1 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
2 Centre for Ecology, Evolution and Conservation, University of East Anglia, Norwich NR4 7TJ, UK
3 Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK
Correspondence
Philip L. Bond
phil.bond{at}uea.ac.uk
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ABSTRACT |
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Present address: Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden.
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INTRODUCTION |
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Micro-organisms use various means to control intracellular copper levels; this includes various influx and efflux mechanisms, and copper complexation by cellular components. The cop operon of Ent. hirae encodes a metal-dependent repressor, an internal Cu+ chaperone, and two membrane-bound P-type ATPases for Cu+ uptake and efflux (Solioz & Stoyanov, 2003). E. coli employs additional mechanisms that include a CBA-type Cu+ efflux Cus system that spans across the inner and outer cell membranes, as well as the plasmid-encoded pco system, by which PcoA is thought to carry Cu+ across the inner membrane, after which it is oxidized in the periplasm (Rensing & Grass, 2003
).
Ferroplasma acidarmanus Fer1 is a mesophilic, extreme acidophile that was isolated from a disused iron mine in Iron Mountain, CA, USA (Edwards et al., 2000). Environments containing high levels of dissolved metals include both active and disused mines, where the production of acid mine drainage and acid rock drainage is catalysed by the action of micro-organisms (Nordstrom & Southam, 1997
). Solutions that flow from the Iron Mountain mine include some of the most acidic and metal-rich mine drainage studied to date (Nordstrom & Alpers, 1999
). The concentrations of iron, zinc, copper and cadmium in Iron Mountain solutions colonized by Fer1 typically exceed 28, 2·5, 0·38 and 0·25 g l1, respectively (Edwards et al., 1998
; Nordstrom & Alpers, 1999
). Acidophiles such as archaeal Ferroplasma species can dominate biofilms that are present in extreme metal contamination (Bond et al., 2000a
), and in these environments metal resistance would be of primary importance. The levels of copper at which metabolic activity has been recorded in various micro-organisms (Table 1
) indicate the high metal-resistance capabilities of acidophiles. At the present time, copper homeostasis is being studied extensively in various neutrophilic bacterial species (see references above). Most archaeal genomes sequenced to date contain sequences with high similarity to copper transport P-type ATPases. However, investigation of the biochemical and genetic metal homeostasis and resistance mechanisms of acidophilic micro-organisms, particularly of the archaea, is virtually in its infancy. These archaea represent challenging yet environmentally relevant systems for an increased understanding of metal resistance and homeostasis (Dopson et al., 2003
).
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In this study, we present initial biochemical, proteomic and molecular characterization of copper resistance in Fer1. Our results show that Fer1 is the most copper-resistant archaeon studied to date, and that transcription of its cop gene cluster is responsive to Cu2+, suggesting an intrinsic role in copper efflux. These findings constitute the first investigative report of an archaeal copper resistance system.
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METHODS |
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Growth of Fer1 in the presence of copper.
Growth was measured after 72 h incubation by detecting the increase in protein (Bradford assay, Bio-Rad) compared to initial protein levels (t=0) in 100 ml batch cultures containing 0, 1, 5, 10 or 20 g Cu2+ l1 (added as CuSO4.5H2O, in addition to the trace element Cu2+ concentration of 0·13 mg l1). All batch-culture toxicity experiments were conducted in triplicate, and the mean and standard deviation (SD) were calculated. Fer1 cells used as inoculum, equivalent to 10 µg of protein, were previously batch cultured in the presence or absence of 1 g Cu2+ l1.
Fe2+-dependent oxygen consumption.
Fer1 cells were harvested from the continuous culture vessel by centrifugation (10 000 g for 20 min) and washed three times in MSM. Resting cells equivalent to 100 µg of protein were resuspended in MSM at 37 °C and equilibrated for 5 min in the presence of various levels of added Cu2+ (0, 1·0, 10, 20, 40 or 80 g l1). Following the addition of FeSO4.7H2O at 2 % (w/v), Fe2+-dependent O2 consumption was assayed (1 ml final volume) using a Clark-type oxygen electrode at 40 °C (Hallberg et al., 1996).
Proteomic analysis of cytoplasmic protein.
For protein expression analyses, replicate one-litre Fer1 cultures were grown to late exponential phase [between 72 and 96 h incubation to a density of 6·1±0·5 (mg protein) l1] in MSM with iron and yeast extract (as described above) in the presence and absence of added Cu2+ at 1 g l1. Cells were harvested by centrifugation (10 000 g for 20 min) at 4 °C and immediately frozen at 20 °C. When required, frozen cells were thawed on ice in 1 ml MSM, centrifuged, resuspended in lysis buffer, and disrupted by sonication (5x3 s bursts at amplitude 7·5 µm). Cell debris was removed by centrifugation (10 000 g for 10 min) and the protein extract was stored in frozen aliquots. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out as previously described (Dopson et al., 2004a).
Gels were stained with EZBlue (Sigma) or silver nitrate (Blum et al., 1987) and scanned using the proEXPRESS proteomic imaging system (PerkinElmer). Images of silver-stained gels from the control and Cu2+-exposed cells were analysed using ProteomWeaver version 1.2 (Definiens). Protein spot matching was initially carried out using the automated matching function and then manually improved by spot land marking. Composite gel images were produced from the replicates for each condition (n=24). Protein spot expression was normalized according to the entire protein complement expressed on the gels (ProteomWeaver), alleviating the need for internal protein standards. After detection of differentially expressed proteins in silver-stained polyacrylamide gels, proteins that could be identified from corresponding EZBlue stained gels were excised using a sterile micropipette tip. Samples were treated by trypsin digestion and analysed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Reflex III; Hesketh et al., 2002
). MALDI-TOF peptide mass fingerprint data were matched against the F. acidarmanus Fer1 genome sequence data (http://genome.ornl.gov/microbial/faci/) using MASCOT (http://www.matrixscience.com) and results were only included when peptide fragment matching was statistically significant (MOWSE score greater than 74).
In vitro DNA manipulations.
Chromosomal DNA was extracted as previously described (Bond et al., 2000b) from 400 ml of Fer1 cells originating from the continuous-culture vessel. Oligonucleotides were synthesized by MWG Biotech (Table 2
). The promoter regions of copY and copZB (dashed boxes, Fig. 1
) were amplified using primer sets copYPfor and copYPrev and copYZBfor and copYZBrev, respectively, by PCR. The resulting PCR products were cloned into pCR4-TOPO using a TOPO TA cloning kit (Invitrogen) according to the manufacturer's instructions. Plasmids containing inserts were transformed into E. coli TOP10. Plasmids containing putative promoter regions were verified by automated DNA sequencing.
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Computational analysis of DNA and protein sequences.
The partial genome sequences for Fer1 and Ferroplasma Type II strains are available from the Oak Ridge National Laboratory (http://genome.ornl.gov/microbial/faci/) and the National Center for Biotechnology Information (http://www.ncbi.nih.gov), respectively. Selected sequences were analysed for protein folding regions, regulator elements and transmembrane spanning regions, using 3D PSSM (http://www.sbg.bio.ic.ac.uk/3dpssm/; Kelley et al., 2000
), NPS@ (http://npsa-pbil.ibcp.fr/cgibin/npsa_automat.pl?page=/NPSA/npsa_hth.html; Dodd & Egan, 1990
), Palindrome (EMBOSS, http://emboss.sourceforge.net/) and HMMTOP (www.enzim.hu/hmmtop/index.html; Tusnady & Simon, 2001
).
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RESULTS |
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Toxicity of Cu2+ towards Fe2+-dependent oxygen consumption
Previously it was shown that Fer1 grows chemomixotrophically, oxidizing Fe2+ in the presence of organic carbon (yeast extract), and the chemomixotrophic growth rates are four times those of chemo-organotrophic growth (oxidizing organic carbon) (Dopson et al., 2004b). Experiments were carried out to determine the effect of Cu2+ on Fe2+ oxidation by Fer1 cells. The abiotic Fe2+ oxidation rate was 1·5 nmol O2 min1, compared to 364±34 nmol O2 min1 (mg Fer1 protein)1 (n=3) in the absence of Cu2+, increasing to 514±40, 456±94 and 441±68 nmol O2 min1 (mg protein)1 (n=3) in the presence of 1, 10 and 20 g Cu2+ l1, respectively. No significant inhibition of the Fe2+ oxidation rate was observed until the addition of 40 g Cu2+ l1, and at 80 g Cu2+ l1 the rate was 266 nmol O2 min1 (mg protein)1 (73 % of the rate in the absence of Cu2+).
Proteomic analysis of Fer1 cells exposed to Cu2+
Fer1 was grown in batch cultures in the presence and absence of 1 g Cu2+ l1 (corresponding to 16 mM) and protein profiles were analysed to identify proteins upregulated in response to Cu2+ (Fig. 3). Higher concentrations of copper significantly affect Fer1 batch-culture growth (Fig. 2
), and therefore would invalidate direct comparisons between copper-exposed and non-exposed cell cultures. None the less, the exposure concentration used (16 mM) would be toxic to many acidophiles and to most neutrophilic bacteria (Table 1
), and is two- to threefold greater than the typical concentrations detected in the Iron Mountain mine solutions. Representative comparisons of protein separations are shown from cells grown in the presence and absence of copper (Fig. 3B, C
). A total of 21 proteins were identified whose synthesis was induced more than 1·5-fold compared to cells grown without additional Cu2+ (Table 3
, Fig. 3
). Proteins identified included molecular chaperones, DNA repair enzymes, enzymes from central metabolic and biosynthetic pathways, and proteins associated with transcription and translation. Certain proteins were detected at multiple locations on our 2D gels (Table 3
, Fig. 3A
), including those with homology to DnaK (spots 20, 36 and 37) and thermosome (spots 27 and 28). High levels of these proteins, detected in the copper-exposed condition (Fig. 3B, 3C
), may have led to detection of degradation products or post-translational modifications. The state of a phosphorylation site, such as that on the E. coli DnaK protein (McCarty & Walker, 1991
), could result in differing migration spots for the Fer1 DnaK homologue.
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Genomic evidence of Fer1 copper transport
A cluster of genes with homology to copper transport systems was previously detected on the annotated genome sequence of Fer1 (Ettema et al., 2003). The putative regulator (designated CopY; Fig. 1
) was previously depicted as being divergently transcribed from the copper chaperone (designated CopZ) and the cation-translocating P-type ATPase (CopB) (Ettema et al., 2003
). However, it appears that copY is transcribed in the same direction as copZ and copB (Fig. 1
). As previously reported by Ettema et al. (2003)
, CopY and CopZ contain Cu-binding domains, known as TRASH (Fig. 1
). CopY has sequence identity (29 %) with a characterized transcriptional regulator of the LrpA family from Pyrococcus furiosus (Brinkman et al., 2000
). This family of regulators is reportedly involved in either positive or negative regulation. copB encodes a putative P-type ATPase Cu transporter (Fig. 1
), and this protein contains predicted sequences for metal binding, ATP binding, phosphorylation and phosphatase sites. Hydropathy profiles revealed numerous transmembrane spanning domains in the P-type ATPase, as expected for a membrane-bound protein. However, primary sequence information is not useful to determine whether the role of this putative transporter is for Cu efflux or uptake.
The Fer1 genome (http://genome.ornl.gov/microbial/faci/) was screened (TBLASTN) for other possible copper transporters using protein sequences from various organisms. This included sequences of ATP7A (human), CopA (Ent. hirae, Helicobacter pylori, Bacillus subtilis and E. coli), CopB (Ent. hirae and Archaeoglobus fulgidus), Ccc2 (Saccharomyces), PacS and CtaA (Synechococcus elongates). One Fer1 gene (number 1580, contig 169) had approximately 25 % sequence identity to these. However, this gene also matched with much higher similarity to known K+ transport systems. Genes encoding other copper-resistance mechanisms, such as the E. coli Cus (transport), Pco (copper oxidation) and Cut (metal sequestration) systems were not detected on the Fer1 genome.
RT-PCR and primer extension analysis of the cop genes
To investigate regulation of the cop genes in response to Cu2+ and other metals, RT-PCR was performed for amplification of regions 1 (copYZB), 2 (copY), 3 (copZB) and 4 (copB; Fig. 1). No RT-PCR products were obtained for any of the primer sets in the absence of Cu2+. In the presence of 1 g Cu2+ l1, no RT-PCR product was obtained for region 1. However, a 2·2 kb RT-PCR product was obtained for region 3, which indicated that the copZ and copB genes are co-transcribed in Fer1, and that copY is transcribed separately. Exposure of Fer1 to other metals (Zn2+, Hg2+, Cd2+, Mg2+, As3+, As5+ and Cu+) did not generate a copB or copZB RT-PCR product, suggesting that these metals did not stimulate transcription under these conditions.
To locate the transcriptional start sites of copZB and copY, primer extension reactions were performed. Two possible transcriptional start sites were detected at 41 and 62 bp upstream from the copY translational start, and a slight increase of copY expression was detected when cells were exposed to Cu2+ (Fig. 4A). Densitometry analysis of the copZB cDNA product revealed a 15-fold increase of the copZB transcript when cells were exposed to 5 g Cu2+ l1 (Fig. 4B
). In these putative promoter regions, possible TATA boxes are indicated (Fig. 4
), and these elements are 25, 22 and 21 bases upstream from suggested transcription start sites of copZB, copY1 and copY2, respectively. Putative BRE (transcription factor B recognition element) regions are also indicated (Fig. 4
), and those for copZB and copY2 are relatively purine rich. Different palindromic sequences 10 bp in size were detected in the TATA-box region of copY2 and 9 bases upstream from the copZB transcription start, possibly indicating protein-binding elements.
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DISCUSSION |
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Previous work strongly suggests that Fer1 obtains energy by iron oxidation (Dopson et al., 2004b). Growth rates are elevated during iron oxidation, and electron transport chain inhibitors decrease both Fe2+ oxidation (azide) and chemomixotrophic growth on Fe2+ (azide or antimycin A; data not shown). Thus, we examined a link between Fer1 energetics and copper exposure by measuring Fe2+-dependent oxygen consumption during short-term exposure to Cu2+. This activity was detected in the presence of extremely high concentrations of Cu2+ (40 and 80 g l1). Moreover, in comparison to no Cu2+ addition, the rate of oxygen consumption increased in the presence of 10 or 20 g Cu2+ l1. Similar stimulation of substrate-dependent oxygen consumption has been observed for arsenite addition to the acidophile Acidithiobacillus caldus (Hallberg et al., 1996
). This increased respiration in the presence of added copper is not attributed to an increase in growth, as the assay period is conducted over a few minutes only, and we already detect lower Fer1 growth rates in the presence of added copper. The increased respiration can be explained as a response to a higher energy demand required to alleviate the affects of increased intracellular levels of copper, in processes such as cation efflux, DNA repair and protein folding.
The putative transcriptional regulator copY is transcribed separately but in the same direction as the putative copper metallochaperone copZ and a P-type ATPase transporter copB. The bacterial metallochaperone CopZ coordinates and transports metals to P-type ATPases (Solioz & Stoyanov, 2003), and this is its suggested role in Fer1. The CopB protein was found to encode the functional consensus motifs necessary to perform as a copper transporter. Our primer-extension results suggest that expression of copZB was strongly upregulated during Cu2+ exposure (Fig. 4
), which was confirmed by RT-PCR. As proposed for a number of other prokaryotic copper-resistance systems, a possible mechanism for Cu-mediated regulation may be via a conformational change in CopY upon binding free cytoplasmic Cu+, resulting in its release from the promoter. Thus, transcription of copZ and copB can proceed, presumably to perform copper efflux. Alternatively, CopY may act as a positive regulator, and its binding (in the presence of copper) may enhance transcription of copZB. Further work, such as footprinting analysis/S1 mapping, is required to determine the transcriptional regulation mechanism of this locus in response to copper stress. So far, our attempts to overexpress CopB have proved unsuccessful, and the cloning suggests that multicopy Fer1 copB is toxic to E. coli (C. Baker-Austin, unpublished results). Regardless of this, the increased expression of the putative chaperone and transporter that we detect in response to increased copper suggests that their purpose is copper efflux.
Effective metal-ion homeostasis requires the balanced activity of metal-ion-uptake, efflux, sequestration and redox systems (Solioz & Stoyanov, 2003; Rensing & Grass, 2003
; Cavet et al., 2003
). So far, our screening of the Ferroplasma genome sequences has failed to reveal the presence of other copper transport mechanisms. There is the possibility that in the acidic metal-leaching environment of strain Fer1, copper-uptake mechanisms are not required. However, other putative cation and multi-drug transport systems are evident on the genome, and future characterization of these and completion of the genome sequence are required to facilitate a more complete picture of copper homeostasis.
The presence of copper can stimulate production of reactive oxygen species, inducing DNA-strand breaks and oxidation of nucleotide bases (Gaetke & Chow, 2003; Kawanishi et al., 1989
). The upregulation of proteins associated with DNA repair suggests that Fer1 undergoes free-radical-induced stress associated with Cu2+ exposure (Table 3
). Cu2+ has also been shown to be toxic due to its ability to bind to histidine residues, and is linked to oxidative damage to lipids and proteins (Gaetke & Chow, 2003
). The repertoire of the so-called heat-shock proteins (HSPs; i.e. thermosome and DnaK) upregulated in Cu2+-exposed Fer1 cells may be directly related to conformational alterations to protein groups within the cell (Table 3
). The paradigm for HSPs, also termed protein chaperones or stress proteins, is to prevent aggregation and maintain proteins folded in native conformations (Fink, 1999
). HSPs are present in all organisms, and are synthesized at increased levels under stress conditions, such as during changes to temperature, salt, pH and redox (Hartl & Hayer-Hartl, 2002
). Additionally, upregulation of HSP in response to metal exposure has been widely reported in numerous eukarya, for example, the effects of cadmium on sea urchins (Roccheri et al., 2004
) and of copper on nematodes (Kammenga et al., 1998
). However, surprisingly, HSP response to metal exposure is not commonly reported in prokaryotes, although it has been linked to the response to Cu2+ in Rhizopus nigricans (Cernila et al., 1999
), Co2+ exposure in Enterobacter liquefaciens (Marrero et al., 2004
) and Cd2+ exposure in E. coli (Ferianc et al., 1998
). It is striking in this instance that five of the 21 Fer1 proteins upregulated in response to Cu2+ are associated with protein stability. It will be of interest to investigate the physiological consequences of exposure of Fer1 to high levels of metals and to see if this is a common response to metal exposure in other prokaryotes.
A number of other proteins differentially synthesized at least 1·5-fold were detected using 2D-PAGE (Table 3). These included metabolic and electron transport proteins, biosynthetic proteins, and enzymes involved in transcription and translation. This is consistent with a proteome-wide toxicological response to metals, as has been demonstrated in other proteomic studies concerned with microbial metal resistance (Noel-Georis et al., 2004
; Vido et al., 2001
). Proteins CopB and CopZ were not detected by 2D-PAGE, although we show these were upregulated during Cu2+ exposure. This may be explained by the fact that hydrophobic membrane proteins (CopB) and very small proteins (CopZ) are difficult to detect using this method (Pradet-Balade et al., 2001
). The Fer1 response to metal exposure will involve features other than protein refolding, DNA repair and active copper efflux. For example, a defining characteristic of extremely acidophilic archaea is the highly impermeable nature of their cell membranes (van de Vossenberg et al., 1998
), and this is likely an important feature of highly metal-resistant organisms.
In conclusion, we have begun characterization of the copper-resistance mechanisms of Ferroplasma acidarmanus Fer1, one of the most metal-resistant organisms studied to date. Responses to copper exposure (at environmentally relevant levels) were detected; these included: an increase in respiration rate, we suggest resulting from increased energy demands; an increase in mRNA transcripts encoding a putative metal chaperone and a P-type ATPase transporter, suggesting their role in copper efflux; and the increased expression of DNA-repair and stress proteins (protein chaperones), to an extent not previously reported in prokaryotes. These findings provide valuable starting points for further investigation of biochemical and molecular mechanisms of extreme metal resistance.
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ACKNOWLEDGEMENTS |
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Received 29 March 2005;
revised 18 May 2005;
accepted 30 May 2005.
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