Protist genetic diversity in the acidic hydrothermal environments of Lassen Volcanic National Park, USA

We examined eukaryote genetic diversity in the hydrothermal environments of Lassen Volcanic National Park (LVNP), Northern California. We sampled hydrothermal areas of the Bumpass Hell, Sulfur Works, Devil's Kitchen, and Boiling Springs Lake sites, all of which included diverse acidic pools, mud pots, and streams with visible algal mats and biofilms. Temperatures varied from 15 to 85 degrees C and pH from 1.7 to 5.8. DNA extraction methods compared by denaturing gradient gel electrophoresis fingerprinting exhibited similar patterns, and showed limited diversity of eukaryotic small subunit (SSU) rRNA genes compared with prokaryotes. We successfully amplified eukaryotic SSU rRNA genes from most environments up to 68 degrees C. Cloned rDNA sequences reveal acidophilic protists dominate eukaryotes in LVNP hydrothermal environments. Most sites showed phototrophic assemblages dominated by chlorophytes and stramenopiles (diatoms and chrysophytes). Heterotrophic taxa, though less abundant, included diverse alveolates (ciliates), amoebae, and flagellates. Fungi were also found at most sites, and metazoans (hexapods, nematodes, platyhelminths) were sometimes detected in less acidic environments, especially in algal mats. While many cloned rDNA sequences showed 95%-99% identity to known acidophilic isolates or environmental clones from other acidic sites (Rio Tinto), sequence diversity generally declined both with decreasing pH and increasing temperature, and both were controlling physical variables on the abundance and distribution of organisms at our sites. However, a pool at 68 degrees C with pH 1.7 yielded the greatest number of distinct sequences. While some were likely contaminants from nearby cooler sites, we suggest that Lassen's acidic hydrothermal features may harbor novel protists.     

Methanogenesis in the sediments of Rio Tinto, an extreme acidic river

Río Tinto (Iberian Pyritic Belt, SW Spain) is well known for its low pH (mean pH 2.3), high redox potential (> +400 mV) and high concentration of heavy metals. In this work we describe and analyse the presence of methanogenic archaea in the extreme acidic and oxidizing environment of the Tinto basin. Methane formation was measured in microcosms inoculated with sediments from the Rio Tinto basin. Methanol, formate, volatile fatty acids and lactate stimulated the production of methane. Methane formation was associated with a decrease of redox potential and an increase in pH. Cores showed characteristic well-defined black bands in which a high acetate concentration was measured among the otherwise reddish-brown sediments with low acetate concentration. Methanosaeta concilii was detected in the black bands. In enrichment cultures, M. concilii (enriched with a complex substrate mixture), Methanobacterium bryantii (enriched with H(2)) and Methanosarcina barkeri (enriched with methanol) were identified. Our results suggest that methanogens thrive in micro-niches with mildly acidic and reducing conditions within Rio Tinto sediments, which are, in contrast, immersed in an otherwise extremely acidic and oxidizing environment.     

An oligonucleotide prokaryotic acidophile microarray: its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA

An oligonucleotide microarray that monitors prokaryotic diversity in extremely acidic environments has been developed. The oligonucleotide probes target most known acidophilic microorganisms, including members of the Nitrospira phylum, Acidithiobacillus genus, acidobacteria, sulfur reducing bacteria, Actinobacteria and Archaea of the Ferroplasma and Thermoplasma genera. The probes were tested for their specificity against the corresponding type strain by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. The microarray was tested and validated against well-established molecular ecology techniques such as molecular cloning and sequencing and FISH by using samples obtained from a natural extremely acidic environment, the Río Tinto (SW Spain). Also, fluorescent labelled total environmental RNA from Río Tinto samples were used as targets for microarray hybridizations. This approach allowed the detection of the most metabolically active prokaryotes of the ecosystem by simultaneously checking probes against 16S and 23S rRNAs as well as other functional genes. Seasonal and spatial variations in the relative expression of specific rRNA genes have been detected between two sampling sites that differ in several physicochemical parameters, mainly iron and sulfur content.     

Predation on prokaryotes in the water column and its ecological implications

The oxic realms of freshwater and marine environments are zones of high prokaryotic mortality. Lysis by viruses and predation by ciliated and flagellated protists result in the consumption of microbial biomass at approximately the same rate as it is produced. Protist predation can favour or suppress particular bacterial species, and the successful microbial groups in the water column are those that survive this selective grazing pressure. In turn, aquatic bacteria have developed various antipredator strategies that range from simply 'outrunning' protists to the production of highly effective cytotoxins. This ancient predator-prey system can be regarded as an evolutionary precursor of many other interactions between prokaryotic and eukaryotic organisms.     

Rio Tinto as a niche for acidophilus enzymes of industrial relevance

Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rıo Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5–5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre-industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.     

Yeast diversity in the acidic Rio Agrio-Lake Caviahue volcanic environment (Patagonia, Argentina)

The Rio Agrio and Lake Caviahue system (RAC), in Northwestern Patagonia, is a natural acidic environment. The aims of this study were to characterize the yeast community and to provide the first ecological assessment of yeast diversity of this extreme aquatic environment. Yeast occurrence and diversity were studied at seven sites where the water pH varied between 1.8 and 6.7. Yeast CFU counts in the river ranged from 30 to 1200 CFU L⁻¹, but in the Lake the values were lower (30–60 CFU L⁻¹). A total of 25 different yeast species were found, 11 of which belonged to undescribed taxa. Among these was an unusual strongly acidophilic Cryptococcus species. The RAC yeast community resembles that of acidic aquatic environments resulting from anthropic activities such as the São Domingos mines in Portugal and the Rio Tinto in Spain, respectively. The isolated yeast species were organized into different grades of adaptation to the RAC aquatic system. Based on the proposed grades, Rhodotorula mucilaginosa , Rhodosporidium toruloides and two novel Cryptococcus species were the most adapted species. These Cryptococcus species are apparently specialists of acidic aquatic environments, and might bear physiological features that possibly account for their ability to thrive in such extreme environments.     

Microbial Community Composition and Ecology of an Acidic Aquatic Environment: The Tinto River, Spain

We studied the correlation between physicochemical and biological characteristics of an acidic river, the Tinto River, in Southwestern Spain. The Tinto River is an extreme environment characterized by its low pH (mean of 2.2) and high concentrations of heavy metals (Fe 2.3 g/L, Zn 0.22 g/L, Cu 0.11 g/L). These extreme conditions are the product of the metabolic activity of chemolithotrophic microorganisms, including iron- and sulfur-oxidizing bacteria, that can be found in high concentrations in its waters. The food chain in the river is very constrained and exclusively microbial. Primary productivity in the Tinto River is the sum of photosynthetic and chemolithotrophic activity. Heterotrophic bacteria and fungi are the major decomposers and protists are the major predators. A correlation analysis including the physicochemical and biological variables suggested a close relationship between the acidic pH values and abundance of both chemolithotrophic bacteria and filamentous fungi. Chemolithotrophic bacteria correlated with the heavy metals found in the river. A principal component analysis of the biotic and abiotic variables suggested that the Tinto River ecosystem can be described as a function of three main groups of variables: pH values, metal concentrations, and biological productivity.     

Extremotolerance in fungi: evolution on the edge

Our planet offers many opportunities for life on the edge: high and low temperatures, high salt concentrations, acidic and basic conditions and toxic environments, to name but a few extremes. Recent studies have revealed the diversity of fungi that can occur in stressful environments that are hostile to most eukaryotes. We review these studies here, with the additional purpose of proposing some mechanisms that would allow for the evolutionary adaptation of eukaryotic microbial life under extreme conditions. We focus, in particular, on life in ice and life at high salt concentrations, as there is a surprising similarity between the fungal populations in these two kinds of environments, both of which are characterized by low water activity. We propose steps of evolution of generalist species towards the development of specialists in extreme habitats. We argue that traits present in some fungal groups, such as asexuality, synthesis of melanin-like pigments and a flexible morphology, are preadaptations that facilitate persistence and eventual adaptation to conditions on the ecological edge, as well as biotope switches. These processes are important for understanding the evolution of extremophiles; moreover, they have implications for the emergence of novel fungal pathogens.     

The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes

Classical studies on protist diversity of freshwater environments worldwide have led to the idea that most species of microbial eukaryotes are known. One exemplary case would be constituted by the ciliates, which have been claimed to encompass a few thousands of ubiquitous species, most of them already described. Recently, molecular methods have revealed an unsuspected protist diversity, especially in oceanic as well as some extreme environments, suggesting the occurrence of a hidden diversity of eukaryotic lineages. In order to test if this holds also for freshwater environments, we have carried out a molecular survey of small subunit ribosomal RNA genes in water and sediment samples of two ponds, one oxic and another suboxic, from the same geographic area. Our results show that protist diversity is very high. The majority of phylotypes affiliated within a few well established eukaryotic kingdoms or phyla, including alveolates, cryptophytes, heterokonts, Cercozoa, Centroheliozoa and haptophytes, although a few sequences did not display a clear taxonomic affiliation. The diversity of sequences within groups was very large, particularly that of ciliates, and a number of them were very divergent from known species, which could define new intra-phylum groups. This suggests that, contrary to current ideas, the diversity of freshwater protists is far from being completely described.     

Characterization of two new thermoacidophilic microalgae: Genome organization and comparison with Galdieria sulphuraria

Abstract Thermoacidophilic algae ( Cyanidiaceae ) constitute a taxonomic group with interesting phylogenetic and ecological implications. In this report, we have classified three thermoacidophilic microalgal isolates from Rio Tinto (Spain) using a combination of classical analysis of phenotypic features and the characterization of their electrophoretically determined karyotypes by means of pulsed-field gel electrophoresis. Using this technique, we have been able to demonstrate that thermoacidophilic algae genomes have the smallest genomes of all photosynthetic eukaryotes studied so far. In addition, we show that two of these Rio Tinto isolates may constitute new species within the genus Galdieria .     

Fossilization of Acidophilic Microorganisms

This study examines fossil microorganisms found in iron-rich deposits in an extreme acidic environment, the Tinto River in SW Spain. Both electron microscopy (SEM and TEM) and non-destructive in situ microanalytical techniques (EDS, EMP and XPS) were used to determine the role of permineralization and encrustation in preserving microorganisms forming biofilms in the sediments. Unicellular algae were preserved by silica permineralization of their cell walls. Bacterial biofilms were preserved as molds by epicellular deposition of schwertmannite around them. In the case of fungi and filamentous algae, we observed permineralization of cell structures by schwertmannite in the sediments. The extracellular polymeric matrix around the cells was also preserved through permineralization of the fibrillar component. The process of permineralization and deposition of iron-rich precipitates present in the acidic waters of Rio Tinto served to preserve many microfossils in an oxidizing environment, in which organic compounds would not normally be expected to persist. Studies of microbial fossil formation mechanisms in modern extreme environments should focus on defining criteria to identify inorganic traces of microbial life in past environments on Earth or other planets.     

Nickel-Resistance Determinants in Acidiphilium sp. PM Identified by Genome-Wide Functional Screening

Acidiphilium spp. are conspicuous dwellers of acidic, metal-rich environments. Indeed, they are among the most metal-resistant organisms; yet little is known about the mechanisms behind the metal tolerance in this genus. Acidiphilium sp. PM is an environmental isolate from Rio Tinto, an acidic, metal-laden river located in southwestern Spain. The characterization of its metal resistance revealed a remarkable ability to tolerate high Ni concentrations. Here we report the screening of a genomic library of Acidiphilium sp. PM to identify genes involved in Ni resistance. This approach revealed seven different genes conferring Ni resistance to E. coli, two of which form an operon encoding the ATP-dependent protease HslVU (ClpQY). This protease was found to enhance resistance to both Ni and Co in E. coli, a function not previously reported. Other Ni-resistance determinants include genes involved in lipopolysaccharide biosynthesis and the synthesis of branched amino acids. The diversity of molecular functions of the genes recovered in the screening suggests that Ni resistance in Acidiphilium sp. PM probably relies on different molecular mechanisms.     

Identification of camelid specific residues in mitochondrial ATP synthase subunits

ATP synthase is an enzyme involved in oxidative phosphorylation from prokaryotic to eukaryotic cells. In mammals it comprises at least 16 subunits from which the mitochondrial encoded ATP6 and ATP8 are essential. Mitochondrial genes variations have been suggested to allow rapid human and animal adaptation to new climates and dietary conditions (Mishmar et al. 2003). Camelidae taxa are uniquely adapted to extremely hot and dry climates of African-Asian territories and to cold and hypoxic environments of the South American Andean region. We sequenced and analyzed ATP6 and ATP8 genes in all camelid species. Based on the available structural data and evolutionary conservation of the deduced proteins we identified features proper of the group. In Old World camels the ATP8, important in the assembly of the F0 complex, showed a number of positively charged residues higher than in the other aligned species. In ATP6 we found the camelid specific substitutions Q47H and I106V that occur in sites highly conserved in other species. We speculate that these changes may have functional importance.     

Diversification of the Microtubule System in the Early Stage of Eukaryote Evolution: Elongation Factor 1α and α-Tubulin Protein Phylogeny of Termite Symbiotic Oxymonad and Hypermastigote Protists

The symbiotic protists of the lower termite have been regarded as a model of early-branched eukaryotes because of their simple cellular systems and morphological features. However, cultivation of these symbiotic protists is very difficult. For this reason, these interesting protists have not been well characterized in terms of their molecular biology. In research on these organisms which have not yet been cultivated, we developed a method for retrieving specific genes from a small number of cells, through micromanipulation without axenic cultivation, and we obtained EF-1 alpha and alpha-tubulin genes from members of the Hypermastigida--the parabasalid protist Trichonympha agilis and the oxymonad protists Pyrsonympha grandis and Dinenympha exilis--from the termite Reticulitermes speratus gut community. Results of phylogenetic analysis of the amino acid sequences of both proteins, EF-1 alpha and alpha-tubulin, indicate that the hypermastigid, parabasalid, and oxymonad protists do not share a close common ancestor. In addition, although the EF-1 alpha phylogeny indicates that these two groups of protists branched at an early stage of eukaryotic evolution, the alpha-tubulin phylogeny indicates that these protists can be assigned to two diversified clades. As shown in a recent investigation of alpha-tubulin phylogeny, eukaryotic organisms can be divided into three classes: an animal--parabasalids clade, a plant--protists clade, and the diplomonads. In this study, we show that parabasalids, including hypermastigids, can be classified as belonging to the animal--parabasalids clade and the early-branching eukaryote oxymonads can be classified as belonging to the plant--protists clade. Our findings suggest that these protists have a cellular microtubule system that has diverged considerably, and it seems that such divergence of the microtubule system occurred in the earliest stage of eukaryotic evolution.     

Extremely acidic, pendulous cave wall biofilms from the Frasassi cave system, Italy

The sulfide-rich Frasassi cave system hosts an aphotic, subsurface microbial ecosystem including extremely acidic (pH 0-1), viscous biofilms (snottites) hanging from the cave walls. We investigated the diversity and population structure of snottites from three locations in the cave system using full cycle rRNA methods and culturing. The snottites were composed primarily of bacteria related to Acidithiobacillus species. Other populations present in the snottites included Thermoplasmata group archaea, bacteria related to Sulfobacillus, Acidimicrobium, and the proposed bacterial lineage TM6, protists, and filamentous fungi. Based on fluorescence in situ hybridization population counts, Acidithiobacillus are key members of the snottite communities, accompanied in some cases by smaller numbers of archaea related to Ferroplasma and other Thermoplasmata. Diversity estimates show that the Frasassi snottites are among the lowest-diversity natural microbial communities known, with one to six prokaryotic phylotypes observed depending on the sample. This study represents the first in-depth molecular survey of cave snottite microbial diversity and population structure, and contributes to understanding of rapid limestone dissolution and cave formation by microbially mediated sulfuric acid speleogenesis.     

A review on the occurrence and taxonomy of heterotrophic protists in extreme acidic environments of pH values ≤ 3

The occurrence and some important taxonomic problems of heterotrophic protists from extremely acidic (pH 3) environments are briefly discussed. Almost all information on the occurrence of heterotrophic protists from extremely acidic environments is restricted to acid mine drainage (AMD) or streams influenced by AMD. Most of the information is provided for ciliates. Very little information is available on the occurrence of flagellates, rhizopods and heliozoa in this environment. Within the ciliates Urotricha, Vorticella and Oxytricha dominate in acidic mining lakes. Actinophrys sp. is the most important heliozoan in these environments. There are many taxonomic problems which are not solved so far. Live observations and taxonomic methods adapted to the extreme chemical matrix are necessary for correct identification.     

Microeukaryotic diversity in the extreme environments of the Iberian Pyrite Belt: a comparison between universal and fungi-specific primer sets, temperature gradient gel electrophoresis and cloning

The Iberian Pyrite Belt extends from Portugal to Spain and is one of the most important pyrite regions in the world. Its aquatic reservoirs display extreme conditions characterized by low pH and high concentrations of heavy metals. In this study, the diversity of microeukaryotes was analysed at the abandoned mines of S?o Domingos (Portugal) and at Rio Tinto (Spain). DNA was extracted from water samples and a set of eukaryotic universal primers directed to the small subunit rRNA genes (rDNA) was used. The amplicons were analysed by molecular cloning and temperature gradient gel electrophoresis (TGGE). In addition, a fungi-specific primer set was also used in TGGE experiments. The fungi-specific primers contributed to a substantial increase in the number of fungal taxa found due, probably, to the relative low density of fungal structures. Several microorganisms, belonging (or closely related) to the ascomycetous yeast Pichia acaciae, the basidiomycetous yeasts Cryptococcus humicola and Cystofilobasidium bisporidii, the green algae Chlamydomonas noctigama and Chlorella protothecoides var. acidicola and some uncultured microeukaryotes were present at both localities, which suggests that specific microorganisms are adapted to the peculiar conditions of the Iberian Pyrite Belt extreme environments. However, in spite of the similarities, a higher algal richness was observed at S. Domingos, whereas for R. Tinto the richness of fungi was more prominent.