Pyrobaculum yellowstonensis Strain WP30 Respires on Elemental Sulfur and/or Arsenate in Circumneutral Sulfidic Geothermal Sediments of Yellowstone National Park.

Thermoproteales (phylum Crenarchaeota) populations are abundant in high-temperature (>70°C) environments of Yellowstone National Park (YNP) and are important in mediating the biogeochemical cycles of sulfur, arsenic, and carbon. The objectives of this study were to determine the specific physiological attributes of the isolate Pyrobaculum yellowstonensis strain WP30, which was obtained from an elemental sulfur sediment (Joseph's Coat Hot Spring [JCHS], 80°C, pH 6.1, 135 µM As) and relate this organism to geochemical processes occurring in situ. Strain WP30 is a chemoorganoheterotroph and requires elemental sulfur and/or arsenate as an electron acceptor. Growth in the presence of elemental sulfur and arsenate resulted in the formation of thioarsenates and polysulfides. The complete genome of this organism was sequenced (1.99 Mb, 58% G+C content), revealing numerous metabolic pathways for the degradation of carbohydrates, amino acids, and lipids. Multiple dimethyl sulfoxide-molybdopterin (DMSO-MPT) oxidoreductase genes, which are implicated in the reduction of sulfur and arsenic, were identified. Pathways for the de novo synthesis of nearly all required cofactors and metabolites were identified. The comparative genomics of P. yellowstonensis and the assembled metagenome sequence from JCHS showed that this organism is highly related (∼95% average nucleotide sequence identity) to in situ populations. The physiological attributes and metabolic capabilities of P. yellowstonensis provide an important foundation for developing an understanding of the distribution and function of these populations in YNP.

Predominant Acidilobus-like populations from geothermal environments in yellowstone national park exhibit similar metabolic potential in different hypoxic microbial communities.

High-temperature (>70°C) ecosystems in Yellowstone National Park (YNP) provide an unparalleled opportunity to study chemotrophic archaea and their role in microbial community structure and function under highly constrained geochemical conditions. Acidilobus spp. (order Desulfurococcales) comprise one of the dominant phylotypes in hypoxic geothermal sulfur sediment and Fe(III)-oxide environments along with members of the Thermoproteales and Sulfolobales. Consequently, the primary goals of the current study were to analyze and compare replicate de novo sequence assemblies of Acidilobus-like populations from four different mildly acidic (pH 3.3 to 6.1) high-temperature (72°C to 82°C) environments and to identify metabolic pathways and/or protein-encoding genes that provide a detailed foundation of the potential functional role of these populations in situ. De novo assemblies of the highly similar Acidilobus-like populations (>99% 16S rRNA gene identity) represent near-complete consensus genomes based on an inventory of single-copy genes, deduced metabolic potential, and assembly statistics generated across sites. Functional analysis of coding sequences and confirmation of gene transcription by Acidilobus-like populations provide evidence that they are primarily chemoorganoheterotrophs, generating acetyl coenzyme A (acetyl-CoA) via the degradation of carbohydrates, lipids, and proteins, and auxotrophic with respect to several external vitamins, cofactors, and metabolites. No obvious pathways or protein-encoding genes responsible for the dissimilatory reduction of sulfur were identified. The presence of a formate dehydrogenase (Fdh) and other protein-encoding genes involved in mixed-acid fermentation supports the hypothesis that Acidilobus spp. function as degraders of complex organic constituents in high-temperature, mildly acidic, hypoxic geothermal systems.

Microbial community structure and sulfur biogeochemistry in mildly-acidic sulfidic geothermal springs in Yellowstone National Park.

Geothermal and hydrothermal waters often contain high concentrations of dissolved sulfide, which reacts with oxygen (abiotically or biotically) to yield elemental sulfur and other sulfur species that may support microbial metabolism. The primary goal of this study was to elucidate predominant biogeochemical processes important in sulfur biogeochemistry by identifying predominant sulfur species and describing microbial community structure within high-temperature, hypoxic, sulfur sediments ranging in pH from 4.2 to 6.1. Detailed analysis of aqueous species and solid phases present in hypoxic sulfur sediments revealed unique habitats containing high concentrations of dissolved sulfide, thiosulfate, and arsenite, as well as rhombohedral and spherical elemental sulfur and/or sulfide phases such as orpiment, stibnite, and pyrite, as well as alunite and quartz. Results from 16S rRNA gene sequencing show that these sediments are dominated by Crenarchaeota of the orders Desulfurococcales and Thermoproteales. Numerous cultivated representatives of these lineages, as well as the Thermoproteales strain (WP30) isolated in this study, require complex sources of carbon and respire elemental sulfur. We describe a new archaeal isolate (strain WP30) belonging to the order Thermoproteales (phylum Crenarchaeota, 98% identity to Pyrobaculum/Thermoproteus spp. 16S rRNA genes), which was obtained from sulfur sediments using in situ geochemical composition to design cultivation medium. This isolate produces sulfide during growth, which further promotes the formation of sulfide phases including orpiment, stibnite, or pyrite, depending on solution conditions. Geochemical, molecular, and physiological data were integrated to suggest primary factors controlling microbial community structure and function in high-temperature sulfur sediments.

Preserving the mucosal barrier during small bowel storage.

BACKGROUND: A major obstacle to successful small bowel transplantation is that of bacterial infection. The aim of this study was to preserve the small bowel mucosal barrier by using oxygenated luminal perfusion with a proven amino acid (AA)-based solution. METHODS: Rat small bowel (n=4) was flushed vascularly with modified University of Wisconsin solution and flushed luminally as follows: group 1, none (control); group 2, AA solution; group 3, 1-hr perfusion then storage with AA; group 4, continuous perfusion with AA. Energetics, malondialdehyde (MDA), glutathione (reduced), and histology were assessed over 24 hr at 4 degrees C. RESULTS: Within 4 hr, adenosine triphosphate (ATP) dropped by 25% to 65% in all groups except for group 4, which remained unchanged from fresh tissue values throughout 12 hr. After 12 hr, ATP in groups 1 through 3 had dropped to 0.5 to 0.9 micromol/g, compared with 1.5 micromol/g for group 4. Even after 24 hr, group 4 levels were more than twofold greater than groups 1 through 3. MDA increased transiently in tissues subjected to simple flush (no perfusion), whereas levels in perfused tissues remained elevated throughout the 24-hr period. Glutathione in group 1 dropped by greater than 50% from fresh tissue values but increased over 24 hr in groups 2 and 3 by 50% to 55%. Overall, histologic injury was markedly less in groups 2 through 4; however, after 24 hr, the lowest injury was observed in group 3 (median, grade 2) compared with groups 1 and 4 (grades 7 and 4). CONCLUSIONS: Our data indicate that perfusion clearly improves tissue energetics. However, mucosal integrity is markedly superior, with only a brief 1-hr period of perfusion; oxidative and mechanical stress are the factors likely responsible for injury resulting from continuous perfusion.

A novel technique of hypothermic luminal perfusion for small bowel preservation.

BACKGROUND: This study improved small bowel preservation using University of Wisconsin (UW) solution in conjunction with hypothermic luminal perfusion. METHODS: Small bowels from Sprague-Dawley rats (n=4) were flushed vascularly with modified UW solution and flushed luminally: group 1, none (clinical control); group 2, UW solution; group 3, 1-hr oxygenated perfusion then static storage with UW; and group 4, 24-hr continuous oxygenated perfusion with UW. Energetics, lipid peroxidation, and histology were assessed during 24 hr at 4 degrees C. RESULTS: After 12 hr, adenosine triphosphate ranged from 0.5 to 0.8 micromol/g in groups 1 to 3 compared with 1.5 micromol/g in group 4. Even after 24 hr, levels in group 4 were more than twofold greater than levels in groups 1 to 3. Energy charge values ([adenosine triphosphate+adenosine diphosphate/2]/total adenylates) decreased from fresh tissue values of 0.69 in all groups except group 4 throughout 24-hr perfusion. Malondialdehyde (MDA; a product of lipid peroxidation) doubled within 4 hr in group 1 and remained high throughout storage. In groups 3 and 4, MDA levels increased as the time of perfusion increased; group 2 showed no elevated MDA levels at any time. After 12 hr, histologic integrity was superior in groups 3 and 4; however after 24 hr, the best Park's grade was observed in group 3 (median grade 4) compared with groups 1 (grade 7) and 4 (grade 6). CONCLUSIONS: Our data indicate that perfusion clearly improves tissue energetics; however, mucosal integrity is superior with only a brief 1-hr period of luminal perfusion, despite limited improvements in energetics.