Thiorhodovibrio frisius and Trv. litoralis spp. nov., Two Novel Members from a Clade of Fastidious Purple Sulfur Bacteria That Exhibit Unique Red-Shifted Light-Harvesting Capabilities.

In the pursuit of cultivating anaerobic anoxygenic phototrophs with unusual absorbance spectra, a purple sulfur bacterium was isolated from the shoreline of Baltrum, a North Sea island of Germany. It was designated strain 970, due to a predominant light harvesting complex (LH) absorption maximum at 963-966 nm, which represents the furthest infrared-shift documented for such complexes containing bacteriochlorophyll a. A polyphasic approach to bacterial systematics was performed, comparing genomic, biochemical, and physiological properties. Strain 970 is related to Thiorhodovibrio winogradskyi DSM 6702T by 26.5, 81.9, and 98.0% similarity via dDDH, ANI, and 16S rRNA gene comparisons, respectively. The photosynthetic properties of strain 970 were unlike other Thiorhodovibrio spp., which contained typical LH absorbing characteristics of 800-870 nm, as well as a newly discovered absorption band at 908 nm. Strain 970 also had a different photosynthetic operon composition. Upon genomic comparisons with the original Thiorhodovibrio strains DSM 6702T and strain 06511, the latter was found to be divergent, with 25.3, 79.1, and 97.5% similarity via dDDH, ANI, and 16S rRNA gene homology to Trv. winogradskyi, respectively. Strain 06511 (=DSM 116345T) is thereby described as Thiorhodovibrio litoralis sp. nov., and the unique strain 970 (=DSM 111777T) as Thiorhodovibrio frisius sp. nov.

Genomic and Phenotypic Characterization of Chloracidobacterium Isolates Provides Evidence for Multiple Species.

Chloracidobacterium is the first and until now the sole genus in the phylum Acidobacteriota (formerly Acidobacteria) whose members perform chlorophyll-dependent phototrophy (i.e., chlorophototrophy). An axenic isolate of Chloracidobacterium thermophilum (strain B T ) was previously obtained by using the inferred genome sequence from an enrichment culture and diel metatranscriptomic profiling analyses in situ to direct adjustments to the growth medium and incubation conditions, and thereby a defined growth medium for Chloracidobacterium thermophilum was developed. These advances allowed eight additional strains of Chloracidobacterium spp. to be isolated from microbial mat samples collected from Mushroom Spring, Yellowstone National Park, United States, at temperatures of 41, 52, and 60°C; an axenic strain was also isolated from Rupite hot spring in Bulgaria. All isolates are obligately photoheterotrophic, microaerophilic, non-motile, thermophilic, rod-shaped bacteria. Chloracidobacterium spp. synthesize multiple types of (bacterio-)chlorophylls and have type-1 reaction centers like those of green sulfur bacteria. Light harvesting is accomplished by the bacteriochlorophyll a-binding, Fenna-Matthews-Olson protein and chlorosomes containing bacteriochlorophyll c. Their genomes are approximately 3.7 Mbp in size and comprise two circular chromosomes with sizes of approximately 2.7 Mbp and 1.0 Mbp. Comparative genomic studies and phenotypic properties indicate that the nine isolates represent three species within the genus Chloracidobacterium. In addition to C. thermophilum, the microbial mats at Mushroom Spring contain a second species, tentatively named Chloracidobacterium aggregatum, which grows as aggregates in liquid cultures. The Bulgarian isolate, tentatively named Chloracidobacterium validum, will be proposed as the type species of the genus, Chloracidobacterium. Additionally, Chloracidobacterium will be proposed as the type genus of a new family, Chloracidobacteriaceae, within the order Blastocatellales, the class Blastocatellia, and the phylum Acidobacteriota.

In-Situ Metatranscriptomic Analyses Reveal the Metabolic Flexibility of the Thermophilic Anoxygenic Photosynthetic Bacterium Chloroflexus aggregans in a Hot Spring Cyanobacteria-Dominated Microbial Mat.

Chloroflexus aggregans is a metabolically versatile, thermophilic, anoxygenic phototrophic member of the phylum Chloroflexota (formerly Chloroflexi), which can grow photoheterotrophically, photoautotrophically, chemoheterotrophically, and chemoautotrophically. In hot spring-associated microbial mats, C. aggregans co-exists with oxygenic cyanobacteria under dynamic micro-environmental conditions. To elucidate the predominant growth modes of C. aggregans, relative transcription levels of energy metabolism- and CO2 fixation-related genes were studied in Nakabusa Hot Springs microbial mats over a diel cycle and correlated with microscale in situ measurements of O2 and light. Metatranscriptomic analyses indicated two periods with different modes of energy metabolism of C. aggregans: (1) phototrophy around midday and (2) chemotrophy in the early morning hours. During midday, C. aggregans mainly employed photoheterotrophy when the microbial mats were hyperoxic (400-800 µmol L-1 O2). In the early morning hours, relative transcription peaks of genes encoding uptake hydrogenase, key enzymes for carbon fixation, respiratory complexes as well as enzymes for TCA cycle and acetate uptake suggest an aerobic chemomixotrophic lifestyle. This is the first in situ study of the versatile energy metabolism of C. aggregans based on gene transcription patterns. The results provide novel insights into the metabolic flexibility of these filamentous anoxygenic phototrophs that thrive under dynamic environmental conditions.

Elioraea tepida, sp. nov., a Moderately Thermophilic Aerobic Anoxygenic Phototrophic Bacterium Isolated from the Mat Community of an Alkaline Siliceous Hot Spring in Yellowstone National Park, WY, USA.

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).

Draft Genome Sequence of a Filamentous Anoxygenic Phototrophic Bacterium, "Candidatus Roseilinea sp. Strain NK_OTU-006," Recovered from Metagenomic Data of a Hot Spring Microbial Mat.

We report here the metagenome-assembled draft genome of an uncultured filamentous anoxygenic phototroph of the phylum Chloroflexota (formerly Chloroflexi), "Candidatus Roseilinea sp. strain NK_OTU-006," recovered from hot spring-associated microbial mats. The 3.6-Mb genome is estimated to be 94% complete and comprises 117 contigs encoding 3,203 predicted genes, including a full-length rRNA operon.

Caldichromatium japonicum gen. nov., sp. nov., a novel thermophilic phototrophic purple sulphur bacterium of the Chromatiaceae isolated from Nakabusa hot springs, Japan.

A novel thermophilic phototrophic purple sulphur bacterium was isolated from microbial mats (56 °C) at Nakabusa hot springs, Nagano prefecture, Japan. Cells were motile, rod-shaped, stain Gram-negative and stored sulphur globules intracellularly. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were the major pigments. Dense liquid cultures were red in colour. Strain No.7T was able to grow photoautotrophically using sulfide, thiosulfate, sulfite and hydrogen (in the presence of sulfide) as electron donors and bicarbonate as the sole carbon source. Optimum growth occurred under anaerobic conditions in the light at 50 °C (range, 40-56 °C) and pH 7.2 (range, pH 7-8). Major fatty acids were C16 : 0 (46.8 %), C16 : 1 ω7c (19.9 %), C18 : 1 ω7c (21.1 %), C14 : 0 (4.6 %) and C18 : 0 (2.4 %). The polar lipid profile showed phosphatidylglycerol and unidentified aminophospholipids to be the major lipids. The only quinone detected was ubiquinone-8. 16S rRNA gene sequence comparisons indicated that the novel bacterium is only distantly related to Thermochromatium tepidum with a nucleotide identity of 90.4 %. The phylogenetic analysis supported the high novelty of strain No.7T with a long-branching phylogenetic position within the Chromatiaceae next to Thermochromatium tepidum. The genome comprised a circular chromosome of 2.99 Mbp (2 989 870 bp), included no plasmids and had a DNA G+C content of 61.2 mol%. Polyphasic taxonomic analyses of the isolate suggested strain No.7T is a novel genus within the Chromatiaceae. The proposed genus name of the second truly thermophilic purple sulphur bacterium is Caldichromatium gen. nov. with the type species Caldichromatium japonicum sp. nov. (DSM 110881=JCM 39101).

Aquabacterium pictum sp. nov., the first aerobic bacteriochlorophyll a-containing fresh water bacterium in the genus Aquabacterium of the class Betaproteobacteria.

A strictly aerobic, bacteriochlorophyll a-containing betaproteobacterium, designated strain W35T, was isolated from a biofilm sampled at Tama River in Japan. The non-motile and rod-shaped cells formed pink-beige pigmented colonies on agar plates containing organic compounds, and showed an in vivo absorption maximum at 871 nm in the near-infrared region, typical for the presence of bacteriochlorophyll a. The new bacterial strain is Gram-negative, and oxidase- and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain W35T was closely related to species in the genus Aquabacterium. The closest phylogenetic relatives of strain W35T were Aquabacterium commune B8T (97.9 % sequence similarity), Aquabacterium citratiphilum B4T (97.2 %) and Aquabacterium limnoticum ABP-4T (97.0 %). The major cellular fatty acids were C16  :  1ω7c (50.4 %), C16  :  0 (22.7 %), summed feature 8 (C18  :  1ω7c/C18  :  1ω6c; 9.7 %), C18  :  3ω6c (5.5 %), C12  :  0 (5.3 %) and C10  :  0 3OH (2.7 %). The respiratory quinone was ubiquinone-8. Predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The G+C content of the genomic DNA was 70.4 mol% (genome data) and 71.4 mol% (HPLC). The genome size of strain W35T is 6.1 Mbp and average nucleotide identity analysis indicated genome similarities of strain W35T and related Aquabacterium type strains to be 78-79 %. The results of polyphasic comparisons showed that strain W35T was clearly distinguishable from other members of the genus Aquabacterium. Therefore, we propose a new species in the genus Aquabacterium, namely, Aquabacterium pictum sp. nov. The type strain is W35T (=DSM 106757T=NBRC 111963T). The description of the genus Aquabacterium is also emended.

Vertical Distribution and Diversity of Phototrophic Bacteria within a Hot Spring Microbial Mat (Nakabusa Hot Springs, Japan).

Phototrophic microbial mats are assemblages of vertically layered microbial populations dominated by photosynthetic microorganisms. In order to elucidate the vertical distribution and diversity of phototrophic microorganisms in a hot spring-associated microbial mat in Nakabusa (Japan), we analyzed the 16S rRNA gene amplicon sequences of the microbial mat separated into five depth horizons, and correlated them with microsensor measurements of O2 and spectral scalar irradiance. A stable core community and high diversity of phototrophic organisms dominated by the filamentous anoxygenic phototrophs, Roseiflexus castenholzii and Chloroflexus aggregans were identified together with the spectral signatures of bacteriochlorophylls (BChls) a and c absorption in all mat layers. In the upper mat layers, a high abundance of cyanobacteria (Thermosynechococcus sp.) correlated with strong spectral signatures of chlorophyll a and phycobiliprotein absorption near the surface in a zone of high O2 concentrations during the day. Deeper mat layers were dominated by uncultured chemotrophic Chlorobi such as the novel putatively sulfate-reducing "Ca. Thermonerobacter sp.", which showed increasing abundance with depth correlating with low O2 in these layers enabling anaerobic metabolism. Oxygen tolerance and requirements for the novel phototroph "Ca. Chloroanaerofilum sp." and the uncultured chemotrophic Armatimonadetes member type OS-L detected in Nakabusa hot springs, Japan appeared to differ from previously suggested lifestyles for close relatives identified in hot springs in Yellowstone National Park, USA. The present study identified various microenvironmental gradients and niche differentiation enabling the co-existence of diverse chlorophototrophs in metabolically diverse communities in hot springs.

Reaction centers of the thermophilic microaerophile, Chloracidobacterium thermophilum (Acidobacteria) I: biochemical and biophysical characterization.

Chloracidobacterium thermophilum is a microaerophilic, anoxygenic member of the green chlorophototrophic bacteria. This bacterium is the first characterized oxygen-requiring chlorophototroph with chlorosomes, the FMO protein, and homodimeric type-1 reaction centers (RCs). The RCs of C. thermophilum are also unique because they contain three types of chlorophylls, bacteriochlorophyll aP esterified with phytol, Chl aPD esterified with Δ2,6-phytadienol, and Zn-BChl aP' esterified with phytol, in the approximate molar ratio 32:24:4. The light-induced difference spectrum of these RCs had a bleaching maximum at 839 nm and also revealed an electrochromic bandshift that is probably derived from a BChl a molecule near P840+. The FX [4Fe-4S] cluster had a midpoint potential of ca. - 581 mV, and the spectroscopic properties of the P+ F X - spin-polarized radical pair were very similar to those of reaction centers of heliobacteria and green sulfur bacteria. The data further indicate that electron transfer occurs directly from A0- to FX, as occurs in other homodimeric type-1 RCs. Washing experiments with isolated membranes suggested that the PscB subunit of these reaction centers is more tightly bound than PshB in heliobacteria. Thus, the reaction centers of C. thermophilum have some properties that resemble other homodimeric reaction centers but also have specific properties that are more similar to those of Photosystem I. These differences probably contribute to protection of the electron transfer chain from oxygen, contributing to the oxygen tolerance of this microaerophile.

Nitrogenase Activity in Thermophilic Chemolithoautotrophic Bacteria in the Phylum Aquificae Isolated under Nitrogen-Fixing Conditions from Nakabusa Hot Springs.

The phylum Aquificae comprises chemolithoautotrophic thermophilic to hyperthermophilic bacteria, in which the nitrogenase reductase gene (nifH) has been reported. However, nitrogen-fixing activity has not yet been demonstrated in members of this deeply branching bacterial phylum. We isolated two thermophilic diazotrophic strains from chemosynthetic microbial communities in slightly alkaline hot springs (≥70°C) in Nakabusa, Nagano Prefecture, Japan. A phylogenetic analysis based on 16S rRNA genes identified these strains as members of the genus Hydrogenobacter within Aquificae. Their NifH sequences showed 96.5 and 97.4% amino acid sequence identities to that from Hydrogenobacter thermophilus TK-6. Nitrogenase activity, measured by acetylene reduction, was confirmed in both strains at 70°C. These novel strains grew under semi-aerobic conditions by using CO2 as the sole carbon source and N2 as the sole nitrogen source in media containing hydrogen and/or thiosulfate. To the best of our knowledge, this is the first demonstration of active nitrogen fixation in thermophilic bacteria at 70°C and in the phylum Aquificae.

First Evidence of Dehydroabietic Acid Production by a Marine Phototrophic Gammaproteobacterium, the Purple Sulfur Bacterium Allochromatium vinosum MT86.

The production of secondary metabolites by a new isolate of the purple sulfur bacterium Allochromatium vinosum, which had shown antibiotic activities during a preliminary study, revealed the production of several metabolites. Growth conditions suitable for the production of one of the compounds shown in the metabolite profile were established and compound 1 was purified. The molecular formula of compound 1 (C20H28O2) was determined by high resolution mass spectra, and its chemical structure by means of spectroscopic methods. The evaluation of these data revealed that the structure of the compound was identical to dehydroabietic acid, a compound known to be characteristically produced by conifer trees, but so far not known from bacteria, except cyanobacteria. The purified substance showed weak antibiotic activities against Bacillus subtilis and Staphylococcus lentus with IC50 values of 70.5 µM (±2.9) and 57.0 µM (±3.3), respectively.

Structural Variations in Chlorosomes from Wild-Type and a bchQR Mutant of Chlorobaculum tepidum Revealed by Single-Molecule Spectroscopy.

Green sulfur bacteria can grow photosynthetically by absorbing only a few photons per bacteriochlorophyll molecule per day. They contain chlorosomes, perhaps the most efficient light-harvesting antenna system found in photosynthetic organisms. Chlorosomes contain supramolecular structures comprising hundreds of thousands of bacteriochlorophyll molecules, which are properly positioned with respect to one another solely by self-assembly and not by using a protein scaffold as a template for directing the mutual arrangement of the monomers. These two features-high efficiency and self-assembly-have attracted considerable attention for developing light-harvesting systems for artificial photosynthesis. However, reflecting the heterogeneity of the natural system, detailed structural information at atomic resolution of the molecular aggregates is not yet available. Here, we compare the results for chlorosomes from the wild type and two mutants of Chlorobaculum tepidum obtained by polarization-resolved, single-particle fluorescence-excitation spectroscopy and theoretical modeling with results previously obtained from nuclear-magnetic resonance spectroscopy and cryo-electron microscopy. Only the combination of information obtained from all of these techniques allows for an unambiguous description of the molecular packing of bacteriochlorophylls within chlorosomes. In contrast to some suggestions in the literature, we find that, for the chlorosomes from the wild type as well as for those from mutants, the dominant secondary structural element features tubular symmetry following a very similar construction principle. Moreover, the results suggest that the various options for methylation of the bacteriochlorophyll molecules, which are a primary source of the structural (and spectral) heterogeneity of wild-type chlorosome samples, are exploited by nature to achieve improved spectral coverage at the level of individual chlorosomes.

15N photo-CIDNP MAS NMR analysis of reaction centers of Chloracidobacterium thermophilum.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by 15N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a' (Zn-BChl a') (Tsukatani et al. in J Biol Chem 287:5720-5732, 2012). Based upon experimental and quantum chemical 15N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a'. Chl a and 81-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.

Diversity of Chlorophototrophic Bacteria Revealed in the Omics Era.

Because of recent advances in omics methodologies, knowledge of chlorophototrophy (i.e., chlorophyll-based phototrophy) in bacteria has rapidly increased. Chlorophototrophs currently are known to occur in seven bacterial phyla: Cyanobacteria, Proteobacteria, Chlorobi, Chloroflexi, Firmicutes, Acidobacteria, and Gemmatimonadetes. Other organisms that can produce chlorophylls and photochemical reaction centers may still be undiscovered. Here we summarize the current status of the taxonomy and phylogeny of chlorophototrophic bacteria as revealed by genomic methods. In specific cases, we briefly describe important ecophysiological and metabolic insights that have been gained from the application of genomic methods to these bacteria. In the 20 years since the completion of the Synechocystis sp. PCC 6803 genome in 1996, approximately 1,100 genomes have been sequenced, which represents nearly the complete diversity of known chlorophototrophic bacteria. These data are leading to new insights into many important processes, including photosynthesis, nitrogen and carbon fixation, cellular differentiation and development, symbiosis, and ecosystem functionality.

"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.

Draft Genome Sequence of Anoxybacillusayderensis Strain MT-Cab (Firmicutes).

The draft genome of the Gram-positive spore-forming Anoxybacillus ayderensis strain MT-Cab (Firmicutes), isolated from an enrichment culture of Chloracidobacterium thermophilum, was sequenced and comprises 2,577,015 bp in 92 contigs. The draft genome is predicted to consist of 2,699 protein-coding genes, 73 tRNA-coding genes, and an estimated 8 rRNA operons.

Polymer-Chlorosome Nanocomposites Consisting of Non-Native Combinations of Self-Assembling Bacteriochlorophylls.

Chlorosomes are one of the characteristic light-harvesting antennas from green sulfur bacteria. These complexes represent a unique paradigm: self-assembly of bacteriochlorophyll pigments within a lipid monolayer without the influence of protein. Because of their large size and reduced complexity, they have been targeted as models for the development of bioinspired light-harvesting arrays. We report the production of biohybrid light-harvesting nanocomposites mimicking chlorosomes, composed of amphiphilic diblock copolymer membrane bodies that incorporate thousands of natural self-assembling bacteriochlorophyll molecules derived from green sulfur bacteria. The driving force behind the assembly of these polymer-chlorosome nanocomposites is the transfer of the mixed raw materials from the organic to the aqueous phase. We incorporated up to five different self-assembling pigment types into single nanocomposites that mimic chlorosome morphology. We establish that the copolymer-BChl self-assembly process works smoothly even when non-native combinations of BChl homologues are included. Spectroscopic characterization revealed that the different types of self-assembling pigments participate in ultrafast energy transfer, expanding beyond single chromophore constraints of the natural chlorosome system. This study further demonstrates the utility of flexible short-chain, diblock copolymers for building scalable, tunable light-harvesting arrays for technological use and allows for an in vitro analysis of the flexibility of natural self-assembling chromophores in unique and controlled combinations.

Complete Genome Sequence of the Photoautotrophic and Bacteriochlorophyll e-Synthesizing Green Sulfur Bacterium Chlorobaculum limnaeum DSM 1677T.

Chlorobaculum limnaeum DSM 1677T is a mesophilic, brown-colored, chlorophototrophic green sulfur bacterium that produces bacteriochlorophyll e and the carotenoid isorenieratene as major pigments. This bacterium serves as a model organism in molecular research on photosynthesis, sulfur metabolism, and bacteriochlorophyll biosynthesis. We report here the complete genome sequence.

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.