School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
Correspondence
Mark Dopson
m.dopson{at}uea.ac.uk
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ABSTRACT |
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Overview |
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Acid-leaching solutions are characterized by high metal concentrations that are toxic to most life and have historically been considered sterile. Solutions that contain the highest recorded natural levels of soluble metals occur at the Iron Mountain site in California, USA. In those solutions, iron has been measured at 1·99 M (111 g l-1) and concentrations of copper, arsenic, cadmium and zinc have all been recorded in the tenths of grams to grams per litre range (Nordstrom & Alpers, 1999). It is well known that AMD solutions are far from sterile and that acidophilic micro-organisms not only tolerate, but thrive in these acidic metal-rich solutions (Hallberg & Johnson, 2001
).
The aim of this communication is to review the knowledge of metal resistance in acidophilic micro-organisms. Considerable insight into general, neutrophilic, microbial resistance mechanisms is currently available (see Nies, 1999). However, micro-organisms surviving in acid-leaching environments should possess the most advanced metal resistance mechanisms, making them ideal systems to study and improve understanding of metal resistance. Here we summarize the current knowledge of this topic, highlighting where gaps and questions are revealed, as well as suggesting some future directions.
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Some environmental and economic aspects of acid leaching |
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Knowledge of metal toxicity and how acidophiles survive in acidic metal-rich environments may provide insights into bioremediation of AMD and ARD sites, the optimization of existing techniques and the development of novel biotechnological processes. A possible example for the optimization of an existing technique is the transfer of metal resistance gene operons between acidophilic micro-organisms, creating multi-metal resistant strains. This would be highly advantageous for bioleaching of metal sulfides and metal sequestration systems for bioremediation of metal-contaminated sites. For instance, bioleaching of arsenic-containing minerals is accelerated at higher temperatures due to increased chemical reaction rates. Unfortunately, typical acidophilic thermophiles (usually archaea) used for bioleaching are not as resistant as moderately thermophilic bacteria to arsenic. Therefore, the ability to transfer arsenic resistance genes to thermophilic archaea could significantly increase the bioleaching efficiency.
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Ecology of acid-leaching environments |
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Micro-organisms inhabiting AMD, ARD and bioleaching environments encounter considerable selective pressure to develop resistance mechanisms to metal ions, providing them with a competitive selective advantage. As a result, the effectiveness of different heavy metal resistance mechanisms would play a significant role in affecting the functional and structural characteristics of microbial communities in acidic environments. Another area of considerable interest is prokaryote metal resistance in the context of surface-attached/biofilm modes of growth. Many micro-organisms grow naturally within biofilms and this form of growth exhibits significant resistance to physio-chemical conditions compared to planktonic modes of growth (LaPlagia & Hartzell, 1997). It is possible that there is a link between acidophile biofilm formation and metal ion concentration, as well as higher metal resistance as compared to planktonic cells. Some examples of the degree of metal resistance in acidophilic micro-organisms are listed in Table 1
; for ease of comparison all concentrations have been converted to molarity (in the text, for those cases where a conversion has been carried out the original values are given in parentheses).
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Metal toxicity and general mechanisms of microbial resistance |
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Acidophile metal resistance mechanisms |
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Neutrophiles are resistant to arsenic via energy-dependent efflux encoded by the ars operon, containing the genes arsRBC. This constitutes an As(V) reductase (ArsC) that reduces As(V) to As(III) prior to efflux via a membrane potential driven pump (ArsB) controlled by a trans-acting repressor (ArsR). In some instances, an ATPase (ArsA) is attached to the ArsB membrane pump that confers a higher level of resistance to arsenic, and a second regulator (ArsD) is sometimes present that controls the upper level of ars expression (Fig. 2; reviewed by Xu et al., 1998
). Acidophilic arsenic resistance operons have been cloned from At. ferrooxidans, At. caldus and A. multivorum (Fig. 2
) and all three operons conferred As(III) resistance in E. coli (Butcher et al., 2000
; de Groot et al., 2001
; Suzuki et al., 1998
). The At. ferrooxidans ArsR protein has been found to be atypical in that it is not conserved in regions formerly shown to be important for As(III) binding and that the arsRC genes were induced irrespective of the form of arsenic added (Butcher & Rawlings, 2002
). The presence of the arsB gene (efflux pump) has been identified by Southern hybridization in various acidophilic micro-organisms; these include At. caldus, Acidithiobacillus thiooxidans (formerly Thiobacillus thiooxidans), At. ferrooxidans, Acidiphilium acidophilum (formerly Thiobacillus acidophilus), Thiomonas cuprina and Acidocella facilis (Dopson et al., 2001
). Neutrophiles have an inside negative membrane potential, whereas acidophiles have a reversed membrane potential (inside positive). Despite the physiological differences and the reversed membrane potential, the At. ferrooxidans ArsB was functional in E. coli (Butcher et al., 2000
). At. ferrooxidans appears to lack a gene homologous to E. coli arsA, but contains the arsRBC genes in an unusual configuration (Fig. 2
; Butcher et al., 2000
). By Southern hybridization two sets of arsenic resistance operons have been identified from At. caldus strains and it has been postulated that all At. caldus strains have a basic set of chromosomal arsenic resistance genes (putatively arsRBC; de Groot et al., 2001
). In addition, three At. caldus strains isolated from a South African biooxidation plant operating for the oxidation of arsenopyrite have been found to contain an additional transposon located set of ars genes (At. caldus #6; Fig. 2
). One of these genes is homologous to the E. coli arsA which when cloned into E. coli confers resistance to As(III) but not to As(V) (de Groot et al., 2001
), although the arsA has not been shown to be functional biochemically. By inference from PFGE of At. ferrooxidans strains Kondratyeva et al. (1995)
suggested that amplification of certain chromosomal fragments in resistant strains, resulting in increased copy number of putative chromosomal resistance genes, was responsible for arsenic resistance. However, no direct evidence that these fragments contained the resistance genes was presented in that study. Although the presence of an arsB-like gene has been identified by Southern hybridization in all of the micro-organisms tested (Dopson et al., 2001
), chromosomally encoded As(V) reduction to As(III) (ArsC activity) followed by energy-dependent efflux of As(III) has only been demonstrated experimentally in a single acidophile, At. caldus KU (Dopson et al., 2001
). At. caldus KU was shown to be resistant to higher concentrations of As(III) and As(V) than E. coli with MICs of 13 and 310 mM, respectively. Although At. caldus KU is resistant to As(V) and As(III), inclusion of 100 and 5 mM, respectively, reduced the growth rate (Hallberg, 1995
).
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In silico studies indicate the presence of an arsB homologue on a number of genomes from acidophilic micro-organisms (Fig. 3), including the archaeon Fp. acidarmanus. In Fp. acidarmanus, a single operon containing genes homologous to arsRB was found, as well as a separate gene encoding a protein similar to ArsA (Gihring et al., 2003
). Unusually, no genes homologous to arsC were identified in Fp. acidarmanus. The in silico data are supported by biochemical results whereby Fp. acidarmanus is resistant to >13·3 mM (>1000 p.p.m.) As(V) and As(III), although no As(V) to As(III) reduction is observed, suggesting that Fp. acidarmanus may have a novel arsenic resistance system. A similar arrangement of arsRB and a partial arsA gene is observed in a related archaeon, Thermoplasma acidophilum (Ruepp et al., 2000
).
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Copper
Copper is used in cytochrome-c oxidase and other related terminal oxygen acceptors and is therefore required in many organisms. Copper is released at AMD, ARD and bioleaching sites and At. ferrooxidans strains adapted to increased levels of Cu(II) have been found to be tolerant to 800 mM (Dew et al., 1999). Other acidophiles shown to be resistant to copper include Leptospirillum ferrooxidans, which shows growth in 5 mM Cu(II) (Johnson et al., 1992
), and those listed in Table 1
. Although At. ferrooxidans was shown to be the most resistant to Cu(II), virtually all of the acidophiles were found to be more resistant than E. coli. Das et al. (1997)
found that At. ferrooxidans copper resistance was inducible and that the resistant strain extracts Cu(II) more rapidly compared to an unadapted strain. Cu(II) inhibition of growth and Fe(II) oxidation has also been demonstrated in Sulfobacillus thermosulfidooxidans subsp. asporogenes via competitive inhibition of Fe(II) oxidation (Vartanyan et al., 1990
).
Two copper resistance systems have been characterized in neutrophiles. The first system consists of P-type ATPases in Gram-positive bacteria that efflux copper out of the cell and the second is the Pseudomonas copper sequestration system that binds copper in the periplasm or close to the outer membrane (reviewed by Nies, 1999). Most investigations of copper resistance in acidophilic micro-organisms have been restricted to At. ferrooxidans. A strain of At. ferrooxidans that took up 700 mg Cu(II) (g dry weight)-1 was adapted to growth in the presence of 600 mM Cu(II) and subsequently Cu(II) uptake decreased to 90 mg Cu(II) (g dry weight)-1 (Boyer et al., 1998
), suggesting that the Cu(II) was excluded from the cell possibly via an inducible efflux system. The copy number of plasmids in At. ferrooxidans decreased with exposure to Cu(II), and increased with subsequent growth in its absence (Pramila et al., 1996
). The change in copy number may simply be a stress response, as At. ferrooxidans plasmid fragments transformed into E. coli did not confer increased Cu(II) resistance (Chisholm et al., 1998
). This suggests that At. ferrooxidans Cu(II) resistance is chromosomal and not plasmid-based. At. ferrooxidans exposure to Cu(II) results in formation of a proteinase-K-sensitive cell-surface component, which upon loss results in decreased Cu(II) adsorption and loss of tolerance, suggesting that some cell-surface components are involved in At. ferrooxidans Cu(II) resistance (Das et al., 1998
). Differential protein expression induced by exposure to Cu(II) in At. ferrooxidans resulted in induction of three proteins (Novo et al., 2000
). Also, RNA arbitrarily primed PCR identified 17 genes induced by Cu(II) in At. ferrooxidans (Paulino et al., 2002
). However, on those occasions no genes with homologies to Cu(II) resistance genes were identified. No reports into archaeal Cu(II) resistance have been published, but Cu(II) resistance has been shown to be inducible in Fp. acidarmanus (unpublished results) and M. sedula is also tolerant to 16 mM Cu(II) (Huber et al., 1989
).
Zinc
Zinc occurs as the divalent cation Zn(II) and although it cannot undergo redox reactions under biological conditions, it is present in a number of enzymes. Zn(II) toxicity is based upon complexation with various cellular components, and in Su. thermosulfidooxidans it has been demonstrated to be due to competitive inhibition of Fe(II) oxidation (Vartanyan et al., 1990). The toxicity of Zn(II) to At. ferrooxidans depends on the growth substrate. One strain resistant to 153 mM (10 g l-1) Zn(II) whilst growing on Fe(II) is sensitive to 92 µM (0·6 mg l-1) when growing on thiosulfate (Trevors et al., 1985
). It was found that At. ferrooxidans strain TFZ was adapted to growth in 1·071 M (70 g l-1) Zn(II) (Kondratyeva et al., 1995
; Table 1
). Other acidophiles resistant to Zn(II) are listed in Table 1
.
The mechanism of zinc resistance in neutrophiles is efflux by P-type ATPases, cation-diffusion facilitator transporters or high-efficiency efflux proteins such as Czc (reviewed by Nies, 1999). Few investigations have gone towards resolving the genetics of acidophile zinc resistance, but from Table 1
it is clear that acidophiles are more resistant than neutrophiles. As was the case for At. ferrooxidans arsenic resistance, based on PFGE Kondratyeva et al. (1995)
suggested zinc resistance to be chromosomally encoded. Adaptation of the strain to increased levels of Zn(II) resulted in an increase in genome fragment size, suggesting increased copy numbers of the operon encoding the putative Zn(II) resistance genes. However, once again no direct evidence that these chromosomal fragments contained the zinc resistance genes was presented. Plasmids isolated from Acidocella strain GS19h have been transformed into sensitive strains of A. multivorum and E. coli. These events conferred increased Zn(II) resistance to the host organisms, suggesting that it is plasmid-based in strain GS19h (Ghosh et al., 1997
). Curing Acidocella strain GS19h of the plasmid resulted in loss of resistance and the MIC fell from 1 M to 5 mM (Ghosh et al., 2000
). Also, Acidiphilium symbioticum KM2 has been shown to harbour three plasmids which, when a mini-plasmid library was created and transformed into E. coli, conferred resistance to Zn(II) and Cd(II). This suggests that Zn(II) and Cd(II) resistance is plasmid-based in A. symbioticum KM2. The genes encoded by the recombinant KM2 plasmid had no sequence similarity to reported metal resistance genes. Therefore, it is likely that a new Zn(II) and Cd(II) resistance mechanism may be in operation (Mahapatra et al., 2002
).
Cadmium
A plethora of studies have demonstrated the toxicity of cadmium to micro-organisms, however, specific mechanisms have yet to be defined. In some micro-organisms, cadmium is taken up via the magnesium or manganese uptake systems (reviewed by Nies, 1999), but the mechanisms in acidophiles have not been elucidated. A possible commercial biotechnological application of acidophiles adapted to high levels of cadmium is for the solubilization of nickel and cadmium from batteries by At. ferrooxidans (Cerruti et al., 1998
). Cd(II) is toxic to micro-organisms through a variety of mechanisms, including binding to thiol groups, protein denaturation, and interaction with calcium and zinc metabolism. The toxicity of Cd(II) to At. ferrooxidans was found to be low in comparison to Hg(II), inhibiting Fe(II) oxidation by 2 % at 8·90 µM (10 mg l-1) whilst similar levels of Hg(II) caused 63 % reduction (De et al., 1997
). However, Cd(II) toxicity is greater than that of zinc and copper to At. ferrooxidans (see previous sections). Cd(II) uptake in At. ferrooxidans has also been reported, and a greater uptake was observed in tolerant strains (Baillet et al., 1997
). Other acidophiles resistant to Cd(II) include a number of Acidiphilum spp. and an Acidocella strain resistant to 700 mM Cd(II), whilst the maximum resistance in archaea was 10 mM in Sulfolobus acidocaldarius and S. solfataricus (Table 1
).
Cadmium ion efflux systems are present in a number of micro-organisms; for example, the Czc system and a P-type ATPase pump (CadA) in Gram-negative and Gram-positive bacteria, respectively (reviewed by Nies, 1999). Ghosh et al. (2000)
found that Acidocella strain GS19h contained three plasmids, one of which when transferred to A. multivorum and E. coli increased the MIC. Also, when GS19h was cured of the plasmids, the MIC dropped, confirming that the plasmids contained genes encoding Cd(II) resistance. The genes contained in these plasmids have not been characterized. As described for Zn(II), the A. symbioticum KM2 Cd(II) resistance mechanism has been cloned into E. coli, conferring Cd(II) resistance (Mahapatra et al., 2002
).
In the manner used to search for acidophile As(III) resistance genes, we used the in silico approach to detect genes involved in acidophile Cd(II) resistance. A number of cadmium resistance operons are present in acidophiles (unpublished data). This is indicated by the presence of gene sequences with homology to the P-type export ATPase gene cadA on many of the sequenced genomes from acidophilic micro-organisms (Fig. 4). The species with the highest homology to the cadA motif was At. ferrooxidans and then the Thermoplasma spp. These high similarities suggest that Cd(II) export may be a common resistance mechanism among acidophiles. However, some care must be exercised with interpretation of this Meta-MEME result, as motif matches may occur with other members of the P-type ATPase family.
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Mercury
Mercury is found in cinnabar (HgS) and its oxidation results in the production of Hg(II) and RISCs. M. sedula has an MIC of 5 µM (Huber et al., 1989) and certain strains of At. ferrooxidans and S. metallicus strain BC have been shown to be sensitive to mercury (Imai et al., 1975
; Mier et al., 1996
) via uncompetitive inhibition of Fe(II) oxidation. This suggests that Hg(II) binds to the Fe(II)-oxidizing enzyme (De et al., 1997
).
A mercury resistance system, analogous to the neutrophilic mechanism, based on an Hg(II)-reducing flavoprotein producing Hg(0) which volatilizes out of the cell, has been found in At. ferrooxidans strains (Booth & Williams, 1984; Olson et al., 1982
). Several different mercury resistance operons have been characterized from strains of At. ferrooxidans and are listed below. The mercury resistance operon from Thiobacillus T3.2 and At. ferrooxidans Tn5037 (Kalyaeva et al., 2001
; Velasco et al., 1999
) consists of: merR, which encodes a unique positive regulatory protein that twists the operator DNA to allow mRNA formation; merT, encoding a mercury uptake protein that may act in association with MerP [a periplasmic Hg(II)-binding protein]; and the final gene, merA, encoding the Hg(II)-reducing flavoprotein (reviewed by Osborn et al., 1997
). The mercury resistance operon from At. ferrooxidans E15 has also been cloned and differs from that of most other Gram-negative bacteria in that it does not start with a merR gene. Instead it consists of a merC gene [Hg(II) transporter into the cell] and a merA gene. In addition, a separate gene cluster occurs containing two functional merR genes, a merC and two non-functional merA genes. The presence of a Tn7 tnsA homologue next to the mer genes suggests that transposition may be responsible for the gene arrangement (Inoue et al., 1991
).
A second type of mercury resistance, not found in neutrophiles, has been identified in At. ferrooxidans SUG 2-2. This strain does not have a Hg(II)-reducing flavoprotein analogous to MerA, but nonetheless catalyses the reduction of Hg(II) to Hg(0) in the presence of Fe(II). The reduction is thought to be catalysed by the cytochrome-c oxidase (Sugio et al., 2001). There is interest in making use of this strain's Hg(II)-reducing capabilities and it has been studied for use in bioremediation of mercury-polluted acidic sites (Takeuchi et al., 2001
). The difference between mercury-sensitive and -resistant strains of At. ferrooxidans has been characterized. It is apparent that purified cytochrome-c oxidase from resistant strains is more tolerant to increased levels Hg(II), which, due to its importance in Fe(II)-oxidizing activity, likely contributes to the resistant strain's increased growth rate (Takeuchi et al., 1999
).
Silver
Silver is released during the oxidation of refractory sulfide ores, where it is trapped as fine particles within the mineral matrix. Silver has been shown to be toxic to M. sedula, L. ferrooxidans and Acidiphilium cryptum at concentrations of 900, 2 and 0·5 µM, respectively (Huber et al., 1989; Johnson et al., 1992
). The toxicity of Ag(I) to At. ferrooxidans growth and oxidation of Fe(II) has also been recorded (Imai et al., 1975
), and Guay et al. (1989)
found that Ag(I) was the most toxic of the metals and ion studied (silver, cadmium, cobalt, copper, zinc, uranyl and As(III)). Silver is toxic to At. ferrooxidans Fe(II) oxidation at a concentration of 0·93 µM (0·10 mg l-1) (De et al., 1996
and references therein), and at 927 µM (100 mg l-1) pyrite oxidation is severely reduced (Norris & Kelly, 1978
). In contrast, after previous silver exposure At. ferrooxidans has been observed to be tolerant to 1 mM (108 mg l-1) Ag(I) (Ehrlich, 1984
). The Ag(I) mode of inhibition of Fe(II) oxidation has been attributed to a mixed mechanism whereby Ag(I) replaces Fe(II) in the active site of the oxidizing enzyme (De et al., 1996
). Inhibition of S. metallicus strain BC (adapted strains were less sensitive) and Sulfolobus rivotincti by Ag(I) has also been reported (Gomez et al., 1999
; Mier et al., 1996
). Silver efflux has been demonstrated in neutrophiles, but the mechanism of Ag(I) resistance in acidophilic micro-organisms has not been elucidated. Bioaccumulation was observed in At. ferrooxidans and At. thiooxidans during biooxidation of sulfide ores (Pooley, 1982
), although it is not known if this conferred higher levels of resistance.
Ferric iron
Iron is the most biologically important metal and is therefore an essential requirement for growth. In most aerated environments, the bioavailability of iron is very low due to chemical oxidation of soluble Fe(II) to Fe(III) in the presence of oxygen and the low solubility of Fe(III) above pH 1·6. However, in acidic environments the solubility of Fe(III) is much greater, and in many cases can reach the g l-1 range. A tolerance to high levels of Fe(III) is required in sulfidic ore leaching environments and one ecological example of this is the competition between At. ferrooxidans and Leptospirillum-like micro-organisms in biooxidation tanks. Both these acidophiles can grow via the oxidation of Fe(II), causing accumulation of Fe(III); however, selection of the Leptospirillum-like micro-organisms can occur and is thought to be due to their greater tolerance to Fe(III) (Rawlings et al., 1999). Another example is the greater tolerance of Sulfobacillus acidophilus than Acidimicrobium ferrooxidans to Fe(III), resulting in dominance of the former in mixed-culture Fe(II)-oxidizing environments (Clark & Norris, 1996
). Fe(III) toxicity has been detected in batch and chemostat studies of Fe(II) oxidation by At. ferrooxidans and chemostat studies of L. ferrooxidans. These show that Fe(III) competitively inhibits Fe(II) oxidation (Boon et al., 1999a
, b
; van Scherpenzel et al., 1988
). The presence of 358 mM (20 g l-1) Fe(III) has been shown to inhibit Fe(II) oxidation and cause cell lysis in At. ferrooxidans (Shrihari & Gandhi, 1990
) and its growth is inhibited by
36 mM (2 g l-1) Fe(III) (Curutchet et al., 1992
). In another study of suspended cells of At. ferrooxidans, Fe(III) inhibition of Fe(II) oxidation occurred above 107 mM (6 g l-1). However, when growing as a biofilm the cells were unaffected by 251 mM (14 g l-1) Fe(III) (Karamanev & Nikolov, 1988
). Also, L. ferrooxidans and A. cryptum have been shown to be resistant to 500 and 300 mM Fe(III), respectively (Johnson et al., 1992
). While acidophiles often tolerate elevated concentrations of iron, no information is available regarding iron resistance mechanisms.
Other metals
Many other metals are found in acidic environments, including uranium, molybdenum and chromium. A number of different acidophiles have been isolated from locations containing these metals, and the following species have been isolated from environments containing uranium: At. ferrooxidans, L. ferrooxidans, At. thiooxidans, Tm. cuprina, cells resembling Sulfolobus/Acidianus spp., Acidiphilium spp. and other heterotrophic iron-oxidizing acidophiles (reviewed by Tuovinen & Bhatti, 1999). In many cases, toxicity studies have been carried out with these metals on acidophiles. Examples are U(VI) toxicity via inhibition of Fe(II) oxidation and CO2 fixation (Dispiroto et al., 1983
) and At. ferrooxidans growth inhibition by >15 mM Cr(III) (Wong et al., 1982
).
A number of mechanisms of resistance to these metals have been identified in acidophiles, including the mode of toxicity to and putative Mo(V) resistance mechanism in At. ferrooxidans strain Funis 2-1 (Yong et al., 1997). Mo(VI) is chemically reduced by Fe(II), and the Mo(V) formed binds to the plasma membrane, probably to the cytochrome-c oxidase (lowering its activity), inhibiting Fe(II) oxidation and consequently growth. Resistance is based on a combination of a cytochrome-c oxidase that is tolerant to higher concentrations of Mo(V) and on an Mo(V)-oxidizing activity sixfold greater than that detected in the sensitive At. ferrooxidans strain AP19-3. A chromium resistance mechanism has been identified in At. ferrooxidans and Acidiphilium rubrum whereby they take up and subsequently precipitate a Cr(VI)-rich compound on the surface of the cell, increasing chromium tolerance (Baillet et al., 1998
; Itoh et al., 1998
).
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Future directions |
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At this time, the genomes of the following six acidophiles have been sequenced: At. ferrooxidans, Fp. acidarmanus, Tp. acidophilum, Tp. volcanium, S. solfataricus and S. tokodaii. This opens the door to a whole new area of research that can be carried out on acidophiles. As can be seen from the unpublished results presented in this review, it is now possible to putatively identify genes associated with metal resistance by in silico studies of the genomes, by searching for analogues of known metal resistance genes. A further strategy that may be employed to elucidate metal resistance systems in acidophilic micro-organisms is experimentally based proteome and transcriptome investigations. These investigations could examine differential expression for detection of metal resistance genes expressed as a response to the addition of metals. Such techniques are increasingly relevant as microbial genome sequence data accumulate.
Presently uncultured acidophiles are detected as important constituents of bioleaching microbial communities (Bond et al., 2000b). Investigation of the metal resistance of these uncultured microbes remains a major challenge. In comparison to other natural microbial communities, acidic environments have a reduced biodiversity as indicated by molecular phylotypes (Bond et al., 2000b
). Such microbial systems perhaps lend themselves to investigation by a mixed-community genome analysis. Another approach is to screen for gene function directly from environmental DNA. This approach has been termed metagenomics and has enormous potential (Rondon et al., 2000
).
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Concluding remarks |
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