[Frontiers in Bioscience 18, 165-189, January 1, 2013]

Responding to toxic compounds: a genomic and functional overview of Archaea

Simonetta Bartolucci1, Patrizia Contursi1, Gabriella Fiorentino1, Danila Limauro1, Emilia Pedone1,2

1Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cinthia, Napoli, Italy,2Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, I-80134 Napoli, Italy

TABLE CONTENTS

1. Abstract
2. Introduction
3. Drug detoxification in Archaea: the state of the art
3.1. Degradative pathways of aromatic compounds in Archaea
3.2. Drug transporters
3.2.1. Primary transporters
3.2.2. Secondary transporters
3.2.2.1. MATE family
3.2.2.2. The DME and SMR families
3.2.2.3. The MFS super family
3.2.2.4. RND super family
3.3. Transcriptional regulation in drug detoxification
4. The dark side of metal ions: toxicity
4.1. Iron
4.2. Nickel and Cobalt
4.3. Copper
4.4. Arsenic
4.5. Mercury
5. Conclusions
6. Acknowledgments
7. References

1. ABSTRACT

Archaea occupy a considerable diversity of niches ranging from extreme of pH, salinity to temperature that cannot be tolerated by other forms of life. There is an increasing consciousness that they have a key role both on the biogeochemical cycling of elements and in the bioremediation of polluted habitat. A greater understanding of metal homeostasis and resistance to toxic compounds in this life domain is required to design new strategies for the bioremediation of contaminated sites. This review describes the strategies developed by Archaea to transform xenobiotic compounds and metal ions present in the environment. The adaptation and/or response to such chemicals and the molecular mechanisms of resistance evolved in Archaea are discussed.

2. INTRODUCTION

The origin of toxic compounds can be various: they can be man-made or can be released by natural environment. Microorganisms have colonized so many different environments that can offer the chance not only to understand the mechanism to survive in harsh habitat but also to contribute to the degradation or metabolic utilization of toxic compounds. Survival and colonisation require the capacity to sense and adapt to environmental changes. Microbial cells respond to such stressful conditions mostly by switching global patterns of gene expression to relieve the environmental stress. Different strategies have been developed by microorganisms to remove toxic compounds such as xenobiotic substances or transition metals.

For many aspects Archaea represent a very interesting model system both for their genetic-molecular characteristics and for their growth conditions. The archaeal microorganisms have colonized habitats mainly characterized by extreme temperature, pH, and salinity; nonetheless the metagenomic analyses have revealed their presence in several types of environments (1). Recent molecular-based studies have shown that Archaea colonize also oil-containing environments, such as petroleum reservoirs, underground crude oil storage cavities and hydrocarbon-polluted aquifers. They are involved in the biodegradation of petroleum hydrocarbons mainly through methanogenesis. Indeed, methanogenes are the most abundant Archaea in the contaminated soils. At same time various archaeal microorganisms are capable to grow in environments contaminated by high concentration of heavy metals. It is clear that deep investigations on the mechanisms involved in homeostasis and resistance to toxic compounds and metal ions are required. Fortunately, new and improved techniques of analysis, combined with an increasing number of genome sequences are rapidly advancing the field of metal ions and xenobiotics metabolism in Archaea. We provide an overview of the strategies developed by Archaea to remove toxic compounds present in the environment. This review will be mainly focused both on different pathways that are involved in the detoxification of xenobiotics/multidrugs and on metal ions with different characteristics regarding their levels of toxicity and their link with fundamental metabolic processes.

3. DRUG DETOXIFICATION IN ARCHAEA: THE STATE OF ART

Toxic chemical compounds are either natural products, which are generated as a consequence of the metabolic activities of living organisms, or xenobiotic compounds, which are mainly produced by human activities (2). Most xenobiotic compounds are recalcitrant to degradation because they contain structures or substituents that are not normally present in natural molecules and hence limit their biodegradability. Owing to their human origin, they have been released into the environment very recently, and therefore, only a small number of organisms, mainly microorganisms, have developed various ways to resist to their toxic effects thus adapting to their new natural ecosystems (3). The majority of non pathogenic microorganisms are also multi-drug resistant and the genes and proteins responsible for resistance are homologous to those found in pathogens, strongly suggesting horizontal gene transfer (4). Several distinct mechanisms account for drug resistance. For example, drugs may be inactivated before reaching their targets by hydrolysis or by formation of inactive derivatives. Hence, multidrug resistance occurs by the accumulation, on resistance plasmids or transposons, of genes, coding for resistance to a specific agent, and/or by the action of multidrug efflux pumps, each of which can pump out more than one drug type (5). Antibiotic resistance starts as a natural phenomenon, but microorganisms can become more resistant due to the massive selection pressure provided by antibiotics themselves.

Aromatic hydrocarbons are pollutants mainly generated by anthropogenic activities associated with the industrial production of dyes, plastics, explosives, detergents, insecticides, and pharmaceuticals. They also constitute one of the three classes of compounds found in petroleum and the majority of them are toxic for human health being mutagenic and carcinogenic, with extent of toxicity mainly depending on the chemical nature of substituent groups and their position in the benzenic ring.

At molecular level, the stress response and the ability to metabolize recalcitrant chemicals and synthetic compounds generally is due to the expression of specific enzymes and biochemical pathways to degrade or transform these compounds (6). If the transformed compounds are still toxic for the cell, coupled multidrug efflux pumps efficiently extrude such molecules. These latter have been demonstrated to be important also for cell homeostasis and intercellular signal trafficking (7).

Metabolism of pollutants, as well as multidrug resistance, is coordinated by the integration of environmental and physiological signals into regulatory systems able to control the expression of target genes in a way that cells can also respond to subtle changes of environmental conditions (8, 9).

These issues will be dealt with in this part of the review mainly focusing on microorganisms of the archaeal domain and discussing on: i) degradative pathways of aromatic compounds ii) xenobiotic/multidrug transporters and iii) transcriptional regulation mechanisms involved in the adaptation and/or stress response to drugs.

3.1. Degradative pathways of aromatic compounds in Archaea

Knowledge on bio-transformation of toxic compounds in this domain of life, even if still at an infancy stage, comes out mainly from the analysis of biodegradation/biotransformation of organic pollutants as well as by "omics" investigation and functional studies.

Catabolic pathways of aromatic compounds in the Archaea have been basically inferred from genomic data comparisons and characterisation of few enzymes. Whole-genome sequencing of Archaea (115 sequenced and annotated to October 2011, http://archaea.ucsc.edu/) and genome comparisons revealed an increasingly complex picture of archaeal phylogeny, evolution, cellular features and processes (10). Archaeal microorganisms harbour many novel enzymes catalyzing reactions and pathways that are not present in Bacteria and Eukarya (11).

Biodegradation of aromatic compounds by thermophilic Archaea has been reported only in a few cases. The first evidence that hyperthermophilic Archaea can anaerobically oxidize aromatic compounds came from a study demonstrating that Ferroglobus placidus could grow at 85�C in anaerobic medium with a variety of aromatic compounds among which benzoate, phenol, 4-hydroxybenzoate, benzaldehyde and 4-hydroxybenzaldehyde as the sole electron donors, whereas two close relatives, Archaeoglobus profundus and A. veneficus could not (12). Some years later, Izzo et al., proved that Sulfolobus solfataricus P2 is able to grow aerobically on phenol as the sole carbon source (13). More recently, it was also shown that another S. solfataricus strain, 98/2, was able to utilize phenol as carbon and energy sources after adaptation on glucose with a small amount of phenol (14). The authors hypothesised the capability for Sulfolobus spp. to metabolize a larger number of aromatic hydrocarbons such as cresols, benzene, toluene, and polycyclic aromatic hydrocarbons (14).

In hyperthermophilic Archaea, the degradation pathways seem to be similar to those found in Bacteria. In the so-called upper pathway, the aromatic ring is first converted by mono- or dioxygenases to dihydroxylated compounds; they undergo ring cleavage reactions producing non-cyclic molecules which are in turn converted, in the lower pathway, into species that can enter in the citric acid cycle. Ring cleavage reactions are catalyzed by extradiol-cleaving dioxygenases (ECDs) and intradiol cleaving dioxygenases (ICDs), which incorporate both atoms of dioxygen into the aromatic substrate and cleave the aromatic ring at positions meta and ortho (15).

A genome analysis of S. solfataricus P2 performed in 2005 by Izzo et al., revealed the existence of: (i) a cluster of orfs coding for the subunits of a hypothetical bacterial multicomponent monooxygenase, (ii) an orf coding for a lower pathway protein of the catechol metabolism, and (iii) an orf coding for a putative catechol 2,3-dioxygenase (Sso1223, EC 1.13.11.2) (13). Two additional orfs, Sso2053 and Sso2054 have been hypothesised to code for enzymes involved in phenylacetate metabolism. Expression and characterization of the putative catechol 2,3-dioxygenase suggested that it could be the enzyme involved in phenol metabolism (13). A catechol 2,3-dioxygenase (C23O) was also characterised from S. solfataricus strain 98/2. The gene was found in the same genomic environment of a gene cluster encoding for a putative multicomponent monooxygenase, matching with the homologous region of S. solfataricus P2 (16). A comparative genomic analysis performed at http://microbes.ucsc.edu indicated that a homologue of the c23o gene is also present in S.islandicus strains LS215, M164 and LD85, but nor in other Chrenarchaea or Euryarchaea, while homologues of Sso2054 have been found in S. tokodaii (13), in S. islandicus strains LS215, M164 and LD85, in S. acidocaldarius, Metallospahera sedula and M. cuprina. To date, no functional characterisation has been reported nor for any of them or forSso2054. We inspected the S. solfataricus KEGG pathways of xenobiotics (www.genome.jp/kegg/pathway.html), (17) and found that C23O (Sso1223 and/or Sso2054) could be involved in the catabolism of different aromatic compounds, like toluene, xylene, benzoate, styrene, 1,4-dichlorobenzene.

Studies on the degradation/transformation of aromatic hydrocarbons in hypersaline environments by halophilic Archaea have shown that Haloarchaea are able to degrade aromatic compounds and to use them as carbon sources. For example, the Haloarchaea strain EH4 was found to be capable of degrading a wide range of n-alkanes and the aromatic hydrocarbons acenaphthene, phenanthrene, anthracene, and 9-metylanthracene (18). Haloferax volcanii D1227 isolated from oil brine-contaminated soil was shown to degrade monoaromatic carboxylic acids, such as benzoate, cinnamate and 3-phenylpropionate (19). Later on, it was shown that this microorganism metabolized these aromatic acids by initial 2-carbon shortening of the side chain to benzoyl-CoA via a mechanism similar to fatty acid beta-oxidation, followed by aromatic degradation using a gentisate pathway. The ring cleavage enzyme is a gentisate 1,2-dioxygenase (EC 1.13.11.4, Figure 1) (20).

Differently from H. volcanii, Haloarcula sp. D1 was shown to be able to metabolize also p-hydroxybenzoic acid through a pathway very similar to that found in H. volcanii involving 2,5-dihydroxybenzoic acid (gentisic acid) as an intermediate (21). Gentisate 1,2-dioxygenases from the two different haloarchaeal genera are closely related belonging to a protein family with members in both Bacteria and Archaea, distinct from other bacterial intra-diol and extra-diol dioxygenases. Adjacent to the gentisate 1,2-dioxygenase genes were also found additional genes, which are highly conserved in both microorganisms, and could participate in the aromatic degradative route (22). More recently, Bonfà et al., isolated ten further Haloferax strains from five hypersaline sites able to metabolize mixtures of aromatic compounds and suggested that this ability is a common widespread feature among the Haloferax spp (23).

The partial genome sequence of Haloterrigena sp. H13, an extreme halophilic archaeon, compared to that of other bacterial and archaeal halophiles has revealed genes that may be involved in biodegradation of several aromatic pollutants, like naphthalene, anthracene,1-/2-methylnaphthalene and genes of the benzoate degradation pathway via benzoyl-CoA formation. Gene homologs of (S)-2-haloacid dehalogenase (EC 3.8.1.2) and salicylate hydroxylase (EC 1.14.13.1), which might be involved in the degradation of dichloroethane and gamma-hexachlorocyclohexane were also found (24).

Among Archaea, methanogens also can metabolise aromatic and polyaromatic compounds; they use aromatic molecules to produce methane in anaerobiosis. For example, toluene and benzene were partially transformed to carbon dioxide and methane by mixed methanogenic cultures derived from ferulate enrichment (25). The metabolic intermediates detected suggested that benzene and toluene degradation occurred via initial oxidation by ring hydroxylation or methyl oxidation respectively, which would result in the production of phenol, cresols, or aromatic alcohols. Furthermore, Chang et al. demonstrated a direct association between anaerobic biodegradation of naphthalene and phenanthrene and methanogenesis using an inhibitor of methanogenesis, bromoethanesulfonic acid (BES), and monitoring the presence of methanogenic populations with 16S rRNA sequencing of isolated microorganisms before and after the addition of BES (26). However, aromatic degradation via methanogenic consortia is likely due to interspecies hydrogen exchange between primary aromatic fermentor, acetogens and methanogens (27)

The potential of proteomics to analyze global protein synthesis has been demonstrated to be a powerful tool to characterize archaeal response to several stresses, especially the response to heat and salt stress, or to analyse protein patterns in cells grown with different substrates, providing a more global view of all of the proteins/enzymes involved.

The proteome of S. solfataricus from cells grown on ethanol as the sole carbon sources (inhibitor of cell growth) has been compared with cells grown on glucose; the global translational responses were investigated; the majority of the changed proteins were either annotated as hypothetical or having energy metabolism related functions. Among these, the well characterised Sso2536 alcohol dehydrogenase was found almost ten fold overexpressed (28).

In H. volcanii the effect of salt stress on the microorganism determines the up-regulation of a homologue of the phage shock protein pspA, also found in other archaeal genomes. In Bacteria this protein has a role in sensing a variety of stresses, including heat shock, osmotic shock and prolonged stationary-phase incubation (29).

Methanosarcina acetivorans is an acetate- and methanol-utilizing methane-producing archaeon. A combination of advanced proteomics and DNA microarray analyses of M. acetivorans grown with either acetate or methanol, showed gene up-regulation for stress-related proteins in acetate- versus methanol-grown cells, including enzymes specific for polyphosphate accumulation and oxidative stress and allowed the identification of putative regulatory proteins belonging to the Multiple antibiotic Resistance Regulators (MarR), and Tetracycline Regulators (TetR) families (30).

In response to environmental stresses, many Archaea are capable of forming biofilm. Cells within the biofilm have an increased tolerance to otherwise toxic environmental conditions (31). Proteomic and transcriptomic analyses to describe physiological and regulatory features associated with biofilms in three Sulfolobus spp, showed alteration in the expression of proteins putatively involved in cellular functions like energy production, energy conversion, adaptation to environmental changes and stress, and substrate transport/binding activities. A small heat shock protein (Hsp20) was found to be biofilm-up-regulated, as well as two other stress related proteins, a thioredoxin and a peroxiredoxin. One gene, encoding 3-oxoacyl-(acyl-carrier-protein) reductase (fabG) was found to be down-regulated in biofilm-associated cells. In P. aeruginosa the enzyme FabG is involved in the production of a quorum sensing autoinducer (32).

Taken together, the reported proteomic/transcriptomic data reveal that adaptation to different lifestyles requires changes in the expression profiles not only of stress related proteins and global regulators, but also of an unpredictable plethora of cellular processes.

3.2. Drug transporters

Prokaryotes and eukaryotes are able to expel actively drugs across the cytoplasmic membrane against their concentration gradients in order to prevent their intracellular accumulation (33, 34). This mechanism is catalysed by transmembrane-proteins, the so-called primary or secondary transporters that couple translocation of substrates to the free energy released upon ATP hydrolysis or to the electrochemical (proton or sodium) gradients across the membrane, respectively (35).

Both primary and secondary transporters are in general pleiotropic towards their substrates. Indeed, they are active not only on drugs but also on a variety of different compounds such as sugars, amino acids, peptides, vitamins, ions, xenobiotics and even polypeptides. Thus, the functions of drug-efflux and metabolite-efflux often overlap, linking this class of proteins to various cellular functions that range from energy supply to osmoregulation, detoxification and virulence (34, 36). The efflux systems that accommodate a wide range of structurally dissimilar drugs, constitute the so-called multidrug transporters family (34, 37). The polyspecific nature of multidrug-resistance efflux pumps is responsible for the "multiple resistance" phenomenon to antibiotics and chemiotherapic agents as well as to natural substances produced by the host (38, 39, 40).

Survey of their genomes revealed that Archaea possess both primary and secondary multidrug transporters and that predicted transporters are more abundant in Euryarchaeal than in Crenarchaeal branch. So far, very few archaeal members have been functionally characterised and no archaea-specific transporter family has been identified. However, a high number of membrane proteins are still categorized as "hypothetical proteins" in the annotated genomes, suggesting that the lack of archaea-specific transporter families might be traced back to their limited functional characterization (2). Indeed, the complexity of the procedures to keep these proteins active has hindered their functional and structural characterisation.

In this study, archaeal sequences encoding for putative multidrug transporters were downloaded from the NCBI Protein database and the relative distribution of primary and secondary transporters in the archaeal domain is showed in table 1. The Transport Classification Database (TCDB) available at http://www.tcdb.org/ as well as the UniProtKB/Swiss-Prot database was also examined and the output data were cross-checked. The general features of archaeal primary and secondary transporters described below are mainly inferred from sequences comparison with the functionally and/or structurally characterised bacterial counterparts. The analysis of the genomic context of individual genes as well as their comparison across multiple archaeal species were performed at the website http://archaea.ucsc.edu/ (9). It must be pointed out that because of the structural differences between archaeal and bacterial membranes, the export function of archaeal transporters may be accomplished only upon association with different membrane components. The representativeness of these efflux transporters in the landscape of archaeal genomes is also discussed in the context of the evolutionary history of Archaea and of their natural environments.

3.2.1. Primary transporters

The ATP Binding Cassette (ABC)-transporter family includes a variety of members which are widely distributed in all domains of life. ABC transporters are involved in different processes such as substrate, chemotherapeutic drugs and antibiotics uptake or export, osmosensing and osmoregulation and play also a role in multidrug resistance. Mutations in these proteins cause diseases such as cystic fibrosis, hyperinsulinemia, macular degeneration (34, 42, 43).

Substrate transport is driven by ATP hydrolysis. Generally, these transporters possess two homologous halves, each containing two domains: a transmembrane domain (TMD) arranged into a six- alfa-helices surrounding and defining the translocation pore and a cytoplasmic nucleotide binding domains (NDB) bound to the cytosolic face of TMD which drive the translocation of the substrate through the hydrolysis ATP (33). ABC transporters branched off during evolution in importers and exporters that transfer substrates across the membrane in opposite directions. Beside this functional difference, their gene structure is also diverse. Indeed, in exporters TMDs and NBDs are encoded as a single polypeptide, whereas in importers TMDs and NBDs reside on separate subunits. Import systems are so far only found in prokaryotes and require an additional substrate binding protein (SBP) to deliver drugs to the transporter. Exporters recruit their substrate directly from the cytoplasm or lipid bilayer (44). Structural and biochemical data of archaeal ABC transporters are available only for ABC importers (42, 45, 46, 47, 48, 49), but none of them is involved in drug detoxification. For the exporters class, there is an indirect experimental evidence for the presence of an ABC-type multidrug transporter in H. vulcanii. Indeed, an anthracycline-resistant mutant of H. vulcanii was shown to transport rhodamine 123 more efficiently than the wild-type. This efflux activity was reduced by ABC-proteins modulators, such as diltiazem and activated by cytotoxic compounds or metabolites derived from aminoacids (50).

3.2.2. Secondary transporters

Secondary multidrug transporters are categorized according to the number of components that the pump has (single or multiple), the number of trans-membrane segment (TMS), the energy source that the pump uses and the type of substrate exported. Some of these families are members of larger super-families of transporters involved in a variety of other physiological functions.

Multidrug secondary transporters belong to the Multidrug/Oligosaccharide/Polysaccharide (MOP), to the drug metabolite transport (DMT), to the major facilitator superfamily (MFS) and to the resistance/nodulation/division (RND) superfamilies.

Among the MOP and DMT families, only those specifically involved in drug/metabolite detoxification will be described, i.e. the Multidrug and Toxic compound extrusion (MATE), the drug metabolite export (DME) and small multidrug Resistance (SMR) families (36, 40, 51, 52, 53).

Secondary transporters show disparate size of their polypeptide chain(s). Indeed, length ranges vary from 1000 amino acids for the RND transporters down to 100 aminoacids for the SMR transporters. MFS and MATE transporters are instead of an intermediate size (400-450 amino acids). Despite the difference in length, two main features are common to members of different families: i) the first and the second halves of secondary transporters are generally homologues, indicating that the corresponding genes might have undergone to duplication events during evolution; ii) nearly all secondary transporters are predicted to adopt a 12-helix structure. This suggests that the 12-TMSs topological organization has been evolutionary selected for its suitability in fulfilling the function of exporting chemically and structurally diverse class of compounds (51, 54).

3.2.2.1. MATE family

Multidrug and toxic compound extrusion (MATE)-type transporters function as exporters of cationic drugs and are widespread in almost all prokaryotes and eukaryotes (55) and are highly represented in the sequence databases. However, being the most recently designated multidrug efflux transport family, only few members (~ 3% of known MATE-proteins in total) have been characterized so far. They use preferentially a solute/cation antiport mechanism and a Na+-chemiosomotic gradient. Among MATE members, one subgroup is archaeal specific and three others include both bacterial and archaeal proteins that appear loosely related, suggesting that the evolutionary relation between them is to track back to vertical transmission from a common ancestor without horizontal transfer (55, 56). In this study, about 200 putative archaeal MATE sequences were retrieved from the NCBI protein database, but none of them has been functionally characterised. Whereas eukaryotic and bacterial genomes possess multiple MATE family paralogues, in archaeal genomes no more than four MATE paralogoues have been found.

3.2.2.2. The DME and SMR families

The DME family includes drugs transporters as well as efflux pumps for aminoacids metabolites and their toxic derivatives. Despite the low sequence conservation, DME members show the same topological organization with ten putative TMSs which clearly exhibit an internal repeat. Few predicted archaeal DME proteins are present in the NCBI and Swiss prot data banks, i.e. Hvo_1714 from H. volcanii, Smar_0735 from Staphylothermus marinus and AF1533 from Archeoglobus fulgidus. Hvo_1714 finds its homologues in the genomes of other euryarchaeal halophiles, as well as in those of several crenarchaeal representatives although they are not annotated as DME members. It has been reported that A. fulgidus has multiple putative DME parologoues on its genome and Pyrococcus horikoshii and P.abyssi contain six homologs in the DME family (57).

SMR members assemble into homo-oligomeric structures (dimers or tetramers), whose subunits are of 100-120 amino acid residues in length and span the membrane as α-helices four times (35, 58, 59, 60). Functionally characterized bacterial members catalyze multidrug efflux driven drug:H+ antiport but the chemical nature of their potential substrates is not well known. SMR members are thought to be the evolutionary building blocks of larger alfa-helical multidrug efflux proteins. Indeed, the structural arrangements of the TM strands of SMR proteins and of other larger multidrug transporters are similar, despite their low sequence similarity and their remarkable difference in length. Based on this evidences, the bigger multidrug transporters might have arisen from subsequent duplication events and/or addition of single TMSs during evolution, i.e. duplication of a 2TMS-encoding genetic element yielded the primordial SMR-type permeases which became first a five-TMSs protein upon addition of one TMS and then a 10 TMS transporter after duplication of the five-TMSs ancestor (61).

Within Archaea, SMR homologues are mainly present in euryarchaeal (Methanogenes and Halophiles) genomes, with only one SMR homologue present on a single genome (61).

. However, we found that in NCBI protein databank, the protein Saci_1019 from S. acidocaldarius is annotated as a putative SMR. BLASTP alignment showed identity (ranging from 25 to 35%) with small multidrug members from Methanomicrobia and Archaeoglobi. Saci_1019 is a 142 aa long protein; secondary structure as well as transmembrane topology prediction suggests that it possesses the typical 4-TMSs topology of SMR members. Furthermore, inspection of its genetic locus revealed that Saci_1019 gene is located next to an aldehyde oxidase, an enzyme characterized by a broad substrate specificity oxidizing aromatic compounds. All together, these observations suggest a functional correlation between detoxification and export of aldehydic drugs at this locus of S. acidocaldarius genome. Interestingly, the chromosome of other Sulfolobus species does not contain a Saci_1019 homologue pointing out to a specie-specific role of this protein.

In the surveyed archaeal genomes, variants of the typical 4-TMSs SMR are present and predicted to bear only two TMDS. This is the case of MM_0735, identified in the genome of M. mazeii and included in the group of Smr-3 (SMR3) in the TCDB. In some sub-members, only homologues of YvaE from Bacillus subtilis, are present in the euryarchaeal sub-phylum of methanonogens. An YvaD gene is commonly located in the same operon of YvaE, but no YvaD homologous are found in Euryarchaea, thus suggesting that YvaE potentially represents a PSMR progenitor (61).

The first and unique archaeal SMR, functionally and structurally characterised is that from the H. salinarum (Hsmr) (62). This exporter shares signature features with the eubacterial counterparts, in terms of the substrates-types transported and distinctive sequence elements. Hsmr finds its homologues in halo-and methano-microbia, with a percentage of identity varying from 61% to 31% and 39% to 33%, respectively. The oligomeric state of Hsmr and residues involved in the oligomerization has also been investigated (60, 63).

Interestingly, different hosts thriving in similar environmental niches bear SMR homologues suggesting that the selective pressure exerted by environment and/or by lifestyle (aerobic/anaerobic) strongly affect the inheritance and/or maintenance of SMR proteins (61).

3.2.2.3. The MFS superfamily

MFS represent the largest group of secondary active membrane transporters that are ubiquitous in the three domains of living organisms and are constituted of 400-450 residues arranged into a 12-helix structure. Individual members show stringent specificity for their substrates. However, as a family, they transport a huge variety of substrates through uniport, symport and/or antiport mechanisms (64-67).

Bacterial MFS members share low sequence identity and a single signature sequences, DRXXRR, conserved in equivalent position in the two homologues halves constituting the MFS proteins (67). Putative archaeal MFS sequences of transporters involved in drug detoxification retrieved from NCBI are 145, with only 1/3 belonging to the crenarchaeal branch. Only one archaeal MFS member from H. salinarium, homologue to the eukaryotic vescicular monoamine transporter (VMAT) has been characterised. VMAT proteins remove neurotransmettitors and toxic compounds from the cytoplasm, thereby conferring resistance to their effects. The H. salinarium protein, likewise the eukaryotic VMAT, confers resistance by expelling actively fluoroquinolones and chloramphenicol, through a proton motive force-dependent transport (68).

3.2.2.4. RND superfamily

Members of the RND superfamily utilize the proton motive force to catalyze substrate efflux. RND proteins are found ubiquitously in Bacteria, Archaea and Eukaryotes. The interest for this class of proteins stems from their pharmaceutical and medical significance, since the intrinsic drug resistance of Gram-negative bacteria is mainly attributable to RND-type drug exporters (69). Most of the RND transport systems consist of large polypeptide chains (700-1300 amino-acid residues) arranged into a 12-helix structure with two inter-regions between helices 1 and 2 and between helices 7 and 8 extended into large cytoplasmatic domains (70). Generally RND proteins arise from an intragenic tandem duplication event with the exception of few predicted RND proteins from some methanogens species that are of half size.

Bacterial RND representatives, such as AcrB from E.coli, work in association with other classes of proteins to exert their drug-efflux function (38, 71, 72). Only few putative AcrB-like members are present in the euryarchaeal genomes and none of them has been functionally characterised.

3.3. Transcriptional regulation in drug detoxification

Microorganisms' capability to biodegrade a wide variety of natural and man-made toxic compounds is orchestrated by the integration of environmental and physiological signals into regulatory systems that tightly control the expression of genes that are able of metabolizing such molecules (8). In the Archaea, the response to drug exposure is finely regulated by local and/or global regulators belonging to the MarR, Mercury Resistance Regulators (MerR), TetR and Arabinose Catabolism (AraC/XylS) families of transcriptional regulators.

MarR family proteins constitute a diverse group of transcriptional regulators that modulate the expression of genes encoding proteins involved in a wide variety of cellular processes including metabolic pathways, stress responses, virulence and degradation or export of harmful chemicals such as phenolic compounds, antibiotics and common household detergents. Adjacent genes located in the genomic locus of marR members are often regulated by the encoded transcription factor (73).

MarR proteins are dimers having a low level of sequence identity and a triangular shape; they bind to their cognate palindromic or pseudopalindromic DNA as homodimers, resulting in either transcriptional repression or activation. The DNA binding domain is a conserved winged helix_turn_helix motif with the two wings located at the corners of the triangle. Another common feature of MarR members is their ability to interact with specific ligands and, upon binding, to modulate DNA recognition. There are more than 12000 MarR-like proteins annotated in bacterial and archaeal genomes to date. In the archaeal domain, the crystal structures of four transcription factors, ST1710 (or StEmrR) from S. tokodaii (74, 75), MTH313 from Methanobacterium thermoautotrophicum (76) PH1061 from P. horikoshii OT3 (77) and BldR from S. solfataricus (78), have been determined.

A coordinate regulatory mechanism to defend against stress by aromatic compounds has been reported for S. solfataricus; this system responds by increasing the expression of a marR operon composed of a permease involved in multidrug efflux and a transcriptional regulator controlling expression of the operon itself and an alcohol dehydrogenase gene (Sso2536adh) responsible for the conversion of the toxic benzaldehyde in the less harmful benzyl alcohol (79, 80). Genomic analyses revealed that such an operon is conserved at least in the genus Sulfolobus. A second MarR member, BldR2, has also been characterised in S. solfataricus. It has been proposed that the protein could be involved in the regulation of aromatic catabolic pathways possibly controlling mechanisms different from those regulated by BldR (81).

The MerR family of transcriptional regulators contain a DNA-binding, winged helix-turn-helix domain of about 70 residues. Most MerR-type transcriptional regulators respond to environmental stimuli, like heavy metals, oxidative stress or antibiotics and a subgroup of metalloregulators are bacterial transcription activators that respond to metal ions. Generally, the helix-turn-helix DNA-binding motif is located in the N-terminal part of these transcriptional regulators and is followed by a coiled-coil region. The C-terminal part of MerR-type regulators contains binding regions that are specific to the effectors recognized (82).

MerR type regulators have been found in euryarchaeal (Methanosarcina, Methanococcus, Thermoplasma, Pyrococcus, Archeoglobus) genomes. Among crenarchaea representatives have been found in Sulfolobales.

TetR is a family of transcriptional repressors found in Bacteria and Archaea. The DNA binding domain is composed of a single helix-turn-helix motif. These proteins are involved in the transcriptional control of catabolic pathways, multidrug efflux pumps, differentiation processes, pathogenicity pathways, the control of the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals. The regulator can be released from the operator sequence upon ligand binding or is modulated by another regulator triggering a cell response to react to environmental insults (83). TetR members have been found both in crenarchaeal and euryarchaeal genomes with almost 200 members retrieved from Swiss Prot; however no functional studies have been conducted on archaeal TetR members. A proteomic and microarray analysis of the M. acetivorans grown with acetate or methanol identified several genes differentially expressed encoding regulatory proteins, among which 2 out of 13 are TetR family members (30).

The AraC/XylS family of transcription activator proteins is defined by a 100-amino-acid region of sequence similarity that forms an independent folding domain containing two helix-turn-helix DNA binding motifs. They are involved in the transcriptional regulation of a variety of cellular processes including carbon metabolism, stress responses and virulence (84). In general, these regulators comprise between 200 and 300 amino acids arranged in two domains: a conserved helix-turn-helix DNA-binding domain located at the C terminus whereas a variable N-terminal domain responsible for both protein dimerization and ligand binding (85). Members of the AraC family have been found only in euryarchaeal genomes. An AraC type DNA binding motif has been identified in the transcriptional regulator Bat from Halobacterium sp. NRC-1. It coordinates the synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen (86).

4. THE DARK SIDE OF METAL IONS: TOXICITY

Some metal ions, many of which are known as heavy metal, have a key role in the physiology of the cell. They can act as cofactors, can be involved in redox reactions or can confer stability to the proteins. At the same time, metal ion concentration has to be strictly controlled; if their homeostasis isn't preserved, the accumulation can produce toxic effects on cell viability. Metal homeostasis has been studied in detail in Bacteria and Eukarya, while in Archaea the study is still in its infancy. Recently, Cvetkovic A. et al (87) have characterized the metalloproteoma of P. furiosus, and compared it with those of E. coli and S. solfataricus revealing a species-specific assimilation of different metals. The comparison of growth inhibition by different metals showed that S. solfataricus was more sensitive to Ag2+, Hg2+, and Ni2+ with respect to E. coli while for Zn2+, Pb2+ and Cu2+ is true the contrary (88). Furthermore, also inside the Sulfolobales there is a great difference in the metal resistance, e.g. for copper, Minimal Inhibitory Concentration (MIC) values range from 1 mM for S. acidocaldarius to 200 mM for S. metallicus (89), suggesting that the different sensibility towards metal ions can be bound to very different habitats.

Heavy metal ions as nickel, cobalt, mercury, copper, arsenic play their toxic role inside the cell in different ways. They can bind to thiol groups of enzymes inhibiting their catalytic activity or they can interact with other divalent cations sequestering the ion and hence inhibiting its physiological role, or they can cause oxidative stress indirectly through uncoupling of electron transport chain, depletion of glutathione (GSH), and accumulation of Reactive Oxygen Species (ROS).

To establish if a metal has a toxic effect, it has to enter inside the cell. Generally the ions transport can be primary or secondary. The main primary metal ions transport systems in Prokaryotes are ABC and Energy-coupling factor (ECF). In addition some ions can enter utilizing specific or unspecific channels (90).

The strategies utilized by microorganisms to defend themselves from toxic ions are essentially based on ATP driven efflux by membrane transporters, detoxification enzymes, and metal sequestration. Here we describe how in Archaea different toxic ions are transported inside the cell and how they respond to metal attack (Table 2). The levels of toxicity of diverse metal ions and their link with fundamental metabolic processes are also analysed. In particular: 1) iron (Fe2+/Fe3+) that is an important trace element with low toxicity; 2) nickel (Ni2+), cobalt (Co2+) and copper (Cu+/Cu2+), that are important cofactors but at same time are toxic elements; 3) arsenic (As(III) /As(V)) and mercury (Hg2+) that have limited beneficial effects, but are highly toxic.

4.1. Iron

The development of oxygenic photosynthesis 2.5 billions of years ago generated a drastic change of life on the Earth. Among the effects produced by oxygen accumulation one regards the reactivity with iron. Iron is the second most abundant element in Earth's crust and is required for the life of almost all organisms because it is involved in the major cellular functions as photosynthesis, respiration, N2 fixation. Iron can be bound in Fe-S cluster or in heme groups to allow the proteins to play their biological functions, or it can modulate the function of transcriptional regulators (91, 92). Iron mainly exists in two different redox forms: the reduced Fe2+ and the oxidised Fe+3.

The oxidation of Fe2+ to Fe+3 makes the iron insoluble and not available for cellular biological processes; on the other hand Fe2+ can be extremely toxic in presence of ROS generating double damage e.g. iron release and protein inactivation (93). Through Fenton reactions (a), Fe2+ generates highly reactive and very dangerous hydroxyl radicals (OHo) that damage DNA, proteins and lipids.

H2O2 +Fe2+ à Fe3+ + OH- + OH. (a)

Furthermore, proteins containing a cluster 4Fe-4S can be damaged during oxidative stress (93). Therefore, it is fundamental to provide the iron necessary for the growth but also to keep it in non toxic form. For these reasons iron homeostasis must be strictly controlled and different strategies have been adopted. They comprise: uptake of free or sequestered iron; intracellular iron storage by proteins such as ferritins; iron-dependent gene regulation; scavenge of free-radicals. Generally, iron transport utilizes siderophores, low-molecular-weight compounds that show a high affinity and selectivity for Fe3+. Siderophore-Fe3+complexes enter in the cell using specific membrane bound receptors and then they are delivered in the cytosol by ABC transporters and released as Fe2+ upon reduction (94). Various uptake processes have been described in Bacteria and Eukaryotes, while a puzzling picture regarding Fe3+ uptake and reduction has been achieved in Archaea (Figure 2).

Inside the cell iron can be stored in different ways that involve mainly iron storage proteins as ferritins and haem-containing bacterioferritins, and DNA binding protein from starved cells (Dps). Ferritins and haem-containing bacterioferritins are composed by 24 identical subunits assembled in a spherical protein with a central cage that acts as iron reservoir; it can host at least 2000-3000 iron atoms in oxidized ferric form. These proteins take the iron in soluble form and store it in the protein cage as oxidized ferric form. The oxidation requires a ferroxidase centre in each subunit.

Dps, a dodecameric protein, can accommodate about 500 iron atoms. Twelve ferroxidase sites are present in the dodecamer, two between each dimer. In several Dps iron has been found bound in these sites characterized by conserved histidine and carboxylate residues. Dps monomers are small proteins with a MW of approximately 20 kDa with a folding of compact four-helix bundle. The fold is essentially similar to that of ferritin and bacterioferritin, suggesting a common ancestor (95, 96). Despite the fold conservation with ferritin and bacterioferritin, Dps-like (Dpsl) proteins exhibit also a variety of activities: they protect from oxidative stress and binds DNA (95). The first Dps was identified in E. coli, it is induced in stationary growth phase by ss factor and binds DNA aspecifically to protect from OHo damage caused by redox stress (97). Furthermore, it was shown its preference to oxidize Fe2+ in presence of H2O2; indicating a preferential role as iron scavenging and DNA shielding against OHo. To date, over a thousand of putative Dps-like proteins have been identified and about 3% have been found in Archaea.

Recently, Dpsl proteins were characterized from S. solfataricus, P. furiousus and H. salinarum (98-100). Dpsl from S. solfataricus maintains the 12-mer organization and a N-terminal extension that mediates the interaction with DNA; Dpsl possesses a different ferroxidase center, located in the four helix bundle monomer, composed by two iron ions coordinated with two histidine and four acidic residues (95, 101). In addition, a pair of cysteine residues (Cys101 and Cys126), juxtaposed between the exterior surface and the channel of the ferroxidase center, can form a disulfide bond. It is possible that such a disulfide bond plays a structural role but a possibility that cysteine residues could be involved in peroxidase activity through the redox cycle of reduced and oxidized forms must be investigated. Like peroxiredoxin (102), SsDpsl could be oxidized by H2O2 forming a disulfide bond, that is reconverted in thiolic form by an electrons redox cascade. As hypothized by Maaty et al. (103) from "omics" results for S. solfataricus, Dpsl together with superoxide dismutase (Sod) and peroxiredoxin could constitute a stressosome complex that would act in a coordinated way to remove ROS.

Through a proteomic approach, the iron metabolism has been investigated in extreme acidophiles Ferroplasma acidophilium spp revealing proteins with a higher iron content (104). This feature could be linked to a role of iron in protein stabilization in a moderately acidic cytoplasm (105). However, the high iron concentration inside the cell makes it highly exposed to oxidative damage. As a consequence, these microorganisms should be equipped with an array of putative antioxidant enzymes, proteins for iron storage and putative proteins involved in iron transport (105). In H. salinarum under low-iron growth conditions, no siderophores were determined in culture supernatants, nonetheless various xenosiderophores can be utilized to transport iron across the membrane; however which is the transport system involved and how the iron is reduced must be still clarified (106). In A. fulgidus a ferric reductase was identified and its structure solved. Unfortunately, it is still unclear, if the enzyme is involved in the assimilation of iron or in the dissimilatory reduction (107-109).

4.2. Nickel and Cobalt

Nickel and cobalt cationic forms (Ni+2 and Co+2) are essential nutrients for microorganisms playing a key role in the metabolism. These metal ions are cofactors of numerous proteins: nickel is fundamental for various metalloenzymes as, NiFe-hydrogenase, carbon monoxide dehydrogenase (Ni-CODH) and NiSod, involved in detoxification of superoxide radical (O2.-). On the other hand, cobalt is mainly found as component of Vitamin B12 (110). To date, the mechanisms of nickel toxicity are poorly understood and possible mechanisms have been suggested to explain the damage of this ion: i) the essential metal of metalloproteins is replaced by nickel, ii) enzyme inactivation or indirect inhibition of oxidative stress response. (111). Cobalt toxicity in E.coli derives from completion with iron on Fe-S clusters, or on protoporphyrin IX of cytochrome and from sulfur assimilation (112). The two ions can be transported both by active ATP driven system and by chemiosmotic gradient. In Archaea three families of nickel and cobalt transporters are identified: NiKABCDE, Nik/CbiMNQO belonging to ABC and ECF transporters family, respectively (113), and NiCoT belonging to a family of secondary metal transporters (114) (Figure 3).

NikABCDE system, likely to ABC cassette system, couples ATP hydrolysis to its uptake. The system is composed by two TMDs that constitute a channel across the membrane and two ATP-hydrolyzing subunits that supply energy for the transport; in addition, a soluble cytoplasmic protein with high affinity to substrate binds and delivers the ion to a transmembrane protein. Putative transporters belonging to the NikABCDE family were found in the genomes of M. acetivorans, M. barkeri and M. mazei (1, 115).

Nik/CbiMNQO belongs to the ECF family of transporters. In particular it is constituted by three functional components: S, T and A units (113). S unit is constituted by a hetero-oligomer that binds the substrate and it is encoded by nik/cbiM and nik/cbiN. NickM and CbiM have seven TMDs with a conserved N-terminus containing a His residue in position 2, essential for the transport (113). Despite the large variations in primary structure, NikN and CbiN have similar three dimensional structures. In the Co2+ transport system CbiM is larger than CbiN and both units are essential for the transport (116). NikM and NikN are often found fused in a single protein or can be replaced by NikK and NikL (113).

The T component is constituted by a conserved transmembrane protein encoded by nik/cbiQ. Nik/CbiQ shows the classical Walker A and B motifs for ATP binding (117). Finally A unit is formed by a couple of ABC ATPases encoded by nik/cbiO. Nik/CbiO is a cytoplasmic ATP-binding protein that interacts with the cytoplasmic loop of Nik/CbiQ.

Comparative and phylogenetic analyses were performed on 400 microbial genomes. Using SEED comparative genomics platform various ECF transporter families were identified and in 39 archaeal genomes nik/cbiMNQO homologues were found (113, 117, 118). The major differences with the ABC system are represented by the lack of an extracytoplasmic solute binding protein and a different subunit assembly.

NiCoT is a secondary transporter of nickel and cobalt also diffused in eukaryotic organisms; it is characterized by eight TMSs and supports high-affinity uptake (114); homologues are present in the thermoacidophilic Archaea S. solfataricus and Thermoplasma acidophilum (1, 115).

In Archaea, the control of nickel and cobalt homeostasis occurs through two different mechanisms: the first is based on the regulation of genes that encode the nickel transporters by the transcriptional repressor NikR and the second regards the active efflux driven by members of the subfamily of P-ATPase named P1Btype-ATPase.

In E. coli the nikABCDE operon is tightly regulated: in anaerobic conditions it is regulated by fumarate and the nitrate reduction regulator (FNR), whereas in presence of excess of nickel its transcription is repressed by NikR (119). NikR has been crystallized and characterized in the anaerobic archaeon P. horikoshii (120, 121). The overall structure is a homotetramer with a central Metal Binding Domain (MBD) and two flanking dimeric ribbon-helix-helix (RHH) domains that bind the DNA. Each MBD contains low and high-affinity nickel binding sites allowing a fine sensing of metal concentration from pico- to nanomolar ranges of concentration (120-122).

Orthologs of E.coli NikR were found in the genomes of different prokaryotes (115). In particular, genome analyses of methanogenic Archaea showed more than four copies of nikR and multiple copies of putative nickel transporters, suggesting the importance to safety nickel homeostasis. Phylogenetical analysis showed that NikR repressors are clustered both in a large group including proteobacteria and Archaea and a smaller group comprising Pyrococcus spp. and Thermococcus kodakarensis. NikR binding sites have been also identified in prokaryotic genomes; they have been grouped in four consensus sequence, two of which are present in Archaea. All sequences contain the same palindromic structure and a distance of 13-14 base pairs, between the two half sites. Using the identified consensus sequence, 28 bacterial and 14 archaeal transporters belonging to NiCoT, NiABCDE and NikMNQ transporters have been identified and repression by NikR has been predicted (115).

The second defence system utilizes a subgroup of P-ATPases: the P1Btype-ATPase. This pump is involved in the efflux across membrane. P1Btype-ATPases are present in all kingdoms of life and have been identified in most archaeal genomes. The structure and function of P1Btype-ATPases for the export of different metals ions were elucidated in different bacteria, plants and fungi (122).

4.3. Copper

Differently by nickel and cobalt that have a medium toxicity, copper is highly toxic for the cell when outside physiological range. Copper catalyses, in Fenton reaction, ROS production (123, 124), it can bind with high affinity to His, Cys and Met residues inactivating the proteins (125, 126) or can damage the iron-sulfur clusters (127). At the same time copper is an important transition element used by cells as cofactor of proteins/enzymes involved in a wide range of biological processes e.g. oxidative phosphorylation and antioxidant defence. Likewise for the other ions described above, every organism also possesses systems to maintain and to regulate the homeostasis of this metal. The different level of copper resistance depends on the environments in which the microorganisms live. For example, E. coli can grow in the presence of 1 mM copper while the acidophiles as F. acidarmanus or S. metallicus colonizing habitats with extreme metal contaminations can grow at copper concentration >200 mM (125).

In Archaea the homeostasis of copper involves mainly two systems: copper efflux and metal sequestration (128) (Figure 4).

Copper efflux is driven by P1B-type ATPases. Those characterized showed high structural stability and different selectivity with respect to Cu+ and Cu2+, the two different oxidation states, (122). Archaeal genomes analyses have allowed the classification in two different subgroups: Cu+-ATPase and Cu2+-ATPase (122).

In the archaeon A. fulgidus the two energetic pumps are named CopA and CopB, able to transport Cu+ and Cu2+ respectively (122, 129). A. fulgidus CopA has been extensively characterized: it consists of eight TMD, an A-domain between helices four and five, an ATP-Binding Domain (BD) between helices six and seven, and a soluble MBD at the N-terminus (N-MBDs). Cu+ isn't transported in its hydrated form to the ATPase subunit but the metal ion is previously bound to the methallochaperone CopZ and then transferred to the N-MBD. The N-MBD binds copper through a conserved CXXC motif determining an allosteric effect on protein conformation that affects the turnover rate of the enzyme (130). The metal ion is then transferred to TM-MBS required for enzyme phosphorylation and for consequent metal translocation (131-134). The residues responsible of ion translocation are two Cys residues in helix six, Asn, Tyr in helix seven and Met, Ser residues in helix eight. In addition A. fulgidus CopA has a MBD at C-terminus, which binds with high affinity Cu+, and characterized by a CHHC motif that was only found in one metallochaperone homolog from Thermosipho melanesiensis BI429. Furthermore C-MDB interacts both with ATP-BD and A domain suggesting a more complex role in vivo of CopA (130).

CopB is rich in His residues that could determine the enzyme selectivity versus Cu2+. The two putative MBSs, in particular the Cys-Pro-His sequence in TM-MBS of helix 6 and the His 17 residue in the N- MBD, could influence the enzyme specificity and be involved in the turnover, respectively (129).

Copper regulation was studied in various bacteria as cyanonabacteria (135, 136), E. coli ( 137) and in the Gram-positive bacterium Enterococcus hirae (138, 139). The operon for copper resistance comprises copY, copZ, copA and copB. copA and copB encode the copper ATPases involved in the uptake and export of Cu+, respectively (138); CopY is a transcriptional regulator and CopZ is a metallochaperone that acts as intracellular copper driver. Inside the cell, Cu+ is transferred from CopA to CopZ that delivers Cu+ to the dimeric CopY repressor. CopY is a Zn containing homodimeric repressor that binds to the copYZAB repressing the transcription (140). In presence of Cu+ CopY releases the zinc, bounds copper ions, looses affinity for its own promoter, and derepresses the operon transcription (138, 141).

Using comparative genomics the copper resistance gene cluster was identified in various Archaea: in F. acidarmanus Fer1 strain, in P2 and 98/2 strains of S. solfataricus and in S. metallicus.

F. acidarmarus Fer1 can grow at high level of copper concentration (~300 mM) indicating high metal-resistance. In this microorganism, copper homeostasis is controlled by a putative transcriptional factor CopY that regulates the expression of a putative metallochaperone (copZ) and P-type ATPase (copB) that are co-transcribed. CopB shows high similarity with both copper uptake and copper export ATPases (125). Upon metallic stress, five of twenty-one proteins associated to protein stability are up-regulated in F. acidarmanus Fer1, suggesting that they could contribute to limit cellular damage (125).

In S. solfataricus two P-type ATPases responsible for copper transport have been identified: CopB (Sso2896) and CopA (Sso2651). CopB catalytic domain is characterized by a phosphatase domain (CopB-A), an ATP-binding and phosphorylation domain (CopB-B) and a heavy metal binding domain (CopB-C). The partial alignment of CopB with E. coli and E. hirae CopA showed the conservation of identical residues in these domains. Furthermore, the capability of CopB-B to hydrolyze ATP, suggests its belonging to the P-type ATPase family (142).

Similarly, a gene copper resistance cluster was also identified in S. solfataricus P2 and a molecular characterization was obtained (143). copR (Sso2652) encodes the transcriptional regulator, copT (Sso10823) encodes the metallochaperone and copA (Sso2651) encodes the P-type ATPase. All these proteins have the signature of TRASH domains characterized by the cysteine motif C-Xaa19-22-Caa3-C predicted to be involved in copper sensing, trafficking and resistance (143-145).

Differently from S. solfataricus P2 strain, 98/2 shows a higher resistance to copper. Like the P2 strain, S. solfataricus 98/2 copRTA operon encodes a transcriptional regulator, a copper binding protein and a P-type ATPase. In order to preserve internal copper homeostasis CopT and CopA levels are maintained through copRTA constitutive expression from the same promoter upstream copR; in the presence of high copper concentration, transcription from a second promoter upstream copTA is induced to remove the copper excess by CopA mediated efflux (143, 145). Sequence analysis in both S. solfataricus strains showed the identity of copR, copT and promoter sequence upstream copTA. It has been suggested that additional trans-acting factors could influence the different copper sensitivity of the two strains.

Copper resistance in Sufolobus spp has also been related to copper sequestration mediated by polyphosphate (polyP). PolyP is a polymer of hundreds of orthophosphate linked by phosphoanhydride bonds whose biosynthesis is catalysed by polyphosphate kinase (PPK) (146) and degradation by an exopolyphosphatase (PPX) (147). In Bacteria a model was proposed in which intracellular cations regulate the activity of PPX releasing Pi from PolyP; the Pi-metal complex is then transported through Phosphate inorganic transport (Pit) outside the cell. In Archaea, polyP accumulation has been described only in Methanosarcina spp and in S. acidocaldarius (148, 149); PPK was identified in S. acidocaldarius and PPX in S. solfataricus (150). pit-like genes have not been found in archaeal genomes (151). Recently, in S. metallicus, Remonnsellez et al. (128) correlated the increase in copper concentration not only to a decrease in the level of polyP, but also to an increase of both PPX enzymatic activity and phosphate efflux, suggesting the existence of a new system of copper detoxification through metal sequestration.

4.4. Arsenic

The arsenic can be found as arsenite, As(III), or as arsenate, As(V). At physiological levels As(III) and As(V) can be involved in the respiratory chain playing a completely different role, indeed while As(III) could function as an electron donor at the start of a membrane respiratory chain, As(V) functions as a terminal electron acceptor for an anaerobic respiratory chain (152- 158). As(V) is a structural analog of phosphate and can inhibit phosphorylation processes by producing unstable arsenylated derivatives. Both in Archaea and Bacteria As(V) can enter cells via two phosphate transport systems: phosphate specific transport (Pst) and Pit (1). Pit system uptakes phosphate and As(V), at similar rates, whereas the Pst is highly specific for phosphate. Considering that As(V) is the thermodynamically favourable form of arsenic under aerobic conditions (159-161), it is likely to be the most common form of arsenic in many environments and in addition it is less labile and toxic than As(III) (159). As(III) results toxic to cells because of its ability to bind to essential sulfhydryl groups of proteins and dithiols such as glutaredoxin. Differently from As(V), As(III), due to its un-ionized form at neutral pH, can passively move across the membrane bilayer or be transported by a carrier protein similar to those that transport un-ionized organic compounds.

The detoxification system involves the reduction of As(V) in As(III) by the cytoplasmic ArsC-type reductase (EC 1.20.4.1) and then it is pumped directly out of the cell by the ArsB protein (Figure 5).

The genes involved in the detoxification are generally clustered in the operon arsRCB: arsR coding the arsenite-responsive transcriptional repressor controlling basal levels of ars expression (162), arsC encoding the arsenate reductase (a) and arsB coding an arsenite-specific transmembrane pump (159, 163, 164) (EC 3.6.3.16) (b)

As(V) + glutaredoxin/thioredoxin à As(III) + glutaredoxin/thioredoxin disulfide + H2O (a)

ATP + H2O + As(III)in à ADP + phosphate + As(III)out (b)

ArsR belongs to a metalloregulatory transcriptional repressors family (SmtB/ArsR), and acts as metal-sensitive DNA binding repressor that controls expression of various metal detoxification genes (165, 166). To define the DNA sequence recognized by this family multiple sequence alignments of putative operator sites and corresponding winged helix-turn-helix (HTH) motifs were used. Using a web application, the Prokaryotic Inter- Genic Exploration Database (PIGED; http://bioinformatics. uwp.edu/~PIGED/home.htm), 60 SmtB/ArsR DNA binding sites or operators, linked to metal detoxification genes, have been predicted among 230 annotated prokaryotic genome sequences (165). Nine archaeal SmtB/ArsR and associated regulons have been predicted by the computational analysis and the binding activity of one of these proteins from M. acetivorans C2A MA4344 was confirmed by biochemical studies (165). In addition, the crystal structure of the product of the ph1932 from P. horikoshii OT3 was solved and it resulted to be an archaeal ArsR (167). In detail, the C-terminal domain is responsible for dimerization and forms a unique hat-shaped helix-bundle; the inside of the hat is a possible effector-binding site of this protein.

Two different bacterial classes of ArsC have been found (152, 168). The first of them is ArsC discovered in Staphylococcus aureus pI258 plasmid and in other Gram-positive bacteria. In pI258 ArsC, the reduction of As(V) involves three Cys residues (158) in a disulfide cascade coupled with Trx, Trx reductase and NADPH as recycling system (169). It is different from the enzyme discovered in enteric bacteria such as E. coli (i.e. from plasmid R773) which has a single active site Cys residue and requires reduced glutathione (GSH) and Grx during the catalytic cycle (170). Both these classes show a low molecular weight, 131 amino acid residues for S. aureus and 141 for E. coli, respectively and are monomeric cytoplasmic enzymes (158).

A detailed study of the phylogenetic distribution of arsC has been performed showing a widespread presence in all three kingdom of life, suggesting that arsenate reductase is an evolutionarily old enzyme that could be present either in the last universal common ancestor or after the Bacteria/Archaea divergence through early HGT events (159).

Recently the structure of ArsC from A. fulgidus Dsm4304 (PDB code 1Y1L) was solved within a structural genomic project (158), showing a Trx fold as other ArsC (171-173). Although the enzyme presents low sequence identity with pI258 ArsC (about 15 %) their structures result to be similar conserving the three catalytic Cys residues. Structurally, it seems evident that the A. fulgidus ArsC, and likely the other archaeal members, could be linked to the Trx-coupled system, but this functional prediction needs to be confirmed by biochemical analyses.

ArsB can function alone or with another As(III)-stimulated ATPase pump. Indeed some microbial strains possess additional genetic determinants, often found on plasmids that confer arsenic resistance. arsA, encoding As(III)-stimulated ATPase, is allosterically activated by As(III) and it functions as catalytic subunit of ArsB (174) increasing As(III) extrusion (175). It is characterized by the signature sequence DTAPTGHTIRLL (176) and it consists of two homologous modules designated as the A1 and A2 loops, a clear result of an ancestral gene duplication and fusion (163, 177, 178); arsD encodes an arsenic chaperone that transfers As(III) to ArsA regulating ars expression (178-180). arsH has been identified but has an uncertain function (181).

In other Archaea like F. acidarmanus, Metallosphaera sedula, S. acidocaldarius and S. solfataricus the lack of some genes involved in the arsenic resistance has been highlighted (182). In particular Gihring et al. investigated the arsenic resistance in arsenic-hypertolerant F. acidarmanus Fer1 (183). In detail, genes encoding putative ArsR and ArsB homologues were found located on a single operon. A gene encoding putative ArsA, located apart from the arsRB operon, was also identified. Arsenate-resistance genes encoding proteins homologous to the ArsC were not found, indicating that additional unknown arsenic-resistance genes exist for arsenate tolerance (183). Later Austin et al., performing transcriptional analyses of the putative arsR and arsB, demonstrated that these genes are co-transcribed, and expressed in response to As(III), but not As(V) (184). In addition in cells exposed to As(III) an enhanced expression of proteins associated with protein refolding, such as the thermosome HSP60 family chaperonin and HSP70 DnaK type heat shock proteins, was detected (184).

Furthermore in silico studies were performed using motif-based searches for identification of genes involved in As(III) resistance (182) suggesting the presence of arsB homologues in other two Archaea T acidophilum and T. volcanium, but no homologues were found for S. solfataricus, S. tokodaii and in S. acidocaldarius strain BC (155).

These Archaea may possess an unpredicted As(V) reductase that does not have significant sequence homology to other ArsC proteins or, finally, completely novel resistance mechanisms may be adopted (182).

An example of a different mechanism of arsenic resistance is represented through methylation and subsequent volatization of As(III) involving the methyltransferase ArsM (EC 2.1.1.137) (c) and (d)  

S-adenosyl-L-methionine + arsenite à S-adenosyl-L-homocysteine + methylarsonate (c)

S-adenosyl-L-methionine + methylarsonite à S-adenosyl-L-homocysteine + dimethylarsinate (d)

ArsM catalyzes the formation of a number of methylated intermediates from As(III) with a consequent loss of arsenic, from both the medium and the cells. Because ArsM homologues are widespread in nature, this microbial-mediated transformation is proposed to have an important impact on the global arsenic cycle (185). In 1971 McBride and Wolfe already observed in Methanobacterium the production of dimethylarsine (186). Other methanogenic Archaea M. bryantii, M. formicicum, and M. barkeri were reported to produce volatile forms of methylated arsenic (1, 187).

Also in the genome sequence of Halobacterium spp. strain NRC-1 genes homologous to those responsible for conferring resistance to arsenic have been annoted (188). These genes occur on both the large extrachromosomal replicon pNRC100 (arsADRC, arsR2 and arsM), and on the chromosome (arsB). In particular on pNRC100 were identified: two additional ars genes located near arsADRC, a second arsR gene (arsR2) and another gene, named arsM encoding for a putative methyltransferase homologous to a recently identified mammalian As(III)-methyltransferase (188). The role of all of these ars genes in arsenic resistance has been studied genetically by gene knockout producing sensitivity to As(III). The authors hypothesize that ArsM could methylate intracellular arsenite, creating a concentration gradient outward representing an alternative resistance mechanism by lowering the intracellular arsenic concentration (188).

4.5. Mercury

Mercury is the heavy metal with the highest toxicity. It can be found in different forms such as elemental (Hg0), ionized (inorganic salts Hg2+and Hg+), organic (typically as methylmercury) or sulfidic (cinnabar) (189). Mercuric ions (Hg2+) and methylmercury are major, human-generated, toxic contaminants present on the earth. Bacteria can adopt different strategies to react to mercury exposure, providing a real mean of bioremediation by taking up these compounds and reducing them to volatile, non-toxic, elemental mercury (Hg0) (190) (Figure 6).

Three types of mercury/ methylmercury transporters have been previously identified in Bacteria and described in detail in the review by Yamaguchi et al.: MerC, MerT and MerF (190). Each of these has distinct topologies in particular MerC is characterized by a four TMS, while most MerT and MerF proteins have three and two TMSs, respectively. MerC has been shown to bind Hg2+ions, but the mode of transport remains unknown while MerT transports Hg2+ into the cytoplasm directly interacting with the periplasmic protein MerP (191). MerP has MBDs homologous to enzymes, chaperones and heavy metal-transporting P-Type ATPases indeed it shares the so named metal binding loop GMTCXX and the typical ferredoxin-like fold (190, 192). In addition, it has been observed that the binding loop undergoes a conformational change in response to metal binding (190). Regarding the last known transporter MerF, its NMR structure was determined and it showed a helix-turn-helix core hairpin loop with the two vicinal pairs of Cys residues, involved in the transport of Hg2+ across the membrane, exposed to the cytoplasm (193). Differently from MerTs, MerF proteins do not function coupled with MerP (190, 191). MerP homologues are absent in eukaryotes but few examples were observed in Archaea, while homologues of merT, merF and merC genes were not found in Archaea. This observation conducted Yamaguchi et al. to hypothesize that these kinds of mercuric resistance systems appeared late in the evolution, at least after the divergence of Archaea and Eukaryotes from Bacteria (190). Nonetheless, they utilized the ubiquitous MBD to derive the MerP homologues.

Once entered in the cells, Hg2+, deriving also by the protonolysis of the organomercurials catalyzed by the organomercurial lyase (MerB) (194), must be converted into the volatile Hg0 (1, 189) to be detoxified. The strategy of detoxification better characterized is mediated by the products of mer operon. In detail merTPCAD operon encodes a group of proteins involved in the binding, transport, and reduction of mercury. As previously cited MerP binds Hg2+ and transfers it to MerT. MerT transports mercuric ion into the cytoplasm where the NADPH-dependent flavoprotein mercuric reductase (MerA) (EC 1.16.1.1) reduces Hg2+ to Hg0 that is released from the cell (a)

Hg2+ + NADPH à Hg0 + NADP(+) + H(+) (a)

MerA belongs to the pyridine nucleotide disulfide oxidoreductase family and its core domains shares significant sequence identity in the redox active disulfide/dithiol active site, NAD(P)H and FAD binding sites with glutathione reductase, trypanthione reductase and lipoamide dehydrogenase (LPD). In particular, Pullikuth and Gill (195) showed by means of phylogenetic reconstructions a common ancestor between LPD and MerA. A peculiar characteristic of MerA is the presence of a short C-terminus extension containing a Cys pair which is involved in delivery of Hg2+ to the inner redox cysteine active site (196, 197).

Phylogenetic reconstructions of MerA indicated its origin in a thermophilic bacterium following the divergence of the Archaea and Bacteria with a subsequent acquisition in Archaea via HGT (197). The majority of the microorganisms possessing MerA homologues live in oxygenic environments suggesting that the presence of MerA is correlated with oxygenation of the biosphere (197).

In Bacteria the expression of the mer operon is regulated by MerD and MerR (189, 198). Both merR and merD gene products bind to the same operator DNA. In the absence of mercury, MerR binds and bends the DNA interacting with the RNA polymerase keeping in an inactive state and consequentially repressing transcription (189, 199-201). In the presence of mercury, MerR undergoes a conformational change favouring mer operon transcription. The coupled action of MerD and MerR was hypothesized suggesting a displacement of MerD on Hg-bound MerR from the mer operator to allow new synthesis of metal-free MerR able to switch off the induction of the mer genes when the external mercury is exhausted. A protein phylogenetic analysis identified two orthologs of merA and merR in the genome of S. solfataricus. The studies conducted by Schelert et al. (189) represented the first report of an archaeal heavy metal resistance system. The archaeal system proved to employ a MerR protein that acts as a negative transcriptional regulator of merA expression, indeed a merR disruption mutant exhibited elevated Hg(II) resistance and at the same time a constitutive synthesis of the merA transcript. Further findings indicated that the toxicity of mercuric ion in S. solfataricus is in part the consequence of transcription inhibition due to transcription factor B inactivation (202). It has been observed that Hg2+ interacts with the regulator MerR (203). Two additional Hg2+ inducible S. solfataricus genes, merH and merI located on either side of merA, were identified by Schelert et al. (203) and constituted the merHAI operon. While a chaperone function for mercury immobilization has been suggested for MerH, the role of MerI is still unknown. Different experiments demonstrated that MerR remained bound to DNA, thus exerting a protective effect over the preinitiation transcription complex. In addition MerR contains a motif resembling a distant ArsR homolog (203). Indeed both ArsR and MerR present the wHTH DNA binding domain (203, 204), but constitute two distinct families. The significant difference between these two families resides in their response to metal ligands; ArsR dissociates from DNA while MerR remains bound. The ArsR motif in MerR, however, is slightly different because it lacks the conserved Cys residue required for ligand-induced DNA release. Despite this divergent feature, the S. solfataricus MerR protein could be the first example of Hg2+responsive ArsR family member.

Another form in which mercuric is found is represented by the methylmercury, a very potent neurotoxin; in Bacteria, the transformation occurs anaerobically, is directly coupled to sulfate respiration and depends on the presence of constitutive and induced methyl transferase pathways (1, 205). Very little is known concerning mercuric methylation in Archaea, making exceptions for some methanogens such as M. maripadulis able to methylate Hg2+ if grown together with Desulfovibrio desulfuricans (1, 206). Bacteria are capable of demethylating methylmercury. This process is regulated by an inducible mer operon and serves as a detoxification mechanism in polluted environments. In Archaea, nothing is known on detoxification by demethylation.

5. CONCLUSIONS

All ecosystems on Earth are submitted to pollution. In particular, a wide range of pollutants and heavy metals are actively biodegraded (mineralized or transformed) in extreme environments characterized by low or elevated temperatures, acidic or alkaline pH, high salinity or high pressure.

Since Archaea often represent the majority of the microbial community in these environments, it is expected that these microorganisms play a leading role in the biogeochemistry of the elements as well as in the degradation of xenobiotics. Nevertheless, the overall degradation mechanisms and the enzymes involved are not completely elucidated so far.

To better understand these processes global approaches such as whole genome-transcriptome-proteome analyses and metagenomics are needed. This could reveal enzymes that can direct novel chemical reactions and/or new catabolic aptitudes as well as new archaeal species or synthropic communities responsible for bioremediation. From a more applied point of view, this knowledge will be critical for developing strategies aimed to remediate polluted habitats.

6. ACKNOWLEDGEMENTS

All the authors equally contributed to the paper. The work was supported in part by research grants from Ministero dell'Istruzione, dell'Università e della Ricerca Scientifica (Progetti di Ricerca di Interesse Nazionale, 2008). Support from the Regional Center of Competence (CRdC ATIBB, Regione Campania, Naples, Italy) is also gratefully acknowledged. We apologize to those authors whose relevant work could not be cited owing to space restrictions.

7. REFERENCES

1. E Bini: Archaeal transformation of metals in the environment. FEMS MicrobiolEcol 73, 1-16 (2010)

2. Rieger P G, H M Meier, M Gerle, U Vogt, T Groth, H J Knackmuss: Xenobiotics in the environment: present and future strategies to obviate the problem of biological persistence. J Biotech 94, 101-123 (2002)
http://dx.doi.org/10.1016/S0168-1656(01)00422-9

3. Roldán M D, E Pérez-Reinado, F Castillo, C Moreno-Vivián: Reduction of polynitroaromatic compounds: the bacterial nitroreductases. FEMS Microbiol Rev 32, 474-500 (2008)
http://dx.doi.org/10.1111/j.1574-6976.2008.00107.x

4. G D Wright: Q&A: Antibiotic resistance: where does it come from and what can we do about it? BMC Biology 8, 123-129 (2010)
http://dx.doi.org/10.1186/1741-7007-8-123

5. H Nikaido: Multidrug Resistance in Bacteria. Annu Rev Biochem 78, 119-46 (2009)
http://dx.doi.org/10.1146/annurev.biochem.78.082907.145923

6. Van Hamme J D, P M Fedorak, J M Foght, M R Gray, H D Dettman: Use of a novel fluorinated organosulfur compound to isolate bacteria capable of carbon-sulfur bond cleavage. Appl Environ Microbiol 70, 1487-93 (2004)
http://dx.doi.org/10.1128/AEM.70.3.1487-1493.2004

7. Martinez J L, M B Sánchez, L Martínez-Solano, A Hernandez, L Garmendia, A Fajardo, C Alvarez-Ortega: Functional role of bacterial multidrug efflux pumps in microbial natural ecosystems. FEMS Microbiol Rev 33, 430-49 (2009)
http://dx.doi.org/10.1111/j.1574-6976.2008.00157.x

8. V Shingler: Integrated regulation in response to aromatic compounds: from signal sensing to attractive behaviour. Environ Microbiol 5, 1226-41 (2003)
http://dx.doi.org/10.1111/j.1462-2920.2003.00472.x

9.Pedone E, S Bartolucci, G Fiorentino: Sensing and adapting to environmental stress: the archaeal tactic. Front Biosci 9, 2909-26 (2004)
http://dx.doi.org/10.2741/1447

10. Brochier-Armanet C, P Forterre, S Gribaldo: Phylogeny and evolution of the Archaea: one hundred genomes later. Curr Opin Microbiol 14, 274-281 (2011)
http://dx.doi.org/10.1016/j.mib.2011.04.015

11. Sato T, H Atomi: Novel metabolic pathways in Archaea. Curr Opin Microbiol 14, 307-314 (2011)
http://dx.doi.org/10.1016/j.mib.2011.04.014

12. Tor J M, D R Lovley: Anaerobic degradation of aromatic compounds coupled to Fe(III) reduction by Ferroglobus placidus. Environ Microbiol 3, 281-7 (2001)
http://dx.doi.org/10.1046/j.1462-2920.2001.00192.x

13. Izzo V, E Notomista, A Picardi, F Pennacchio, A Di Donato: The thermophilic archaeon Sulfolobus solfataricus is able to grow on phenol. Res Microbiol 156, 677-89 (2005)
http://dx.doi.org/10.1016/j.resmic.2005.04.001

14. Christen P, S Davidson, Y Combet-Blanc, RAuria: Phenol biodegradation by the thermoacidophilic archaeon Sulfolobus solfataricus 98/2 in a fed-batch bioreactor. Biodegradation 22, 475-484 (2011)
http://dx.doi.org/10.1007/s10532-010-9420-6

15. J B Broderick: Catechol dioxygenases. Essays Biochem 34, 173-189 (1999)

16. Chae J C, E Kim, E Bini, G J Zylstra: Comparative analysis of the catechol 2,3-dioxygenase gene locus in thermoacidophilic archaeon Sulfolobus solfataricus strain 98/2. Biochem Biophys Res Commun 357, 815-9 (2007)
http://dx.doi.org/10.1016/j.bbrc.2007.04.027

17. Kanehisa M, S Goto, M Furumichi, M Tanabe, M Hirakawa: KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res 38, D355-60 (2010)
http://dx.doi.org/10.1093/nar/gkp896

18. Le Borgne S, D Paniagua, R Vazquez-Duhalt: Biodegradation of organic pollutants by halophilic bacteria and archaea. J Mol Microb Biotech 15, 74-92 (2008)
http://dx.doi.org/10.1159/000121323

19. Emerson D, S Chauhan, P Oriel, J A Breznak: Haloferax sp. D 1227, a halophilicarchaeon capable of growth on aromatic compounds. Arch Microbiol 161, 445-452 (1994)
http://dx.doi.org/10.1007/BF00307764

20. Fu W, P Oriel: Gentisate 1,2-dioxygenase from Haloferax sp.D1227. Extremophiles 4, 439-46 (1998)
http://dx.doi.org/10.1007/s007920050090

21. Fairley D J, D R Boyd, N D Sharma, C C Allen, P Morgan, M J Larkin: Aerobic metabolism of 4-hydroxybenzoic acid in Archaea via an unusual pathway involving an intramolecular migration (NIH shift). Appl Environ Microbiol 68, 6246-6255 (2002)
http://dx.doi.org/10.1128/AEM.68.12.6246-6255.2002

22. Fairley D J, G Wang, C Rensing, I L Pepper, M J Larkin: Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera. Appl Microbiol Biotechnol 73, 691-695 (2006)
http://dx.doi.org/10.1007/s00253-006-0509-0

23. Bonfá M R, M J Grossman, E Mellado, L R Durrant: Biodegradation of aromatic hydrocarbons by Haloarchaea and their use for the reduction of the chemical oxygen demand of hypersaline petroleum produced water. Chemosphere 84, 1671-6 (2011)
http://dx.doi.org/10.1016/j.chemosphere.2011.05.005

24. Ding J Y, M C Lai: The biotechnological potential of the extreme halophilic archaea Haloterrigena sp. H13 in xenobiotic metabolism using a comparative genomics approach. Environ Technol 31, 905-14 (2010)
http://dx.doi.org/10.1080/09593331003734210

25. Grbić-Galić D, T M Vogel: Transformation of toluene and benzene by mixed methanogenic cultures. Appl Environ Microbiol 53, 254-60 (1987)

26. Chang W, Y Um, T R Holoman: Polycyclic aromatic hydrocarbon (PAH) degradation coupled to methanogenesis. Biotech Lett 28, 425-430 (2006)
http://dx.doi.org/10.1007/s10529-005-6073-3

27.Qiu Y L, S Hanada A Ohashi, H Harada, Y Kamagata, Y Sekiguchi: Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the first cultured anaerobe capable of degrading phenol to acetate in obligate syntrophic associations with a hydrogenotrophic methanogen. Appl Environ Microbiol 74, 2051-8 (2008)
http://dx.doi.org/10.1128/AEM.02378-07

28. Chong P K, A M Burja, H Radianingtyas, A Fazeli, P C Wright: Proteome and transcriptional analysis of ethanol-grown Sulfolobus solfataricus P2 reveals ADH2, a potential alcohol dehydrogenase. J Proteome Res 6, 3985-94 (2007)
http://dx.doi.org/10.1021/pr070232y

29. Bidle K A, P A Kirkland, J L Nannen, J A Maupin-Furlow: Proteomic analysis of Haloferax volcanii reveals salinity-mediated regulation of the stress response protein PspA. Microbiology154, 1436-43 (2008)
http://dx.doi.org/10.1099/mic.0.2007/015586-0

30. Li L, Q Li, L Rohlin, U Kim, K Salmon, T Rejtar, R P Gunsalus, B L Karger, J G Ferry: Quantitative proteomic and microarray analysis of the archaeon Methanosarcina acetivorans grown with acetate versus methanol. J Proteome Res 6, 759-71 (2007)
http://dx.doi.org/10.1021/pr060383l

31. Lapaglia C, P LHartzell: Stress-induced production of biofilm in the hyperthermophile Archaeoglobus fulgidus. Appl Environ Microbiol 63, 3158-63 (1997)

32. Koerdt A, A Orell, T K Pham, J Mukherjee, A Wlodkowski, E Karunakaran, C A Biggs, P C Wright, S V Albers: Macromolecular fingerprinting of Sulfolobus species in biofilm: a transcriptomic and proteomic approach combined with spectroscopic analysis. J Proteome Res 10, 4105-19 (2011)
http://dx.doi.org/10.1021/pr2003006

33. K Mc Keegan: The structure and function of drug pumps: an update. Trends Microbiol 11, 21-29 (2003)
http://dx.doi.org/10.1016/S0966-842X(02)00010-0

34. Lubelski J, W N Konings, A J Driessen: Distribution and physiology of ABC-type transporters contributing to multidrug resistance in Bacteria. Microbiol and MolBiol Rev 71, 463-476 (2007)
http://dx.doi.org/10.1128/MMBR.00001-07

35. Putman M, H W van Veen, W N Konings: Molecular properties of bacterial multidrug transporters. Microbiol and MolecBiol Rev 64, 672-693 (2000)
http://dx.doi.org/10.1128/MMBR.64.4.672-693.2000

36.Yen M R, J S Chen, J L Marquez, E I Sun, M H Saier: Multidrug resistance: phylogenetic characterization of superfamilies of secondary carriers that include drug exporters. Meth Mol Biol 637, 47-64 (2010)
http://dx.doi.org/10.1007/978-1-60761-700-6_3

37. Saier M H Jr, I T Paulsen: Phylogeny of multidrug transporters. Seminars in Cell & Develop Biol 12, 205-213 (2001)
http://dx.doi.org/10.1006/scdb.2000.0246

38. Nikaido H, Y Takatsuka: Mechanisms of RND multidrug efflux pumps. Biochim Biophys Acta 1794, 769-81 (2009)

39. K Lewis: Multidrug resistance: Versatile drug sensors of bacterial cells. Curr Biol 9, 403-407 (1999)
http://dx.doi.org/10.1016/S0960-9822(99)80254-1

40L J Piddock: Multidrug-resistance efflux pumps - not just for resistance. Nat Rev Microbiol 4, 629-36 (2006)
http://dx.doi.org/10.1038/nrmicro1464

41. Schneider K L, K S Pollard, R Baertsch, A Pohl, T M Lowe: The UCSC Archaeal Genome Browser. Nucleic Acid Res 34, D407-D410 (2006)
http://dx.doi.org/10.1093/nar/gkj134

42. Albers S V, S M Koning, W N Konings, A J Driessen: Insights into ABC transport in archaea. Bioenerg Biomembr 36, 5-15 (2004)
http://dx.doi.org/10.1023/B:JOBB.0000019593.84933.e6

43. Biemans-Oldehinke E, M K Doeven, B Poolman: ABC transporter architecture and regulatory roles of accessory domains. FEBS Lett 580, 1023-1035(2006)
http://dx.doi.org/10.1016/j.febslet.2005.11.079

44. Oldham M L, A L Davidson, J Chen: Structural insights into ABC transporter mechanism. Curr Opin Struct Biol 18, 726-733 (2008)
http://dx.doi.org/10.1016/j.sbi.2008.09.007

45. Chung Y J, M H Saier Jr: SMR-type multidrug resistance pumps. Curr Opin Drug Disc Dev 4, 237-245 (2001)

46. Lee S J, A Böhm, M Krug, W Boos: The ABC of binding-protein-dependent transport in Archaea. Trends Microbiol 15: 389-397 (2007)
http://dx.doi.org/10.1016/j.tim.2007.08.002

47. Hollenstein K R, J P Dawson, K P Locher: Structure and mechanism of ABC transporter proteins. Curr Opin Struct Biol 17, 412-418 (2007)
http://dx.doi.org/10.1016/j.sbi.2007.07.003

48. Oliveira A S, A M Baptista, C M Soares: Insights into the molecular mechanism of an ABC transporter: conformational changes in the NBD dimer of MJ0796. J Phys Chem B 114, 5486-96 (2010)
http://dx.doi.org/10.1021/jp905735y

49. Mulligan C, M Fischer, G H Thomas: Tripartite ATP-independent periplasmic (TRAP) transporters in bacteria and archaea. FEMS Microbiol Rev 35, 68-86 (2011)
http://dx.doi.org/10.1111/j.1574-6976.2010.00236.x

50. Kaidoh K, S Miyauchi, A Abe, S Tanabu, T Nara, N Kamo: Rhodamine 123 efflux transporter in Haloferaxvolcanii is induced when cultured under 'metabolic stress' by amino acids: the efflux system resembles that in a doxorubicin-resistant mutant. Biochem J 314, 355-359 (1996)

51. Borges-Walmsley M I, K S McKeegan, A R Walmsley: Structure and function of efflux pumps that confer resistance to drugs. Biochem J 376, 313-38 (2003)
http://dx.doi.org/10.1042/BJ20020957

52. I T Paulsen: Multidrug efflux pumps and resistance: regulation and evolution. Curr Opin Microbiol 6, 446-51 (2003)
http://dx.doi.org/10.1016/j.mib.2003.08.005

53. Li X Z, H Nikaido: Efflux-Mediated Drug Resistance in Bacteria. Drugs 64,159-204 (2004)
http://dx.doi.org/10.2165/00003495-200464020-00004

54. Walmsley A R, B P Rosen, D L Mayers: Transport mechanism of resistance to drugs and toxic metals. In: Antimicrobial Drug Resistance, volume 1 Mechanisms of Drug Resistance. Eds: D L Mayers (2009)

DOI 10.1007/978-1-59745-180-2_10 © Humana Press

55. Hvorup R N, B Winnen, A B Chang, Y Jiang, X F Zhou, M H Saier: The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily. Eur J Biochem 270, 799-813 (2003)
http://dx.doi.org/10.1046/j.1432-1033.2003.03418.x

56. Omote H, M Hiasa, T Matsumoto, M Otsuka, Y. Moriyama. The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations. Trends Pharmacol Sci 27, 587-93 (2006)
http://dx.doi.org/10.1016/j.tips.2006.09.001

57. Jack D L, N M Yang, M H Saier Jr: The drug/metabolite transporter superfamily. Eur J Biochem 268, 3620-3639 (2001)
http://dx.doi.org/10.1046/j.1432-1327.2001.02265.x

58. Paulsen I T, M H Brown, R A Skurray: Proton-dependent multidrug efflux systems. Microbiol Rev 60, 575-608 (1996)

59. Bay D C, K L Rommens, R J Turner: Small multidrug resistance proteins: a multidrug transporter family that continues to grow. Biochim Biophys Acta 1778, 1814-38 (2008)
http://dx.doi.org/10.1016/j.bbamem.2007.08.015

60. Poulsen B E, A Rath, C M Deber: The Assembly Motif of a Bacterial Small Multidrug Resistance Protein. J Biol Chem 284, 9870-9875 (2009)
http://dx.doi.org/10.1074/jbc.M900182200

61. Bay D C, R J Turner: Diversity and evolution of the small multidrug resistance protein family, BMC Evol Biol 9, 140 (2009)
http://dx.doi.org/10.1186/1471-2148-9-140

62. Ninio S, S Schuldiner: Characterization of an archaeal multidrug transporter with a unique amino acid composition. J Biol Chem 278, 12000-5 (2003)
http://dx.doi.org/10.1074/jbc.M213119200

63. Rath A, R A Melnyk, C M Deber: Evidence for Assembly of Small Multidrug Resistance Proteins by a "Two-faced" Transmembrane Helix. J Biol Chem 281, 15546-15553 (2006)
http://dx.doi.org/10.1074/jbc.M600434200

64. Pao S, S I T Paulsen, M H Jr Saier: Major facilitator superfamily. Microbiol Mol Biol 62, 1-34 (1998)

65. Chang A B, R Lin, W K Studley, C V Tran, M H Jr Saier: Phylogeny as a guide to structure and function of membrane transport proteins. Mol Membr Biol 2, 171-81 (2004)
http://dx.doi.org/10.1080/09687680410001720830

66. Ren Q, I T Paulsen: Comparative Analyses of Fundamental Differences in Membrane Transport Capabilities in Prokaryotes and Eukaryotes. PLoS Comput Biol, 1: e27 (2005)
http://dx.doi.org/10.1371/journal.pcbi.0010027

67. Law C J, P C Maloney, D N Wang: Ins and outs of major facilitator superfamily antiporters. Annu Rev Microbiol 62, 289-305 (2008)
http://dx.doi.org/10.1146/annurev.micro.61.080706.093329

68. Vardy E, S Steiner-Mordoch, S Schuldiner: Characterization of bacterial drug antiporters homologous to mammalian neurotransmitter transporters. J Bacteriol 187, 7518-25 (2005)
http://dx.doi.org/10.1128/JB.187.21.7518-7525.2005

69. Murakami S, A Yamaguchi: Multidrug-exporting secondary transporters. Curr Opin Struct Biol 13, 443-52 (2003)
http://dx.doi.org/10.1016/S0959-440X(03)00109-X

70. Tseng T T, K S Gratwick, J Kollman, D Park, D H Nies, A Goffeau & M H Jr Saier: The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J Mol Microbiol Biotechnol 1, 107-25 (1999)

71. H Nikaido.: Multidrug efflux pumps of gram-negative bacteria. J Bacteriol 178, 5853-9 (1996)

72. Kim E, H D H Nies, M M McEvoy, C Rensing: Switch or funnel: how RND-type transport systems control periplasmic metal homeostasis. J Bacteriol 193, 2381-7 (2011)
http://dx.doi.org/10.1128/JB.01323-10

73. Perera I C, A Grove: Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators. J Mol Cell Biol 2, 243-254 (2010)
http://dx.doi.org/10.1093/jmcb/mjq021

74. Miyazono K, M Tsujimura, Y Kawarabayasi, M Tanokura: Crystal structure of an archaeal homologue of multidrug resistance repressor protein, EmrR, from hyperthermophilic archaea Sulfolobus tokodaii strain 7. Proteins 67, 1138-1146 (2007)
http://dx.doi.org/10.1002/prot.21327

75. Kumarevel T, T Tanaka, M Nishio, S C Gopinath, K Takio, A Shinkai, P K Kumar, S. Yokoyama: Crystal structure of the MarR family regulatory protein, ST1710, from Sulfolobus tokodaii strain 7. J Struct Biol 161, 9-17 (2008)
http://dx.doi.org/10.1016/j.jsb.2007.08.017

76. Saridakis V, D Shahinas, X Xu, D Christendat: Structural insight on the mechanism of regulation of the MarR family of proteins: high-resolution crystal structure of a transcriptional repressor from Methanobacterium thermoautotrophicum. J Mol Biol 377, 655-667 (2008)
http://dx.doi.org/10.1016/j.jmb.2008.01.001

77. Okada U, N Sakai, M Yao, N Watanabe, I Tanaka: Structural analysis of the transcriptional regulator homolog protein from Pyrococcus horikoshii OT3. Proteins 63, 1084-1086 (2006)
http://dx.doi.org/10.1002/prot.20913

78. Di Fiore A, G Fiorentino, R M Vitale, R Ronca, P Amodeo, C Pedone, S Bartolucci, G. De Simone: Structural analysis of BldR from Sulfolobus solfataricus provides insights into the molecular basis of transcriptional activation in archaea by MarR family proteins. J Mol Biol 388, 559-569 (2009)
http://dx.doi.org/10.1016/j.jmb.2009.03.030

79. Fiorentino G, R Cannio, M Rossi, S. Bartolucci: Transcriptional regulation of the gene encoding an alcohol dehydrogenase in the archaeon Sulfolobus solfataricus involves multiple factors and control elements. J Bacteriol 185, 3926-34 (2003)
http://dx.doi.org/10.1128/JB.185.13.3926-3934.2003

80. Fiorentino G, R Ronca, R Cannio, M Rossi, S. Bartolucci: MarR-like transcriptional regulator involved in detoxification of aromatic compounds in Sulfolobus solfataricus. J Bacteriol 189, 7351-7360 (2007)
http://dx.doi.org/10.1128/JB.00885-07

81. Fiorentino G, I Del Giudice, S Bartolucci, L Durante, L Martino, P Del Vecchio: Identification and Physicochemical Characterization of BldR2 from Sulfolobus solfataricus, a Novel Archaeal Member of the MarR Transcription Factor Family. Biochemistry 50, 6607-21 (2011)
http://dx.doi.org/10.1021/bi200187j

82. Brown N L, J V Stoyanov, S P Kidd, J L Hobman: The MerR family of transcriptional regulators. FEMS Microbiol Rev 27, 145-63 (2003)
http://dx.doi.org/10.1016/S0168-6445(03)00051-2

83. Ramos J L, M Martínez-Bueno, A J Molina-Henares, W Terán, K. Watanabe, X Zhang, M T Gallegos, R Brennan, R. Tobes: The TetR family of transcriptional repressors. Microbiol Mol Biol Rev 69, 326-56 (2005)
http://dx.doi.org/10.1128/MMBR.69.2.326-356.2005

84. Gallegos M T, R Schleif, A Bairoch, K Hofmann, J L Ramos: AraC/XylS Family of Transcriptional Regulators. Microbiol Mol Biol Rev 61, 393-410 (1997)

85. Yang J, M Tauschek, R M Robins-Browne: Control of bacterial virulence by AraC-like regulators that respond to chemical signals. Trends Microbiol 19, 128-135 (2011)
http://dx.doi.org/10.1016/j.tim.2010.12.001

86. Baliga N S, S P Kennedy, W V Ng, L Hood, S Das Sarma: Genomic and genetic dissection of an archaeal regulon. Proc Natl Acad Sci U S A 98, 2521-5 (2001)
http://dx.doi.org/10.1073/pnas.051632498

87. Cvetkovic A, A L Menon, M P Thorgersen, J W Scott, F L 2nd Poole, F E Jr Jenney, W A Lancaster, J L Praissman, S Shanmukh, B J Vaccaro, S A Trauger, E Kalisiak, J V Apon, G Siuzdak, S M Yannone, J A Tainer, M W Adams: Microbial metalloproteomes are largely uncharacterized. Nature 466, 779-82 (2010)
http://dx.doi.org/10.1038/nature09265

88. D H Nies: Microbial heavy-metal resistance. Appl Microbiol Biotechnol 51, 730-50 (1999)
http://dx.doi.org/10.1007/s002530051457

89. Orell A, C A Navarro, R Arancibia, J C Mobarec, C A Jerez : Life in blue: copper resistance mechanisms of bacteria and archaea used in industrial biomining of minerals. Biotechnol Adv 28, 839-48 (2010)
http://dx.doi.org/10.1016/j.biotechadv.2010.07.003

90. Bun-ya M, K Shikata, S Nakade, C Yompakdee, S Harashima, Y Oshima: Two new genes, PHO86 and PHO87, involved in inorganic phosphate uptake in Saccharomyces cerevisiae. Curr Genet 29, 344-51 (1996)

91. Zheng M, B Doan, T D Schneider, G Storz: OxyR and SoxRS regulation of fur. J Bacteriol 181, 4639-4643 (1999)

92. Schmid A K, M Pan, K Sharma, N S Baliga: Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon. Nucleic Acids Res 39, 2519-2533 (2010)
http://dx.doi.org/10.1093/nar/gkq1211

93. J A Imlay: Pathways of oxidative damage. Annu Rev Microbiol 57, 395-418 (2003)
http://dx.doi.org/10.1146/annurev.micro.57.030502.090938

94. Andrews S C, A K Robinson, F Rodríguez-Quiñones: Bacterial iron homeostasis FEMS Microbiol Rev 27, 215-37 (2003)
http://dx.doi.org/10.1016/S0168-6445(03)00055-X

95. Haikarainen T, A C Papageorgiou: Dps-like proteins: structural and functional insights into a versatile protein family. Cell Mol Life Sci 67, 341-51 (2010)
http://dx.doi.org/10.1007/s00018-009-0168-2

96. Peña M M, G S Bullerjahn: The DpsA protein of Synechococcus sp. strain PCC7942 is a DNA-binding hemoprotein. Linkage of the Dps and bacterioferritin protein families. J Biol Chem 270, 22478-82 (1995)
http://dx.doi.org/10.1074/jbc.270.38.22478

97. Almirón M, A J Link, D Furlong, R Kolter: A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli. Genes Dev 6, 2646-54 (1992)
http://dx.doi.org/10.1101/gad.6.12b.2646

98. Wiedenheft B, J Mosolf, D Willits, M Yeager, K A Dryden, M Young, T Douglas: An archaeal antioxidant: characterization of a Dps-like protein from Sulfolobus solfataricus. Proc Natl Acad Sci U S A 102, 10551-6 (2005)
http://dx.doi.org/10.1073/pnas.0501497102

99. Ramsay B, B Wiedenheft, M Allen, G H Gauss, C M Lawrence, M Young, T Douglas: Dps-like protein from the hyperthermophilic archaeon Pyrococcus furiosus. J Inorg Biochem 100, 1061-8 (2006)
http://dx.doi.org/10.1016/j.jinorgbio.2005.12.001

100. Reindel S, C L Schmidt, S Anemüller, B F Matzanke: Characterization of a non-haem ferritin of the Archaeon Halobacterium salinarum, homologous to Dps (starvation-induced DNA-binding protein). Biochem Soc Trans 30, 713-5 (2002)
http://dx.doi.org/10.1042/BST0300713

101. Gauss G H, P Benas, B Wiedenheft, M Young, T Douglas, C M Lawrence: Structure of the DPS-like protein from Sulfolobus solfataricus reveals a bacterioferritin-like dimetal binding site within a DPS-like dodecameric assembly. Biochemistry 45, 10815-27 (2006)
http://dx.doi.org/10.1021/bi060782u

102. Limauro D, E Pedone, I Galdi, S Bartolucci: Peroxiredoxins as cellular guardians in Sulfolobus solfataricus: characterization of Bcp1, Bcp3 and Bcp4. FEBS J 275, 2067-77 (2008)
http://dx.doi.org/10.1111/j.1742-4658.2008.06361.x

103. Maaty W S, B Wiedenheft, P Tarlykov, N Schaff, J Heinemann, J Robison-Cox, J Valenzuela, A Dougherty, P Blum, C M Lawrence, T Douglas, M J Young, B Bothner: Something Old, Something New, Something Borrowed; How the Thermoacidophilic Archaeon Sulfolobus solfataricus Responds to Oxidative Stress. PLoS One 4, e6964 (2009)
http://dx.doi.org/10.1371/journal.pone.0006964

104. Dopson M, C Baker-Austin, A Hind, J P Bowman, P L Bond: Characterization of Ferroplasma isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments. Appl Environ Microbiol 70, 2079-2088 (2004)
http://dx.doi.org/10.1128/AEM.70.4.2079-2088.2004

105. Potrykus J, V R Jonna, M Dopson: Iron homeostasis and responses to iron limitation in extreme acidophiles from the Ferroplasma genus. Proteomics 11, 52-63 (2011)
http://dx.doi.org/10.1002/pmic.201000193

106. Hubmacher D, B F Matzanke, S Anemüller: Iron-uptake in the Euryarchaeon Halobacterium salinarum. Biometals 20, 539-47 (2007)
http://dx.doi.org/10.1007/s10534-006-9064-5

107. Schröder I, E Johnson, S de Vries: Microbial ferric iron reductases. FEMS Microbiol Rev 27, 427-47 (2003)
http://dx.doi.org/10.1016/S0168-6445(03)00043-3

108. Chiu H J, E Johnson, I Schröder, D C Rees: Crystal structures of a novel ferric reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus and its complex with NADP+. Structure 9, 311-9 (2001)
http://dx.doi.org/10.1016/S0969-2126(01)00589-5

109. Vadas A, H G Monbouquette, E Johnson, I Schröder: Identification and characterization of a novel ferric reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus. J Biol Chem 274, 36715-21 (1999)
http://dx.doi.org/10.1074/jbc.274.51.36715

110. Mulrooney S B, R P Hausinger: Nickel uptake and utilization by microorganisms. FEMS Microbiol Rev 27, 239-261 (2003)
http://dx.doi.org/10.1016/S0168-6445(03)00042-1

111. Macomber L, R P Hausinger: Mechanisms of nickel toxicity in microorganisms. Metallomics 3, 1153-1162 (2011)
http://dx.doi.org/10.1039/c1mt00063b

112. Majtan T, F E Frerman, J P Kraus: Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation. Biometals 24, 335-47 (2011)
http://dx.doi.org/10.1007/s10534-010-9400-7

113. Eitinger T, D A Rodionov, M Grote, E Schneider: Canonical and ECF-type ATP-binding cassette importers in prokaryotes: diversity in modular organization and cellular functions. FEMS Microbiol Rev 35, 3-67 (2011)
http://dx.doi.org/10.1111/j.1574-6976.2010.00230.x

114. Zhang Y, D A Rodionov, M S Gelfand, V N Gladyshev: Comparative genomic analyses of nickel, cobalt and vitamin B12 utilization. BMC Genomics 10, 78 (2009)
http://dx.doi.org/10.1186/1471-2164-10-78

115. Rodionov D A, P Hebbeln, M S Gelfand, T Eitinger: Comparative and functional genomic analysis of prokaryotic nickel and cobalt uptake transporters: evidence for a novel group of ATP-binding cassette transporters. J Bacteriol 188, 317-27 (2006)
http://dx.doi.org/10.1128/JB.188.1.317-327.2006

116. Siche S, O Neubauer, P Hebbeln, T Eitinger: A bipartite S unit of an ECF-type cobalt transporter. Res Microbiol 161, 824-9 (2010)
http://dx.doi.org/10.1016/j.resmic.2010.09.010

117. Rodionov D A, P Hebbeln, A Eudes, J ter Beek, I A Rodionova, G B Erkens, D J Slotboom, M S Gelfand, A L Osterman, A D Hanson, T Eitinger: A novel class of modular transporters for vitamins in prokaryotes. J Bacteriol 191, 42-51 (2009)
http://dx.doi.org/10.1128/JB.01208-08

118. Overbeek R, T Begley, R M Butler, J V Choudhuri, H Y Chuang, M Cohoon, V de Crécy-Lagard, N Diaz, T Disz, R Edwards, M Fonstein, E D Frank, S Gerdes, E M Glass, A Goesmann, A Hanson, D Iwata-Reuyl, R Jensen, N Jamshidi, L Krause, M Kubal, N Larsen, B Linke, A C McHardy, F Meyer, H Neuweger, G Olsen, R Olson, A Osterman, V Portnoy, G D Pusch, D A Rodionov, C Rückert, J Steiner, R Stevens, I Thiele, O Vassieva, Y Ye, O Zagnitko, V Vonstein: The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res 33, 5691-702 (2005)
http://dx.doi.org/10.1093/nar/gki866

119. Phillips C M, E R Schreiter, Y Guo, S C Wang, D B Zamble, C L Drennan: Structural basis of the metal specificity for nickel regulatory protein NikR. Biochemistry 47, 1938-46 (2008)
http://dx.doi.org/10.1021/bi702006h

120. Chivers P T, T H Tahirov: Structure of Pyrococcus horikoshii NikR: nickel sensing and implications for the regulation of DNA recognition. J Mol Biol 348, 597-607 (2005)
http://dx.doi.org/10.1016/j.jmb.2005.03.017

121. Sindhikar D J, A E Roitberg, K M Jr. Merz: Apo and nickel-bound forms of the Pyrococcus horikoshii species of the metalloregulatory protein: NikR characterized by molecular dynamics simulations. Biochemistry 48, 12024-33 (2009)
http://dx.doi.org/10.1021/bi9013352

122. Argüello J M, E Eren, M González-Guerrero: The structure and function of heavy metal transport P1B-ATPases.Biometals 20, 233-48 (2007)
http://dx.doi.org/10.1007/s10534-006-9055-6

123. Lloyd D R, D H Phillips: Oxidative DNA damage mediated by copper_II/, iron_II/ and nickel_II/ Fenton reactions: evidence for site-specific mechanisms in the formation of double-strand breaks,8-hydroxydeoxyguanosine and putative intrastrand cross-links. Mut Res 424, 23-36 (1999)

124. J A Imlay: Cellular defenses against superoxide and hydrogen peroxide. Annu Rev Biochem 77, 755-76 (2008)
http://dx.doi.org/10.1146/annurev.biochem.77.061606.161055

125. Baker-Austin C, M Dopson, M Wexler, R G Sawers, P L Bond: Molecular insight into extreme copper resistance in the extremophilic archaeon 'Ferroplasma acidarmanus' Fer1. Microbiology 151, 2637-46 (2005)
http://dx.doi.org/10.1099/mic.0.28076-0

126. Camakaris J, I Voskoboinik, J F Mercer: Molecular mechanisms of copper homeostasis Biochem Biophys Res Commun 261, 225-32 (1999)
http://dx.doi.org/10.1006/bbrc.1999.1073

127. Macomber L, J A Imlay: The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity. Proc Natl Acad Sci U S A 106, 8344-9 (2009)
http://dx.doi.org/10.1073/pnas.0812808106

128. Remonsellez F, A Orell, C A Jerez: Copper tolerance of the thermoacidophilic archaeon Sulfolobus metallicus: possible role of polyphosphate metabolism. Microbiology 152, 59-66 (2006)
http://dx.doi.org/10.1099/mic.0.28241-0

129. Mana-Capelli S, A K Mandal, J M Argüello: Archaeoglobus fulgidus CopB is a thermophilic Cu2+ ATPase: functional role of its histidine-rich-N-terminal metal binding domain. J Biol Chem 278, 40534-41 (2003)
http://dx.doi.org/10.1074/jbc.M306907200

130. Agarwal S, D Hong, N K Desai, M H Sazinsky, J M Argüello, A C Rosenzweig: Structure and interactions of the C-terminal metal binding domain of Archaeoglobus fulgidus CopA. Proteins 78, 2450-8 (2010)

131. Yang Y, A K Mandal, L M Bredeston, F L González-Flecha, J M Argüello: Activation of Archaeoglobus fulgidus Cu(+)-ATPase CopA by cysteine. Biochim Biophys Acta 1768, 495-501 (2007)
http://dx.doi.org/10.1016/j.bbamem.2006.09.013

132. Wu C C, W J Rice, D L Stokes: Structure of a copper pump suggests a regulatory role for its metal-binding domain. Structure 16, 976-85 (2008)
http://dx.doi.org/10.1016/j.str.2008.02.025

133. Rice W J, A Kovalishin, D L Stokes, W J Rice, A Kovalishin, D L Stokes: Role of metal-binding domains of the copper pump from Archaeoglobus fulgidus. Biochem Biophys Res Commun 348, 124-31 (2006)
http://dx.doi.org/10.1016/j.bbrc.2006.07.012

134. González-Guerrero M, J M Argüello: Mechanism of Cu+-transporting ATPases: soluble Cu+ chaperones directly transfer Cu+ to transmembrane transport sites. Proc Natl Acad Sci U S A 105, 5992-7 (2008)
http://dx.doi.org/10.1073/pnas.0711446105

135. Castielli O, B De la Cerda, J A Navarro, M Hervás, M A De la Rosa: Proteomic analyses of the response of cyanobacteria to different stress conditions. FEBS Lett 583, 1753-8 (2009)
http://dx.doi.org/10.1016/j.febslet.2009.03.069

136. Cavet J S, G P Borrelly, N J Robinson: Zn, Cu and Co in cyanobacteria: selective control of metal availability. FEMS Microbiol Rev 27, 165-81 (2003)
http://dx.doi.org/10.1016/S0168-6445(03)00050-0

137. Rensing C, G Grass: Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS Microbiol Rev 27, 197-213 (2003)
http://dx.doi.org/10.1016/S0168-6445(03)00049-4

138. Solioz M, H K Abicht, M Mermod, S J Mancini: Response of gram-positive bacteria to copper stress. Biol Inorg Chem 15, 3-14 (2010)
http://dx.doi.org/10.1007/s00775-009-0588-3

139. Solioz M, J V Stoyanov: Copper homeostasis in Enterococcus hirae. FEMS Microbiol Rev 27, 183-95 (2003)
http://dx.doi.org/10.1016/S0168-6445(03)00053-6

140. Strausak D, M Solioz: CopY is a copper-inducible repressor of the Enterococcus hirae copper ATPases. J Biol Chem 272, 8932-6 (1997)
http://dx.doi.org/10.1074/jbc.272.14.8932

141. Magnani D, M Solioz: Copper chaperone cycling and degradation in the regulation of the cop operon of Enterococcus hirae. Biometals 18, 407-12 (2005)
http://dx.doi.org/10.1007/s10534-005-3715-9

142. Deigweiher K, T L 4th Drell, A Prutsch, A J Scheidig, M Lübben: Expression, isolation, and crystallization of the catalytic domain of CopB, a putative copper transporting ATPase from the thermoacidophilic archaeon Sulfolobus solfataricus. J Bioenerg Biomembr 36, 151-9 (2004)
http://dx.doi.org/10.1023/B:JOBB.0000019607.05233.4c

143. Ettema T J, A B Brinkman, P P Lamers, N G Kornet, W M de Vos, J van der Oost: Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2. Microbiology 152, 1969-79 (2006)
http://dx.doi.org/10.1099/mic.0.28724-0

144. Ettema T J, M A Huynen, W M de Vos, J van der Oost: TRASH: a novel metal-binding domain predicted to be involved in heavy-metal sensing, trafficking and resistance. Trends Biochem Sc 28, 170-3 (2003)
http://dx.doi.org/10.1016/S0968-0004(03)00037-9

145. Villafane A A, Y Voskoboynik, M Cuebas, I Ruhl, E Bini: Response to excess copper in the hyperthermophile Sulfolobus solfataricus strain 98/2. Biochem Biophys Res Commun 385, 67-71 (2009)
http://dx.doi.org/10.1016/j.bbrc.2009.05.013

146. Akiyama M, E Crooke, A Kornberg: The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and membrane location of the protein. J Biol Chem 267, 22556-61 (1992)

147. Akiyama M, E Crooke, A Kornberg: An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon. J Biol Chem 268, 633-9 (1993)

148. Scherer P A, H P Bochem: Ultrastructural investigation of 12 Methanosarcinae and related species grown on methanol for occurrence of polyphosphatelike inclusions. Canadian J Microbiol 29,1190-1199 (1983)
http://dx.doi.org/10.1139/m83-182

149. Sko� rko R, J Osipiuk, K O Stetter: Glycogen-boundpolyphosphate kinase from the archaebacterium Sulfolobus acidocaldarius. J Bacteriol 171, 5162-5164 (1989)

150. Cardona S T, F P Cha� vez, C A Jerez: The exopolyphosphatasegene from Sulfolobus solfataricus: characterization of the first gene found to be involved in polyphosphate metabolism in Archaea. Appl Environ Microbiol 68, 4812-4819 (2002)
http://dx.doi.org/10.1128/AEM.68.10.4812-4819.2002

151. Persson B L, J O Lagerstedt, J R Pratt, J Pattison-Granberg, K Lundh, S Shokrollahzadeh, F Lundh: Regulation of phosphate acquisition in Saccharomyces cerevisiae. Curr Genet 43, 225-244 (2003)
http://dx.doi.org/10.1007/s00294-003-0400-9

152. Mukhopadhyay R, B P Rosen, L T Phung, S Silver: Microbial arsenic: from geocycles to genes and enzymes. FEMS Microbiol Rev 26, 311-25 (2002)
http://dx.doi.org/10.1111/j.1574-6976.2002.tb00617.x

153. Lebrun E, M Brugna, F Baymann, D Muller, D Lièvremont, M C Lett, W Nitschke: Arsenite oxidase, an ancient bioenergetic enzyme. Mol Biol Evol 20, 686-93 (2003)
http://dx.doi.org/10.1093/molbev/msg071

154. Silver S, L T Phung: Genes and enzymes involved in bacterial oxidation and reduction of inorganic arsenic. Appl Environ Microbiol 71, 599-608 (2005)
http://dx.doi.org/10.1128/AEM.71.2.599-608.2005

155. Sehlin M, E Börje Lindström: Oxidation and reduction of arsenic by Sulfolobus acidocaldarius strain BC H. FEMS Microbiol Lett 93, 87-92 (1992)
http://dx.doi.org/10.1111/j.1574-6968.1992.tb05045.x

156. Duval S, A L Ducluzeau, W Nitschke, B Schoepp-Cothenet: Enzyme phylogenies as markers for the oxidation state of the environment: the case of respiratory arsenate reductase and related enzymes. BMC Evol Biol 8, 206 (2008)
http://dx.doi.org/10.1186/1471-2148-8-206

157. Cozen A E, M T Weirauch, K S Pollard, D L Bernick, J M Stuart, T M. Lowe: Transcriptional map of respiratory versatility in the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. J Bacteriol 191, 782-94 (2009)
http://dx.doi.org/10.1128/JB.00965-08

158. Messens J, S Silver: Arsenate reduction: thiol cascade chemistry with convergent evolution. J Mol Biol 362, 1-17 (2006)
http://dx.doi.org/10.1016/j.jmb.2006.07.002

159. Jackson C R, S L Dugas: Phylogenetic analysis of bacterial and archaeal arsC gene sequences suggests an ancient, common origin for arsenate reductase. BMC Evol Biol 3, 18 (2003)
http://dx.doi.org/10.1186/1471-2148-3-18

160. Cullen W R, K J Reimer: Arsenic speciation in the environment. Chem Rev 89, 713-764 (1989)
http://dx.doi.org/10.1021/cr00094a002

161. Ferguson J F, J Gavis: A review of the arsenic cycle in natural waters. Water Res 6, 1259-1274 (1972)
http://dx.doi.org/10.1016/0043-1354(72)90052-8

162. Wu J, B P Rosen: The ArsR protein is a trans-acting regulatory protein. Mol Microbiol 5, 1331-6 (1991)
http://dx.doi.org/10.1111/j.1365-2958.1991.tb00779.x

163. Cervantes C, G Ji, J Ramirezc, S Silver: Resistance to arsenic compounds in microorganisms. FEMS Microbiol Rev 15, 355-367 (1994)
http://dx.doi.org/10.1111/j.1574-6976.1994.tb00145.x

164. Ji G, S Silver, E A E Garber, H Ohtake, C Cervantes, P Corbisier: Bacterial molecular genetics and enzymatic transformations of arsenate, arsenite and chromate, p. 529-539. In: Biohydrometallurgical technologies, Vol. 2 The Minerals, Metals, & Materials Society. Eds: Warrendale, Pa. AE Torma, ML Apel, CL Brierley (1993)

165. Bose M, D Slick, M J Sarto, P Murphy, D Roberts, J Roberts, R D Barber: Identification of SmtB/ArsR cis elements and proteins in archaea using the Prokaryotic InterGenic Exploration Database (PIGED). Archaea 2, 39-49 (2006)
http://dx.doi.org/10.1155/2006/837139

166. Busenlehner L S, M A Pennella, D P Giedroc: The SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural insights into prokaryotic metal resistance. FEMS Microbiol Rev 27, 131-43 (2003)
http://dx.doi.org/10.1016/S0168-6445(03)00054-8

167. Itou H, M Yao, N Watanabe, I Tanaka: Crystal structure of the PH1932 protein, a unique archaeal ArsR type winged-HTH transcription factor from Pyrococcus horikoshii OT3. Proteins 70, 1631-4 (2008)
http://dx.doi.org/10.1002/prot.21851

168. Mukhopadhyay R, B P Rosen: Arsenate Reductases in Prokaryotes and Eukaryotes. Environ Health Persp 110, 745-748 (2002)
http://dx.doi.org/10.1289/ehp.02110s5745

169. Roos G, L Buts, K Van Belle, E Brosens, P Geerlings, R Loris, L Wyns, J. Messens: Interplay between ion binding and catalysis in the thioredoxin-coupled arsenate reductase family. J Mol Biol 360, 826-38 (2006)
http://dx.doi.org/10.1016/j.jmb.2006.05.054

170. Shi J, A Vlamis-Gardikas, F Aslund, A Holmgren, B P Rosen: Reactivity of glutaredoxins 1, 2, and 3 from Escherichia coli shows that glutaredoxin 2 is the primary hydrogen donor to ArsC-catalyzed arsenate reduction. J Biol Chem 274, 36039-42 (1999)
http://dx.doi.org/10.1074/jbc.274.51.36039

171. Pedone E, D Limauro, K D'Ambrosio, G De Simone, S Bartolucci: Multiple catalytically active thioredoxin folds: a winning strategy for many functions. Cell Mol Life Sci 67, 3797-814 (2010)
http://dx.doi.org/10.1007/s00018-010-0449-9

172. Kinch L N, D Baker, N V Grishin: Deciphering a novel thioredoxin-like fold family. Proteins 52, 323-331 (2003)
http://dx.doi.org/10.1002/prot.10425

173. Martin P, S De Mel, J Shi, T Gladysheva, D L Gatti, B P Rosen, B F Edwards: Insights into the structure, solvation, and mechanism of ArsC arsenate reductase, a novel arsenic detoxification enzyme. Structure 9, 1071-81 (2001)
http://dx.doi.org/10.1016/S0969-2126(01)00672-4

174. Rosen B P: Families of arsenic transporters. Trends Microbiol 7, 207-212 (1999)
http://dx.doi.org/10.1016/S0966-842X(99)01494-8

175. Zhou T, S Radaev, B P Rosen, D L Gatti.: Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump. The EMBO Journal 19, 4838-4845 (2000)
http://dx.doi.org/10.1093/emboj/19.17.4838

176. Rosen B P: Biochemistry of arsenic detoxification. FEBS Lett 529, 86-92 (2002)
http://dx.doi.org/10.1016/S0014-5793(02)03186-1

177. Chen C M, T K Misra, S Silver, B P Rosen: Nucleotide sequence of the structural genes for an anion pump. The plasmid-encoded arsenical resistance operon. J Biol Chem 261, 15030-15 (1986)

178. Rensing C, M Ghosh, B P Rosen: Families of Soft-Metal-Ion-Transporting ATPases. J Bacteriol 181, 5891-5897 (1999)

179. Wu J, B P Rosen: The arsD gene encodes a second trans-acting regulatory protein of the plasmid-encoded arsenical resistance operon. Mol Microbiol 8, 615-623 (1993)
http://dx.doi.org/10.1111/j.1365-2958.1993.tb01605.x

180. Yang J, S Rawat, T L Stemmler, B P Rosen: Arsenic Binding and Transfer by the ArsD As(III) Metallochaperone. Biochemistry 49, 3658-3666 (2010)
http://dx.doi.org/10.1021/bi100026a

181. Neyt C N, M Iriarte, V H Thi, G R Cornelis: Virulence and arsenic resistance in Yersiniae. J Bacteriol 179, 612-619 (1997)

182. Dopson M, C Baker-Austin, P R Koppineedi, P L Bond: Growth in sulfidic mineral environments: metal resistance mechanisms in acidophilic micro-organisms. Microbiology 149, 1959-1970 (2003)
http://dx.doi.org/10.1099/mic.0.26296-0

183. Gihring T M, P L Bond, S C Peters, J F Banfield: Arsenic resistance in the archaeon "Ferroplasma acidarmanus": new insights into the structure and evolution of the ars genes. Extremophiles 7, 123-130 (2003)

184. Baker-Austin C, M Dopson, M Wexler, R G Sawers, A Stemmler, B P Rosen, P L Bond: Extreme arsenic resistance by the acidophilic archaeon 'Ferroplasma acidarmanus' Fer1. Extremophiles 11, 425-434 (2007)
http://dx.doi.org/10.1007/s00792-006-0052-z

185. Qin J, B P Rosen, Y Zhang, G Wang, S Franke, C Rensing: Arsenic detoxification and evolution of trimethylarsine gas by a microbial arsenite S-adenosylmethionine methyltransferase Proc Natl Acad Sci U S A 103, 2075-2080 (2006)
http://dx.doi.org/10.1073/pnas.0506836103

186. McBride B C, R S Wolfe: Biosynthesis of dimethylarsine by Methanobacterium. Biochemistry 10, 4312-4317 (1971)
http://dx.doi.org/10.1021/bi00799a024

187. Michalke K, E B Wickenheiser, M Mehring, A V Hirner, R Hensel: Production of volatile derivatives of metal(loid)s by microflora involved in anaerobic digestion of sewage sludge. Appl Environ Microbiol 66, 2791-6 (2000)
http://dx.doi.org/10.1128/AEM.66.7.2791-2796.2000

188. Wang G, S P Kennedy, S Fasiludeen, C Rensing, S. Das Sarma: Arsenic Resistance in Halobacterium sp. Strain NRC-1 Examined by Using an Improved Gene Knockout System. J Bacteriol 186, 3187-3194 (2004)
http://dx.doi.org/10.1128/JB.186.10.3187-3194.2004

189. Schelert J, V Dixit, V Hoang, J Simbahan, M Drozda, P Blum: Occurrence and Characterization of Mercury Resistance in the Hyperthermophilic Archaeon Sulfolobus solfataricus by Use of Gene Disruption. J Bacteriol 186, 427-437 (2004)
http://dx.doi.org/10.1128/JB.186.2.427-437.2004

190. Yamaguchi A, D G Tamang, M H Jr Saier: Mercury Transport in Bacteria. Water Air Soil Pollut 182, 219-234 (2007)
http://dx.doi.org/10.1007/s11270-007-9334-z

191. Wilson J R, C Leang, A P Morby, J L Hobman, N L Brown: MerF is a mercury transport protein: different structures but a common mechanism for mercuric ion transporters? FEBS Lett 472, 78-82 (2000)
http://dx.doi.org/10.1016/S0014-5793(00)01430-7

192. Serre L, E Rossy, E Pebay-Peyroula, C Cohen-Addad, J Covès: Crystal structure of the oxidized form of the periplasmic mercury-binding protein MerP from Ralstonia metallidurans CH34. J Mol Biol 339, 161-71 (2004)
http://dx.doi.org/10.1016/j.jmb.2004.03.022

193. Howell S C, M F Mesleh, S J Opella: NMR Structure Determination of a Membrane Protein with Two Transmembrane Helices in Micelles: MerF of the Bacterial Mercury Detoxification System. Biochemistry 44, 5196-5206 (2005)
http://dx.doi.org/10.1021/bi048095v

194. Di Lello P, G C Benison, H Valafar, K E Pitts, A O Summers, P Legault, J G Omichinski: NMR structural studies reveal a novel protein fold for MerB, the organomercurial lyase involved in the bacterial mercury resistance system. Biochemistry 43, 8322-32 (2004)
http://dx.doi.org/10.1021/bi049669z

195. Pullikuth A K, S S Gill: Primary structure of an invertebrate dihydrolipoamide dehydrogenase with phylogenetic relationship to vertebrate and bacterial disulfide oxidoreductases. Gene 200, 163-172 (1997)
http://dx.doi.org/10.1016/S0378-1119(97)00413-7

196. Engst S, S M Miller: Alternative routes for entry of HgX2 into the active site of mercuric ion reductase depend on the nature of the X ligands. Biochemistry 38, 3519-3529 (1999)
http://dx.doi.org/10.1021/bi982680c

197. Barkay T, K Kritee, E Boyd, G Geesey: A thermophilic bacterial origin and subsequent constraints by redox, light and salinity on the evolution of the microbial mercuric reductase. Environ Microbiol 12, 2904-2917 (2010)
http://dx.doi.org/10.1111/j.1462-2920.2010.02260.x

198. Champier L, V Duarte, I Michaud-Soret, J Covès: Characterization of the MerD protein from Ralstonia metallidurans CH34: a possible role in bacterial mercury resistance by switching off the induction of the mer operon. Mol Microbiol 52, 1475-85 (2004)
http://dx.doi.org/10.1111/j.1365-2958.2004.04071.x

199. Ansari A Z, M L Chael, T V O'Halloran: Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature 355, 87-89 (1992)
http://dx.doi.org/10.1038/355087a0

200. Ansari A Z, J E Bradner, T V O'Halloran: DNA-bend modulation in a repressor-to-activator switching mechanism. Nature 374, 371- 375 (1995)
http://dx.doi.org/10.1038/374370a0

201. Brown N L, J V Stoyanov, S P Kidd, J L Hobman: The MerR family of transcriptional regulators. FEMS Microbiol Rev 27, 145-163 (2003)
http://dx.doi.org/10.1016/S0168-6445(03)00051-2

202. Dixit V, E Bini, M Drozda, P Blum: Mercury inactivates transcription and the generalized transcription factor TFB in the archaeon Sulfolobus solfataricus. Antimicrob Agents Chemother 48, 1993-1999 (2004)
http://dx.doi.org/10.1128/AAC.48.6.1993-1999.2004

203. Schelert J, M Drozda, V Dixit, A Dillman, P Blum: Regulation of Mercury Resistance in the Crenarchaeote Sulfolobus solfataricus. J Bacteriol 188, 7141-7150 (2006)
http://dx.doi.org/10.1128/JB.00558-06

204. Aravind L, V Anantharaman, S Balaji, M M Babu, L M Iyer: The many faces of the helix-turn-helix domain: transcription regulation and beyond. FEMS Microbiol Rev 29, 231-262 (2005)

205. King J K, J E Kostka, M E Frischer, F M Saunders: Sulfate-Reducing Bacteria Methylate Mercury at Variable Rates in Pure Culture and in Marine Sediments. Appl Environ Microbiol 66, 2430-2437 (2000)
http://dx.doi.org/10.1128/AEM.66.6.2430-2437.2000

206. Pak K R, R Bartha: Mercury Methylation by Interspecies Hydrogen and Acetate Transfer between Sulfidogens and Methanogens. Appl Environ Microbiol 64, 1987-1990 (1998)

Key Words: Archaea, Xenobiotic Compounds, Heavy Metal, Pollution, Review

Send correspondence to: Patrizia Contursi-Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cinthia, Napoli, Italy. Tel: 39081679166, Fax: 39081679053, E-mail: contursi@unina.it