Recent discoveries of novel microorganisms have transformed paradigms in biological research. One example involves the remarkable change in our understanding of prokaryotic contributions to global biogeochemical cycles, based on the discovery of ubiquitous and abundant mesophilic members of the division Crenarchaeota. This group was previously thought to be comprised exclusively of thermophilic and hyperthermophilic archaea, but is now known to include a broad membership of mesophilic and low-temperature archaea living in non-extreme habitats. Currently, there is only one representative of this important group in pure culture: a marine isolate that fixes inorganic carbon into cellular biomass under aerobic conditions, using ammonia for energy. The ammonia oxidation process is the first step in microbially mediated nitrification, which links the mineralization of organic matter (formation of NH4+) to the recycling of N to the atmosphere (denitrification of NO3-). The metabolism of this isolate, together with results from environmental studies, suggest that autotrophic ammonia oxidation in archaea, rather than bacteria, may be responsible for the majority of nitrification in both marine and soil ecosystems. The diversity of habitats colonized by nonthermophilic crenarchaeotes and results from in situ metabolic studies suggest these organisms possess additional metabolic capabilities that may further impact elemental cycling on Earth. This project is a first step in understanding niche diversification in nonthermophilic crenarchaeotes. Fine-scale microbiological and geochemical analyses will be coupled with genome mining, gene expression studies and laboratory cultivation to test the hypothesis that aerobic chemoautotrophic ammonia oxidation is an important metabolic strategy for nonthermophilic crenarchaeotes in freshwater sediments, while also investigating the importance of a suite of other metabolic strategies for these archaea. This work is expected to broaden our understanding of the genomics and ecological properties of these enigmatic archaea in significant ways.
Situated at the crossroads of microbiology, genomics, ecology and geochemistry, the research in this project will involve students from high school, undergraduate, graduate and postdoctoral levels in department- and university-wide efforts to forge new, interdisciplinary scientific partnerships. Three specific objectives will be addressed in the development of the education components of the project: (i) Creation of an interdisciplinary graduate curriculum emphasizing molecules to global ecosystems; (ii) Development of an EBS degree program focused on interfaces between environmental systems and human health; and (iii) Integration of high school and undergraduate students into the research goals of the project. The development of the EBS graduate degree program (Objectives i and ii) will help Oregon to become a leader in cross-scale, interdisciplinary research on environmental systems and human health. Addressing the latter objective, a collaborative program for undergraduate research experience in genomics is proposed between OHSU and Pacific University, a 4-year undergraduate institution. The goals of this program are to provide undergraduates with hands-on experience in genomics through the annotation and analysis of emergent crenarchaeal genome sequence data. The development of research and education programs that seek to identify novel metabolic properties of nonthermophilic crenarchaeotes will help elucidate the ecological roles and importance in global elemental cycles of members of this recently-discovered archaeal group.
Anthropogenic perturbation of the global nitrogen cycle, from fossil fuel combustion and excessive fertilizer use, has led to a number of environmental problems including eutrophication, acidification, loss of biodiversity, and increased emission of greenhouse gases. Nitrogen cycling is governed by a variety of microbially-mediated processes, including nitrification, denitrification, ammonification, dissimilatory nitrate reduction to ammonium (DNRA) and anammox. Aerobic ammonia oxidation, the first and rate-limiting step in nitrification (the conversion of ammonium to nitrate), was thought for over a century to be carried out exclusively by ammonia oxidizing bacteria (AOB). Recent studies, however, have shown that ammonia oxidizing archaea (AOA) also exist. These organisms are members of the newly designated phylum Thaumarchaeota, While Thaumarchaeota appear to have substantive roles in nitrification in marine environments, their contributions to nitrification in terrestrial environments are not well understood. Our research, therefore, examined the metabolic properties, phylogenetic diversity, and ecological functions of Thaumarchaeota in terrestrial environments using both cultivation-dependent and independent approaches. Two terrestrial systems were investigated: freshwater sediments and the plant rhizosphere. In order to evaluate the contributions of archaea and bacteria to nitrification in Columbia River freshwater sediments, we identified environmental factors associated with their abundance, distribution and activities. We used a two-pronged approach: (i) fine-scale characterization of the sediment microenvironment using electrochemical probes and solution chemistry to measure variables; and (ii) biomolecular analysis of microbial communities at high-resolution in fractionated sediment cores. Measured variables included oxygen, Eh, pH, ammonium, nitrite and nitrate, as well as gene and transcript abundance of the ammonia oxidation biomarker amoA. AOA amoA genes and gene transcripts were generally present at higher abundance compared to those of β-AOB, regardless of season and depth, suggesting potentially important roles by AOA in nitrification. Composition changes in AOA populations across steep sediment O2 and Eh gradients were also observed. A combination of environmental factors including oxygen, nitrite+nitrate, and Eh were significantly correlated with archaeal amoA gene abundance. To determine nitrification potential by AOA and AOB, nitrification-coupled growth assays were performed in sediment slurry incubations. Results showed that β-AOB were the dominant ammonia oxidizers in sediment incubations with either organic or inorganic amendments, despite the fact that AOA were more abundant than β-AOB in natural sediments. The possibility of a low rate of undetected ammonia oxidation by AOA could not be ruled out, however. Results suggested that β-AOB populations responded more dramatically than AOA to environmental variability, which is significant in the dynamic Columbia River ecosystem. The relative resistance to change by AOA may be a factor in their generally higher abundance in natural sediments. Using a rhizosphere enrichment culture system, we characterized the diversity and metabolic potential of mesophilic soil Thaumarchaeota. Comparative analysis of 16S rRNA and amoA genes indicated that specific archaeal clades were selected under different conditions. Three amoA-containing archaeal clades were identified, while a fourth clade identified by 16S rRNA gene analysis alone, designated the "root clade," had no associated amoA gene. Analysis of archaeal community composition by PCR-SSCP under different culture conditions revealed that the root clade was present only in media with organic amendment, while amoA–containing clades were present in media with either organic or inorganic amendments. We proposed that the root clade may consist of heterotrophic nitrifiers, possibly containing an amoA gene not yet detected. Furthermore, results from gene abundance and expression analyses, together with nitrification potential assays suggested differential contributions by the clades to measured nitrification potential in our system. Taken together, our results indicate that both sediment and soil Thaumarchaeota exhibit diverse metabolic lifestyles. This project gsignificantly impacted the scientific education of one Ph.D. and one M.S. student from OHSU, and a number of undergraduate students from Pacific University. Five interns (some for multiple summers) were recruited to take part in the associated program for "Environmental Research Experience in Genomics at OHSU" (EREGO). Students learned to plan and execute experiments, and to interpret and communicate scientific results. All gave formal presentations on their work at the conclusion of their internships. Furthermore, this project supported the professional development of a collaborative faculty member at Pacific University, through funding to attend a genomics workshop held by the Department of Energy’s Joint Genome Institute. Attendance at the workshop led to a successful grant to incorporate annotation of one of JGI's 'orphan' genomes into the curriculum at Pacific University. The classes incorporating the JGI IMG system and bioinformatics analyses include Microbiology, Genetics, Molecular Biology, and Bioinformatics, taught by a variety of faculty members. Because of this, the topics of genomics and bioinformatics are becoming familiar to a wide range of students at Pacific University, which enrolls a relatively high proportion of Pacific Islanders, an under-represented group in STEM disciplines. Since students from non-biology majors also take these courses, the new curriculum is introducing a broader section of the public to genomic science.
