Inorganic Polyphosphate, Exopolyphosphatase, and Pho84-Like Transporters May Be Involved in Copper Resistance in Metallosphaera sedula DSM 5348T

Polyphosphates (PolyP) are linear polymers of orthophosphate residues that have been proposed to participate in metal resistance in bacteria and archaea. In addition of having a CopA/CopB copper efflux system, the thermoacidophilic archaeon Metallosphaera sedula contains electron-dense PolyP-like granules and a putative exopolyphosphatase (PPXMsed, Msed_0891) and four presumed pho84-like phosphate transporters (Msed_0846, Msed_0866, Msed_1094, and Msed_1512) encoded in its genome. In the present report, the existence of a possible PolyP-based copper-resistance mechanism in M. sedula DSM 5348T was evaluated. M. sedula DSM 5348T accumulated high levels of phosphorous in the form of granules, and its growth was affected in the presence of 16 mM copper. PolyP levels were highly reduced after the archaeon was subjected to an 8 mM CuSO4 shift. PPXMsed was purified, and the enzyme was found to hydrolyze PolyP in vitro. Essential residues for catalysis of PPXMsed were E111 and E113 as shown by a site-directed mutagenesis of the implied residues. Furthermore, M. sedula ppx, pho84-like, and copTMA genes were upregulated upon copper exposure, as determined by qRT-PCR analysis. The results obtained support the existence of a PolyP-dependent copper-resistance system that may be of great importance in the adaptation of this thermoacidophilic archaeon to its harsh environment.


Introduction
Acid mine drainage (AMD) and acid rock drainage (ARD) are the major environmental problems caused by biomining.Diverse biotic and abiotic strategies have been developed to treat AMD [1].Bioremediation of metal-polluted soils consists of two steps: (i) the solubilization of metals from the matrix into the liquid phase and (ii) the extraction and concentration of metals [2].Consortiums of thermophilic bacteria have been proposed as possible candidates for bioremediation of metal-contaminated sites due to their capacity to adsorb metals [3].Thermoacidophilic archaea, such as Sulfolobus metallicus and Acidianus brierleyi, are important microorganisms used in bioleaching and can live in the hostile environmental conditions present in AMD.These archaea are iron and sulfur oxidizers, living in acidic and high-temperature environments (>60 °C), and are generally very resistant to high metal concentrations [4].Although these unique characteristics make thermoacidophiles possible efficient candidates for bioremediation of AMD, so far no strategies have been proposed using these microorganisms.A better understanding of how thermoacidophilic archaea can survive in AMD-conditions is still needed [5].
Metallosphaera sedula is a thermoacidophilic archaeon, first isolated from a continental solfataric field in Italy [6].This microorganism is able to grow heterotrophically, autotrophically, and mixotrophically [7] and at high concentrations of metals such as copper [6].Genomic analyses of strain DSM 5348 T [8] revealed some genes that could be involved in copper resistance in this microorganism.A P-type ATPase CopA (Msed_0490), a metallochaperone CopM (Msed_0491), and a transcriptional regulator CopT (Msed_0492) constitute a functional copper efflux system [9].Cross-species complementation of a copper-sensitive Sulfolobus solfataricus copR mutant with these genes from M. sedula DSM 5348 T increased its copper resistance by 2fold.Interestingly, copper resistance of a M. sedula strain mutant in copA was almost 2 times reduced compared to the wild-type strain.However, the resistance of M. sedula copA mutant was 4 times higher than that of S. solfataricus copR strain complemented with copTMA genes from M. sedula.That residual resistance present in the M. sedula copA mutant indicates that this archaeon may possess additional determinants to cope with copper stress [9].
Another possible metal resistance mechanism that has been proposed for the acidophilic S. metallicus and Acidithiobacillus ferrooxidans involves inorganic polyphosphate (PolyP) [10,11].These inorganic molecules are lineal polymers of orthophosphate residues linked by anhydride bonds [12].In some acidophilic microorganisms, PolyP accumulates in the form of granules in the cytoplasm and it is rapidly hydrolyzed to inorganic phosphate (P i ) after copper exposure [13].The enzyme that synthesizes these polymers ("polyP polymerase") has not yet been described for Crenarchaeota [4].The exopolyphosphatase (PPX) catalyzes the hydrolysis of the terminal P i residue of PolyP in E. coli [14], and a functional archaeal PPX has been characterized in S. solfataricus [15].It has been suggested that metals could form metalphosphate complexes that might be transported out of the cell by H + -phosphate symporters [16,17].In E. coli, P i was expelled from the cell by a PitA H + -phosphate symporter following a copper shock, immediately after PolyP was degraded [18].However, the PitA-based mechanism seems not to be ubiquitous, since other species, such as A. ferrooxidans and S. metallicus, lack orthologous genes to pitA and P i is also supposed to be exported after a copper shift in those species.It has been proposed that a putative pho84-like gene, homolog to the yeast Pho84 H + -phosphate symporter, could carry out that role [10].
M. sedula DSM 5348 T also accumulates electron-dense granules most likely composed of PolyP [13], and its gene Msed_0891 encodes for a putative PPX Msed [8].Although only a fragment of a PitA homolog is encoded in its genome, it contains four genes coding for putative Pho84-like phosphate transporters: Msed_0846, Msed_0866, Msed_1094, and Msed_1512 [8].It is possible that these proteins carry out the role of the lacking PitA in a putative PolyP-based copper-resistance mechanism in this strain.Recently, an extremely high copper-resistant (>200 mM) M. sedula strain CuR1 was isolated [9].The sequencing of its genome revealed that a frameshift mutation had restored a complete pitA gene, encoded partially by the pseudogene Msed_1517 of strain DSM 5348 T [19].Those findings suggested that M. sedula could also have a copper-resistance mechanism involving PolyP, the putative PPX gene, and H + -phosphate symporters.In the present report, the PPX Msed enzyme from M. sedula DSM 5348 T was purified and partially characterized in its capacity to hydrolyze PolyP.The effects of copper on growth and PolyP levels, and the increased transcriptional levels of ppx, copTMA, and the four pho84-like genes in the presence of copper, further support the existence of a PolyP-dependent copper-resistance mechanism in the archaeon M. sedula DSM 5348 T .Four different growth conditions were analyzed: (i) "control": microorganisms were grown in DSMZ medium 88 with no other additions until late exponential phase.(ii) "Nonadapted": microorganisms never exposed to copper before were grown in DSMZ medium 88 and were added to 8 mM CuSO 4 upon inoculation.(iii) "Adapted": microorganisms were grown in DSMZ medium 88 supplemented with increasing copper concentrations (2,4,8,12, and up to 16 mM).Thereafter, adapted cells were grown at 16 mM copper upon inoculation.(iv) "Shift": microorganisms grown in DSMZ medium 88 up to the late exponential phase were abruptly exposed to 8 mM CuSO 4 in the same medium.

Materials and Methods
Commercial E. coli TOP 10 (Invitrogen) and BL21 (DE3) strains were used for cloning and protein expression, respectively.These transformed bacteria were grown at 37 °C in Luria-Bertani (LB) medium supplemented with 50 μg/mL kanamycin.For solid plate cultures, 1.5% agar was added to the medium.
2.2.qRT-PCR.M. sedula DSM 5348 T cells were collected by centrifugation, washed twice with 10 mL of M88 medium, and resuspended in 60 μL of TEN buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA, and 100 mM NaCl).Sixty μL of TENST buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 100 mM NaCl, 1.6% sodium n-lauroylsarcosine, and 0.12% Triton X-100) was added to the suspension before RNA extraction.Total RNA from M. sedula was extracted with TRIzol Reagent (Ambion) and cDNA was synthesized as described previously [20,21].Transcriptional gene expression levels were quantified by qRT-PCR as described before [22].M. sedula 16S rRNA was selected as a reference gene, since its expression was found to be the most stable under the experimental conditions used.Primers used are listed in Table S1.
2.3.PolyP Extraction and Quantification.PolyP was extracted from 1 mL M. sedula cultures, essentially as described by Ault-Riché et al. [23].To quantify PolyP, 30 μL of the extracted samples were mixed with 30 μL of 2 N HCl, and polyP was hydrolyzed by incubation for 30 min at 95 °C.Finally, the solution was neutralized with 1.5 M Tris and inorganic phosphate was quantified using the EnzChek Phosphate Assay kit (Invitrogen).

Cloning of PPX Msed and Generation of Site-Directed
Mutants.The gene Msed_0981 was amplified from the genomic DNA from M. sedula DSM 5348 T and subcloned in the pGEM-T Easy vector (Promega).After digestion with NdeI 2 Archaea and XhoI, the fragment obtained was cloned in the pET28a-TEV expression vector, which is a derivative of pET28a (Invitrogen) containing the recognition and cut sites for the TEV protease from Tobacco Mosaic Virus.E111A, E112A, and E113A PPX Msed mutants were carried out by sitedirected ligase-independent mutagenesis (SLIM) as described previously [24].All primers used are listed in Table S1.To purify the wild-type and modified proteins, E. coli BL21 (DE3) was transformed with the corresponding plasmids.PPX activity was determined by analyzing the release of P i from PolyP.P i liberated was measured by using the EnzCheck phosphate assay kit (Invitrogen), and the amount of phosphate present in a reaction done in the absence of the enzyme was subtracted as a blank.Enzymatic polyP degradation was visualized by UREA-PAGE as described before [25].
Electrophoresis was run at 300 V for 1.5 h.The remaining PolyP was stained with a solution containing 0.05% toluidine blue, 25% methanol, and 5% glycerol for 20 min.Finally, the gel was washed with 25% methanol and 5% glycerol to eliminate the excess of toluidine blue.

Homology Protein Modeling of PPX Msed .
A model for the PPX Msed structure was built including residues 1 to 298, by using homology modeling as implemented in Modeller v9.16 [26].Aquifex aeolicus PPX structure 1T6C was used as the template, and Clustal X was used to align the protein sequences [27].The coordinates for residues G143, S146, and E148 in the 1T6C structure (G136, S139, and E141 in PPX Msed ) were fixed during the modeling, to maintain their configuration in the final PPX Msed model.

Transmission Electron Microscopy and Energy-Dispersive X-Ray Microanalysis (EDX).
To visualize M. sedula DSM 5348 T cells and their electron-dense granules, samples were prepared for electron transmission microscopy as described before [28].M. sedula cells were harvested by centrifugation for 15 min at 1500 ×g and washed by centrifugation for 1 min at 10,000 ×g, with the M88 medium.The pellet was resuspended in M88 medium to a concentration of 1 × 10 9 cells/mL, and 10 μL of this suspension was placed over nickel grids.The excess of liquid was absorbed with filter paper and grids were vacuum-dried.Unstained cells were observed with a Titan 80-300 kV S/TEM transmission electronic microscope.To determine the elemental composition of the samples, the grids were analyzed by energy-dispersive X-ray microanalysis (EDX), as described before [10,11].
2.8.Statistical Analysis.Statistically significant differences of transcriptional data, comparing means regardless of time and treatments, were determined using two-way ANOVA and the post hoc Tukey HSD test (GraphPad Software Inc.).Enzymatic data were analyzed using a one-way ANOVA, followed by Tukey's test with the same program.P values of <0.05 were considered as significant.

Results and Discussion
3.1.Response of Adapted and Nonadapted M. sedula DSM 5348 T to a Copper Challenge.PolyP has been associated with copper resistance in A. ferrooxidans [10] and S. metallicus [11], since their high PolyP levels decreased when cells were shifted to a medium containing CuSO 4 .Although it has been previously shown that M. sedula DSM 5348 T accumulates electron-dense granules [13], the chemical composition of these structures has not been confirmed.Therefore, an elemental analysis of these granules was carried out by electron microscopy coupled with EDX, and the percentages of phosphorous were estimated in different areas of the cell (Figure S1).The 9.43% of phosphorous present in the granule area, compared with 0.07% in the cytoplasm, strongly suggests that these electron-dense granules contain PolyP.Consequently, M. sedula DSM 5348 T could also have a PolyP-based copper-resistance mechanism as that one proposed for S. metallicus and A. ferrooxidans [10,11].
To evaluate whether PolyP levels changed when M. sedula DSM 5348 T cells were exposed to copper ions, copper-adapted and nonadapted cells were grown in the presence or absence of the metal.Figure 1(a) shows that growth of nonadapted M. sedula DMS 5348 T cells was 50% inhibited when exposed to 8 mM copper, as it has been previously described [29].In addition, the growth of adapted cells exposed to 16 mM was also half of that observed for cells not exposed to copper.
The levels of PolyP were quantified at different times of growth (Figure 1(b)).M. sedula DSM 5348 T cells accumulated high amounts of PolyP, reaching up to 450 nmol of PolyP/mg of protein.A 20% decrease in PolyP levels was observed in the nonadapted cells exposed to 8 mM CuSO 4 compared to control cells.Moreover, PolyP levels of the adapted cells were very low (less than 50 nmol of P i /mg of 3 Archaea protein), and these levels remained unchanged during the entire growth curve (Figure 1(b)).Therefore, synthesis and hydrolysis of PolyP could be continuously occurring in copper-adapted cells, and as a consequence, the polymer molecules would not accumulate under those conditions.Previously, it was reported that when Sulfolobus metallicus (another member of the Sulfolobales) cells are shifted to copper sulfate concentrations up to 100 mM, there was a rapid decrease in their exopolyphosphatase (PPX) activity.This was concomitant in time with a decrease in their polyP levels and a stimulation of a phosphate efflux.In addition, copper in the range of 10 micromolars greatly stimulated PPX activity in cell-free extracts from S. metallicus [11].These results supported a possible role of polyphosphate metabolism in copper resistance, in the members of the genus Sulfolobus.It is expected that M. sedula has a similar behaviour, although this does not discard a possible copper damage in the unknown archaeal PPK enzyme.
The basal PolyP level remaining in the cells, in the adapted condition, may be required to respond as an inorganic protein chaperone, a role recently described for PolyP in cells subjected to oxidative stress [30].On the contrary, PolyP levels decreased sharply to around 40% by 60 min after an 8 mM CuSO 4 shock (Figure 1(c)), similar to what was described before for S. metallicus [11] and A. ferrooxidans [10].The results in Figure 1 suggest that PolyP and PPX may also have a role in copper homeostasis in M. sedula DSM 5348 T .2(a)).The optimal temperature for the enzyme was 65 °C (data not shown), and it had a preference for long-chain PolyP of 700 residues of P i (Figure 2(b)).It has been reported that the PPXs from E. coli (PPX Eco ) and P. aeruginosa require Mg 2+ as a cofactor for their activity [14,31], whereas the S. solfataricus enzyme requires Mn 2+ [15].The activity of PPX Msed increased 3.6-fold in the presence of 20 mM MgCl 2 (Figure 2(c)), and no effect on its activity was observed when using MnCl 2 (Figure 2(c)).
The active site of PPX Eco is formed by a catalytic E121 and four residues that coordinate the Mg 2+ metal cofactor in a metal-binding site: D141, G143, S146, and E148 (Figure 3(a)) [32,33].An alignment between the PPXs from E. coli, S. solfataricus, and M. sedula revealed that all the amino acid residues of their putative active sites were conserved, except for D141 that was substituted by a glutamic acid in some of the crenarchaeal proteins (Figure 3(a) and Figure S2).These residues corresponded to E113, E134, G136, S139, and E141 in PPX Msed .Additionally, E112 was also conserved in the three species, and E111 was only conserved in the PPXs of crenarchaeota.To evaluate the possible role of the residues in the active site, E111, E112, and E113 were replaced by alanine by site-directed mutagenesis, and the activity of the mutants was evaluated.Mutants E111A and E113A had no PPX activity, suggesting that these residues are essential for catalysis in PPX Msed (Figure 3(b)).On the contrary, the PPX activity of mutant E112A was the same compared to that of the wild-type enzyme (not shown).These results suggest that both E111 and E113 could be catalytic residues in PPX Msed .Theorizing about a structural connection between E111, E113, and a possible catalytic mechanism, a homology model of PPX Msed was generated and compared to the crystallized PPX Eco (PDB: 1U6Z).The Aquifex aeolicus PPX (PPX Aaeo ) (PDB: 1T6C) was used as the template because it has 28% identity with PPX Msed , and the structure has a Ca 2+ ion in the metal-binding site of the active site, resembling the divalent cation expected for PPX Msed .Considering the difference in the number of amino acid residues between the two proteins (315 in PPX Aaeo versus 420 in PPX Msed ) and the fact that the sequences and structures of the active sites are conserved in most of the crystallized PPXs so far, solely the first 298 residues of PPX Msed were modeled, and only the active site was analyzed (Figure 3(c)).The model anticipated that E113 was the closest residue to the metal-binding site in PPX Msed and it could be the catalytic residue.E111 was predicted to be located 2.66 Å from H155 in PPX Msed .E111 could indirectly stabilize the metal-binding site, by preventing the interaction between H155 and E141 that could disturb the proper coordination of the metal.On the other hand, the side chain of E112 points in the opposite direction of the metal-binding site.This could structurally explain why the mutation of this residue did not alter the activity of PPX Msed .Further biophysical analyses should be carried out to prove these hypotheses.6 Archaea In terms of transcriptional regulation, it would be possible that ppx levels increase after a copper shock, since PolyP amounts were sharply reduced under that stressing condition (Figure 1(c)).It was previously reported that transcriptional levels of ppx did not change significantly, after 1 h shock of M. sedula DSM 5348 T, in the presence of metals such as copper, zinc, nickel, uranium, and cobalt as determined by microarray analyses [29].However, it was not assessed whether ppx was induced at earlier times after the shock.Accordingly, transcriptional levels of ppx were quantified by qRT-PCR at different times after an 8 mM CuSO 4 shock (Figure 4).It was found that ppx was upregulated during the first 75 min after the metal shift, with a peak in its expression 30 min after the shift.The results presented here suggest that PPX Msed could play an important role in short-term copper resistance after a metal shock, possibly being one of the first copper determinants used to respond to copper stress.
It is known that PPX Eco is inhibited in vitro by 20 mM P i [14].PPX Msed was also inhibited in vitro in the presence of 1 mM P i (data not shown).This inhibition may be very important in vivo, since PolyP hydrolysis stimulated by the presence of the metal could come to an end.

Relative Transcriptional Expression of copTMA Genes in
Adapted and Nonadapted M. sedula DSM 5348 T Challenged to Copper.Other proteins that are involved in copper resistance in M. sedula DSM 5348 T are CopA (Msed_0490), CopM (Msed_0491), and CopT (Msed_0492) [8].CopA is a P-type ATPase efflux pump, CopM is a putative metallochaperone, and CopT is a transcriptional regulator homolog to CopR.All these genes are functional in this archaeon [9].In S. solfataricus, these genes are cotranscribed constitutively, as an operon.However, only the transcription of copM and copA was also upregulated in the presence of copper, due to the activation of a second promoter upstream of the constitutive copTMA promoter [34,35].While in S. solfataricus, the three genes form a cluster in the same direction; copT in M. sedula DSM 5348 T is orientated opposite to the other genes (Figure 5(a)) [9].In terms of regulation, microarray analyses revealed that the expression levels of copA and copM increased and copT levels decreased after 60 min of a 4 or 8 mM CuSO 4 shift in M. sedula DSM 5348 T [29].This kinetic behaviour is equivalent to that observed for ppx (Figure 4).
Short-term copper effects can be analyzed with a shock condition, since the microorganism is exposed to the metal for briefer periods compared with its generation time.On the contrary, long-term effects can be analyzed during the growth of nonadapted cells for intervals longer than a generation time, in adapted cells, in which the microorganisms have been exposed to the metal for several generations.To assess for a possible differential timing in the expression of these genes, during long-term copper exposure in M. sedula DSM 5348 T , relative transcriptional levels of genes Msed_0490, Msed_0491, and Msed_0492 were quantified by qRT-PCR in copper-adapted and nonadapted cells grown at exponential (60 h), late exponential (72 h), and stationary 7 Archaea (84 h) phases (Figure 5).In general, gene expression of these determinants was higher at the exponential phase (Figures 5(b) and 5(c)).In the nonadapted condition, when cells were exposed for the first time to 8 mM CuSO 4 , the expression of copM increased 4.5-fold at 60 and 72 h in exponential phase (Figure 5(b)).In cells adapted to copper exposure, the transcriptional levels of copM were up to three times higher than that of the other genes in exponential phase and had a peak in its expression at 72 h (Figure 5(c)).These results suggest that the genes analyzed are regulated differently in response to copper ions and that their expression might also be controlled by distinct internal copper-responsive promoters and transcription regulators, as it occurs in S. solfataricus [35].

Possible Pho84-Like Phosphate Transporters Are
Upregulated Differently in M. sedula DSM 5348 T Exposed to Copper.The model for copper resistance in microorganisms mediated by PolyP proposes that the P i released from PolyP by PPX could complex metal ions.These complexes would be transported out of the cell by H + -phosphate symporters [13,16,17], such as the low-affinity phosphate transporter PitA from E. coli [18].Recently, a pitA-homolog gene was found in M. sedula CuR1, which is truncated in the "wild- 8 Archaea type" strain DSM 5348 T [19].As already mentioned, there are no pitA-homologs in some acidophilic species, such as the bacterium A. ferrooxidans and the archaeons S. metallicus and F. acidarmanus [4].In those microorganisms, it has been proposed that proteins orthologous to the Pho84 phosphate transporter from Saccharomyces cerevisiae could carry out the role of PitA, transporting metal-phosphate complexes outside the cell [10,11].The copper MIC value (8-16 mM) of M. sedula DSM 5348 T is much lower than that of CuR1 mutant (160 mM Cu) [19].Therefore, the pho84-like phosphate transporters present in the strain DSM 5348 T would not entirely replace its lack of PitA, in eliminating Cuphosphate complexes.As suggested before, some unidentified transport protein could be responsible for excluding these complexes instead of PitA [19].
It is also important to consider that an excess of Pi entering the cells through the PitA system or other phosphate transporters would be toxic to the microorganisms.The pho84-like phosphate transporters present in M. sedula DSM 5348 T may also have a role in eliminating the extra Pi generated from polyP.
Saccharomyces cerevisiae Pho84 is a high-affinity H +phosphate symporter functional only at acidic pH [36].In the genome of M. sedula DSM 5348 T , four genes have been annotated as putative Pho84-like phosphate transporters: Msed_1512, Msed_1094, Msed_0866, and Msed_0846 [8].To evaluate whether the transcription of these genes is copper-responsive, their transcriptional levels were quantified by qRT-PCR in M. sedula DSM 5348 T cells shifted to an 8 mM CuSO 4 shock (Figure 6(a)).The four putative Pho84like genes were upregulated after the metal shift, with a maximum expression at 30 min after the shock.Msed_0846 showed the highest increase after the shift, suggesting it could be the most important transporter in an immediate copper Figure 7: Cartoon working model for copper resistance in M. sedula.In M. sedula DSM 5348 T , copper ions could bind to the transcriptional repressor CopT (Msed_0492) and prevent the binding of this protein to the DNA to activate the transcription of copA (Msed_0490), copT, and copM (Msed_0491) genes.CopM is a possible Cu-binding protein that could sequester copper ions in the cytoplasm.CopA is a putative P-type metal-efflux ATPase that could couple the efflux of copper ions from the cytoplasm and the energy from ATP hydrolysis.On the other hand, PPX (Msed_0891) could hydrolyze PolyP to generate P i that could complex copper ions.These complexes could be transported outside the cell, together with H + by the H + -phosphate symporters, such as the Pho84-like phosphate transporters (Msed_0846, Msed_0866, Msed_1094, and Msed_1512) in both strains DSM 5348 T and CuR1.This last strain has an additional PitA phosphate transporter that could enhance the efflux of complexes and would explain its higher copper resistance.This model is based on the results of the present work and previously reported proposals [8,9,11,13,19,29,34,35]. response in M. sedula DSM 5348 T .This behaviour is similar to that of the ppx gene already seen in Figure 4, suggesting a possible common regulation mechanism for this group of genes.
Moreover, it has not been previously reported whether this copper-resistance PolyP-based mechanism has a role in long-term copper resistance in M. sedula.To evaluate the possible role of pho84-like genes in long-term copper response in M. sedula DSM 5348 T , the transcriptional expression of these genes was assessed by qRT-PCR during exponential (60 h), late exponential (72 h), and stationary (84 h) phases of nonadapted and adapted cells.Nonadapted cells exposed to 8 mM CuSO 4 showed differences in the expression of the putative phosphate transporters during growth (Figure 6(b)).Transcriptional levels of Msed_0866 and Msed_1512 were maximal at 60 h and 72 h of exposure to the metal (in exponential phase), whereas Msed_1094 had its peak in stationary phase at 84 h of copper exposure.The expression of Msed_0846 did not change significantly in both nonadapted and adapted cells.In M. sedula DSM 5348 T cells adapted to 16 mM CuSO 4 , the transcriptional levels of the putative transporter encoded by Msed_1512 increased 15-fold at exponential phase (Figure 6(c)).The transcriptional levels of the other possible transporters were not significantly different in the adapted cells.These results suggest that the 4 genes are upregulated by the presence of copper, but their "transcriptional timing" could be different in the nonadapted growth condition.Possibly, Msed_0866 and Msed_1512 could have predominant roles during exponential phase and Msed_1094 could be the most important putative transporter in stationary phase.Msed_0846 might have a role in the immediate defense mechanism against a copper stress, as in the case of a metal shock.On the contrary, under a constant high copper exposure, such as 16 mM CuSO 4 in the adapted condition, Msed_1512 could be by far the most important copper-responsive transporter during the exponential phase of growth.

Conclusions
The experimental evidence obtained indicates that M. sedula strain DSM 5348 T can live in 16 mM CuSO 4 and could have a polyP-based copper-resistance mechanism.The higher transcriptional expression of the four putative pho84-like phosphate transporter genes (Msed_0846, Msed_0866, Msed_1094, and Msed_1512) in M. sedula DSM 5348 T grown in the presence of copper suggests that the proteins they code for could carry out the same function of PitA present in other species.Figure 7 shows a working cartoon model with the possible copper-resistance determinants known in M. sedula.The experiments presented here suggest that this model would be a dynamic one and that not all the indicated proteins are needed in the same levels during the different stages of copper resistance.After an immediate exposure to copper ions, as it occurs in the case of a sudden metal shift, all possible determinants studied were overexpressed after the shift.In the nonadapted phase, when the archaea have grown less than 90 h under a copper stress, CopM could have an important role possibly in sequestering metal ions within the cell.Pho84-like phosphate transporters have differential expression: while Msed_0866 and Msed_1512 seem to be needed preferentially during exponential phase, Msed_1094 could have a major contribution during stationary phase.In the very late stages of copper resistance, when the cell is adapted to higher concentrations of the metal, Msed_1512 could be the most important Pho84-like phosphate transporter and CopA and CopM could be involved in copper efflux and sequestration of the metal, respectively.The results presented suggest there might be a tight regulation network behind the expression of these genes.Nevertheless, further work will be required to understand better the suggested role for the components involved in copper resistance in M. sedula.

Figure 1 :
Figure1: Effect of copper ions in the growth and PolyP levels of M. sedula DSM 5348 T .Growth curves (a) and polyP levels (b) were determined at the indicated copper concentrations added upon inoculation.Cells previously grown up to the late exponential phase were shifted to grow at 8 mM CuSO 4 at time 0, and polyP levels were immediately determined thereafter at the indicated times after the shift (c).

Figure 2 :
Figure 2: The gene Msed_0891 encodes for a functional exopolyphosphatase.(a) In vitro enzymatic PPX activity assay of the protein encoded by Msed_0891.PPX Msed was incubated with PolyP 700 as described in Materials and Methods at 65 °C for the times indicated.The remaining PolyP in the samples was visualized by UREA-PAGE.(b) Substrate preference of PPX Msed .The enzyme was incubated with the PolyP of different chain lengths indicated, as described in Materials and Methods.(c) Effect of MgCl 2 and MnCl 2 in PPX activity.PPX Msed activity was determined at the indicated metal concentrations ( * * P < 0 01).

Figure 3 :Figure 4 :
Figure 3: Catalytic residues of the possible active site of PPX Msed .(a) Protein sequence alignment of the residues in the potential active site of M. sedula, S. solfataricus, A. aeolicus, and E. coli PPXs.The residues that have been proven essential for catalysis in PPX Eco are highlighted in bold [32, 33] (asterisk indicates fully conserved residue, colon indicates strongly similar properties, and period indicates weakly similar properties).(b) Activity of PPX Msed and the E111A and E113A mutants.(c) Cartoon representation of the possible active site of PPX Msed .Amino acids in the metal binding site are shown in pink, the mutated residues are shown in cyan, and residues near E111 are shown in yellow.

Figure 5 :
Figure 5: Transcriptional levels of copA, copM, and copT genes at different points in the growth phase of M. sedula DSM 5348 T cells grown in the presence of copper ions.(a) Diagram of the copTMA in M. sedula DSM 5348 T .Relative gene expression was quantified by qRT-PCR.(b) Nonadapted cells exposed to 8 mM CuSO 4 .(c) Adapted cells exposed to 16 mM CuSO 4 .Error bars indicate standard deviations based on three different experimental values.Values with different letters differ significantly (P < 0 05).

Figure 6 :
Figure 6: Differential transcriptional expression of putative pho84-like genes in M. sedula DSM 5348 T cells subjected to copper.(a) Nonadapted cells exposed to an 8 mM copper shift during the indicated times.(b) Nonadapted cells exposed to 8 mM CuSO 4 during the indicated culture times.(c) Adapted cells exposed to 16 mM CuSO 4 .Values with different letters differ significantly (P < 0 05).
[8]was suggested that PolyP hydrolysis could be carried out by a putative PPX Msed encoded by M. sedula DSM 5348 T gene Msed_0891[8].The decrease in PolyP levels after a copper shock is most likely due to its hydrolysis by this putative enzyme.To evaluate whether Msed_0891 encoded for a functional PPX, the gene was expressed in E. coli and the corresponding protein was purified.PPX Msed was a functional PPX, able to hydrolyze PolyP in vitro, as seen by its disappearance from the reaction by using gel electrophoresis (Figure