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The identification and characterization of genes involved in the microbial oxidation of arsenite will contribute to our understanding of factors controlling As cycling in natural systems. Towards this goal, we recently characterized the widespread occurrence of aerobic arsenite oxidase genes ( aroA-like) from pure-culture bacterial isolates, soils, sediments and geothermal mats, but were unable to detect these genes in all geothermal systems where we have observed microbial arsenite oxidation. Consequently, the objectives of the current study were to measure arsenite-oxidation rates in geochemically diverse thermal habitats in Yellowstone National Park (YNP) ranging in pH from 2.6 to 8, and to identify corresponding 16S rRNA and aroA genotypes associated with these arsenite-oxidizing environments. Geochemical analyses, including measurement of arsenite-oxidation rates within geothermal outflow channels, were combined with 16S rRNA gene and aroA functional gene analysis using newly designed primers to capture previously undescribed aroA-like arsenite oxidase gene diversity. The majority of bacterial 16S rRNA gene sequences found in acidic (pH 2.6–3.6) Fe-oxyhydroxide microbial mats were closely related to Hydrogenobaculum spp. (members of the bacterial order Aquificales), while the predominant sequences from near-neutral (pH 6.2–8) springs were affiliated with other Aquificales including Sulfurihydrogenibium spp., Thermocrinis spp. and Hydrogenobacter spp., as well as members of the Deinococci, Thermodesulfobacteria and β- Proteobacteria. Modified primers designed around previously characterized and newly identified aroA-like genes successfully amplified new lineages of aroA-like genes associated with members of the Aquificales across all geothermal systems examined. The expression of Aquificales aroA-like genes was also confirmed in situ, and the resultant cDNA sequences were consistent with aroA genotypes identified in the same environments. The aroA sequences identified in the current study expand the phylogenetic distribution of known Mo-pterin arsenite oxidase genes, and suggest the importance of three prominent genera of the order Aquificales in arsenite oxidation across geochemically distinct geothermal habitats ranging in pH from 2.6 to 8. [ABSTRACT FROM AUTHOR]

The source waters of acid-sulphate-chloride (ASC) geothermal springs located in Norris Geyser Basin, Yellowstone National Park contain several reduced chemical species, including H2, H2S, As(III), and Fe(II), which may serve as electron donors driving chemolithotrophic metabolism. Microorganisms thriving in these environments must also cope with high temperatures, low pH (∼3), and high concentrations of sulphide, As(III), and boron. The goal of the current study was to correlate the temporal and spatial distribution of bacterial and archaeal populations with changes in temperature and geochemical energy gradients occurring throughout a newly formed (redirected) outflow channel of an ASC spring. A suite of complimentary analyses including aqueous geochemistry, microscopy, solid phase identification, and 16S rDNA sequence distribution were used to correlate the appearance of specific microbial populations with biogeochemical processes mediating S, Fe, and As cycling and subsequent biomineralization of As(V)-rich hydrous ferric oxide (HFO) mats. Rapid As(III) oxidation (maximum first order rate constants ranged from 4 to 5 min−1, t1/2 = 0.17 − 0.14 min) was correlated with the appearance ofHydrogenobaculumandThiomonas–like populations, whereas the biogenesis of As(V)-rich HFO microbial mats (mole ratios of As:Fe∼0.7) was correlated with the appearance ofMetallosphaera,Acidimicrobium, andThiomonas–like populations. Several 16S sequences detected near the source were closely related to sequences of chemolithotrophic hyperthermophilic populations includingStygiolobusandHydrogenobaculumorganisms that are known H2 oxidizers. The use of H2, reduced S(–II,0), Fe(II) and perhaps As(III) by different organisms represented throughout the outflow channel was supported by thermodynamic calculations, confirming highly exergonic redox couples with these electron donors. Results from this work demonstrated that chemical energy gradients play an important role in establishing distinct community structure as a function of distance from geothermal spring discharge. [ABSTRACT FROM AUTHOR]

"Metallosphaera yellowsionensis" is a thermoacidophilic archaeon isolated from Yellowstone National Park that is capable of autotrophic growth using Fe(II), elemental S, or pyrite as electron donors. Analysis of the draft genome sequence from M. yellowsionensis strain MK1 revealed seven different copies of heme copper oxidases (subunit I) in a total of five different terminal oxidase complexes, including doxBCEF, foxABCDEF- GHIJ, soxABC, and the soxM supercomplex, as well as a novel hypothetical two-protein doxB-like polyferredoxin complex. Other genes found in M. yellowstonensis with possible roles in S and or Fe cycling include a thiosulfate oxidase (tqoAB), a sulfite oxidase (som), a cbsA cytochrome b5581566, several small blue copper proteins, and a novel gene sequence coding for a putative multicopper oxidase (Mco). Results from gene expression studies, including reverse transcriptase (RT) quantitative PCR (qPCR) of cultures grown autotrophically on either Fe(II), pyrite, or elemental S showed that thefox gene cluster and mco are highly expressed under conditions where Fe(II) is an electron donor. Metagenome sequence and gene expression studies of Fe-oxide mats confirmed the importance offox genes (e.g.,foxA andfoxC) and mco under Fe(II)-oxidizing conditions. Protein modeling of FoxC suggests a novel lysine-lysine or lysine-arginine heme B binding domain, indicating that it is likely the cytochrome component of a heterodimer complex with foxG as a ferredoxin subunit. Analysis of mco shows that it encodes a novel multicopper blue protein with two plastocyanin type I copper domains that may play a role in the transfer of electrons within the Fox protein complex. An understanding of metabolic pathways involved in aerobic iron and sulfur oxidation in Sulfolobales has broad implications for understanding the evolution and niche diversification of these thermophiles as well as practical applications in fields such as bioleaching of trace metals from pyritic ores. [ABSTRACT FROM AUTHOR]