Thermoplasmatales and sulfur-oxidizing bacteria dominate the microbial community at the surface water of a CO2-rich hydrothermal spring located in Tenorio Volcano National Park, Costa Rica.

Here we report the chemical and microbial characterization of the surface water of a CO2-rich hydrothermal vent known in Costa Rica as Borbollones, located at Tenorio Volcano National Park. The Borbollones showed a temperature surrounding 60 °C, a pH of 2.4 and the gas released has a composition of ~ 97% CO2, ~ 0.07% H2S, ~ 2.3% N2 and ~ 0.12% CH4. Other chemical species such as sulfate and iron were found at high levels with respect to typical fresh water bodies. Analysis by 16S rRNA gene metabarcoding revealed that in Borbollones predominates an archaeon from the order Thermoplasmatales and one bacterium from the genus Sulfurimonas. Other sulfur- (genera Thiomonas, Acidithiobacillus, Sulfuriferula, and Sulfuricurvum) and iron-oxidizing bacteria (genera Sideroxydans, Gallionella, and Ferrovum) were identified. Our results show that CO2-influenced surface water of Borbollones contains microorganisms that are usually found in acid rock drainage environments or sulfur-rich hydrothermal vents. To our knowledge, this is the first microbiological characterization of a CO2-dominated hydrothermal spring from Central America and expands our understanding of those extreme ecosystems.

Cuniculiplasmataceae, their ecogenomic and metabolic patterns, and interactions with 'ARMAN'.

Recently, the order Thermoplasmatales was expanded through the cultivation and description of species Cuniculiplasma divulgatum and corresponding family Cuniculiplasmataceae. Initially isolated from acidic streamers, signatures of these archaea were ubiquitously found in various low-pH settings. Eight genomes with various levels of completeness are currently available, all of which exhibit very high sequence identities and genomic conservation. Co-existence of Cuniculiplasmataceae with archaeal Richmond Mine acidophilic nanoorganisms ('ARMAN')-related archaea representing an intriguing group within the "microbial dark matter" suggests their common fundamental environmental strategy and metabolic networking. The specific case of "Candidatus Mancarchaeum acidiphilum" Mia14 phylogenetically affiliated with "Ca. Micrarchaeota" from the superphylum "Ca. Diapherotrites" along with the presence of other representatives of 'DPANN' with significantly reduced genomes points at a high probability of close interactions between the latter and various Thermoplasmatales abundant in situ. This review critically assesses our knowledge on specific functional role and potential of the members of Cuniculiplasmataceae abundant in acidophilic microbiomes through the analysis of distribution, physiological and genomic patterns, and their interactions with 'ARMAN'-related archaea.

The novel extremely acidophilic, cell-wall-deficient archaeon Cuniculiplasma divulgatum gen. nov., sp. nov. represents a new family, Cuniculiplasmataceae fam. nov., of the order Thermoplasmatales.

Two novel cell-wall-less, acidophilic, mesophilic, organotrophic and facultatively anaerobic archaeal strains were isolated from acidic streamers formed on the surfaces of copper-ore-containing sulfidic deposits in south-west Spain and North Wales, UK. Cells of the strains varied from 0.1 to 2 µm in size and were pleomorphic, with a tendency to form filamentous structures. The optimal pH and temperature for growth for both strains were 1.0-1.2 and 37-40 °C, with the optimal substrates for growth being beef extract (3 g l- 1) for strain S5T and beef extract with tryptone (3 and 1 g l- 1, respectively) for strain PM4. The lipid composition was dominated by intact polar lipids consisting of a glycerol dibiphytanyl glycerol tetraether (GDGT) core attached to predominantly glycosidic polar headgroups. In addition, free GDGT and small relative amounts of intact and core diether lipids were present. Strains S5T and PM4 possessed mainly menaquinones with minor fractions of thermoplasmaquinones. The DNA G+C content was 37.3 mol% in strain S5T and 37.16 mol% for strain PM4. A similarity matrix of 16S rRNA gene sequences (identical for both strains) showed their affiliation to the order Thermoplasmatales, with 73.9-86.3 % identity with sequences from members of the order with validly published names. The average nucleotide identity between genomes of the strains determined in silico was 98.75 %, suggesting, together with the 16S rRNA gene-based phylogenetic analysis, that the strains belong to the same species. A novel family, Cuniculiplasmataceae fam. nov., genus Cuniculiplasma gen. nov. and species Cuniculiplasma divulgatum sp. nov. are proposed based on the phylogenetic, chemotaxonomic analyses and physiological properties of the two isolates, S5T and PM4 ( = JCM 30641 = VKM B-2940). The type strain of Cuniculiplasma divulgatum is S5T ( = JCM 30642T = VKM B-2941T).

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.

Environmental, biogeographic, and biochemical patterns of archaea of the family Ferroplasmaceae.

About 10 years ago, a new family of cell wall-deficient, iron-oxidizing archaea, Ferroplasmaceae, within the large archaeal phylum Euryarchaeota, was described. In this minireview, I summarize the research progress achieved since then and report on the current status of taxonomy, biogeography, physiological diversity, biochemistry, and other research areas involving this exciting group of acidophilic archaea.

Uncultured archaea dominate in the thermal groundwater of Uzon Caldera, Kamchatka.

The thermoacidophilic microbial community inhabiting the groundwater with pH 4.0 and temperature 50°C at the East Thermal Field of Uzon Caldera, Kamchatka, was examined using pyrosequencing of the V3 region of the 16S rRNA gene. Bacteria comprise about 30% of microorganisms and are represented primarily by aerobic lithoautotrophs using the energy sources of volcanic origin--thermoacidophilic methanotrophs of the phylum Verrucomicrobia and Acidithiobacillus spp. oxidising metals and reduced sulfur compounds. More than 70% of microbial population in this habitat were represented by archaea, in majority affiliated with "uncultured" lineages. The most numerous group (39% of all archaea) represented a novel division in the phylum Euryarchaeota related to the order Thermoplasmatales. Another abundant group (33% of all archaea) was related to MCG1 lineage of the phylum Crenarchaeota, originally detected in the Yellowstone hot spring as the environmental clone pJP89. The organisms belonging to these two groups are widely spread in hydrothermal environments worldwide. These data indicate an important environmental role of these two archaeal groups and should stimulate the investigation of their metabolism by cultivation or metagenomic approaches.

Pantothenate kinase from the thermoacidophilic archaeon Picrophilus torridus.

Pantothenate kinase (CoaA) catalyzes the first step of the coenzyme A (CoA) biosynthetic pathway and controls the intracellular concentrations of CoA through feedback inhibition in bacteria. An alternative enzyme found in archaea, pantoate kinase, is missing in the order Thermoplasmatales. The PTO0232 gene from Picrophilus torridus, a thermoacidophilic euryarchaeon, is shown to be a distant homologue of the prokaryotic type I CoaA. The cloned gene clearly complements the poor growth of the temperature-sensitive Escherichia coli CoaA mutant strain ts9, and the recombinant protein expressed in E. coli cells transfers phosphate to pantothenate at pH 5 and 55 degrees C. In contrast to E. coli CoaA, the P. torridus enzyme is refractory to feedback regulation by CoA, indicating that in P. torridus cells the CoA levels are not regulated by the CoaA step. These data suggest the existence of two subtypes within the class of prokaryotic type I CoaAs.

Acidiplasma aeolicum gen. nov., sp. nov., a euryarchaeon of the family Ferroplasmaceae isolated from a hydrothermal pool, and transfer of Ferroplasma cupricumulans to Acidiplasma cupricumulans comb. nov.

A novel acidophilic, cell-wall-less archaeon, strain V(T), was isolated from a hydrothermal pool on Vulcano Island, Italy. The morphology of cells was observed to vary from pleomorphic to coccoid. The temperature range for growth of strain V(T) was 15-65 degrees C with an optimum at 45 degrees C. The pH for growth ranged from pH 0 to 4 with an optimal at pH 1.4-1.6. Strain V(T) was able to grow aerobically and anaerobically, oxidizing ferrous iron and reducing ferric iron, respectively. The isolate grew chemo-organotrophically with yeast extract and yeast extract with glucose as the sources of energy and carbon. The molar G+C content in the DNA was 36 mol%. 16S rRNA gene sequence analysis demonstrated that strain V(T) was a member of the family Ferroplasmaceae, order Thermoplasmatales, phylum Euryarchaeota, showing sequence identities of 100 % with Ferroplasma cupricumulans BH2(T), 95.4 % with Ferroplasma acidiphilum Y(T), 94 % with Picrophilus torridus DSM 9790(T) and 92 % with Picrophilus oshimae DSM 9789(T). 16S rRNA gene sequence-based phylogenetic analysis showed that strain V(T) formed a monophyletic cluster together with F. cupricumulans BH2(T) and all other thermophilic isolates with available 16S rRNA gene sequences, whereas F. acidiphilum Y(T) formed another cluster with mesophilic isolates within the family Ferroplasmaceae. DNA-DNA hybridization values between strain V(T) and F. cupricumulans BH2(T) were well below 70 %, indicating that the two strains belong to separate species. Principal membrane lipids of strain V(T) were dibiphytanyl-based tetraether lipids containing pentacyclic rings. The polar lipids were dominated by a single phosphoglycolipid derivative based on a galactosyl dibiphytanyl phosphoglycerol tetraether, together with smaller amounts of monoglycosyl and diglycosyl dibiphytanyl ether lipids and the corresponding phosphoglycerol derivatives. The major respiratory quinones present were naphthoquinone derivatives. Given the notable physiological and chemical differences as well as the distinct phylogenetic placement of the new isolate relative to the type species of the genus Ferroplasma, we propose strain V(T) as a member of a new genus and species, Acidiplasma aeolicum gen. nov., sp. nov. The type strain of Acidiplasma aeolicum is strain V(T) (=DSM 18409(T) =JCM 14615(T)). In addition, we propose to transfer Ferroplasma cupricumulans Hawkes et al. 2008 to the genus Acidiplasma as Acidiplasma cupricumulans comb. nov. (type strain BH2(T) =DSM 16551(T) =JCM 13668(T)).

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).

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.

Genetic exchange across a species boundary in the archaeal genus ferroplasma.

Speciation as the result of barriers to genetic exchange is the foundation for the general biological species concept. However, the relevance of genetic exchange for defining microbial species is uncertain. In fact, the extent to which microbial populations comprise discrete clusters of evolutionarily related organisms is generally unclear. Metagenomic data from an acidophilic microbial community enabled a genomewide, comprehensive investigation of variation in individuals from two coexisting natural archaeal populations. Individuals are clustered into species-like groups in which cohesion appears to be maintained by homologous recombination. We quantified the dependence of recombination frequency on sequence similarity genomewide and found a decline in recombination with increasing evolutionary distance. Both inter- and intralineage recombination frequencies have a log-linear dependence on sequence divergence. In the declining phase of interspecies genetic exchange, recombination events cluster near the origin of replication and are localized by tRNAs and short regions of unusually high sequence similarity. The breakdown of genetic exchange with increasing sequence divergence could contribute to, or explain, the establishment and preservation of the observed population clusters in a manner consistent with the biological species concept.

Molecular insight into extreme copper resistance in the extremophilic archaeon 'Ferroplasma acidarmanus' Fer1.

'Ferroplasma acidarmanus' strain Fer1 is an extremely acidophilic archaeon involved in the genesis of acid mine drainage, and was isolated from copper-contaminated mine solutions at Iron Mountain, CA, USA. Here, the initial proteomic and molecular investigation of Cu(2+) resistance in this archaeon is presented. Analysis of Cu(2+) toxicity via batch growth experiments and inhibition of oxygen uptake in the presence of ferrous iron demonstrated that Fer1 can grow and respire in the presence of 20 g Cu(2+) l(-1). The Fer1 copper resistance (cop) loci [originally detected by Ettema, T. J. G., Huynen, M. A., de Vos, W. M. & van der Oost, J. Trends Biochem Sci 28, 170-173 (2003)] include genes encoding a putative transcriptional regulator (copY), a putative metal-binding chaperone (copZ) and a putative copper-transporting P-type ATPase (copB). Transcription analyses demonstrated that copZ and copB are co-transcribed, and transcript levels were increased significantly in response to exposure to high levels of Cu(2+), suggesting that the transport system is operating for copper efflux. Proteomic analysis of Fer1 cells exposed to Cu(2+) revealed the induction of stress proteins associated with protein folding and DNA repair (including RadA, thermosome and DnaK homologues), suggesting that 'Ferroplasma acidarmanus' Fer1 uses multiple mechanisms for resistance to high levels of copper.

Characterization of Ferroplasma isolates and Ferroplasma acidarmanus sp. nov., extreme acidophiles from acid mine drainage and industrial bioleaching environments.

Three recently isolated extremely acidophilic archaeal strains have been shown to be phylogenetically similar to Ferroplasma acidiphilum Y(T) by 16S rRNA gene sequencing. All four Ferroplasma isolates were capable of growing chemoorganotrophically on yeast extract or a range of sugars and chemomixotrophically on ferrous iron and yeast extract or sugars, and isolate "Ferroplasma acidarmanus" Fer1(T) required much higher levels of organic carbon. All four isolates were facultative anaerobes, coupling chemoorganotrophic growth on yeast extract to the reduction of ferric iron. The temperature optima for the four isolates were between 35 and 42 degrees C and the pH optima were 1.0 to 1.7, and "F. acidarmanus" Fer1(T) was capable of growing at pH 0. The optimum yeast extract concentration for "F. acidarmanus" Fer1(T) was higher than that for the other three isolates. Phenotypic results suggested that isolate "F. acidarmanus" Fer1(T) is of a different species than the other three strains, and 16S rRNA sequence data, DNA-DNA similarity values, and two-dimensional polyacrylamide gel electrophoresis protein profiles clearly showed that strains DR1, MT17, and Y(T) group as a single species. "F. acidarmanus" Fer1(T) groups separately, and we propose the new species "F. acidarmanus" Fer1(T) sp. nov.

Enumeration and characterization of acidophilic microorganisms isolated from a pilot plant stirred-tank bioleaching operation.

Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45 degrees C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.

Bacterial origin for the isoprenoid biosynthesis enzyme HMG-CoA reductase of the archaeal orders Thermoplasmatales and Archaeoglobales.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase or HMGR) fulfills an essential role in archaea, as it is required for the synthesis of isoprenoid ethers, the main component of archaeal cell membranes. There are two clearly homologous but structurally different classes of the enzyme, one found mainly in eukaryotes and archaea (class 1), and the other found in bacteria (class 2). This feature facilitated the identification of several cases of interdomain lateral gene transfer (LGT), in particular, the bacterial origin for the HMGR gene from the archaeon Archaeoglobus fulgidus. In order to investigate if this LGT event was recent and limited in its scope or had a broad and long-term impact on the recipient and its related lineages, the HMGR gene was amplified and sequenced from a variety of archaea. The survey covered close relatives of A. fulgidus, the only archaeon known prior to this study to possess a bacterial-like HMGR; representatives of each main euryarchaeal group were also inspected. All culturable members of the archaeal group Archaeoglobales were found to display an HMGR very similar to the enzyme of the bacterium Pseudomonas mevalonii. Surprisingly, two species of the genus Thermoplasma also harbor an HMGR of bacterial origin highly similar to the enzymes found in the Archaeoglobales. Phylogenetic analyses of the HMGR gene and comparisons to reference phylogenies from other genes confirm a common bacterial origin for the HMGRs of Thermoplasmatales and Archaeoglobales. The most likely explanation of these results includes an initial bacteria-to-archaea transfer, followed by a another event between archaea. Their presence in two divergent archaeal lineages suggests an important adaptive role for these laterally transferred genes.

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.