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Bacterial lipids are well-preserved in ancient rocks and certain ones have been used as indicators of specific bacterial metabolisms or environmental conditions existing at the time of rock deposition. Here we show that an anaerobic bacterium produces 3-methylhopanoids, pentacyclic lipids previously detected only in aerobic bacteria and widely used as biomarkers for methane-oxidizing bacteria. Both Rhodopila globiformis, a phototrophic purple nonsulfur bacterium isolated from an acidic warm spring in Yellowstone, and a newly isolated Rhodopila species from a geochemically similar spring in Lassen Volcanic National Park (USA), synthesized 3-methylhopanoids and a suite of related hopanoids and contained the genes encoding the necessary biosynthetic enzymes. Our results show that 3-methylhopanoids can be produced under anoxic conditions and challenges the use of 3-methylhopanoids as biomarkers of oxic conditions in ancient rocks and as prima facie evidence that methanotrophic bacteria were active when the rocks were deposited. [ABSTRACT FROM AUTHOR]

Summary: Photosynthetic bacteria are abundant in alkaline, terrestrial hot springs and there is a long history of research on phototrophs in Yellowstone National Park (YNP). Hot springs provide a framework to examine the ecophysiology of phototrophs in situ because they provide natural gradients of geochemistry, pH and temperature. Phototrophs within the Cyanobacteria and Chloroflexi groups are frequently observed in alkaline hot springs. Decades of research has determined that temperature constrains Cyanobacteria in alkaline hot springs, but factors that constrain the distribution of phototrophic Chloroflexi remain unresolved. Using a combination of 16S rRNA gene sequencing and photoassimilation microcosms, we tested the hypothesis that temperature would constrain the activity and composition of phototrophic Cyanobacteria and Chloroflexi. We expected diversity and rates of photoassimilation to decrease with increasing temperature. We report 16S rRNA amplicon sequencing along with carbon isotope signatures and photoassimilation from 45 to 72°C in two alkaline hot springs. We find that Roseiflexus, Chloroflexus (Chloroflexi) and Leptococcus (Cyanobacteria) operational taxonomic units (OTUs) have distinct distributions with temperature. This distribution suggests that, like phototrophic Cyanobacteria, temperature selects for specific phototrophic Chloroflexi taxa. The richness of phototrophic Cyanobacteria decreased with increasing temperature along with a decrease in oxygenic photosynthesis, whereas Chloroflexi richness and rates of anoxygenic photosynthesis did not decrease with increasing temperature, even at temperatures approaching the upper limit of photosynthesis (~72–73°C). Our carbon isotopic data suggest an increasing prevalence of the 3‐hydroxypropionate pathway with decreasing temperature coincident with photoautotrophic Chloroflexi. Together these results indicate temperature plays a role in defining the niche space of phototrophic Chloroflexi (as has been observed for Cyanobacteria), but other factors such as morphology, geochemistry, or metabolic diversity of Chloroflexi, in addition to temperature, could determine the niche space of this highly versatile group. [ABSTRACT FROM AUTHOR]

A novel thermophilic, microaerophilic, anoxygenic, and chlorophototrophic member of the phylum Acidobacteria, Chloracidobacterium thermophilum strain BT, was isolated from a cyanobacterial enrichment culture derived from microbial mats associated with Octopus Spring, Yellowstone National Park, Wyoming. C. thermophilum is strictly dependent on light and oxygen and grows optimally as a photoheterotroph at irradiance values between 20 and 50 μmol photons m-2 s-1. C. thermophilum is unable to synthesize branched-chain amino acids (AAs), L-lysine, and vitamin B12, which are required for growth. Although the organism lacks genes for autotrophic carbon fixation, bicarbonate is also required. Mixtures of other AAs and 2-oxoglutarate stimulate growth. As suggested from genomic sequence data, C. thermophilum requires a reduced sulfur source such as thioglycolate, cysteine, methionine, or thiosulfate. The organism can be grown in a defined medium at 51°C (Topt; range 44-58°C) in the pH range 5.5-9.5 (pHopt = ~7.0). Using the defined growth medium and optimal conditions, it was possible to isolate new C. thermophilum strains directly from samples of hot spring mats in Yellowstone National Park, Wyoming. The new isolates differ from the type strain with respect to pigment composition, morphology in liquid culture, and temperature adaptation. [ABSTRACT FROM AUTHOR]

Strain MS-P2T was isolated from microbial mats associated with Mushroom Spring, an alkaline siliceous hot spring in Yellowstone National Park, WY, USA. The isolate grows chemoheterotrophically by oxygen-dependent respiration, and light stimulates photoheterotrophic growth under strictly oxic conditions. Strain MS-P2T synthesizes bacteriochlorophyll a and the carotenoid spirilloxanthin. However, photoautotrophic growth did not occur under oxic or anoxic conditions, suggesting that this strain should be classified as an aerobic anoxygenic phototrophic bacterium. Strain MS-P2T cells are motile, curved rods about 0.5 to 1.0 μm wide and 1.0 to 1.5 μm long. The optimum growth temperature is 45–50 °C, and the optimum pH for growth is circum-neutral (pH 7.0–7.5). Sequence analysis of the 16S rRNA gene revealed that strain MS-P2T is closely related to Elioraea species, members of the class Alphaproteobacteria, with a sequence identity of 96.58 to 98%. The genome of strain MS-P2T is a single circular DNA molecule of 3,367,643 bp with a mol% guanine-plus-cytosine content of 70.6%. Based on phylogenetic, physiological, biochemical, and genomic characteristics, we propose this bacteriochlorophyll a-containing isolate is a new species belonging to the genus Elioraea, with the suggested name Elioraeatepida. The type-strain is strain MS-P2T (= JCM33060T = ATCC TSD-174T). [ABSTRACT FROM AUTHOR]

Environmental gradients are expected to promote the diversification and coexistence of ecological specialists adapted to local conditions. Consistent with this view, genera of phototrophic microorganisms in alkaline geothermal systems generally appear to consist of anciently divergent populations which have specialized on different temperature habitats. At White Creek (Lower Geyser Basin, Yellowstone National Park), however, a novel, 16S rRNA-defined lineage of the filamentous anoxygenic phototroph Chioroflexus (OTU 10, phylum Chloroflexi) occupies a much wider thermal niche than other 16S rRNA-defined groups of phototrophic bacteria. This suggests that Chioroflexus OTU lOis either an ecological generalist or, alternatively, a group of cryptic thermal specialists which have recently diverged. To distinguish between these alternatives, we first isolated laboratory strains of Chioroflexus OTU 10 from along the White Creek temperature gradient. These strains are identical for partial gene sequences encoding the 16S rRNA and malonyl coenzyme A (CoA) reductase. However, strains isolated from upstream and downstream samples could be distinguished based on sequence variation at pcs, which encodes the propionyl-CoA synthase of the 3-hydroxypropionate pathway of carbon fixation used by the genus Chloroflexus. We next demonstrated that strains have diverged in temperature range for growth. Specifically, we obtained evidence for a positive correlation between thermal niche breadth and temperature optimum, with strains isolated from lower temperatures exhibiting greater thermal specialization than the most thermotolerant strain. The study has implications for our understanding of both the process of niche diversification of microorganisms and how diversity is organized in these hot spring communities. [ABSTRACT FROM AUTHOR]

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium ' Candidatus Chloracidobacterium thermophilum' were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats. [ABSTRACT FROM AUTHOR]

Chlorophotoautotrophy, the use of chlorophylls to convert light energy into chemical energy for carbon dioxide fixation, is the primary metabolic process linking the inorganic and organic carbon pools on Earth. To understand the potential effects of various environmental constraints on the evolution of chlorophototrophy better, we studied the distribution, diversity, and abundance of chlorophylls and genes involved in their synthesis along geothermal gradients in Yellowstone National Park, Wyoming. Genes involved in chlorophyll biosynthesis were constrained to temperatures of less than ∼70 °C and were only detected at this elevated temperature when the pH was in the circumneutral to alkaline range. The upper temperature limit for the detection of chlL/ bchL1 and bchY2 decreased systematically with increasingly acidic pH, an observation likely attributable to sulfide, which upon oxidation, generates acidic spring water and reduces the availability of bicarbonate the preferred source of inorganic carbon for phototrophs. Spring pH was also the best predictor of the phylogenetic diversity of chlL/ bchL communities. The phylogenetic similarity of chlL/ bchL genes between sites was significantly correlated with that of chlorophylls. The predominance of chlorophyll a and bacteriochlorophyll a among extracted pigments was consistent with predominance of chlL/ bchL genes affiliated with the Cyanobacteria and Chloroflexiales, respectively, and might be related to the fact that the majority of these organisms are photoautotrophs. Together, these results suggest that a combination of temperature, pH, and/or sulfide influences the distribution, diversity, and evolution of chlorophotrophs and the chlorophylls that they synthesize. [ABSTRACT FROM AUTHOR]

Genes encoding nitrogenase ( nifH) were amplified from sediment and photosynthetic mat samples collected in the outflow channel of Mound Spring, an alkaline thermal feature in Yellowstone National Park. Results indicate the genetic capacity for nitrogen fixation over the entire range of temperatures sampled (57.2°C to 80.2°C). Amplification of environmental nifH transcripts revealed in situ expression of nifH genes at temperatures up to 72.7°C. However, we were unable to amplify transcripts of nifH at the higher-temperature locations (> 72.7°C). These results indicate that microbes at the highest temperature sites contain the genetic capacity to fix nitrogen, yet either do not express nifH or do so only transiently. Field measurements of nitrate and ammonium show fixed nitrogen limitation as temperature decreases along the outflow channel, suggesting nifH expression in response to the downstream decrease in bioavailable nitrogen. Nitrogen stable isotope values of Mound Spring sediment communities further support geochemical and genetic data. DNA and cDNA nifH amplicons form several unique phylogenetic clades, some of which appear to represent novel nifH sequences in both photosynthetic and chemosynthetic microbial communities. This is the first report of in situ nifH expression in strictly chemosynthetic zones of terrestrial (non-marine) hydrothermal systems, and sets a new upper temperature limit (72.7°C) for nitrogen fixation in alkaline, terrestrial hydrothermal environments. [ABSTRACT FROM AUTHOR]

In this study, glass rods suspended at the air-water interface in the runoff channel of Fairy Geyser, Yellowstone National Park, WY, were used as a substratum to promote the development of biofllms that resembled multilayered mat communities in the splash zone at the geyser's source. This approach enabled the establishment of the temporal relationship between the appearance of Cyanobacteria, which ultimately formed the outer green layer, and the development of a red underlayer containing Rose(flexus-like Chioroflexi. This is the first study to define time-dependent successional events involved in the development of differently colored layers within microbial mats associated with many thermal features in Yellowstone National Park. Initial (1-month) biofilms were localized below the air-water interface (60 to 70°C), and the majority of retrieved bacterial sequence types were similar to Synechococcus and Thermus isolates. Biofllms then shifted, becoming established at and above the air-water interface after 3 months. During winter sampling (6 to 8 months), distinct reddish orange microcolonies were observed, consistent with the appearance of Roseiflexus-Iike sequences and bacteriochlorophyll a pigment signatures. Additionally, populations of Cyanobacteria diversified to include both unicellular and filamentous cell and sequence types. Distinct green and red layers were observed at 13 months. Planctomycetes-like sequences were also retrieved in high abundance from final biofllm layers and winter samples. Finally, biomass associated with geyser vent water contained Roseiflexus-like sequence types, in addition to other high-abundance sequence types retrieved from biofllm samples, supporting the idea that geothermal water serves as an inoculum for these habitats. [ABSTRACT FROM AUTHOR]

Alkaline siliceous hot spring microbial mats in Yellowstone National Park are composed of two dominant phototropic groups, cyanobacteria and green non-sulfur-like bacteria (GNSLB). While cyanobacteria are thought to cross-feed low-molecular-weight organic compounds to support photoheterotrophic metabolism in GNSLB, it is unclear how this could lead to the heavier stable carbon isotopic signatures in GNSLB lipids compared with cyanobacterial lipids found in previous studies. The two groups of phototrophs were separated using percoll density gradient centrifugation and subsequent lipid and stable carbon isotopic analysis revealed that we obtained fractions with a ∼60-fold enrichment in cyanobacterial and an approximately twofold enrichment in GNSLB biomass, respectively, compared with the mat itself. This technique was used to study the diel cycling and 13C content of the glucose pools in and the uptake of 13C-bicarbonate by the cyanobacteria and GNSLB, as well as the transfer of incorporated 13C from cyanobacteria to GNSLB. The results show that cyanobacteria have the highest bicarbonate uptake rates and accumulate glucose during the afternoon in full light conditions. In contrast, GNSLB have relatively higher bicarbonate uptake rates compared with cyanobacteria in the morning at low light levels. During the night GNSLB take up carbon that is likely derived through fermentation of cyanobacterial glucose enriched in 13C. The assimilation of 13C-enriched cyanobacterial carbon may thus lead to enriched 13C-contents of GNSLB cell components. [ABSTRACT FROM AUTHOR]

Molecular and culture based methods were used to survey endolithic, photosynthetic communities from hot spring-formed travertine rocks of various ages, ranging from<10 to greater than 300 000 years. Much of this travertine contained a 1–3-mm-thick greenish band composed mainly of cyanobacteria 1–5 mm below the rock surface. The travertine rocks experienced desiccation in summer and freezing in winter. A total of 83 environmental 16S rRNA gene sequences were obtained from clone libraries and denaturing gradient gel electrophoresis. Small subunit rRNA gene sequences and cell morphology were determined for 36 cyanobacterial culture isolates from these samples. Phylogenetic analysis showed that the 16S rRNA gene sequences fell into 15 distinct clusters, including several novel lineages of cyanobacteria. [ABSTRACT FROM AUTHOR]

Novel red, filamentous, gliding bacteria formed deep red layers in several alkaline hot springs in Yellowstone National Park. Filaments contained densely layered intracellular membranes and bacteriochlorophyll a. The in vivo absorption spectrum of the red layer filaments was distinct from other phototrophs, with unusual bacteriochlorophyll a signature peaks in the near-infrared (IR) region (807 nm and 911 nm). These absorption peaks were similar to the wavelengths penetrating to the red layer of the mats as measured with in situ spectroradiometry. The filaments also demonstrated maximal photosynthetic uptake of radiolabeled carbon sources at these wavelengths. The red layer filaments displayed anoxygenic photoheterotrophy, as evidenced by the specific incorporation of acetate, not bicarbonate, and by the absence of oxygen production. Photoheterotrophy was unaffected by sulfide and oxygen, but was diminished by high-intensity visible light. Near-IR radiation supported photoheterotrophy. Morphologically and spectrally similar filaments were observed in several springs in Yellowstone National Park, including Octopus Spring. Taken together, these data suggest that the red layer filaments are most similar to the photoheterotroph, Heliothrix oregonensis. Notable differences include mat position and coloration, absorption spectra, and prominent intracellular membranes. [ABSTRACT FROM AUTHOR]