2422 CDSs were detected, and consensus promotor logos were derived. Interestingly, many leaderless messenger RNAs (mRNAs) were found. In addition, many mRNAs were transcribed from multiple alternative TSSs. We observed pervasive intragenic TSSs both in sense and antisense to CDSs. Antisense transcripts were enriched near the 5' end of mRNAs, indicating a functional role in post-transcriptional regulation. In total, 578 TSSs were detected in intergenic regions, of which 35 were identified as putative small regulatory RNAs. Finally, we provide a detailed analysis of the main copper resistance clusters in CH34, which include many intragenic and antisense transcripts. These results clearly highlight the ubiquity of noncoding transcripts in the CH34 transcriptome, many of which are putatively involved in the regulation of metal resistance.
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dna binding and transcription activation by unphosphorylated response regulator agrr from Cupriavidus metallidurans involved in silver resistance
Frontiers in Microbiology, 2020Co-Authors: Muntasir Ali, K. Mijnendonckx, A. Provoost, Rob Van Houdt, Daniel CharlierAbstract:Even though silver and silver nanoparticles at low concentrations are considered safe for human health, their steadily increasing use and associated release in nature is not without risk since it may result in the selection of silver-resistant microorganisms, thus impeding the utilization of silver as antimicrobial agent. Furthermore, increased resistance to metals may be accompanied by increased antibiotic resistance. Inactivation of the histidine kinase and concomitant upregulation of the cognate response regulator (RR) of the AgrRS two-component system was previously shown to play an important role in the increased silver resistance of laboratory adapted mutants of Cupriavidus metallidurans. However, binding of AgrR, a member of the OmpR/PhoP family of RRs with a conserved phosphoreceiver aspartate residue, to potential target promoters has never been demonstrated. Here we identify differentially expressed genes in the silver-resistant mutant NA4S in non-selective conditions by RNA-seq and demonstrate sequence-specific binding of AgrR to six selected promoter regions of upregulated genes and divergent operons. We delimit binding sites by DNase I and in gel copper-phenanthroline footprinting of AgrR-DNA complexes, and establish a high resolution base-specific contact map of AgrR-DNA interactions using premodification binding interference techniques. We identified a 16-bp core AgrR binding site (AgrR box) arranged as an imperfect inverted repeat of 6 bp (ATTACA) separated by 4 bp variable in sequence (6-4-6). AgrR interacts with two major groove segments and the intervening minor groove, all aligned on one face of the helix. Furthermore, an additional in phase imperfect direct repeat of the half-site may be observed slightly up and/or downstream of the inverted repeat at some operators. Mutant studies indicated that both inverted and direct repeats contribute to AgrR binding in vitro and AgrR-mediated activation in vivo. From the position of the AgrR box it appears that AgrR may act as a Type II activator for most investigated promoters, including positive autoregulation. Furthermore, we show in vitro binding and in vivo activation with dephosphomimetic AgrR mutant D51A, indicating that unphosphorylated AgrR is the active form of the RR in mutant NA4S.
Natalie Leys - One of the best experts on this subject based on the ideXlab platform.
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phenotypic and genetic characterization of temperature induced mutagenesis and mortality in Cupriavidus metallidurans
Frontiers in Microbiology, 2021Co-Authors: Rob Van Houdt, Pieter Monsieurs, A. Provoost, Natalie Leys, Joachim Vandecraen, Wietse Heylen, Abram AertsenAbstract:Cupriavidus metallidurans strains display a decreased viability when incubated in rich medium at a temperature of 37°C compared to their normal growth temperature of 30°C. A phenomenon coined “temperature-induced mortality and mutagenesis” (TIMM). To scrutinize this aberrant phenotype further, the contribution of specific inducers and protective agents were determined. Different growth media, including Lysogeny Broth (LB) and Schatz, and components, including casamino acids, particular amino acids (proline, cysteine, glycine, glutamine, leucine, histidine and phenylalanine) and ammonium, were found to induce TIMM at 37°C. Sorbitol was found to counteract TIMM. Furthermore, although TIMM is well-conserved within the C. metallidurans species, multiple and strain-specific TIMM-inducers exist. Twenty nine percent of the TIMM survivors inherited resistance to TIMM. Whole-genome sequencing of two resistant derivatives revealed an important role of an uncharacterized oxidoreductase, indicating putative metabolic poisoning when grown in high concentration nitrogen-containing media at 37°C.
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adaptation of Cupriavidus metallidurans ch34 to toxic zinc concentrations involves an uncharacterized abc type transporter
Microorganisms, 2021Co-Authors: Rob Van Houdt, Pieter Monsieurs, Natalie Leys, Joachim Vandecraen, Abram AertsenAbstract:Cupriavidus metallidurans CH34 is a well-studied metal-resistant β-proteobacterium and contains a battery of genes participating in metal metabolism and resistance. Here, we generated a mutant (CH34ZnR) adapted to high zinc concentrations in order to study how CH34 could adaptively further increase its resistance against this metal. Characterization of CH34ZnR revealed that it was also more resistant to cadmium, and that it incurred seven insertion sequence-mediated mutations. Among these, an IS1088 disruption of the glpR gene (encoding a DeoR-type transcriptional repressor) resulted in the constitutive expression of the neighboring ATP-binding cassette (ABC)-type transporter. GlpR and the adjacent ABC transporter are highly similar to the glycerol operon regulator and ATP-driven glycerol importer of Rhizobium leguminosarum bv. viciae VF39, respectively. Deletion of glpR or the ABC transporter and complementation of CH34ZnR with the parental glpR gene further demonstrated that loss of GlpR function and concomitant derepression of the adjacent ABC transporter is pivotal for the observed resistance phenotype. Importantly, addition of glycerol, presumably by glycerol-mediated attenuation of GlpR activity, also promoted increased zinc and cadmium resistance in the parental CH34 strain. Upregulation of this ABC-type transporter is therefore proposed as a new adaptation route towards metal resistance.
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the transcriptomic landscape of Cupriavidus metallidurans ch34 acutely exposed to copper
Genes, 2020Co-Authors: Laurens Maertens, Natalie Leys, Jeanyves Matroule, Rob Van HoudtAbstract:Bacteria are increasingly used for biotechnological applications such as bioremediation, biorecovery, bioproduction, and biosensing. The development of strains suited for such applications requires a thorough understanding of their behavior, with a key role for their transcriptomic landscape. We present a thorough analysis of the transcriptome of Cupriavidus metallidurans CH34 cells acutely exposed to copper by tagRNA-sequencing. C. metallidurans CH34 is a model organism for metal resistance, and its potential as a biosensor and candidate for metal bioremediation has been demonstrated in multiple studies. Several metabolic pathways were impacted by Cu exposure, and a broad spectrum of metal resistance mechanisms, not limited to copper-specific clusters, was overexpressed. In addition, several gene clusters involved in the oxidative stress response and the cysteine-sulfur metabolism were induced. In total, 7500 transcription start sites (TSSs) were annotated and classified with respect to their location relative to coding sequences (CDSs). Predicted TSSs were used to re-annotate 182 CDSs. The TSSs of 2422 CDSs were detected, and consensus promotor logos were derived. Interestingly, many leaderless messenger RNAs (mRNAs) were found. In addition, many mRNAs were transcribed from multiple alternative TSSs. We observed pervasive intragenic TSSs both in sense and antisense to CDSs. Antisense transcripts were enriched near the 5' end of mRNAs, indicating a functional role in post-transcriptional regulation. In total, 578 TSSs were detected in intergenic regions, of which 35 were identified as putative small regulatory RNAs. Finally, we provide a detailed analysis of the main copper resistance clusters in CH34, which include many intragenic and antisense transcripts. These results clearly highlight the ubiquity of noncoding transcripts in the CH34 transcriptome, many of which are putatively involved in the regulation of metal resistance.
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copper resistance mediates long term survival of Cupriavidus metallidurans in wet contact with metallic copper
Frontiers in Microbiology, 2020Co-Authors: Laurens Maertens, Natalie Leys, Ilse Coninx, Jeanyves Matroule, Jurgen Claesen, Rob Van HoudtAbstract:Metallic copper to combat bacterial proliferation in drinking water systems is being investigated as an attractive alternative to existing strategies. A potential obstacle to this approach is the induction of metal resistance mechanisms in contaminating bacteria, that could severely impact inactivation efficacy. Thus far, the role of these resistance mechanisms has not been studied in conditions relevant to drinking water systems. Therefore, we evaluated the inactivation kinetics of Cupriavidus metallidurans CH34 in contact with metallic copper in drinking water. Viability and membrane permeability were examined for 9 days through viable counts and flow cytometry. After an initial drop in viable count, a significant recovery was observed starting after 48 h. This behavior could be explained by either a recovery from an injured/viable-but-non-culturable state or regrowth of surviving cells metabolizing lysed cells. Either hypothesis would necessitate an induction of copper resistance mechanisms, since no recovery was seen in a CH34 mutant strain lacking metal resistance mechanisms, while being more pronounced when copper resistance mechanisms were pre-induced. Interestingly, no biofilms were formed on the copper surface, while extensive biofilm formation was observed on the stainless steel control plates. When CH34 cells in water were supplied with CuSO4, a similar initial decrease in viable counts was observed, but cells recovered fully after 7 days. In conclusion, we have shown that long-term bacterial survival in the presence of a copper surface is possible upon the induction of metal resistance mechanisms. This observation may have important consequences in the context of the increasing use of copper as an antimicrobial surface, especially in light of potential co-selection for metal and antimicrobial resistance.
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spontaneous mutation in the agrrs two component regulatory system of Cupriavidus metallidurans results in enhanced silver resistance
Metallomics, 2019Co-Authors: K. Mijnendonckx, Pieter Monsieurs, A. Provoost, Natalie Leys, Max Mergeay, Paul Janssen, Muntasir Ali, Daniel Charlier, Rob Van HoudtAbstract:The uncontrolled and widespread use of (nano)silver compounds has led to the increased release of these compounds into the environment, raising concerns about their negative impact on ecosystems. Concomitantly, silver resistance determinants are widely spread among environmental and clinically relevant bacteria although the underlying mechanisms are not yet fully understood. We show that Cupriavidus metallidurans is able to adapt to toxic silver concentrations. However, none of the known silver resistance determinants present in C. metallidurans are involved in the adaptative response. Instead, increased silver resistance is achieved by the concerted action of a two-component system AgrR–AgrS, previously not associated with metal resistance, and two periplasmic proteins PrsQ1 and PrsQ2. Both proteins belong to an unique group of small, uncharacterized, secreted proteins restricted to the genera Cupriavidus and Ralstonia. This system gives C. metallidurans the ability to withstand much higher silver concentrations. The latter could be facilitated by the accumulation of silver ions and the formation of silver nanoparticles.
Martin Herzberg - One of the best experts on this subject based on the ideXlab platform.
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interplay between the zur regulon components and metal resistance in Cupriavidus metallidurans
Journal of Bacteriology, 2019Co-Authors: Lucy Bütof, Martin Herzberg, Cornelia Grose, Hauke Lilie, Dietrich H. NiesAbstract: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 G3E-type 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.
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the third pillar of metal homeostasis in Cupriavidus metallidurans ch34 preferences are controlled by extracytoplasmic function sigma factors
Metallomics, 2019Co-Authors: Cornelia Grose, Martin Herzberg, Grit Schleuder, Claudia Schwarzenberger, Anja Poehlein, Kathrin Blank, Dietrich H. NiesAbstract:The role of extracytoplasmic function (ECF) sigma factors in multiple metal homeostasis of the metallophilic bacterium Cupriavidus metallidurans was studied. RNA sequencing was used to predict 3084 operons in the genome of this bacterium, including 11 for ECF sigma factors, and to measure transcript abundances. Mutants carrying multiple deletions in genes for ECF sigma factors were constructed and characterized. Mutants and parent were challenged with a metal mix, changes in the global gene expression profile and the overall metal content determined. All 11 ECF sigma factors were involved in metal homeostasis. The three ECF sigma factors RpoI, RpoJ and RpoK synchronized iron homeostasis with that of other divalent metal cations, RpoO, RpoL and RpoM magnesium and phosphorous homeostasis with that of zinc and with cadmium resistance. Factors RpoE, CnrH and RpoP controlled the response to nickel and cobalt, RpoQ and RpoR may be assigned to the thiol and sulfide metabolism. All 11 ECF sigma factors overlap in their function and control gene expression involved in metal homeostasis, however, except CnrH, no other ECF sigma factor was needed for up-regulation of 63 predicted operons responding to metal shock, 48 of these encoding metal efflux pumps. Moreover, disturbance of the cellular metal content resulting from missing sigma factors also affected silencing and un-silencing of genomic islands. Together, these data demonstrate on a global and systemic level how a robust network of ECF sigma factors and other regulators allow C. metallidurans to handle a mixture of toxic transition metal cations, which are conditions the bacterium faces in its natural environment. Iron homeostasis is to be maintained at any cost, followed by the necessity for magnesium, phosphorous and zinc homeostasis on the second level, and cobalt plus nickel coming last.
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synergistic gold copper detoxification at the core of gold biomineralisation in Cupriavidus metallidurans
Metallomics, 2018Co-Authors: Lucy Bütof, Nicole Wiesemann, Martin Herzberg, Frank Reith, M Altzschner, Alexander W Holleitner, Dietrich H. NiesAbstract:The bacterium Cupriavidus metallidurans is capable of reducing toxic Au(I/III)-complexes into metallic gold (Au) nano-particles, thereby mediating the (trans)formation of Au nuggets in Earth surface environments. In this study we describe a novel detoxification pathway, which prevents synergistic copper (Cu)/Au-toxicity. Gold-complexes and Cu-ions exert cooperative toxicity, because cellular uptake of Au(I/III)-complexes blocks Cu(I) export from the cytoplasm by the Cu-efflux pump CupA. Using a combination of micro-analytical and biochemical methods we show that inducible resistance to these Cu/Au mixtures is mediated by the periplasmic Cu(I)-oxidase CopA, which functions as an oxygen-consuming Au(I)-oxidase. With high Au-complex loads the enzymatic activity of CopA detoxifies the reduction pathway of Au(III)-complexes via Au(I)-intermediates to Au(0) nanoparticles in the periplasm. Thereby the concentration of highly toxic Au(I) in the cytoplasm is diminished, while allowing direct reduction of Au(III) to Au nanoparticles in the periplasm. This permits C. metallidurans to thrive in Au-rich environments and biomineralise metallic Au.
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Synergistic Toxicity of Copper and Gold Compounds in Cupriavidus metallidurans.
Applied and Environmental Microbiology, 2017Co-Authors: Nicole Wiesemann, Lucy Bütof, Lutz Berthold, Joël Brugger, Martin Herzberg, Gema Martínez-criado, Barbara Etschmann, Dirk Dobritzsch, Gerd Hause, Sacha BaginskyAbstract:The bacterium Cupriavidus metallidurans can reduce toxic gold(I/III) complexes and biomineralize them into metallic gold (Au) nanoparticles, thereby mediating the (trans)formation of Au nuggets. In Au-rich soils, most transition metals do not interfere with the resistance of this bacterium to toxic mobile Au complexes and can be removed from the cell by plasmid-encoded metal efflux systems. Copper is a noticeable exception: the presence of Au complexes and Cu ions results in synergistic toxicity, which is accompanied by an increased cytoplasmic Cu content and formation of Au nanoparticles in the periplasm. The periplasmic Cu-oxidase CopA was not essential for formation of the periplasmic Au nanoparticles. As shown with the purified and reconstituted Cu efflux system CupA, Au complexes block Cu-dependent release of phosphate from ATP by CupA, indicating inhibition of Cu transport. Moreover, Cu resistance of Au-inhibited cells was similar to that of mutants carrying deletions in the genes for the Cu-exporting PIB1-type ATPases. Consequently, Au complexes inhibit export of cytoplasmic Cu ions, leading to an increased cellular Cu content and decreased Cu and Au resistance. Uncovering the biochemical mechanisms of synergistic Au and Cu toxicity in C. metallidurans explains the issues this bacterium has to face in auriferous environments, where it is an important contributor to the environmental Au cycle.IMPORTANCE C. metallidurans lives in metal-rich environments, including auriferous soils that contain a mixture of toxic transition metal cations. We demonstrate here that copper ions and gold complexes exert synergistic toxicity because gold ions inhibit the copper-exporting P-type ATPase CupA, which is central to copper resistance in this bacterium. Such a situation should occur in soils overlying Au deposits, in which Cu/Au ratios usually are ≫1. Appreciating how C. metallidurans solves the problem of living in environments that contain both Au and Cu is a prerequisite to understand the molecular mechanisms underlying gold cycling in the environment, and the significance and opportunities of microbiota for specific targeting to Au in mineral exploration and ore processing.
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the components of the unique zur regulon of Cupriavidus metallidurans mediate cytoplasmic zinc handling
Journal of Bacteriology, 2017Co-Authors: Lucy Bütof, Martin Herzberg, Cornelia Grose, Christopher Schmidtvogler, Dietrich H. NiesAbstract:Zinc is an essential trace element, yet it is toxic at high concentrations. In the betaproteobacterium Cupriavidus metallidurans, the highly efficient removal of surplus zinc from the periplasm is responsible for the outstanding metal resistance of the organism. Rather than having a typical Zur-dependent, high-affinity ATP-binding cassette transporter of the ABC protein superfamily for zinc uptake at low concentrations, C. metallidurans has the secondary zinc importer ZupT of the zinc-regulated transporter, iron-regulated transporter (ZRT/IRT)-like protein (ZIP) family. It is important to understand, therefore, how this zinc-resistant bacterium copes with exposure to low zinc concentrations. Members of the Zur regulon in C. metallidurans were identified by comparing the transcriptomes of a Δzur mutant and its parent strain. The consensus sequence of the Zur-binding box was derived for the zupTp promoter-regulatory region by use of a truncation assay. The motif was used to predict possible Zur boxes upstream of Zur regulon members. The binding of Zur to these boxes was confirmed. Two Zur boxes upstream of the cobW1 gene, encoding a putative zinc chaperone, proved to be required for complete repression of cobW1 and its downstream genes in cells cultivated in mineral salts medium. A Zur box upstream of each of zur-cobW2, cobW3, and zupT permitted both low expression levels of these genes and their upregulation under conditions of zinc starvation. This demonstrates a compartmentalization of zinc homeostasis in C. metallidurans, where the periplasm is responsible for the removal of surplus zinc, cytoplasmic components are responsible for the management of zinc as an essential cofactor, and the two compartments are connected by ZupT.IMPORTANCE Elucidating zinc homeostasis is necessary for understanding both host-pathogen interactions and the performance of free-living bacteria in their natural environments. Escherichia coli acquires zinc under conditions of low zinc concentrations via 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 C. metallidurans achieves high tolerance to zinc through 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 the management of zinc by cytoplasmic zinc chaperones, whose synthesis is controlled by the Zur regulator. This demonstrates a new mechanism, involving compartmentalization, for organizing zinc homeostasis.
Jacques Covès - One of the best experts on this subject based on the ideXlab platform.
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Response of CnrX from Cupriavidus metallidurans CH34 to nickel binding.
Metallomics : integrated biometal science, 2015Co-Authors: Antoine P. Maillard, Cornelia Grosse, Dietrich H. Nies, Sandra Künnemann, Anne Volbeda, Grit Schleuder, Isabelle Petit-härtlein, Eve De Rosny, Jacques CovèsAbstract:Resistance to high concentration of nickel ions is mediated in Cupriavidus metallidurans by the CnrCBA transenvelope efflux complex. Expression of the cnrCBA genes is regulated by the transmembrane signal transduction complex CnrYXH. Together, the metal sensor CnrX and the transmembrane antisigma factor CnrY control the availability of the extracytoplasmic function sigma factor CnrH. Release of CnrH from sequestration by CnrY at the cytoplasmic side of the membrane depends essentially on the binding of the agonist metal ion Ni(ii) to the periplasmic metal sensor domain of CnrX. CnrH availability leads to transcription initiation at the promoters cnrYp and cnrCp and to the expression of the genes in the cnrYXHCBA nickel resistance determinant. The first steps of signal propagation by CnrX rely on subtle metal-dependent allosteric modifications. To study the nickel-mediated triggering process by CnrX, we have altered selected residues, F66, M123, and Y135, and explored the physiological consequences of these changes with respect to metal resistance, expression of a cnrCBA-lacZ reporter fusion and protein production. M123C- and Y135F-CnrXs have been further characterized in vitro by metal affinity measurements and crystallographic structure analysis. Atomic-resolution structures of metal-bound M123C- and Y135F-CnrXs showed that Ni(ii) binds two of the three canonical conformations identified and that Ni(ii) sensing likely proceeds by conformation selection.
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spectroscopic characterization of the metal binding sites in the periplasmic metal sensor domain of cnrx from Cupriavidus metallidurans ch34
Biochemistry, 2011Co-Authors: Juliette Trepreau, Olivier Proux, Geraldine Sarret, Antoine P. Maillard, Eve De Rosny, Carole Duboc, Isabelle Petithartlein, Anne Imberty, Jacques CovèsAbstract:CnrX, the dimeric metal sensor of the three-protein transmembrane signal transduction complex CnrYXH of Cupriavidus metallidurans CH34, contains one metal-binding site per monomer. Both Ni and Co elicit a biological response and bind the protein in a 3N2O1S coordination sphere with a nearly identical octahedral geometry as shown by the X-ray structure of CnrXs, the soluble domain of CnrX. However, in solution CnrXs is titrated by 4 Co-equiv and exhibits an unexpected intense band at 384 nm that was detected neither by single-crystal spectroscopy nor under anaerobiosis. The data from a combination of spectroscopic techniques (spectrophotometry, electron paramagnetic resonance, X-ray absorption spectroscopy) showed that two sites correspond to those identified by crystallography. The two extra binding sites accommodate Co(II) in an octahedral geometry in the absence of oxygen and are occupied in air by a mixture of low-spin Co(II) as well as EPR-silent Co(III). These extra sites, located at the N-terminus o...
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copk from Cupriavidus metallidurans ch34 binds cu i in a tetrathioether site characterization by x ray absorption and nmr spectroscopy
Journal of the American Chemical Society, 2010Co-Authors: Geraldine Sarret, Max Mergeay, Jacques Covès, Jean-louis Hazemann, Adrien Favier, Beate BerschAbstract:Cupriavidus metallidurans CH34 is a bacterium that is resistant to high metal concentrations in the environment. Increased copper resistance is associated with the cop cluster on the large plasmid pMOL30 that is composed of at least 21 genes. The copK gene encodes a 74 residue periplasmic protein whose expression is strongly upregulated in the presence of copper. CopK was previously shown to cooperatively bind Cu(I) and Cu(II) in distinct, specific sites. The solution structure of Cu(I)−CopK and the characterization of the Cu(I) site by X-ray absorption spectroscopy and NMR are reported here. EXAFS spectra are in agreement with a tetrathioether Cu(I) site, providing so far unique spectral information on a 4S-coordinated Cu(I) in a protein. The methionine residues forming the Cu(I) site, M28, M38, M44, and M54, are identified by NMR. We propose the chemical shift of the methionine Ce as a new and sensitive probe for the detection of Cu(I) bound to thioether groups. The solution structure of Cu(I)−CopK demo...
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Evidence for Conformational Changes upon Copper Binding to Cupriavidus metallidurans CzcE
Biochemistry, 2010Co-Authors: Isabelle Petit-härtlein, Geraldine Sarret, Richard Kahn, Eric Girard, Jean-louis Hazemann, Patrick Gourhant, Jacques CovèsAbstract:CzcE is a periplasmic protein from Cupriavidus metallidurans CH34 that can bind four copper atoms per dimer. We have crystallized the apo form of the protein and determined its structure at 1.85 A ° resolution. Three Cu atoms were localized by soaking apo-CzcE crystals into a CuCl2 solution. We identified His24 as a Cu(II) ligand in each protomer and Asp100 as a key residue for Cu binding at the interface of the dimer. The role of these amino acids was confirmed by site-directed mutagenesis and UV-visible spectroscopy. The fourth Cu atom was not located. The oxidized form of CzcE contains four Cu(II) atoms, while the reduced form contains four Cu(I) atoms. Average coordination spheres of four N or O atoms for Cu(II) and of oneNorOatom and two S atoms for Cu(I) were determined by X-ray absorption spectroscopy. As there is no evidence for preformed metal-binding sites in apo-CzcE, we suggest that different conformational changes occurred upon Cu(II) or Cu(I) binding. These changes were further demonstrated by digestion experiments that gave different proteolysis patterns depending not only on the presence of the metal but also on its speciation. The ability of CzcE to bind copper and to adapt its conformation to different copper oxidation states could be related to a role in copper sensing for this protein.
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site directed mutagenesis reveals a conservation of the copper binding site and the crucial role of his24 in coph from Cupriavidus metallidurans ch34
Journal of Inorganic Biochemistry, 2009Co-Authors: Veronique Sendra, Beate Bersch, Serge Gambarelli, Jacques CovèsAbstract:CopH is a periplasmic copper-binding protein from Cupriavidus metallidurans CH34 that contains two histidine residues. Both His24 and His26 contribute to the formation of two high-affinity copper-binding sites in wild-type CopH and are likely involved in a 2N2O coordination sphere in the equatorial plane. We have used site-directed mutagenesis, and a series of spectroscopic and calorimetric studies to further characterize the copper-binding sites in CopH. While His24 plays a predominant role in copper affinity, one Cu-binding site was lost when either histidine residue was mutated. However, as shown by NMR and EPR, the mutation of the His residues does not affect the structural organization of the Cu-binding site nor the number of nitrogen ligands involved in copper ligation. In the absence of structural data, we propose a model that conciliates most of the spectroscopic data recorded during this study.
