Importing statistical measures into Artemis enhances gene identification in the <Emphasis Type="Italic">Leishmania</Emphasis> genome project

Background

Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces.

Results

Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODON USAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence.

Conclusion

An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

     

Decadal warming causes a consistent and persistent shift from heterotrophic to autotrophic respiration in contrasting permafrost ecosystems

Soil carbon in permafrost ecosystems has the potential to become a major positive feedback to climate change if permafrost thaw increases heterotrophic decomposition. However, warming can also stimulate autotrophic production leading to increased ecosystem carbon storage—a negative climate change feedback. Few studies partitioning ecosystem respiration examine decadal warming effects or compare responses among ecosystems. Here, we first examined how 11 years of warming during different seasons affected autotrophic and heterotrophic respiration in a bryophyte‐dominated peatland in Abisko, Sweden. We used natural abundance radiocarbon to partition ecosystem respiration into autotrophic respiration, associated with production, and heterotrophic decomposition. Summertime warming decreased the age of carbon respired by the ecosystem due to increased proportional contributions from autotrophic and young soil respiration and decreased proportional contributions from old soil. Summertime warming's large effect was due to not only warmer air temperatures during the growing season, but also to warmer deep soils year‐round. Second, we compared ecosystem respiration responses between two contrasting ecosystems, the Abisko peatland and a tussock‐dominated tundra in Healy, Alaska. Each ecosystem had two different timescales of warming (<5 years and over a decade). Despite the Abisko peatland having greater ecosystem respiration and larger contributions from heterotrophic respiration than the Healy tundra, both systems responded consistently to short‐ and long‐term warming with increased respiration, increased autotrophic contributions to ecosystem respiration, and increased ratios of autotrophic to heterotrophic respiration. We did not detect an increase in old soil carbon losses with warming at either site. If increased autotrophic respiration is balanced by increased primary production, as is the case in the Healy tundra, warming will not cause these ecosystems to become growing season carbon sources. Warming instead causes a persistent shift from heterotrophic to more autotrophic control of the growing season carbon cycle in these carbon‐rich permafrost ecosystems.     

Fungal Diversity in Permafrost and Tallgrass Prairie Soils under Experimental Warming Conditions

Soil fungi play a major role in terrestrial ecosystem functioning through interactions with soil structure, plants, micro- and mesofauna, and nutrient cycling through predation, pathogenesis, mutualistic, and saprotrophic roles. The diversity of soil fungi was assessed by sequencing their 28S rRNA gene in Alaskan permafrost and Oklahoma tallgrass prairie soils at experimental sites where the effect of climate warming is under investigation. A total of 226,695 reads were classified into 1,063 genera, covering 62% of the reference data set. Using the Bayesian Classifier offered by the Ribosomal Database Project (RDP) with 50% bootstrapping classification confidence, approximately 70% of sequences were returned as “unclassified” at the genus level, although the majority (∼65%) were classified at the class level, which provided insight into these lesser-known fungal lineages. Those unclassified at the genus level were subjected to BLAST analysis against the ARB-SILVA database, where ∼50% most closely matched nonfungal taxa. Compared to the more abundant sequences, a higher proportion of rare operational taxonomic units (OTU) were successfully classified to genera at 50% bootstrap confidence, indicating that the fungal rare biosphere in these sites is not composed of sequencing artifacts. There was no significant effect after 1 year of warming on the fungal community structure at both sites, except perhaps for a few minor members, but there was a significant effect of sample depth in the permafrost soils. Despite overall significant community structure differences driven by variations in OTU dominance, the prairie and permafrost soils shared 90% and 63% of all fungal sequences, respectively, indicating a fungal “seed bank” common between both sites.     

Nitrogen Isotope Patterns in Alaskan Black Spruce Reflect Organic Nitrogen Sources and the Activity of Ectomycorrhizal Fungi

Global patterns in soil, plant, and fungal stable isotopes of N (δ¹⁵N) show promise as integrated metrics of N cycling, particularly the activity of ectomycorrhizal (ECM) fungi. At small spatial scales, however, it remains difficult to differentiate the underlying causes of plant δ¹⁵N variability and this limits the application of such measurements to better understand N cycling. We conducted a landscape-scale analysis of δ¹⁵N values from 31 putatively N-limited monospecific black spruce (Picea mariana) stands in central Alaska to assess the two main hypothesized sources of plant δ¹⁵N variation: differing sources and ECM fractionation. We found roughly 20% of the variability in black spruce foliar N and δ¹⁵N values to be correlated with the concentration and δ¹⁵N values of soil NH₄ ⁺ and dissolved organic N (DON) pools, respectively. However, ¹⁵N-based mixing models from 24 of the stands suggested that fractionation by ECM fungi obscures the ¹⁵N signature of soil N pools. Models, regressions, and N abundance data all suggested that increasing dependence on soil DON to meet black spruce growth demands predicates increasing reliance on ECM-derived N and that black spruce, on average, received 53% of its N from ECM fungi. Future research should partition the δ¹⁵N values within the soil DON pool to determine how choice of soil δ¹⁵N values influence modeled ECM activity. The C balance of boreal forests is tightly linked to N cycling and δ¹⁵N values may be useful metrics of changes to these connections.     

Characterization of 13 newly isolated strains of anaerobic, cellulolytic, thermophilic bacteria

Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have several positive implications with respect to future development of a transformation system for cellulolytic thermophiles. Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280. Received 12 September 2000/ Accepted in revised form 20 November 2000     

Nutrient Addition Prompts Rapid Destabilization of Organic Matter in an Arctic Tundra Ecosystem

Nutrient availability in the arctic is expected to increase in the next century due to accelerated decomposition associated with warming and, to a lesser extent, increased nitrogen deposition. To explore how changes in nutrient availability affect ecosystem carbon (C) cycling, we used radiocarbon to quantify changes in belowground C dynamics associated with long-term fertilization of graminoid-dominated tussock tundra at Toolik Lake, Alaska. Since 1981, yearly fertilization with nitrogen (N) and phosphorus (P) has resulted in a shift to shrub-dominated vegetation. These combined changes have altered the quantity and quality of litter inputs, the vertical distribution and dynamics of fine roots, and the decomposition rate of soil organic C. The loss of C from the deep organic and mineral soil has more than offset the C accumulation in the litter and upper organic soil horizons. In the litter and upper organic horizons, radiocarbon measurements show that increased inputs resulted in overall C accumulation, despite being offset by increased decomposition in some soil pools. To reconcile radiocarbon observations in the deeper organic and mineral soil layers, where most of the ecosystem C loss occurred, both a decrease in input of new root material and a dramatic increase of decomposition rates in centuries-old soil C pools were required. Therefore, with future increases in nutrient availability, we may expect substantial losses of C which took centuries to accumulate.