How eukaryotic algae can adapt to the Spain's Rio Tinto: a neo-Darwinian proposal for rapid adaptation to an extremely hostile ecosystem

Microalgae contributed 60% of the total biomass in the extremely hostile (pH 2 and metal-rich waters) environment of Rio Tinto (which is used as a model for the astrobiology of Mars). These algae are closely related to nonextreme lineages, suggesting that adaptation to Rio Tinto water (RTW) must occur rapidly. Fitness from both the microalga Dictyosphaerium chlorelloides and the cyanobacterium Microcystis aeruginosa was inhibited when they were cultured in RTW. After further incubation for several weeks, D. chlorelloides survived, as a result of the growth of a variant that was resistant to RTW, but RTW-resistant cells did not appear in M. aeruginosa. A Luria-Delbrück fluctuation test revealed that D. chlorelloides RTW-resistant cells arose randomly by rare spontaneous mutations before the RTW exposure (1.38 x 10(-6) mutants per cell division). The mutants with a diminished fitness are maintained in nonextreme waters as the result of a balance between new RTW-resistant cells arising by mutation and RTW-resistant mutants eliminated by natural selection (equilibrium at c. 15 RTW-resistant per 10(7) wild-type cells). Rapid adaptation of eukaryotic algae to RTW could be the result of selection of RTW-resistant mutants occurring spontaneously in nonextreme populations that arrived fortuitously at the river in the past, or in the present continuously.     

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.     

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.     

From genes to genomes: beyond biodiversity in Spain's Rio Tinto

Spain's Rio Tinto, or Red River, an example of an extremely acidic (pH 1.7-2.5) environment with a high metal content, teems with prokaryotic and eukaryotic microbial life. Our recent studies based on small-subunit rRNA genes reveal an unexpectedly high eukaryotic phylogenetic diversity in the river when compared to the relatively low prokaryotic diversity. Protists can therefore thrive in and dominate extremely acidic, heavy-metal-laden environments. Further, because we have discovered protistan acidophiles closely related to neutrophiles, we can hypothesize that the transition from neutral to acidic environments occurs rapidly over geological time scales. How have these organisms adapted to such environments? We are currently exploring the alterations in physiological mechanisms that might allow for growth of eukaryotic microbes at acid extremes. To this end, we are isolating phylogenetically diverse protists in order to characterize and compare ion-transporting ATPases from cultured acidophiles with those from neutrophilic counterparts. We predict that special properties of these ion transporters allow protists to survive in the Rio Tinto.     

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.     

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.     

Comparative microbial ecology study of the sediments and the water column of the Río Tinto, an extreme acidic environment

Due to its highly metalliferous waters and low pH, the Rio Tinto has shown its potential for modelling both acid mine drainage systems and biohydrometallurgical operations. Most geomicrobiological studies of these systems have focused on the oxic water column. A sequence-based approach in combination with in situ detection techniques enabled us to examine the composition and structure of the microbial communities associated with the suboxic and anoxic sediments along the river course and to compare them with the planktonic communities inhabiting the water column. The results obtained with the different approaches were consistent and revealed some major patterns: higher cell density and higher richness (75 vs. 48 operational taxonomic units) in the sediments than in the water column. The microbial communities were related but the river sediments appear to be enriched in certain populations, some of which have not previously been reported in the Rio Tinto basin. The differences detected between sampling stations along the river correlate with certain environmental parameters (e.g. iron concentration gradient). The biological and geochemical data show the importance of the sediments as representing a phase of particular high diversity, probably related to key metabolic processes within both the iron and the sulfur cycles.     

FLORIDOSIDES IN CYANIDIUM CALDARIUM,CYANIDIOSCHYZON MEROLAE AND GALDIERIA SULPHURARIA(RHODOPHYTA,CYANIDIOPHYCEAE)1

Cyanidium caldarium Geitler, Cyanidioschyzon merolae De Luca, Taddei & Varano and Galdieria sulphuraria (Galdieri) Merola are the three thermoacidophilic algae characterized by a chloroplast which is bounded by a single membrane. The presence of this atypical chloroplast made the inclusion of these algae in the Rhodophyta difficult. The discovery in the three algae of floridoside and isofloridoside, typical storage products of red algae, in compatible with their inclusion in the Rhodophyta     

Plastid‐encoded rbcL phylogeny suggests widespread distribution of Galdieria phlegrea (Cyanidiophyceae,Rhodophyta)

The rare microscopic red alga Galdieria phlegrea (Cyanidiohyceae, Rhodophyta) has been discovered in the extremely acid Tinto River in Spain and this occurrence is here related to previous knowledge about the distribution and ecology of this enigmatic alga. The taxonomic affiliation of the new isolate of G. phlegrea was revealed by reconstructing the phylogeny of plastid‐encoded rbcL. According to this phylogeny, the Tinto River alga is closely related to other G. phlegrea strains originating from extreme habitats in Czechia, Italy and Turkey, suggesting a wider distribution and higher ecological versatility than previously thought. The results suggest that G. phlegrea, and then possibly also other cyanidiophycean algae, are not as restricted to strongly acidic and hot microhabitats as previously believed, which, in turn, may indicate that they may commonly have been overlooked and possibly are much more widespread than is currently believed.     

Further study on polyamines in primitive unicellular eukaryotic algae

The possible usefulness of polyamines as chemotaxonomic markers has been investigated in eukaryotic algae. Polyamines were analyzed in 12 species of primitive unicellular eukaryotic algae including some anomalous species. Norspermidine and norspermine in addition to putrescine and spermidine are widely distributed in most unicellular species of the algae. However, neither norspermidine nor norspermine was found in the taxonomically conflicting algae, Cyanophora and Glaucocystis, which contain cyanellae, or in a primitive red alga, Porphyridium. A thermoacidophilic eukaryotic alga, Cyanidium, is rich in both norspermidine and norspermine. Appreciable amounts of spermine and sym-homospermidine were detected only in the species belonging to the Rhodophyta (red algae).     

Phylogenetic characterization of a new thermoacidophilic bacterium isolated from hot springs in Japan

Three strains of thermophilic-acidophilic bacteria isolated previously from different hot springs in Japan were characterized by molecular genetic methods. The strategy taken involved PCR amplification, sequencing and restriction pattern analysis of 16S rDNA, 16S-23S rDNA spacer polymorphism analysis and genomic DNA-DNA hybridization. A phylogenetic analysis based on 16S rDNA sequences showed that the new thermoacidophilic isolates formed a genetically coherent group at the species level and fell into a major cluster together with members of the genera Alicyclobacillus and Sulfobacillus with A. acidocaldarius and A. acidoterrestris as their closest relatives. The levels of binary sequence similarity between the isolates and the two Alicyclobacillus species were 97.6 to 97.9%, values considered low enough to warrant placement of the isolates in a distinct species of the genus Alicyclobacillus. The 16S rDNA restriction pattern analysis, but not 16S-23S rDNA spacer polymorphism analysis, was useful for differentiating the isolates from the established Alicyclobacillus species. DNA-DNA hybridization assays demonstrated a distinct phylogenetic position of our isolates as a genospecies within the genus Alicyclobacillus. On the basis of these results, the thermoacidophilic isolates should be classified into a new species of Alicyclobacillus. The results of this study suggest that this new genospecies of Alicyclobacillus is widely distributed in hot springs in Japan.     

DNA barcoding of a new record of epi-endophytic green algae Ulvella leptochaete (Ulvellaceae,Chlorophyta) in India

Epi-endophytic green algae comprise one of the most diverse and phylogenetically primitive groups of green algae and are considered to be ubiquitous in the world’s oceans; however, no reports of these algae exist from India. Here we report the serendipitous discovery of Ulvella growing on intertidal green algae Cladophora glomerata and benthic red algae Laurencia obtusa collected from India. DNA barcodes at nuclear ribosomal DNA Internal Transcriber Spacer (nrDNA ITS) 1 and 2 regions for Indian isolates from the west and east coasts have been generated for the first time. Based on morphology and DNA barcoding, isolates were identified as Ulvella leptochaete. Phylogenetic reconstruction of concatenated dataset using Maximum Likelihood method differentiated Indian isolates from other accessions of this alga available in Genbank, albeit with low bootstrap support. Monophyly of Ulvella leptochaete was obvious in both of our phylogenetic analyses. With this first report of epi-endophytic algae from Indian territorial waters, the dire need to catalogue its cryptic diversity is highlighted and avenues of future research are discussed.     

Microbial ecology of an extreme acidic environment,the Tinto River

The Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals. The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt. Molecular ecology techniques were used to analyze the diversity of this microbial community. The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification. A good correlation between the two approaches was found. Comparative sequence analysis of DGGE bands showed the presence of organisms related to Leptospirillum spp., Acidithiobacillus ferrooxidans, Acidiphilium spp., "Ferrimicrobium acidiphilum," Ferroplasma acidiphilum, and Thermoplasma acidophilum. The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes. More than 80% of the cells were affiliated with the domain Bacteria, with only a minor fraction corresponding to ARCHAEA: Members of Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans, and Acidiphilium spp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected. Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined. Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested.     

Yeast Diversity in the Extreme Acidic Environments of the Iberian Pyrite Belt

In the Iberian Pyrite Belt (IPB), acid rock drainage gives rise to aquatic habitats with low pH and high concentrations of heavy metals, a situation that causes important environmental problems. We investigated the occurrence and diversity of yeasts in two localities of the IPB: São Domingos (Portugal) and Rio Tinto (Spain). Yeast isolation was performed on conventional culture media (MYP), acidified (pH 3) media (MYP3), and on media prepared with water from the study sites (MYPw). The main goal of the study was to determine the structure of the yeast community; a combination of molecular methods was used for accurate species identifications. Our results showed that the largest fraction of the yeast community was recovered on MYPw rather than on MYP and MYP3. Twenty-seven yeast species were detected, 48% of which might represent undescribed taxa. Among these, an undescribed species of the genus Cryptococcus required low pH for growth, a property that has not been observed before in yeasts. The communities of S. Domingos and R. Tinto showed a considerable resemblance, and eight yeast species were simultaneously found in both localities. Taking into consideration the physicochemical parameters studied, we propose a hierarchic organization of the yeast community in terms of high-, intermediate-, or low-stress conditions of the environment. According to this ranking, the acidophile yeast Cryptococcus sp. 5 is considered the most tolerant species, followed by Cryptococcus sp. 3 and Lecytophora sp. Species occurring in situations of intermediate environmental stress were Candida fluviatilis, Rhodosporidium toruloides, Williopsis californica, and three unidentified yeasts belonging to Rhodotorula and Cryptococcus.     

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.     

Multilocus sequence typing performed on Campylobacter coli isolates from humans, broilers, pigs and cattle originating in Denmark

AIMS: To assess whether Campylobacter coli isolated from different sources in Denmark constitute separate populations. METHODS AND RESULTS: Multilocus sequence typing (MLST) was applied to 160 C. coli isolates from animal origin, food products and human cases of gastroenteritis. The isolates were collected in Denmark over a 2-year period. In total, 84 sequence types (STs) were obtained and 57 of these STs were novel to this study. Ten per cent of the isolates possessed STs that were found in both human, poultry and pig isolates. Only 10% of the isolates from pigs shared ST with isolates from humans, and these shared STs were found in poultry isolates as well. CONCLUSIONS: Great genetic diversity was seen within the Danish C. coli population. Furthermore, we found that the C. coli types isolated from Danish pigs constitute a small fraction of the C. coli causing human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: C. coli isolates from pigs is presumably not a significant source of human campylobacteriosis in Denmark. The Danish C. coli isolates include 68% STs novel to this study, showing a great diversity compared with studies from other countries.     

Genetic diversity of Neisseria meningitidis strains isolated in Rio de Janeiro, Brazil, evaluated by multilocus enzyme electrophoresis

AIMS: To analyse Neisseria meningitidis isolates from meningococcal meningitis cases in Rio de Janeiro (Brazil) from 1990 to 1993 and 1999-2002, to determine the genetic and relatedness with hypervirulent and epidemic strains. METHODS AND RESULTS: The isolates were analysed by multilocus enzyme electrophoresis (MEE) clustering into 83 electrophoretic types (ET). All isolates from 1999 to 2002, formed a cluster which included one strain of the ET-5 complex worldwide associated with epidemics. CONCLUSIONS: The overall results suggested a panmictic structure probably because of recombination events. The observation of a separated cluster including isolates from 1999 to 2002 and an ET-5 complex strain, also suggested the introduction of strains genetically related with this hypervirulent complex in the State of Rio de Janeiro (Brazil) over the last 5 years. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of strains related to the ET-5 complex in several states of Brazil was already described elsewhere, but this is the first time it was reported in the State of Rio de Janeiro. Our findings reinforce the necessity to genetically determine the clones which should be considered to produce a national vaccine against meningococcal meningitis.     

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.     

Activity time of male Euglossina and preference for artificial fragrances in forest and dunes in the Environmental Protection Area of the Mamanguape River Bar, Rio Tinto, Paraíba, Brazil

Euglossina bees form a group with about 200 species known, widespread in Neotropical Region. Males collect aromatic compounds on flowers and by using synthesized analogs it is possible to accomplish faunistic inventories and study several ecological aspects of these bees. In this work we studied the activity hours and the preference of aromatic compounds by Euglossina males in forest and dunes at Environmental Protection Area of the Mamanguape River Bar, Rio Tinto, Paraíba State, between August 2002 and July 2004. Six artificial compounds were used as attractive: benzyl acetate, ionone beta, skatole, eucalyptol, eugenol and vanillin. Nine species were sampled, being two of them exclusive of the forest. Euglossa cordata (L.) and Eulaema nigrita Lepeletier were the most abundant species as in the forest as on dunes. Males were more active between 8:00 a.m. and 10:00 a.m. in both areas, with no variation throughout the year. Eucalyptol and eugenol attracted most number of species. E. cordata demonstrated preference by ionone beta and eucalyptol and E. nigrita by skatole and eucalyptol.     

Molecular and biological diversity of HIV-1 in Brazil.

To determine the genomic polymorphism and biological properties present in HIV-1 Brazilian isolates, we analyzed five viral isolates obtained from patients residing in Rio de Janeiro (P1 and P5), S?o Paulo (P3) and Bahia (P2 and P4) states. For each viral isolate in vitro characteristics such as replication rate, syncytium-inducing capacity and cell death were observed in lymphoblastoid (H9, CEM and peripheral blood mononuclear cells) as well as monocytoid (U937) cells. In addition, the evaluation of the restriction fragment length polymorphism of these isolates was also performed using a panel of endonucleases such as Hind III, Bgl II, Sac I, Pst I, Kpn I and Eco RI. One of the isolates (P1), showed the highest phenotypic and genotypic divergence, when compared to others. The results found suggest a HIV heterogeneity in Brazil similar to that already described in other regions of the world.