The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.

The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma.

Extreme zinc tolerance in acidophilic microorganisms from the bacterial and archaeal domains.

Zinc can occur in extremely high concentrations in acidic, heavy metal polluted environments inhabited by acidophilic prokaryotes. Although these organisms are able to thrive in such severely contaminated ecosystems their resistance mechanisms have not been well studied. Bioinformatic analysis of a range of acidophilic bacterial and archaeal genomes identified homologues of several known zinc homeostasis systems. These included primary and secondary transporters, such as the primary heavy metal exporter ZntA and Nramp super-family secondary importer MntH. Three acidophilic model microorganisms, the archaeon 'Ferroplasma acidarmanus', the Gram negative bacterium Acidithiobacillus caldus, and the Gram positive bacterium Acidimicrobium ferrooxidans, were selected for detailed analyses. Zinc speciation modeling of the growth media demonstrated that a large fraction of the free metal ion is complexed, potentially affecting its toxicity. Indeed, many of the putative zinc homeostasis genes were constitutively expressed and with the exception of 'F. acidarmanus' ZntA, they were not up-regulated in the presence of excess zinc. Proteomic analysis revealed that zinc played a role in oxidative stress in At. caldus and Am. ferrooxidans. Furthermore, 'F. acidarmanus' kept a constant level of intracellular zinc over all conditions tested whereas the intracellular levels increased with increasing zinc exposure in the remaining organisms.

An archaeal iron-oxidizing extreme acidophile important in acid mine drainage.

A new species of Archaea grows at pH approximately 0.5 and approximately 40 degrees C in slime streamers and attached to pyrite surfaces at a sulfide ore body, Iron Mountain, California. This iron-oxidizing Archaeon is capable of growth at pH 0. This species represents a dominant prokaryote in the environment studied (slimes and sediments) and constituted up to 85% of the microbial community when solution concentrations were high (conductivity of 100 to 160 millisiemens per centimeter). The presence of this and other closely related Thermoplasmales suggests that these acidophiles are important contributors to acid mine drainage and may substantially impact iron and sulfur cycles.

Application of real-time PCR to monitor population dynamics of defined mixed cultures of moderate thermophiles involved in bioleaching of chalcopyrite.

To compare oxidative dissolution rates of chalcopyrite by different consortia of moderately thermophilic acidophiles, various defined mixed cultures of three bacteria Acidithiobacillus caldus s2, Leptospirillum ferriphilum YSK, and Sulfobacillus sp. LN and one archaeon Ferroplasma thermophilum L1 were studied in batch shake flask cultures incubated at 45 degrees C. Chalcopyrite dissolution was determined by measuring variations of soluble copper, ferric iron, and pH. Microbial population dynamics involved in bioleaching process were monitored using real-time quantitative polymerase chain reaction (PCR) technology. The complex consortia containing both chemoautotrophic (L. ferriphilum and At. caldus) and chemomixotrophic (Sulfobacillus LN and F. thermophilum) moderate thermophiles were found to be the most efficient in all of those tested. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, involving transformations of iron and sulfur and transfer of organic compound, were considered to play a critical role in promoting chalcopyrite dissolution. The real-time PCR assay was reliable to analyze population dynamics of moderate thermophiles in bioleaching systems, and the analysis results were consistent with physiological characteristics of these strains.

Characterisation of a stable laboratory co-culture of acidophilic nanoorganisms.

This study describes the laboratory cultivation of ARMAN (Archaeal Richmond Mine Acidophilic Nanoorganisms). After 2.5 years of successive transfers in an anoxic medium containing ferric sulfate as an electron acceptor, a consortium was attained that is comprised of two members of the order Thermoplasmatales, a member of a proposed ARMAN group, as well as a fungus. The 16S rRNA identity of one archaeon is only 91.6% compared to the most closely related isolate Thermogymnomonas acidicola. Hence, this organism is the first member of a new genus. The enrichment culture is dominated by this microorganism and the ARMAN. The third archaeon in the community seems to be present in minor quantities and has a 100% 16S rRNA identity to the recently isolated Cuniculiplasma divulgatum. The enriched ARMAN species is most probably incapable of sugar metabolism because the key genes for sugar catabolism and anabolism could not be identified in the metagenome. Metatranscriptomic analysis suggests that the TCA cycle funneled with amino acids is the main metabolic pathway used by the archaea of the community. Microscopic analysis revealed that growth of the ARMAN is supported by the formation of cell aggregates. These might enable feeding of the ARMAN by or on other community members.

Adhesion of Ferroplasma acidiphilum onto pyrite calculated from the extended DLVO theory using the van Oss-Good-Chaudhury approach.

The adhesion behavior of Ferroplasma acidiphilum archaeon to pyrite mineral was investigated experimentally and theoretically. F. acidiphilum showed high affinity to adhere to pyrite surface at acidic regions, however low affinity was observed at neutral and alkaline regions. The microbe-mineral adhesion was assessed by the extended DLVO theory. Hamaker constants, electron donors, electron acceptors and surface charges for the microbe and the mineral were experimentally determined. The extended DLVO theory was used to explain the adhesion results. Significant changes to the pyrite surface properties after being treated with the microbial cells were observed. Pyrite lost its hydrophobic nature and became hydrophilic, the contact angle of untreated pyrite was 61 degrees and this decreased to 36 degrees after the treatment. As a consequence, the flotation experiment results showed that F. acidiphilum strain could act as a good depressant for pyrite in xanthat flotation; where in absence of F. acidiphilum cells, over 95% of pyrite can be recovered as a float. However, when the mineral was pretreated with F. acidiphilum cells, less than 20% can be recovered as a float.

Molybdate treatment and sulfate starvation decrease ATP and DNA levels in Ferroplasma acidarmanu.

Sulfate is a primary source of sulfur for most microbes and in some prokaryotes it is used an electron acceptor. The acidophile Ferroplasma acidarmanus (strain fer1) requires a minimum of 150 mM of a sulfate-containing salt for growth. Sulfate is assimilated by F. acidarmanus into proteins and reduced to form the volatile organic sulfur compounds methanethiol and dimethyldisulfide. In the absence of sulfate, cell death occurs by an unknown mechanism. In this study, cell viability and genomic DNA and ATP contents of F. acidarmanus were monitored in response to the absence of sulfate or the presence of sulfate and the sulfate analog molybdate (MoO(4) (2-)). Cellular DNA and ATP contents were monitored as markers of cell viability. The absence of sulfate led to a decrease in viable cell numbers of greater than 7 log(10 )within 5 days, a > 99% reduction in genomic DNA within 3 days, and a > 60% decrease in ATP within 6 h. Likewise, cells incubated with MoO(4) (2-) lost viability (decreased by > 2 log(10) in 5 days), extractable genomic DNA (reduction of > 60% in 2 days), and ATP (reduction of > 70 % in 2 hours). These results demonstrate that sulfate deprivation or the presence of molybdate have similar impacts on cell viability and essential biomolecules. Sulfate was coupled to cellular ATP content and maintenance of DNA integrity in F. acidarmanus, a finding that may be applicable to other acidophiles that are typically found in sulfate-rich biotopes.

Prokaryotes that grow optimally in acid have purine-poor codons in long open reading frames.

In nucleic acids the N-glycosyl bonds between purines and their ribose sugar moities are broken under acid conditions. If one strand of a duplex DNA segment were more vulnerable to mutation than the other, then the archaeon Picrophilus torridus, with an optimum growth pH near zero, could have adapted by decreasing the purine content of that strand. Yet, P. torridus has an optimum growth temperature near 60 degrees C, and thermophiles prefer purine-rich codons. We found that, as in other thermophiles, high growth temperature correlates with the use of purine-rich codons. The extra purines are often in third, non-amino acid determining, codon positions. However, as in other acidophiles, as open reading frame lengths increase, there is increased use of purine-poor codons, particularly those without purines in second, amino acid-determining, codon positions. Thus, P. torridus can be seen as adapting (a) to temperature by increasing its purines in all open reading frames without greatly impacting protein amino acid compositions, and (b) to pH by decreasing purines in longer open reading frames, thereby potentially impacting protein amino acid compositions. It is proposed that longer open reading frames, being larger mutational targets, have become less vulnerable to depurination by virtue of pyrimidine for purine substitutions.

Production of methanethiol and volatile sulfur compounds by the archaeon "Ferroplasma acidarmanus".

Acidophiles are typically isolated from sulfate-rich ecological niches yet the role of sulfur metabolism in their growth and survival is poorly defined. Studies of heterotrophically grown "Ferroplasma acidarmanus" showed that its growth requires a minimum of 100 mM of a sulfate-containing salt. Headspace gas analyses by GC/MS determined that the volatile sulfur compound emitted by active "F. acidarmanus" cultures is methanethiol. In "F. acidarmanus" cultures grown either heterotrophically or chemolithotrophically, methanethiol was produced constitutively. Radiotracer studies with (35)S-labeled methionine, cysteine, and sulfate showed that all three were used in methanethiol production. Additionally, (3)H-labeled methionine was incorporated into methanethiol and was probably used as a methyl-group donor. Methanethiol production in whole cell lysates supplied with SO (3) (2-) indicated that NADPH-dependant sulfite reductase and methyltransferase activities were present. Cell lysates also contained enzymatic activity for methionine-gamma-lyase that cleaved the side chain of either methionine to form methanethiol or cysteine to produce H(2)S. Since methanethiol was detected from the degradation of cysteine, it is likely that sulfide was methylated by a thiol methyltransferase. Collectively, these data demonstrate that "F. acidarmanus" produces methanethiol through the metabolism of methionine, cysteine, or sulfate. This is the first report of a methanethiol-producing acidophile, thus identifying a new contributor to the global sulfur cycle.

Thermogymnomonas acidicola gen. nov., sp. nov., a novel thermoacidophilic, cell wall-less archaeon in the order Thermoplasmatales, isolated from a solfataric soil in Hakone, Japan.

A novel thermoacidophilic, cell wall-less archaeon, strain IC-189T, was isolated from a solfataric field in Ohwaku-dani, Hakone, Japan. The cells were irregular cocci, sometimes lobed, club-shaped or catenated, and were highly variable in size, ranging from 0.8 to 8.0 microm in diameter. The strain grew at temperatures in the range 38-68 degrees C (optimally at 60 degrees C) and at pH 1.8-4.0 (optimally at around pH 3.0). Strain IC-189T was obligately aerobic and heterotrophic, requiring yeast extract for growth. Yeast extract, glucose and mannose served as carbon and energy sources. The polar lipids consisted mainly of cyclic or acyclic glycerol-bisdiphytanyl-glycerol tetraethers, and the predominant quinone was a menaquinone with seven isoprenoid units (MK-7). The G+C content of total DNA was 56.1 mol%. 16S rRNA gene sequence analysis revealed that strain IC-189T was a member of the order Thermoplasmatales, but diverged from the hitherto known species of the genera Thermoplasma, Picrophilus and Ferroplasma (86.2-91.0% sequence similarity). These phenotypic and phylogenetic properties clearly support a separate taxonomic status for this strain. Therefore, strain IC-189T represents a novel genus (order Thermoplasmatales) and species, for which the name Thermogymnomonas acidicola gen. nov., sp. nov. is proposed, with type strain IC-189T (=JCM 13583T=DSM 18835T).

Ferroplasma and relatives, recently discovered cell wall-lacking archaea making a living in extremely acid, heavy metal-rich environments.

For several decades, the bacterium Acidithiobacillus (previously Thiobacillus) has been considered to be the principal acidophilic sulfur- and iron-oxidizing microbe inhabiting acidic environments rich in ores of iron and other heavy metals, responsible for the metal solubilization and leaching from such ores, and has become the paradigm of such microbes. However, during the last few years, new studies of a number of acidic environments, particularly mining waste waters, acidic pools, etc., in diverse geographical locations have revealed the presence of new cell wall-lacking archaea related to the recently described, acidophilic, ferrous-iron oxidizing Ferroplasma acidiphilum. These mesophilic and moderately thermophilic microbes, representing the family Ferroplasmaceae, were numerically significant members of the microbial consortia of the habitats studied, are able to mobilize metals from sulfide ores, e.g. pyrite, arsenopyrite and copper-containing sulfides, and are more acid-resistant than iron and sulfur oxidizing bacteria exhibiting similar eco-physiological properties. Ferroplasma cell membranes contain novel caldarchaetidylglycerol tetraether lipids, which have extremely low proton permeabilities, as a result of the bulky isoprenoid core, and which are probably a major contributor to the extreme acid tolerance of these cell wall-less microbes. Surprisingly, several intracellular enzymes, including an ATP-dependent DNA ligase have pH optima close to that of the external environment rather than of the cytoplasm. Ferroplasma spp. are probably the major players in the biogeochemical cycling of sulfur and sulfide metals in highly acidic environments, and may have considerable potential for biotechnological applications such as biomining and biocatalysis under extreme conditions.

Analysis of differential protein expression during growth states of Ferroplasma strains and insights into electron transport for iron oxidation.

To investigate the metabolic biochemistry of iron-oxidizing extreme acidophiles, a proteomic analysis of chemomixotrophic and chemo-organotrophic growth, as well as protein expression in the absence of organic carbon, was carried out in Ferroplasma species. Electron transport chain inhibitor studies, spectrophotometric analysis and proteomic results suggest that oxidation of ferrous iron may be mediated by the blue copper-haem protein sulfocyanin and the derived electron passes to a cbb3 terminal electron acceptor. Despite previous suggestions of a putative carbon dioxide fixation pathway, no up-regulation of proteins typically associated with carbon dioxide fixation was evident during incubation in the absence of organic carbon. Although a lack of known carbon dioxide fixation proteins does not constitute proof, the results suggest that these strains are not autotrophic. Proteins putatively involved in central metabolic pathways, a probable sugar permease and flavoproteins were up-regulated during chemo-organotrophic growth in comparison to the protein complement during chemomixotrophic growth. These results reflect a higher energy demand to be derived from the organic carbon during chemo-organotrophic growth. Proteins with suggested function as central metabolic enzymes were expressed at higher levels during chemomixotrophic growth by Ferroplasma acidiphilum Y(T) compared to 'Ferroplasma acidarmanus' Fer1. This study addresses some of the biochemical and bioenergetic questions fundamental for survival of these organisms in extreme acid-leaching environments.

Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum, Picrophilus torridus and Picrophilus oshimae.

The thermoacidophilic Archaea Thermoplasma acidophilum (optimal growth at 60 degrees C and pH 1-2), Picrophilus torridus and Picrophilus oshimae (optimal growth at 60 degrees C and pH 0.7) were able to utilize starch as sole carbon source. During growth these microorganisms secreted heat and acid-stable glucoamylases into the culture fluid. Applying SDS gel electrophoresis activity bands were detected with appearent molecular mass (Mw) of 141.0, 95.0 kDa for T. acidophilum, 133.0, 90.0 kDa for P. torridus and 140.0, 85.0 kDa for P. oshimae. The purified enzymes were incubated with various polymeric substrates such as starch, pullulan, panose and isomaltose. The product pattern, analyzed by HPLC, showed that in all cases glucose was formed as the sole product of hydrolysis. The purified glucoamylases were optimally active at pH 2.0 and 90 degrees C and have an isoelectric points (pI) between 4.5 and 4.8. Enzymatic activity was detected even at pH 1.0 and 100 degrees C. The glucoamylases were thermostable at elevated temperature with a half-life of 24 h at 90 degrees C for both P. torridus and T acidophilum, and 20 h at 90 degrees C for P oshimae. The enzyme system of T acidophilum has a lower Km value for soluble starch (1.06 mg/ml) than the enzymes from P. oshimae and P. torridus (4.35 mg/ml and 2.5 mg/ml), respectively. Enzyme activity was not affected by Na+, Mg++, Ca++, Ni++, Zn++, Fe++, EDTA and DTT.

Ferroplasma acidiphilum gen. nov., sp. nov., an acidophilic, autotrophic, ferrous-iron-oxidizing, cell-wall-lacking, mesophilic member of the Ferroplasmaceae fam. nov., comprising a distinct lineage of the Archaea.

An isolate of an acidophilic archaeon, strain YT, was obtained from a bioleaching pilot plant. The organism oxidizes ferrous iron as the sole energy source and fixes inorganic carbon as the sole carbon source. The optimal pH for growth is 1.7, although growth is observed in the range pH 1.3 to 2.2. The cells are pleomorphic and without a cell wall. 16S rRNA gene sequence analysis showed this strain to cluster phylogenetically within the order 'Thermoplasmales' sensu Woese, although with only 89.9 and 87.2% sequence identity, respectively, to its closest relatives, Picrophilus oshimae and Thermoplasma acidophilum. Other principal differences from described species of the 'Thermoplasmales' are autotrophy (strain YT is obligately autotrophic), the absence of lipid components typical of the ' Thermoplasmales' (no detectable tetraethers) and a lower temperature range for growth (growth of strain YT occurs between 15 and 45 degrees C). None of the sugars, amino acids, organic acids or other organic compounds tested was utilized as a carbon source. On the basis of the information described above, the name Ferroplasma acidiphilum gen. nov., sp. nov. is proposed for strain YT within a new family, the Ferroplasmaceae fam. nov. Strain YT is the type and only strain of F. acidiphilum. This is the first report of an autotrophic, ferrous-iron-oxidizing, cell-wall-lacking archaeon.

Arsenic resistance in the archaeon "Ferroplasma acidarmanus": new insights into the structure and evolution of the ars genes.

Arsenic resistance in the acidophilic iron-oxidizing archaeon " Ferroplasma acidarmanus" was investigated. F. acidarmanus is native to arsenic-rich environments, and culturing experiments confirm a high level of resistance to both arsenite and arsenate. Analyses of the complete genome revealed protein-encoding regions related to known arsenic-resistance genes. Genes encoding for ArsR (arsenite-sensitive regulator) and ArsB (arsenite-efflux pump) homologues were found located on a single operon. A gene encoding for an ArsA relative (anion-translocating ATPase) located apart from the arsRB operon was also identified. Arsenate-resistance genes encoding for proteins homologous to the arsenate reductase ArsC and the phosphate-specific transporter Pst were not found, indicating that additional unknown arsenic-resistance genes exist for arsenate tolerance. Phylogenetic analyses of ArsA-related proteins suggest separate evolutionary lines for these proteins and offer new insights into the formation of the arsA gene. The ArsB-homologous protein of F. acidarmanus had a high degree of similarity to known ArsB proteins. An evolutionary analysis of ArsB homologues across a number of species indicated a clear relationship in close agreement with 16S rRNA evolutionary lines. These results support a hypothesis of arsenic resistance developing early in the evolution of life.