Synergistic gold-copper detoxification at the core of gold biomineralisation in Cupriavidus metallidurans.

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.

Interplay between seven secondary metal uptake systems is required for full metal resistance of Cupriavidus metallidurans.

The beta-proteobacterium Cupriavidus metallidurans is able to grow in metal-contaminated environments due to having sophisticated metal efflux systems. Here, the contribution of all seven known secondary metal uptake systems (ZupT, PitA, CorA1, CorA2, CorA3, ZntB, HoxN) to metal resistance is characterized. In a strategic deletion approach, all ten double deletion mutants, a variety of triple and quadruple mutants, and from the Δ4 mutant (ΔzupT ΔcorA1 ΔcorA2 ΔcorA3) the mutants Δ5 (=Δ4 ΔpitA), Δ6 (=Δ4 ΔpitA ΔzntB), and finally Δ7 (ΔzupT ΔcorA1 ΔcorA2 ΔcorA3 ΔpitA ΔzntB ΔhoxN) were constructed. Metal resistance, metal content, and regulation of expression of these genes were characterized in these mutants. The ΔzupT single deletion strain exhibited an extended lag phase in Tris-buffered liquid mineral salts medium (TMM) compared to its parent strain AE104, indicating a decreased fitness level. Further deletions up to Δ6 did not influence growth in TMM without added metals but fitness of the Δ7 strain dropped to a lower level compared to Δ6, Δ5 and ΔzupT. The cells of the Δ7 multiple deletion strain still contained all essential metals, demonstrating that additional metal import systems must exist in C. metallidurans. PitA was an important contributor of metal:phosphate complexes to C. metallidurans. Up to Δ5 no evidence was found for increased expression of the transporter genes to recruit substitutes for the deleted importers. Only the hoxN-lacZ reporter gene fusion displayed a changed expression pattern in the Δ6 strain, indicating recruitment of HoxN. Metal resistance of the deletion strains decreased along the deletion series although all strains still contained metal efflux systems: up to the Δ6 mutant the overall fitness was kept at the ΔzupT mutant strain level at the cost of a diminished competence to handle µM concentrations of transition metals. Together, these data demonstrated an important contribution of the seven secondary metal import systems to metal homeostasis in this bacterium.

Deletion of the zupT gene for a zinc importer influences zinc pools in Cupriavidus metallidurans CH34.

Cupriavidus metallidurans strain CH34 accomplishes a high level of transition metal resistance by a combination of rather unspecific transition metal import and controlled efflux of surplus metals. Using the plasmid-free mutant strain AE104 that possesses only a limited number of metal efflux systems, cellular metal pools were identified as counterparts of these transport reactions. At low zinc concentrations strain AE104 took up Zn(II) until the zinc content reached an optimum level of 70,000 Zn(II) per cell in the exponential phase of growth, whereas a ΔzupT mutant lacking the zinc importer ZupT contained only 20,000 Zn(II)/cell, possibly the minimum zinc content. Mutant and parent cells accumulated up to 125,000 Zn(II) per cell at high (100 µM) external zinc concentrations (optimum zinc content). When the mutant strain Δe4, which has all the known genes for zinc efflux systems deleted, was cultivated in the presence of zinc concentrations close to its upper tolerance level (10 µM), these cells contained 250,000 Zn(II) per cell, probably the maximum zinc content. Instead of zinc, 120,000 cobalt or cadmium ions could also fill-up parts of this zinc pool, showing that it is in fact an undefined pool of divalent transition metal cations bound with low substrate specificity. Even when the cells contained sufficient numbers of total zinc, the zinc importer ZupT was required for important cellular processes, indicating the presence of a pool of tightly bound zinc ions, which depends on ZupT for efficient replenishment. The absence of ZupT led to the formation of inclusion bodies, perturbed oxidative stress resistance and decreased efficiency in the synthesis of the zinc-dependent subunit RpoC of the RNA polymerase, leading to RpoC accumulation. Moreover, when a czc allele for a zinc-exporting transenvelope efflux system CzcCBA was constitutively expressed in a ΔzupT mutant, this led to the disappearance of the CzcA protein and the central subunit of the protein complex, and to the loss of metal resistance. This phenomenon occurred only if the ΔzupT cells had been cultivated for several transfers in solid culture medium, which generated severe zinc starvation. Thus, two zinc pools appear to exist in C. metallidurans: one pool of at least 20,000 tightly bound Zn(II) ions, in addition to a second pool of up to 200,000 cations bound loosely and with low substrate specificity.

ZitB (YbgR), a member of the cation diffusion facilitator family, is an additional zinc transporter in Escherichia coli.

The Escherichia coli zitB gene encodes a Zn(II) transporter belonging to the cation diffusion facilitator family. ZitB is specifically induced by zinc. ZitB expression on a plasmid rendered zntA-disrupted E. coli cells more resistant to zinc, and the cells exhibited reduced accumulation of (65)Zn, suggesting ZitB-mediated efflux of zinc.

Heavy metal-resistant bacteria as extremophiles: molecular physiology and biotechnological use of Ralstonia sp. CH34.

In contrast to thermophilic or psychrophilic organisms, heavy metal-resistant bacteria do not supply enzymes that are active under harsh conditions, but are themselves tools for the evaluation and remediation of heavy metal-contaminated environments. Ralstonia sp. CH34 is a gram-negative bacterium with a remarkable set of resistance determinants, allowing this bacterium to live in extreme environments that are heavily contaminated with toxic metal ions. These heavy metal ions are mostly detoxified by inducible ion efflux systems that reduce the intracellular concentration of a given ion by active export. Because all metal resistance determinants in this bacterium are inducible, their regulatory systems can be used to develop biosensors that measure the biologically important concentrations of heavy metals in an environment. Resistance based on metal ion efflux detoxifies only the cytoplasm of the respective cell. Therefore, this resistance mechanism cannot be used directly to develop biotechnological procedures; however, metal ion efflux can protect a cell in a metal-contaminated environment. Thus, the cell can be enabled to mediate biochemical reactions such as precipitation of heavy metals with the carbon dioxide produced during growth or degradation of xenobiotics.

Regulation of the cnr cobalt and nickel resistance determinant from Ralstonia sp. strain CH34.

Ralstonia sp. strain CH34 is resistant to nickel and cobalt cations. Resistance is mediated by the cnr determinant located on plasmid pMOL28. The cnr genes are organized in two clusters, cnrYXH and cnrCBA. As revealed by reverse transcriptase PCR and primer extension, transcription from these operons is initiated from promoters located upstream of the cnrY and cnrC genes. These two promoters exhibit conserved sequences at the -10 (CCGTATA) and -35 (CRAGGGGRAG) regions. The CnrH gene product, which is required for expression of both operons, is a sigma factor belonging to the sigma L family, whose activity seems to be governed by the membrane-bound CnrY and CnrX gene products in response to Ni(2+). Half-maximal activation from the cnrCBA operon was determined by using appropriate lacZ gene fusions and was shown to occur at an Ni(2+) concentration of about 50 microM.

CzcD is a heavy metal ion transporter involved in regulation of heavy metal resistance in Ralstonia sp. strain CH34.

The Czc system of Ralstonia sp. strain CH34 mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB(2)A cation-proton antiporter. The CzcD protein is involved in the regulation of the Czc system. It is a membrane-bound protein with at least four transmembrane alpha-helices and is a member of a subfamily of the cation diffusion facilitator (CDF) protein family, which occurs in all three domains of life. The deletion of czcD in a Ralstonia sp. led to partially constitutive expression of the Czc system due to an increased transcription of the structural czcCBA genes, both in the absence and presence of inducers. The czcD deletion could be fully complemented in trans by CzcD and two other CDF proteins from Saccharomyces cerevisiae, ZRC1p and COT1p. All three proteins mediated a small but significant resistance to cobalt, zinc, and cadmium in Ralstonia, and this resistance was based on a reduced accumulation of the cations. Thus, CzcD appeared to repress the Czc system by an export of the inducing cations.

Energetics and topology of CzcA, a cation/proton antiporter of the resistance-nodulation-cell division protein family.

The membrane-bound CzcA protein, a member of the resistance-nodulation-cell division (RND) permease superfamily, is part of the CzcCB(2)A complex that mediates heavy metal resistance in Ralstonia sp. CH34 by an active cation efflux mechanism driven by cation/proton antiport. CzcA was purified to homogeneity after expression in Escherichia coli, reconstituted into proteoliposomes, and the kinetics of heavy metal transport by CzcA was determined. CzcA is composed of 12 transmembrane alpha-helices and two large periplasmic domains. Two conserved aspartate and a glutamate residue in one of these transmembrane spans are essential for heavy metal resistance and proton/cation antiport but not for facilitated diffusion of cations. Generalization of the resulting model for the function of CzcA as a two-channel pump might help to explain the functions of other RND proteins in bacteria and eukaryotes.

Microbial heavy-metal resistance.

We are just beginning to understand the metabolism of heavy metals and to use their metabolic functions in biotechnology, although heavy metals comprise the major part of the elements in the periodic table. Because they can form complex compounds, some heavy metal ions are essential trace elements, but, essential or not, most heavy metals are toxic at higher concentrations. This review describes the workings of known metal-resistance systems in microorganisms. After an account of the basic principles of homoeostasis for all heavy-metal ions, the transport of the 17 most important (heavy metal) elements is compared.

Transcriptional organization of the czc heavy-metal homeostasis determinant from Alcaligenes eutrophus.

The Czc system of Alcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. DNA sequencing of the region upstream of the czcNICBADRS determinant located on megaplasmid pMOL30 revealed the 5' end of czcN and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of czc. Additional open reading frames upstream of czc had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide czcN message and a 400-nucleotide czcI message were found, and the presence of 6, 200-nucleotide czcCBA message (D. Van der Lelie et al., Mol. Microbiol. 23:493-503, 1997) was confirmed. Induction of czcN, czcI, czcCBA, and czcDRS followed a similar pattern: transcription was induced best by 300 microM zinc, less by 300 microM cobalt, and only slightly by 300 microM cadmium. Reverse transcription-PCR gave evidence for additional continuous transcription from czcN to czcC and from czcD to czcS, but not between czcA and czcD nor between czcS and a 131-amino-acid open reading frame following czcS. The CzcR putative response regulator was purified and shown to bind in the 5' region of czcN. A reporter strain carrying a czcNIC-lacZ-czcBADRS determinant on plasmid pMOL30 was constructed, as were DeltaczcR and DeltaczcS mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn2+, (ii) induction of the beta-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of czcCBA mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.

The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins.

A previous report identified and classified a small family of gram-negative bacterial drug and heavy metal efflux permeases, now commonly referred to as the RND family (TC no. 2.6). We here show that this family is actually a ubiquitous superfamily with representation in all major kingdoms. We report phylogenetic analyses that define seven families within the RND superfamily as follows: (1) the heavy metal efflux (HME) family (gram negative bacteria), (2) the hydrophobe/amphiphile efflux-1 (HAE1) family (gram negative bacteria), (3) the nodulation factor exporter (NFE) family (gram negative bacteria), (4) the SecDF protein-secretion accessory protein (SecDF) family (gram negative and gram positive bacteria as well as archaea), (5) the hydrophobe/amphiphile efflux-2 (HAE2) family (gram positive bacteria), (6) the eukaryotic sterol homeostasis (ESH) family, and (7) the hydrophobe/amphiphile efflux-3 (HAE3) family (archaea and spirochetes). Functionally uncharacterized proteins were identified that are members of the RND superfamily but fall outside of these seven families. Some of the eukaryotic homologues function as enzymes and receptors instead of (or in addition to) transporters. The sizes and topological patterns exhibited by members of all seven families are shown to be strikingly similar, and statistical analyses establish common descent. Multiple alignments of proteins within each family allow derivation of family-specific signature sequences. Structural, functional, mechanistic and evolutionary implication of the reported results are discussed.

CHR, a novel family of prokaryotic proton motive force-driven transporters probably containing chromate/sulfate antiporters.

We describe a small family of proteins, CHR, which contains members that function in chromate and/or sulfate transport. CHR proteins occur in bacteria and archaea. They consist of about 400 amino acyl residues, appear to have 10 transmembrane alpha-helical segments in an unusual 4+6 arrangement, and arose by an intragenic duplication event.

Alcaligenes eutrophus as a bacterial chromate sensor.

In Alcaligenes eutrophus CH34, determinants encoding inducible resistance to chromate (chr) and to cobalt and nickel (cnr) are located adjacent to each other on plasmid pMOL28. To develop metal-sensing bacterial strains, a cloned part of plasmid pMOL28, which contains both determinants, was mutated with Tn5-lacZ. The chr::lacZ fusions were specifically induced by chromium; cnr was induced best by Ni2+ but was also induced by Co2+, Mn2+, chromate, Cu2+, Cd2+, and Zn2+. The broad-host-range IncP1 plasmid pEBZ141, which contains a chr::lux fusion, was constructed. A. eutrophus AE104(pEBZ141), carrying a chr::lux transcriptional fusion, could be used as a biosensor for chromate when cultivated in glycerol as an optimal carbon source. Chromate and bichromate were the best inducers; induction by Cr3+ was 10 times lower, and other ions induced only a little or not at all. Interactions among induction of the chr resistance determinant, chromate reduction, chromate accumulation, and the sulfate concentration of the growth medium were demonstrated.

New functions for the three subunits of the CzcCBA cation-proton antiporter.

The membrane-bound CzcCBA protein complex mediates heavy metal resistance in Alcaligenes eutrophus by an active cation efflux mechanism driven by cation-proton antiport. The CzcA protein alone is able to mediate weak resistance to zinc and cobalt and is thus the central antiporter subunit. The two histidine-rich motifs in the CzcB subunit are not essential for zinc resistance; however, deletion of both motifs led to a small but significant loss of resistance to this cation. Translation of the czcC gene encoding the third subunit of the CzcCBA complex starts earlier than predicted, and CzcC is probably a periplasmic protein, as judged by the appearance of two bands after expression of czcC in Escherichia coli under control of the phage T7 promoter. Fusions of CzcC and CzcB with alkaline phosphatase and beta-galactosidase are in agreement with a periplasmic location of most parts of both proteins. Both CzcC and CzcB are bound to a membrane, probably the outer membrane, by themselves and do not need either CzcA or each other as an anchoring protein. Based on these data, a new working model for the function of the Czc system is discussed.

Two-component regulatory system involved in transcriptional control of heavy-metal homoeostasis in Alcaligenes eutrophus.

The czc determinant, which mediates resistance to Co2+, Zn2+ and Cd2+ in Alcaligenes eutrophus CH34 by cation efflux, is regulated by a two-component regulatory system composed of the sensor histidine kinase CzcS and the response activator CzcR (in addition to other components previously described). Regulatory genes are arranged in an upstream regulatory region (URR) and a downstream regulatory region (DRR). Transcription of czcCBA and of the URR was regulated by heavy-metal cations. DNA sequencing of the region downstream of czcD revealed the presence of the czcR and czcS genes which together with czcD form the DRR. Regulation of the DRR was studied with a czcD::lacZ translational fusion and a czcS::lux transcriptional fusion. Expression of both genes is also regulated by heavy metals. The genes of the URR yielded three mRNAs of approx. 1200, 500 and 200 nucleotides, respectively. The genes czcCBA for the cation/proton antiporter CzcCBA were transcribed by one operon as a transcript of 6200 nucleotides.

The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutrophus functions as a cation-proton antiporter in Escherichia coli.

The function of the CzcABC protein complex, which mediates resistance to Co2+, Zn2+, and Cd2+ in Alcaligenes eutrophus by cation efflux, was investigated by using everted membrane vesicles of Escherichia coli and an acridine orange fluorescence quenching assay. Since metal cation uptake could not be measured with inside-out membrane vesicles prepared from A. eutrophus and since available E. coli strains did not express the Czc-mediated resistance to cobalt, zinc, and cadmium salts, mutants of E. coli which exhibited a Czc-dependent increase in heavy metal resistance were isolated. E. coli mutant strain EC351 constitutively accumulated Co2+, Zn2+, and Cd2+. In the presence of Czc, net uptake of these heavy metal cations was reduced to the wild-type level. Inside-out vesicles prepared from E. coli EC351 cells displayed a Czc-dependent uptake of Co2+, Zn2+, and Cd2+ and a cation-triggered acridine orange fluorescence increase. The czc-encoded protein complex CzcABC was shown to be a zinc-proton antiporter.

Ion efflux systems involved in bacterial metal resistances.

Studying metal ion resistance gives us important insights into environmental processes and provides an understanding of basic living processes. This review concentrates on bacterial efflux systems for inorganic metal cations and anions, which have generally been found as resistance systems from bacteria isolated from metal-polluted environments. The protein products of the genes involved are sometimes prototypes of new families of proteins or of important new branches of known families. Sometimes, a group of related proteins (and presumedly the underlying physiological function) has still to be defined. For example, the efflux of the inorganic metal anion arsenite is mediated by a membrane protein which functions alone in Gram-positive bacteria, but which requires an additional ATPase subunit in some Gram-negative bacteria. Resistance to Cd2+ and Zn2+ in Gram-positive bacteria is the result of a P-type efflux ATPase which is related to the copper transport P-type ATPases of bacteria and humans (defective in the human hereditary diseases Menkes' syndrome and Wilson's disease). In contrast, resistance to Zn2+, Ni2+, Co2+ and Cd2+ in Gram-negative bacteria is based on the action of proton-cation antiporters, members of a newly-recognized protein family that has been implicated in diverse functions such as metal resistance/nodulation of legumes/cell division (therefore, the family is called RND). Another new protein family, named CDF for 'cation diffusion facilitator' has as prototype the protein CzcD, which is a regulatory component of a cobalt-zinc-cadmium resistance determinant in the Gram-negative bacterium Alcaligenes eutrophus. A family for the ChrA chromate resistance system in Gram-negative bacteria has still to be defined.

CzcR and CzcD, gene products affecting regulation of resistance to cobalt, zinc, and cadmium (czc system) in Alcaligenes eutrophus.

The czcR gene, one of the two control genes responsible for induction of resistance to Co2+, Zn2+, and Cd2+ (czc system) in the Alcaligenes eutrophus plasmid pMOL30, was cloned and characterized. The 1,376-bp sequence upstream of the czcCBAD structural genes encodes a 41.4-kDa protein, the czcR gene product, transcribed in the opposite direction of that of the czcCBAD genes. The putative CzcR polypeptide (355 amino acid residues) contains 11 cysteine and 14 histidine residues which might form metal cation-binding sites. A czcC::lacZ reporter gene translational fusion was constructed, inserted into plasmid pMOL30 in A. eutrophus, and expressed under the control of CzcR. Zn2+, Co2+, and Cd2+, as well as Ni2+, Cu2+, Hg2+, and Mn2+ and even Al3+, served as inducers of beta-galactosidase activity. Besides the CzcR protein, the membrane-bound CzcD protein was essential for induction of czc. The CzcR and CzcD proteins display no sequence similarity to two-component regulatory systems of a sensor and a response activator type; however, CzcD has 34% identity with the ZRC-1 protein, which mediates zinc resistance in Saccharomyces cerevisiae (A. Kamizomo, M. Nishizawa, Y. Teranishi, K. Murata, and A. Kimura, Mol. Gen. Genet. 219:161-167, 1989).

Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae.

The gene merA coding for bacterial mercuric ion reductase was cloned under the control of the yeast promoter for alcohol dehydrogenase I in the yeast-Escherichia coli shuttle plasmid pADH040-2 and transformed into Saccharomyces cerevisiae AH22. The resulting transformant harbored stable copies of the merA-containing hybrid plasmid, displayed a fivefold increase in the MIC of mercuric chloride, and synthesized mercuric ion reductase activity.

Resistance to cadmium, cobalt, zinc, and nickel in microbes.

The divalent cations of cobalt, zinc, and nickel are essential nutrients for bacteria, required as trace elements at nanomolar concentrations. However, at micro- or millimolar concentrations, Co2+, Zn2+, and Ni2+ (and "bad ions" without nutritional roles such as Cd2+) are toxic. These cations are transported into the cell by constitutively expressed divalent cation uptake systems of broad specificity, i.e., basically Mg2+ transport systems. Therefore, in case of a heavy metal stress, uptake of the toxic ions cannot be reduced by a simple down-regulation of the transport activity. As a response to the resulting metal toxicity, metal resistance determinants evolved which are mostly plasmid-encoded in bacteria. In contrast to that of the cation Hg2+, chemical reduction of Co2+, Zn2+, Ni2+, and Cd2+ by the cell is not possible or sensible. Therefore, other than mutations limiting the ion range of the uptake system, only two basic mechanisms of resistance to these ions are possible (and were developed by evolution): intracellular complexation of the toxic metal ion is mainly used in eucaryotes; the cadmium-binding components are phytochelatins in plant and yeast cells and metallothioneins in animals, plants, and yeasts. In contrast, reduced accumulation based on an active efflux of the cation is the primary mechanism developed in procaryotes and perhaps in Saccharomyces cerevisiae. All bacterial cation efflux systems characterized to date are plasmid-encoded and inducible but differ in energy-coupling and in the number and types of proteins involved in metal transport and in regulation. In the gram-positive multiple-metal-resistant bacterium Staphylococcus aureus, Cd2+ (and probably Zn2+) efflux is catalyzed by the membrane-bound CadA protein, a P-type ATPase. However, a second protein (CadC) is required for full resistance and a third one (CadR) is hypothesized for regulation of the resistance determinant. The czc determinant from the gram-negative multiple-metal-resistant bacterium Alcaligenes eutrophus encodes proteins required for Co2+, Zn2+, and Cd2+ efflux (CzcA, CzcB, and CzcC) and regulation of the czc determinant (CzcD). In the current working model CzcA works as a cation-proton antiporter, CzcB as a cation-binding subunit, and CzcC as a modifier protein required to change the substrate specificity of the system from Zn2+ only to Co2+, Zn2+, and Cd2+.