Geographic and Ecological Diversity of Green Sulfur Bacteria in Hot Spring Mat Communities.

Three strains of thermophilic green sulfur bacteria (GSB) are known; all are from microbial mats in hot springs in Rotorua, New Zealand (NZ) and belong to the species Chlorobaculum tepidum. Here, we describe diverse populations of GSB inhabiting Travel Lodge Spring (TLS) (NZ) and hot springs ranging from 36.1 °C to 51.1 °C in the Republic of the Philippines (PHL) and Yellowstone National Park (YNP), Wyoming, USA. Using targeted amplification and restriction fragment length polymorphism analysis, GSB 16S rRNA sequences were detected in mats in TLS, one PHL site, and three regions of YNP. GSB enrichments from YNP and PHL mats contained small, green, nonmotile rods possessing chlorosomes, chlorobactene, and bacteriochlorophyll c. Partial 16S rRNA gene sequences from YNP, NZ, and PHL mats and enrichments from YNP and PHL samples formed distinct phylogenetic clades, suggesting geographic isolation, and were associated with samples differing in temperature and pH, suggesting adaptations to these parameters. Sequences from enrichments and corresponding mats formed clades that were sometimes distinct, increasing the diversity detected. Sequence differences, monophyly, distribution patterns, and evolutionary simulation modeling support our discovery of at least four new putative moderately thermophilic Chlorobaculum species that grew rapidly at 40 °C to 44 °C.

The Vitamin D Metabolite Ratio (VMR) is a Biomarker of Vitamin D Status That is Not Affected by Acute Changes in Vitamin D Binding Protein.

BACKGROUND: 25-hydroxyvitamin D[25(OH)D] may be a poor marker of vitamin D status due to variability in levels of vitamin D binding protein (VDBP). The vitamin D metabolite ratio (VMR) is the ratio of 24,25-dihydroxyvitamin D[24,25(OH)2D3] to 25(OH)D3 and has been postulated to reflect vitamin D sufficiency independent of variability in VDBP. Therapeutic plasma exchange (TPE) is a procedure that removes plasma, including VDBP, and may lower bound vitamin D metabolite concentrations. Effects of TPE on the VMR are unknown. METHODS: We measured 25(OH)D, free 25(OH)D, 1,25-dihydroxyvitamin D[1,25(OH)2D], 24,25(OH)2D3, and VDBP in persons undergoing TPE, before and after treatment. We used paired t-tests to assess changes in these biomarkers during a TPE procedure. RESULTS: Study participants (n = 45) had a mean age of 55 ± 16 years; 67% were female; and 76% were white. Compared to pretreatment concentrations, TPE caused a significant decrease in total VDBP by 65% (95%CI 60,70%), as well as all the vitamin D metabolites-25(OH)D by 66% (60%,74%), free 25(OH)D by 31% (24%,39%), 24,25(OH)2D3 by 66% (55%,78%) and 1,25(OH)2D by 68% (60%,76%). In contrast, there was no significant change in the VMR before and after a single TPE treatment, with an observed mean 7% (-3%, 17%) change in VMR. CONCLUSIONS: Changes in VDBP concentration across TPE parallel changes in 25(OH)D, 1,25(OH)2D, and 24,25(OH)2D3, suggesting that concentrations of these metabolites reflect underlying VDBP concentrations. The VMR is stable across a TPE session despite a 65% reduction in VDBP. These findings suggest that the VMR is a marker of vitamin D status independent of VDBP levels.

Identification of a Functionally Efficient and Thermally Stable Outward Sodium-Pumping Rhodopsin (BeNaR) from a Thermophilic Bacterium.

Rhodopsins are transmembrane proteins with retinal chromophores that are involved in photo-energy conversion and photo-signal transduction in diverse organisms. In this study, we newly identified and characterized a rhodopsin from a thermophilic bacterium, Bellilinea sp. Recombinant Escherichia coli cells expressing the rhodopsin showed light-induced alkalization of the medium only in the presence of sodium ions (Na+), and the alkalization signal was enhanced by addition of a protonophore, indicating an outward Na+ pump function across the cellular membrane. Thus, we named the protein Bellilinea Na+-pumping rhodopsin, BeNaR. Of note, its Na+-pumping activity is significantly greater than that of the known Na+-pumping rhodopsin, KR2. We further characterized its photochemical properties as follows: (i) Visible spectroscopy and HPLC revealed that BeNaR has an absorption maximum at 524 nm with predominantly (>96%) the all-trans retinal conformer. (ii) Time-dependent thermal denaturation experiments revealed that BeNaR showed high thermal stability. (iii) The time-resolved flash-photolysis in the nanosecond to millisecond time domains revealed the presence of four kinetically distinctive photointermediates, K, L, M and O. (iv) Mutational analysis revealed that Asp101, which acts as a counterion, and Asp230 around the retinal were essential for the Na+-pumping activity. From the results, we propose a model for the outward Na+-pumping mechanism of BeNaR. The efficient Na+-pumping activity of BeNaR and its high stability make it a useful model both for ion transporters and optogenetics tools.

Therapeutic plasma exchange in the intensive care unit: Rationale, special considerations, and techniques for combined circuits.

Therapeutic plasma exchange (TPE) is an extracorporeal blood purification technique with proven efficacy in a variety of conditions, including in the intensive care setting. It is not uncommon for a critically ill patient to require more than one extracorporeal procedure in addition to TPE. This review focuses on the combination of TPE with other extracorporeal circuits in a critical care setting via a single vascular access (either in-series, parallel, or a hybrid mode) which is often referred to as performing procedures "in tandem." Authors performed literature review via pubmed.gov using search terms: plasma exchange, plasmapheresis, apheresis, tandem circuits, combined circuits, critical care, ICU, CRRT, hemodialysis, and ECMO. Thirty-eight English-language, peer-reviewed papers were appraised that satisfied the content of this review on techniques for combining circuits with plasma exchange, as well as describing the advantages of tandem procedures and potential complications that can arise. Performing these procedures simultaneously can be advantageous in reducing total procedure and staffing time, avoiding placement of additional central lines, reducing overall need for anticoagulation, and limiting multiple blood primes in certain populations. However, the described combined circuits are complex, associated with higher complications, and require a skilled team to understand and mitigate the potential complications associated with these combined procedures.

Allochromatium tepidum, sp. nov., a hot spring species of purple sulfur bacteria.

We describe a new species of purple sulfur bacteria (Chromatiaceae, anoxygenic phototrophic bacteria) isolated from a microbial mat in the sulfidic geothermal outflow of a hot spring in Rotorua, New Zealand. This phototroph, designated as strain NZ, grew optimally near 45 °C but did not show an absorption maximum at 915 nm for the light-harvesting-reaction center core complex (LH1-RC) characteristic of other thermophilic purple sulfur bacteria. Strain NZ had a similar carotenoid composition as Thermochromatium tepidum, but unlike Tch. tepidum, grew photoheterotrophically on acetate in the absence of sulfide and metabolized thiosulfate. The genome of strain NZ was significantly larger than that of Tch. tepidum but slightly smaller than that of Allochromatium vinosum. Strain NZ was phylogenetically more closely related to mesophilic purple sulfur bacteria of the genus Allochromatium than to Tch. tepidum. This conclusion was reached from phylogenetic analyses of strain NZ genes encoding 16S rRNA and the photosynthetic functional gene pufM, from phylogenetic analyses of entire genomes, and from a phylogenetic tree constructed from the concatenated sequence of 1090 orthologous proteins. Moreover, average nucleotide identities and digital DNA:DNA hybridizations of the strain NZ genome against those of related species of Chromatiaceae supported the phylogenetic analyses. From this collection of properties, we describe strain NZ here as the first thermophilic species of the genus Allochromatium, Allochromatium tepidum NZT, sp. nov.

Relationship between Microorganisms Inhabiting Alkaline Siliceous Hot Spring Mat Communities and Overflowing Water.

The compositions of Octopus Spring and Mushroom Spring (Yellowstone National Park, Wyoming, USA) microbial mats have been thoroughly studied, but the compositions of the effluent waters that flow above the mats have not. In this study, cells in the mats and overflowing waters of both springs were investigated at multiple sites where Synechococcus spp. are the dominant cyanobacteria (ca. 72°C to ca. 50°C), and on several dates. In addition to microscopic analyses of stained and autofluorescent cells, 16S rRNA gene sequencing was used to characterize the major taxa present and a protein-encoding gene (psaA) was sequenced and analyzed by ecotype simulation to predict species of Synechococcus The mats of both springs were similar in terms of the downstream distribution of predominant taxa detected previously. However, waters above these mats were predominated by taxa that reside in upstream mats or communities above the upper-temperature limit of the mat. A disturbance/recolonization study was performed at a site normally predominated by Synechococcus species adapted to low temperatures. After removing indigenous Synechococcus cells, Synechococcus species adapted to higher temperatures, which were predominant in the water overflowing this site, colonized the newly forming mat. Differences in recolonization under reduced and UV-screened irradiance suggested that, in addition to physical transport, environmental conditions likely select for species that are better adapted to these different conditions and can influence mat recovery. A transport model was developed and used to predict that, in Mushroom Spring, erosion predominates in the narrower and deeper upstream effluents and deposition predominates over erosion in wider and shallower downstream effluents.IMPORTANCE In flowing aquatic systems, cell erosion and deposition are important to the dispersal of cells from one location to another. Very little is known about microbial dispersal and the physical processes that underlie it. This study demonstrates its importance to colonization of downstream surfaces and especially to the recolonization and functioning of disturbed sites. Ecological systems in flowing environments are often, roughly speaking, pseudosteady, in that nutrients enter the system and by-products leave at relatively steady rates. Over time, material inputs and outputs must balance. Measurements of input fluxes (e.g., growth rates and proxies, such as photosynthesis rates) are frequent. However, erosion and deposition of cells are seldom measured and ecological significance is sometimes neglected. The importance of these parameters is immediately evident in any attempt to construct a model of long-time community behavior, as spatial ecological structure is significantly impacted and can be dominated by migration of organisms, even in small numbers.

Biogeography of American Northwest Hot Spring A/B'-Lineage Synechococcus Populations.

Previous analyses have shown how diversity among unicellular cyanobacteria inhabiting island-like hot springs is structured relative to physical separation and physiochemical differences among springs, especially at local to regional scales. However, these studies have been limited by the low resolution provided by the molecular markers surveyed. We analyzed large datasets obtained by high-throughput sequencing of a segment of the photosynthesis gene psaA from samples collected in hot springs from geothermal basins in Yellowstone National Park, Montana, and Oregon, all known from previous studies to contain populations of A/B'-lineage Synechococcus. The fraction of identical sequences was greater among springs separated by <50 km than among springs separated by >50 km, and springs separated by >800 km shared sequence variants only rarely. Phylogenetic analyses provided evidence for endemic lineages that could be related to geographic isolation and/or geochemical differences on regional scales. Ecotype Simulation 2 was used to predict putative ecotypes (ecologically distinct populations), and their membership, and canonical correspondence analysis was used to examine the geographical and geochemical bases for variation in their distribution. Across the range of Oregon and Yellowstone, geographical separation explained the largest percentage of the differences in distribution of ecotypes (9.5% correlated to longitude; 9.4% to latitude), with geochemical differences explaining the largest percentage of the remaining differences in distribution (7.4-9.3% correlated to magnesium, sulfate, and sulfide). Among samples within the Greater Yellowstone Ecosystem, geochemical differences significantly explained the distribution of ecotypes (6.5-9.3% correlated to magnesium, boron, sulfate, silicon dioxide, chloride, and pH). Nevertheless, differences in the abundance and membership of ecotypes in Yellowstone springs with similar chemistry suggested that allopatry may be involved even at local scales. Synechococcus populations have diverged both by physical isolation and physiochemical differences, and populations on surprisingly local scales have been evolving independently.

Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat.

The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria (Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (Ci), but some evidence suggests that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of Ci incorporation into different taxa, mat samples were incubated with [13C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of 13C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with 13C. Most 13C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins involved in photosynthesis, Ci fixation, and N2 fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for Ci fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to Ci fixation. Other taxa showed much lower [13C]bicarbonate incorporation.IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven Ci fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in Ci fixation. Because this pathway imparts a lower degree of selection of isotopically heavy Ci than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of 13C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the 13C/12C ratios of mat biomass will help geochemists interpret the 13C/12C ratios of organic carbon in the fossil record.

"Candidatus Thermonerobacter thiotrophicus," A Non-phototrophic Member of the Bacteroidetes/Chlorobi With Dissimilatory Sulfur Metabolism in Hot Spring Mat Communities.

In this study we present evidence for a novel, thermophilic bacterium with dissimilatory sulfur metabolism, tentatively named "Candidatus Thermonerobacter thiotrophicus," which is affiliated with the Bacteroides/Ignavibacteria/Chlorobi and which we predict to be a sulfate reducer. Dissimilatory sulfate reduction (DSR) is an important and ancient metabolic process for energy conservation with global importance for geochemical sulfur and carbon cycling. Characterized sulfate-reducing microorganisms (SRM) are found in a limited number of bacterial and archaeal phyla. However, based on highly diverse environmental dsrAB sequences, a variety of uncultivated and unidentified SRM must exist. The recent development of high-throughput sequencing methods allows the phylogenetic identification of some of these uncultured SRM. In this study, we identified a novel putative SRM inhabiting hot spring microbial mats that is a member of the OPB56 clade ("Ca. Kapabacteria") within the Bacteroidetes/Chlorobi superphylum. Partial genomes for this new organism were retrieved from metagenomes from three different hot springs in Yellowstone National Park, United States, and Japan. Supporting the prediction of a sulfate-reducing metabolism for this organism during period of anoxia, diel metatranscriptomic analyses indicate highest relative transcript levels in situ for all DSR-related genes at night. The presence of terminal oxidases, which are transcribed during the day, further suggests that these organisms might also perform aerobic respiration. The relative phylogenetic proximity to the sulfur-oxidizing, chlorophototrophic Chlorobi further raises new questions about the evolution of dissimilatory sulfur metabolism.

The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. II. Metabolic Functions of Abundant Community Members Predicted from Metagenomic Analyses.

Microbial mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin of Yellowstone National Park have been extensively characterized. Previous studies have focused on the chlorophototrophic organisms of the phyla Cyanobacteria and Chloroflexi. However, the diversity and metabolic functions of the other portion of the community in the microoxic/anoxic region of the mat are poorly understood. We recently described the diverse but extremely uneven microbial assemblage in the undermat of Mushroom Spring based on 16S rRNA amplicon sequences, which was dominated by Roseiflexus members, filamentous anoxygenic chlorophototrophs. In this study, we analyzed the orange-colored undermat portion of the community of Mushroom Spring mats in a genome-centric approach and discuss the metabolic potentials of the major members. Metagenome binning recovered partial genomes of all abundant community members, ranging in completeness from ~28 to 96%, and allowed affiliation of function with taxonomic identity even for representatives of novel and Candidate phyla. Less complete metagenomic bins correlated with high microdiversity. The undermat portion of the community was found to be a mixture of phototrophic and chemotrophic organisms, which use bicarbonate as well as organic carbon sources derived from different cell components and fermentation products. The presence of rhodopsin genes in many taxa strengthens the hypothesis that light energy is of major importance. Evidence for the usage of all four bacterial carbon fixation pathways was found in the metagenome. Nitrogen fixation appears to be limited to Synechococcus spp. in the upper mat layer and Thermodesulfovibrio sp. in the undermat, and nitrate/nitrite metabolism was limited. A closed sulfur cycle is indicated by biological sulfate reduction combined with the presence of genes for sulfide oxidation mainly in phototrophs. Finally, a variety of undermat microorganisms have genes for hydrogen production and consumption, which leads to the observed diel hydrogen concentration patterns.

The Dark Side of the Mushroom Spring Microbial Mat: Life in the Shadow of Chlorophototrophs. I. Microbial Diversity Based on 16S rRNA Gene Amplicons and Metagenomic Sequencing.

Microbial-mat communities in the effluent channels of Octopus and Mushroom Springs within the Lower Geyser Basin at Yellowstone National Park have been studied for nearly 50 years. The emphasis has mostly focused on the chlorophototrophic bacterial organisms of the phyla Cyanobacteria and Chloroflexi. In contrast, the diversity and metabolic functions of the heterotrophic community in the microoxic/anoxic region of the mat are not well understood. In this study we analyzed the orange-colored undermat of the microbial community of Mushroom Spring using metagenomic and rRNA-amplicon (iTag) analyses. Our analyses disclosed a highly diverse community exhibiting a high degree of unevenness, strongly dominated by a single taxon, the filamentous anoxygenic phototroph, Roseiflexus spp. The second most abundant organisms belonged to the Thermotogae, which have been hypothesized to be a major source of H2 from fermentation that could enable photomixotrophic metabolism by Chloroflexus and Roseiflexus spp. Other abundant organisms include two members of the Armatimonadetes (OP10); Thermocrinis sp.; and phototrophic and heterotrophic members of the Chloroflexi. Further, an Atribacteria (OP9/JS1) member; a sulfate-reducing Thermodesulfovibrio sp.; a Planctomycetes member; a member of the EM3 group tentatively affiliated with the Thermotogae, as well as a putative member of the Arminicenantes (OP8) represented ≥1% of the reads. Archaea were not abundant in the iTag analysis, and no metagenomic bin representing an archaeon was identified. A high microdiversity of 16S rRNA gene sequences was identified for the dominant taxon, Roseiflexus spp. Previous studies demonstrated that highly similar Synechococcus variants in the upper layer of the mats represent ecological species populations with specific ecological adaptations. This study suggests that similar putative ecotypes specifically adapted to different niches occur within the undermat community, particularly for Roseiflexus spp.

In Situ Hydrogen Dynamics in a Hot Spring Microbial Mat during a Diel Cycle.

UNLABELLED: Microbes can produce molecular hydrogen (H2) via fermentation, dinitrogen fixation, or direct photolysis, yet the H2 dynamics in cyanobacterial communities has only been explored in a few natural systems and mostly in the laboratory. In this study, we investigated the diel in situ H2 dynamics in a hot spring microbial mat, where various ecotypes of unicellular cyanobacteria (Synechococcus sp.) are the only oxygenic phototrophs. In the evening, H2 accumulated rapidly after the onset of darkness, reaching peak values of up to 30 µmol H2 liter(-1) at about 1-mm depth below the mat surface, slowly decreasing to about 11 µmol H2 liter(-1) just before sunrise. Another pulse of H2 production, reaching a peak concentration of 46 µmol H2 liter(-1), was found in the early morning under dim light conditions too low to induce accumulation of O2 in the mat. The light stimulation of H2 accumulation indicated that nitrogenase activity was an important source of H2 during the morning. This is in accordance with earlier findings of a distinct early morning peak in N2 fixation and expression of Synechococcus nitrogenase genes in mat samples from the same location. Fermentation might have contributed to the formation of H2 during the night, where accumulation of other fermentation products lowered the pH in the mat to less than pH 6 compared to a spring source pH of 8.3. IMPORTANCE: Hydrogen is a key intermediate in anaerobic metabolism, and with the development of a sulfide-insensitive microsensor for H2, it is now possible to study the microdistribution of H2 in stratified microbial communities such as the photosynthetic microbial mat investigated here. The ability to measure H2 profiles within the mat compared to previous measurements of H2 emission gives much more detailed information about the sources and sinks of H2 in such communities, and it was demonstrated that the high rates of H2 formation in the early morning when the mat was exposed to low light intensities might be explained by nitrogen fixation, where H2 is formed as a by-product.

The molecular dimension of microbial species: 2. Synechococcus strains representative of putative ecotypes inhabiting different depths in the Mushroom Spring microbial mat exhibit different adaptive and acclimative responses to light.

Closely related strains of thermophilic Synechococcus were cultivated from the microbial mats found in the effluent channels of Mushroom Spring, Yellowstone National Park (YNP). These strains have identical or nearly identical 16S rRNA sequences but are representative of separate, predicted putative ecotype (PE) populations, which were identified by using the more highly resolving psaA locus and which predominate at different vertical positions within the 1-mm-thick upper-green layer of the mat. Pyrosequencing confirmed that each strain contained a single, predominant psaA genotype. Strains differed in growth rate as a function of irradiance. A strain with a psaA genotype corresponding to a predicted PE that predominates near the mat surface grew fastest at high irradiances, whereas strains with psaA genotypes representative of predominant subsurface populations grew faster at low irradiance and exhibited greater sensitivity to abrupt shifts to high light. The high-light-adapted and low-light-adapted strains also exhibited differences in pigment content and the composition of the photosynthetic apparatus (photosystem ratio) when grown under different light intensities. Cells representative of the different strains had similar morphologies under low-light conditions, but under high-light conditions, cells of low-light-adapted strains became elongated and formed short chains of cells. Collectively, the results presented here are consistent with the hypothesis that closely related, but distinct, ecological species of Synechococcus occupy different light niches in the Mushroom Spring microbial mat and acclimate differently to changing light environments.

The molecular dimension of microbial species: 1. Ecological distinctions among, and homogeneity within, putative ecotypes of Synechococcus inhabiting the cyanobacterial mat of Mushroom Spring, Yellowstone National Park.

Based on the Stable Ecotype Model, evolution leads to the divergence of ecologically distinct populations (e.g., with different niches and/or behaviors) of ecologically interchangeable membership. In this study, pyrosequencing was used to provide deep sequence coverage of Synechococcus psaA genes and transcripts over a large number of habitat types in the Mushroom Spring microbial mat. Putative ecological species [putative ecotypes (PEs)], which were predicted by an evolutionary simulation based on the Stable Ecotype Model (Ecotype Simulation), exhibited distinct distributions relative to temperature-defined positions in the effluent channel and vertical position in the upper 1 mm-thick mat layer. Importantly, in most cases variants predicted to belong to the same PE formed unique clusters relative to temperature and depth in the mat in canonical correspondence analysis, supporting the hypothesis that while the PEs are ecologically distinct, the members of each ecotype are ecologically homogeneous. PEs responded differently to experimental perturbations of temperature and light, but the genetic variation within each PE was maintained as the relative abundances of PEs changed, further indicating that each population responded as a set of ecologically interchangeable individuals. Compared to PEs that predominate deeper within the mat photic zone, the timing of transcript abundances for selected genes differed for PEs that predominate in microenvironments closer to upper surface of the mat with spatiotemporal differences in light and O2 concentration. All of these findings are consistent with the hypotheses that Synechococcus species in hot spring mats are sets of ecologically interchangeable individuals that are differently adapted, that these adaptations control their distributions, and that the resulting distributions constrain the activities of the species in space and time.

The molecular dimension of microbial species: 3. Comparative genomics of Synechococcus strains with different light responses and in situ diel transcription patterns of associated putative ecotypes in the Mushroom Spring microbial mat.

Genomes were obtained for three closely related strains of Synechococcus that are representative of putative ecotypes (PEs) that predominate at different depths in the 1 mm-thick, upper-green layer in the 60°C mat of Mushroom Spring, Yellowstone National Park, and exhibit different light adaptation and acclimation responses. The genomes were compared to the published genome of a previously obtained, closely related strain from a neighboring spring, and differences in both gene content and orthologous gene alleles between high-light-adapted and low-light-adapted strains were identified. Evidence of genetic differences that relate to adaptation to light intensity and/or quality, CO2uptake, nitrogen metabolism, organic carbon metabolism, and uptake of other nutrients were found between strains of the different putative ecotypes. In situ diel transcription patterns of genes, including genes unique to either low-light-adapted or high-light-adapted strains and different alleles of an orthologous photosystem gene, revealed that expression is fine-tuned to the different light environments experienced by ecotypes prevalent at various depths in the mat. This study suggests that strains of closely related PEs have different genomic adaptations that enable them to inhabit distinct ecological niches while living in close proximity within a microbial community.

Diel metabolomics analysis of a hot spring chlorophototrophic microbial mat leads to new hypotheses of community member metabolisms.

Dynamic environmental factors such as light, nutrients, salt, and temperature continuously affect chlorophototrophic microbial mats, requiring adaptive and acclimative responses to stabilize composition and function. Quantitative metabolomics analysis can provide insights into metabolite dynamics for understanding community response to such changing environmental conditions. In this study, we quantified volatile organic acids, polar metabolites (amino acids, glycolytic and citric acid cycle intermediates, nucleobases, nucleosides, and sugars), wax esters, and polyhydroxyalkanoates, resulting in the identification of 104 metabolites and related molecules in thermal chlorophototrophic microbial mat cores collected over a diel cycle in Mushroom Spring, Yellowstone National Park. A limited number of predominant taxa inhabit this community and their functional potentials have been previously identified through metagenomic and metatranscriptomic analyses and in situ metabolisms, and metabolic interactions among these taxa have been hypothesized. Our metabolomics results confirmed the diel cycling of photorespiration (e.g., glycolate) and fermentation (e.g., acetate, propionate, and lactate) products, the carbon storage polymers polyhydroxyalkanoates, and dissolved gasses (e.g., H2 and CO2) in the waters overlying the mat, which were hypothesized to occur in major mat chlorophototrophic community members. In addition, we have formulated the following new hypotheses: (1) the morning hours are a time of biosynthesis of amino acids, DNA, and RNA; (2) photo-inhibited cells may also produce lactate via fermentation as an alternate metabolism; (3) glycolate and lactate are exchanged among Synechococcus and Roseiflexus spp.; and (4) fluctuations in many metabolite pools (e.g., wax esters) at different times of day result from species found at different depths within the mat responding to temporal differences in their niches.

Draft Genome Sequence of the Deinococcus-Thermus Bacterium Meiothermus ruber Strain A.

The draft genome sequence of the Deinococcus-Thermus group bacterium Meiothermus ruber strain A, isolated from a cyanobacterial enrichment culture obtained from Octopus Spring (Yellowstone National Park, WY), comprises 2,968,099 bp in 170 contigs. It is predicted to contain 2,895 protein-coding genes, 44 tRNA-coding genes, and 2 rRNA operons.

Recombination Does Not Hinder Formation or Detection of Ecological Species of Synechococcus Inhabiting a Hot Spring Cyanobacterial Mat.

Recent studies of bacterial speciation have claimed to support the biological species concept-that reduced recombination is required for bacterial populations to diverge into species. This conclusion has been reached from the discovery that ecologically distinct clades show lower rates of recombination than that which occurs among closest relatives. However, these previous studies did not attempt to determine whether the more-rapidly recombining close relatives within the clades studied may also have diversified ecologically, without benefit of sexual isolation. Here we have measured the impact of recombination on ecological diversification within and between two ecologically distinct clades (A and B') of Synechococcus in a hot spring microbial mat in Yellowstone National Park, using a cultivation-free, multi-locus approach. Bacterial artificial chromosome (BAC) libraries were constructed from mat samples collected at 60°C and 65°C. Analysis of multiple linked loci near Synechococcus 16S rRNA genes showed little evidence of recombination between the A and B' lineages, but a record of recombination was apparent within each lineage. Recombination and mutation rates within each lineage were of similar magnitude, but recombination had a somewhat greater impact on sequence diversity than mutation, as also seen in many other bacteria and archaea. Despite recombination within the A and B' lineages, there was evidence of ecological diversification within each lineage. The algorithm Ecotype Simulation identified sequence clusters consistent with ecologically distinct populations (ecotypes), and several hypothesized ecotypes were distinct in their habitat associations and in their adaptations to different microenvironments. We conclude that sexual isolation is more likely to follow ecological divergence than to precede it. Thus, an ecology-based model of speciation appears more appropriate than the biological species concept for bacterial and archaeal diversification.