Microbial Nitrogen cycling in Nevada Geothermal Springs

Hot spring habitats above maximum photosynthetic temperature (73 ºC) are not well understood with respect to nitrogen (N) cycling. Few predictions have been made, and even fewer measurements of in situ activities have been reported. Thermodynamic calculations based on in situ chemical and temperature measurements will be used to predict the occurrence of the specific N-cycling reactions. In addition, these measurements in two springs will aid in an attempt to cultivate ammonia oxidizing species

Novel thermophilic cellulolytic isolates belonging to the phylum Chloroflexi

Current biofuel technologies utilize valuable foodstuffs, such as corn kernels and cane sugar, as sources of easily metabolized sugars. Microbes are used to ferment these sugars into bioethanol, a first-generation biofuel. However, in order to avoid diverting foodstuffs from the food supply, the development of second-generation biofuels technology is necessary. Second-generation biofuels are produced by converting structurally complex lignocellulosic biomass, such as agricultural and municipal wastes, to fermentable sugars or directly to biofuels. The major technological hurdle limiting the mass production of second-generation biofuels is the difficulty in efficiently converting structurally complex lignocellulosic materials to fermentable sugars or directly to biofuels. The discovery of novel thermophilic microorganisms and enzymes that have high activities or broad substrate ranges on plant polymers addresses this challenge

A Spatial and temporal analysis of microbial communities in Great Boiling Spring, Nevada, U.S.A.

Great Boiling Spring (GBS) is a large, circumneutral, long residence time geothermal spring in the US Great Basin. Twelve samples were taken from four different sediment sites and the planktonic community in the bulk water of GBS on up to four different dates. Microbial community composition and diversity was assessed by using a barcoded, improved universal primer set targeting the V8 portion of the 16S rRNA gene and PCR. Over 200,000 products were sequenced using the Roche 454 GS FLX Titanium System. Sediment and planktonic microbial communities were distinct with very little overlap, regardless of the sampling location or temperature. Planktonic communities were extremely uneven and were dominated by a single phylotype related to Thermocrinis in the Aquificales. Benthic microbial communities grouped according to temperature and sampling location. Two locations, Site A (80-87°C) and Site B (79°C), were predominantly composed of the crenarchaeal class Thermoprotei, the novel archaeal lineage pSL4, and the novel bacterial lineage GAL35. Populations of the ammonia oxidizing archaeon “Candidatus Nitrosocaldus yellowstonii” comprised 5-15% of all samples when Site A was cooler than normal (80°C) and at cooler sites throughout the spring (76-62°C). At cooler temperature sites (76-62°C), the phylum-level diversity and evenness were significantly higher, and bacteria made up a significantly higher percentage of the population. To our knowledge, this is the most detailed study of the spatial and temporal variation in any geothermal spring. The study underscores the distinctness of planktonic and benthic communities and the importance of temperature in driving the spatial variation of microbial phylotypes throughout the mineralogically homogenous source pool. 8

Nitrogen cycle in Great Basin hot springs

Nitrification and denitrification are two important steps in the nitrogen cycle . Nitrification, a two step process, leads to the production of NO3-, (Fig. 1). In the first step, ammonia oxidation, NH3 is oxidized to NO2-, and in the second step, nitrite oxidation, NO2- is oxidized to NO3-. Until recently, very little was know about nitrification in high temperature environments. However, in 2008 a thermophilic archaeon, named “Candidatus Nitrosocaldus yellowstonii”, was shown to mediate ammonia oxidation up to 74°C. More recently , NO2- oxidizing bacteria were discovered that are active in temperatures up to 48°C(4). While NH3 oxidation is generally considered to be the rate limiting step, this may not be the case at high temperatures since accumulation of NO2- has been reported in some hot springs where NH3 is the dominant form of inorganic nitrogen (1)

Isolation of Diverse Members of the Aquificales from Geothermal Springs in Tengchong, China

The order Aquificales (phylum Aquificae) consists of thermophilic and hyperthermophilic bacteria that are prominent in many geothermal systems, including those in Tengchong, Yunnan Province, China. However, Aquificales have not previously been isolated from Tengchong. We isolated five strains of Aquificales from diverse springs (temperature 45.2–83.3°C and pH 2.6–9.1) in the Rehai Geothermal Field from sites in which Aquificales were abundant. Phylogenetic analysis showed that four of the strains belong to the genera Hydrogenobacter, Hydrogenobaculum, andSulfurihydrogenibium, including strains distant enough to likely justify new species ofHydrogenobacter and Hydrogenobaculum. The additional strain may represent a new genus in theHydrogenothermaceae. All strains were capable of aerobic respiration under microaerophilic conditions; however, they had variable capacity for chemolithotrophic oxidation of hydrogen and sulfur compounds and nitrate reduction

Exploring diversity of Nitrate reducing thermophiles in Nevada hot springs

High rates of denitrification have been measured in Nevada geothermal hot springs, but little is known about the thermophiles that contribute to this activity. We hypothesize that heterotrophic bacteria in the genus Thermus are the most important denitrifiers in the springs. Alternatively, other microorganisms including chemolithotrophs may also be important. To test these hypotheses, several different strategies will be used to try to enrich and isolate nitrate-reducing microorganisms. Isolates will be identified by 16S rRNA gene PCR and sequencing. Subsequently, representative isolates will be chosen for nitrate reductase gene (narG) sequencing and for studies on the kinetics of nitrate reduction at high temperature. These data will provide information on how these microorganisms may behave in situ and how their activities may affect nitrogen cycling in the hot springs

Family-level sampling of mitochondrial genomes in coleoptera: compositional heterogeneity and phylogenetics

Mitochondrial genomes are readily sequenced with recent technology and thus evolutionary lineages can be sampled more densely. This permits better phylogenetic estimates and assessment of potential biases resulting from heterogeneity in nucleotide composition and rate of change. We gathered 245 mitochondrial sequences for the Coleoptera representing all 4 suborders, 15 superfamilies of Polyphaga, and altogether 97 families, including 159 newly sequenced full or partial mitogenomes. Compositional heterogeneity greatly affected 3rd codon positions, and to a lesser extent the 1st and 2nd positions, even after RY coding. Heterogeneity also affected the encoded protein sequence, in particular in the nad2, nad4, nad5 and nad6 genes. Credible tree topologies were obtained with the nhPhyML (‘non-homogeneous’) algorithm implementing a model for branch-specific equilibrium frequencies. Likelihood searches using RAxML were improved by data partitioning by gene and codon position. Finally, the PhyloBayes software, which allows different substitution processes for amino acid replacement at various sites, produced a tree that best matched known higher-level taxa and defined basal relationships in Coleoptera. After rooting with Neuropterida outgroups, suborder relationships were resolved as (Polyphaga (Myxophaga (Archostemata + Adephaga))). The infraorder relationships in Polyphaga were (Scirtiformia (Elateriformia (Staphyliniformia + Scarabaeiformia (Bostrichiformia (Cucujiformia)))). Polyphagan superfamilies were recovered as monophyla except Staphylinoidea (paraphyletic for Scarabaeiformia) and Cucujoidea, which can no longer be considered a valid taxon. The study shows that, whilst compositional heterogeneity is not universal, it cannot be eliminated for some mitochondrial genes, but dense taxon sampling and the use of appropriate Bayesian analyses can still produce robust phylogenetic trees

Researching nitrite oxidation at high temperatures

15N-nitrate (NO3 -) pool dilution experiments show that ammonia (NH3) is oxidized to nitrate in geothermal springs up to at least 85C; however, nitrite (NO2 -)- oxidizing microorganisms are only known to grow up to 66°C. We hypothesize that thermophilic microorganisms oxidize nitrite to nitrate at high temperatures. Alternatively, it is possible that nitrite is oxidized abiotically. We propose to test these hypotheses by setting up microbial enrichments designed to grow thermophilic nitrite oxidizing bacteria by varying incubation temperature (50, 65, 80°C), oxygen concentration (20% and 5%), and cultivation media. A negative control consisting of filtered spring water (0.1 μm) will be used to determine whether nitrite is oxidized abiotically. Enrichments will be monitored for nitrite oxidation activity by using colorimetric assays for nitrite and nitrate. Enrichments showing activity will be used as a source to try to isolate and/or identify responsible microorganisms and to study the kinetics of nitrite oxidation at high temperature

Genomic foundations of carbon fixation in bacteria living in hot springs

Photosynthesis does not occur above 73°C, so organisms living above this temperature must obtain useable carbon by some other mechanism. It is generally assumed that carbon is fixed by thermophiles through the process of chemolithoautotrophy; however, primary production has never been demonstrated to occur in hot springs \u3e73°C. We have shown that two organisms, Thermocrinis and Pyrobaculum, make up more than 90% of the cells in an 80°C Great Basin hot spring, Great Boiling Spring. We hypothesize that these organisms fix carbon in the hot spring via the reverse tricarboxylic acid (rTCA) cycle. To test this hypothesis we will: i) confirm that Thermocrinis and Pyrobaculum dominate in water from the spring; ii) determine whether key genes for the rTCA cycle, citryl co-A lyase (ccl), 2-oxoglutarate:ferredoxin oxidoreductase (korA), pyruvate:ferredoxin oxidoreductase (porA), are present and expressed in the spring; and iii) measure rates of carbon fixation in the spring. Linkage of the genetic data with carbon fixation rate data may help to provide an image of carbon fixation and cycling in Great Basin hot springs

Genome-wide repeat dynamics reflect phylogenetic distance in closely related allotetraploid Nicotiana (Solanaceae)

Nicotiana sect. Repandae is a group of four allotetraploid species originating from a single allopolyploidisation event approximately 5 million years ago. Previous phylogenetic analyses support the hypothesis of N. nudicaulis as sister to the other three species. This is concordant with changes in genome size, separating those with genome downsizing (N. nudicaulis) from those with genome upsizing (N. repanda, N. nesophila, N. stocktonii). However, a recent analysis reflecting genome dynamics of different transposable element families reconstructed greater similarity between N. nudicaulis and the Revillagigedo Island taxa (N. nesophila and N. stocktonii), thereby placing N. repanda as sister to the rest of the group. This could reflect a different phylogenetic hypothesis or the unique evolutionary history of these particular elements. Here we re-examine relationships in this group and investigate genome-wide patterns in repetitive DNA, utilising high-throughput sequencing and a genome skimming approach. Repetitive DNA clusters provide support for N. nudicaulis as sister to the rest of the section, with N. repanda sister to the two Revillagigedo Island species. Clade-specific patterns in the occurrence and abundance of particular repeats confirm the original (N. nudicaulis (N. repanda (N. nesophila ? N. stocktonii))) hypothesis. Furthermore, overall repeat dynamics in the island species N. nesophila and N. stocktonii confirm their similarity to N. repanda and the distinctive patterns between these three species and N. nudicaulis. Together these results suggest that broad-scale repeat dynamics do in fact reflect evolutionary history and could be predicted based on phylogenetic distance