An arsenite-oxidizing Hydrogenobaculum strain was isolated from a geothermal spring in Yellowstone National Park, Wyo., that was previously shown to contain microbial populations engaged in arsenite oxidation. The isolate was sensitive to both arsenite and arsenate and behaved as an obligate chemolithoautotroph that used H 2 as its sole energy source and had an optimum temperature of 55 to 60°C and an optimum pH of 3.0. The arsenite oxidation in this organism displayed saturation kinetics and was strongly inhibited by H 2 S.Arsenite [As(III)] is often the predominant valence of inorganic arsenic in geothermal source waters, although arsenate [As(V)] can also be present, with As(V)/As(III) ratios varying among different springs due to mixing with meteoric surface waters prior to discharge (3,12,19). However, subsequent to discharge, As(V)/As(III) ratios in the spring water can also be significantly influenced by redox transformations (10, 12), which are well documented for microorganisms (2,4,5,6,10,11,16,17). As(V) reduction is widespread among prokaryotes, occurring when As(V) is utilized as an electron acceptor for anaerobic or microaerobic respiration (13) or as part of a detoxification strategy (8). As(III) oxidation has likewise been observed in various organisms, where it has also been viewed as an apparent detoxification mechanism (1, 14, 15) or as a source of energy to support chemolithoautotrophic growth (1, 17).We previously documented rapid microbial oxidation of As(III) in an acid-sulfate-chloride-type geothermal spring in Norris Geyser Basin, Yellowstone National Park (9). This shallow spring is fed by a nearly constant geothermal source water (63°C, pH 3.1) containing ϳ35 M As(III). The prokaryote microbial community in this spring forms visually and chemically distinguishable mats. A filamentous yellow microbial mat containing visible amounts of S 0 (63 to 60°C) is present 0 to ϳ3.5 m from the spring source and changes to a brown, Fe(III) oxyhydroxide filamentous microbial mat (51 to 55°C) at ϳ3.5 to 5 m from the spring source (9). Chemical analysis of the aqueous and solid phases documented high rates of As(III) oxidation in the brown mat region, and the role of microorganisms in As(III) oxidation was confirmed in assays that showed no As(III) oxidation in the formaldehyde-killed samples (9). The PCR-generated 16S ribosomal DNA clone libraries representing the yellow and brown mat regions were dominated by Hydrogenobaculum-and Desulfurella-like sequences (7). However, since the phylogenetic data could not predict which population(s) was involved in the As(III) oxidation, the present study was conducted to initiate isolation and characterization of the As(III)-oxidizing microorganism(s) in this spring for use in modeling important and dominant biogeochemical features found in this spring type.Sampling, enrichment, and isolation. Brown microbial mat material was aseptically sampled and transferred to sterile 70-ml serum bottles and submerged with 35 ml of spring water sampled from above the mat. Th...
Geothermal waters contain numerous potential electron donors capable of supporting chemolithotrophybased primary production. Thermodynamic predictions of energy yields for specific electron donor and acceptor pairs in such systems are available, although direct assessments of these predictions are rare. This study assessed the relative importance of dissolved H 2 and H 2 S as energy sources for the support of chemolithotrophic metabolism in an acidic geothermal spring in Yellowstone National Park. H 2 S and H 2 concentration gradients were observed in the outflow channel, and vertical H 2 S and O 2 gradients were evident within the microbial mat. H 2 S levels and microbial consumption rates were approximately three orders of magnitude greater than those of H 2 . Hydrogenobaculum-like organisms dominated the bacterial component of the microbial community, and isolates representing three distinct 16S rRNA gene phylotypes (phylotype ؍ 100% identity) were isolated and characterized. Within a phylotype, O 2 requirements varied, as did energy source utilization: some isolates could grow only with H 2 S, some only with H 2 , while others could utilize either as an energy source. These metabolic phenotypes were consistent with in situ geochemical conditions measured using aqueous chemical analysis and in-field measurements made by using gas chromatography and microelectrodes. Pure-culture experiments with an isolate that could utilize H 2 S and H 2 and that represented the dominant phylotype (70% of the PCR clones) showed that H 2 S and H 2 were used simultaneously, without evidence of induction or catabolite repression, and at relative rate differences comparable to those measured in ex situ field assays. Under in situ-relevant concentrations, growth of this isolate with H 2 S was better than that with H 2 . The major conclusions drawn from this study are that phylogeny may not necessarily be reliable for predicting physiology and that H 2 S can dominate over H 2 as an energy source in terms of availability, apparent in situ consumption rates, and growth-supporting energy.Thermophiles dominate the deepest and shortest branches of the Bacteria and Archaea domains in the tree of life, suggesting that they are likely ancestors of Earth's contemporary microbial populations (8,35). Consequently, these organisms have attracted considerable attention due to interest in the origin of enzymes and metabolic pathways that are thought to have evolved from such organisms. Chemolithotrophic metabolism is foundational to primary productivity in geothermal environments where temperatures exceed the limit of photosynthesis. The bioenergetics of such systems have been examined from the perspective of theoretical energy yield as a way of discussing the relative importance of the various electron donors and acceptors that could support primary productivity (3)(4)(5)22). Other studies have sought to link the inferred physiology of microbial populations with the predicted energy yields obtainable from the inorganic constituents present (4...
The unicellular eukaryotic algae Cyanidium, Galdieria, and Cyanidioschyzon (herein referred to as ''cyanidia'') are the only photoautotrophs occurring in acidic (pH<4.0) geothermal environments at temperatures above 401C. In Yellowstone National Park (YNP), we examined an annual event we refer to as ''mat decline,'' where cyanidial mats undergo a seasonably defined color fading. Monthly sampling of chemical, physical, and biological features revealed that spring aqueous chemistry was essentially invariant over the 1-year sampling period. However, multiple regression analysis suggested that a significant proportion of algal most probable number (MPN) count variation could be explained by water temperature and UV-visible (VIS) light exposure. Irradiance manipulations (filtering) were then coupled with 14 CO 2 incorporation experiments to directly demonstrate UV inhibition of photosynthesis. Population dynamics were also evident in 18S rDNA PCR clone libraries, which were different in composition at MPN maxima and minima, and again evident in PCR-amplified chloroplast genomic short sequence repeat (SSR) analysis. PCR-cloned SSRs of the YNP isolates and mats were very similar to Cyanidioschyzon merolae Luca, Taddei et Varano, although distance analysis could distinguish the YNP cyanidia from the genome sequenced C. merolae that was isolated in Italy. Unexpectedly, while phylogenetic analysis of 18S rDNA sequences and SSR sequences derived from YNP cyanidial mats and pure cultures suggested these algae are most closely related to C. merolae (99.7% identity), cell morphology was consistent with the genera Galdieria and Cyanidium.
Previous studies in an acid-sulfate-chloride spring in Yellowstone National Park found that microbial arsenite [As(III)] oxidation is absent in regions of the spring outflow channel where H 2 S exceeds ϳ5 M and served as a backdrop for continued efforts in the present study. Ex situ assays with microbial mat samples demonstrated immediate As(III) oxidation activity when H 2 S was absent or at low concentrations, suggesting the presence of As(III) oxidase enzymes that could be reactivated if H 2 S is removed. Cultivation experiments initiated with mat samples taken from along the H 2 S gradient in the outflow channel resulted in the isolation of an As(III)-oxidizing chemolithotroph from the low-H 2 S region of the gradient. The isolate was phylogenetically related to Acidicaldus and was characterized in vitro for spring-relevant properties, which were then compared to its distribution pattern in the spring as determined by denaturing gradient gel electrophoresis and quantitative PCR. While neither temperature nor oxygen requirements appeared to be related to the occurrence of this organism within the outflow channel, H 2 S concentration appeared to be an important constraint. This was verified by in vitro pure-culture modeling and kinetic experiments, which suggested that H 2 S inhibition of As(III) oxidation is uncompetitive in nature. In summary, the studies reported herein illustrate that H 2 S is a potent inhibitor of As(III) oxidation and will influence the niche opportunities and population distribution of As(III) chemolithotrophs. (38). Progress toward understanding the genetics and physiology of As(III) oxidation is at an early stage, being limited to a few definitive papers that describe the biochemical and structural features of one of the two identified As(III) oxidases (4,11,37) and recent studies that have identified the structural genes that encode these As(III) oxidases (31, 40).Even less is known about the ecology of As(III) chemolithotrophs. Drainage waters originating from commercial mining operations often contain appreciable amounts of As(III) and thus are potential habitats for organisms capable of using As(III) as an energy source. Such was the case for the As(III) chemolithotrophs isolated from gold mines (39, 40). Also, waters originating from geothermal sources often carry significant As(III) (5, 28, 45). The facultative anaerobic As(III) chemolithotroph isolated by Oremland et al. (34) was obtained from anaerobic bottom waters of Mono Lake, a meromictic lake containing high concentrations of As(III) derived from geothermal inputs (33). Thermophiles capable of oxidizing As(III) have been isolated (10,14), and a recent PCR-based survey documented the presence and expression of As(III) oxidase structural genes in geothermal springs in Yellowstone National Park (YNP), WY (21). However, to date, the lone documentation of a thermophile capable of As(III) chemolithotrophy is a brief notation that Sulfurihydrogenibium azorense can use As(III) as an electron donor (1).Our previous studies in the...
We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the ␣-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaeabiased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.
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