Zodletone Spring Research Articles

Metagenomic sequencing of a Patescibacteria-containing enrichment from Zodletone spring in Oklahoma, USA.

An enrichment of sulfidic sediments from Zodletone spring was sequenced as a metagenome. Draft genomes representing Cloacimonadota, Deltabacterota, Firmicutes, and Patescibacteria were binned and annotated and will aid functional genomics and cultivation efforts.

Complete Genome Sequence of Desulfomicrobium sp. Strain ZS1 from Zodletone Spring in Oklahoma, USA.

Desulfomicrobium sp. strain ZS1 is an obligate anaerobic, sulfate-reducing member of the Desulfobacterota from Zodletone Spring, an anoxic sulfide-rich spring in southwestern Oklahoma. Its complete genome was sequenced using a combination of Illumina and Oxford Nanopore platforms and encodes 3,364 proteins and 81 RNAs on a single chromosome.

Microbial Diversity and Sulfur Cycling in an Early Earth Analogue: From Ancient Novelty to Modern Commonality.

ABSTRACTLife emerged and diversified in the absence of molecular oxygen. The prevailing anoxia and unique sulfur chemistry in the Paleo-, Meso-, and Neoarchean and early Proterozoic eras may have supported microbial communities that differ from those currently thriving on the earth’s surface. Zodletone spring in southwestern Oklahoma represents a unique habitat where spatial sampling could substitute for geological eras namely, from the anoxic, surficial light-exposed sediments simulating a preoxygenated earth to overlaid water column where air exposure simulates oxygen intrusion during the Neoproterozoic era. We document a remarkably diverse microbial community in the anoxic spring sediments, with 340/516 (65.89%) of genomes recovered in a metagenomic survey belonging to 200 bacterial and archaeal families that were either previously undescribed or that exhibit an extremely rare distribution on the current earth. Such diversity is underpinned by the widespread occurrence of sulfite, thiosulfate, tetrathionate, and sulfur reduction and the paucity of sulfate reduction machineries in these taxa. Hence, these processes greatly expand lineages mediating reductive sulfur-cycling processes in the tree of life. An analysis of the overlaying oxygenated water community demonstrated the development of a significantly less diverse community dominated by well-characterized lineages and a prevalence of oxidative sulfur-cycling processes. Such a transition from ancient novelty to modern commonality underscores the profound impact of the great oxygenation event on the earth’s surficial anoxic community. It also suggests that novel and rare lineages encountered in current anaerobic habitats could represent taxa that once thrived in an anoxic earth but have failed to adapt to earth’s progressive oxygenation.

Five Metagenome-Assembled Genomes of the Rare Phylum CSSED10-310 from Zodletone Spring (Oklahoma, USA).

ABSTRACTWe analyzed five metagenome-assembled genomes (MAGs) belonging to the rare, yet-uncultured phylum CSSED10-310 recovered from the anoxic sediments of Zodletone Spring (Oklahoma). Our analysis suggests their potential involvement in sulfite respiration.

Candidatus Krumholzibacterium zodletonense gen. nov., sp nov, the first representative of the candidate phylum Krumholzibacteriota phyl. nov. recovered from an anoxic sulfidic spring using genome resolved metagenomics

The accumulation of genomes of uncultured organisms has highlighted the need for devising a taxonomic and nomenclature scheme to validate names and prevent redundancies. We here report on the recovery and analysis of four phylogenetically related genomes recovered from an anoxic sulfide and sulfur-rich spring (Zodletone spring) in southwestern Oklahoma. Phylogenetic analysis based on 120 single copy markers attested to their position as a novel distinct bacterial phylum. Genomic analysis suggests Gram-negative flagellated organisms that possess type IV pili. The organisms are predicted to be rod-shaped, slow-growers, with an anoxic, heterotrophic, and fermentative lifestyle. Predicted substrate utilization pattern includes multiple amino acids, dipeptides, tripeptides, and oligpopeptides; as well as few sugars. Predicted auxotrophies include proline, vitamin B6, lipoic acid, biotin, and vitamin B12. Assessment of the putative global distribution pattern of this novel lineage suggests its preference to anoxic marine, terrestrial, hydrocarbon-impacted, and freshwater habitats. We propose the candidatus name Krumholzibacterium zodletonense gen. nov, sp. nov. for Zgenome0171T, with the genome serving as the type material for the novel family Krumholzibacteriaceae fam. nov., order Krumholzibacteriales ord. nov., class Krumholzibacteria class nov., and phylum Krumholzibacteriota phyl. nov. The type material genome assembly is deposited in GenBank under accession number QTKG01000000.

Controls on Barite Crystal Morphology during Abiotic Precipitation

Barite (BaSO4) is a stable and widely distributed mineral in Earth’s crust. As such, barite has the potential to preserve specific geochemical and morphological characteristics representative of conditions at the time of its formation, which could be useful for interpretations of Earth’s ancient rocks and paleoenvironments. In this study, we used variations in saturation index, solution temperature, solution chemistry, presence of organics, and Mg2+ and Ca2+ ions to investigate variations in barite crystal morphology. Through 42 experiments, we simulated poorly understood, low temperature spring settings similar to Zodletone Spring in Anadarko Basin, Oklahoma. Using SEM/EDS, we identified barite rosettes, rounded barite, euhedral/square-shaped barite, and elliptical barite as the crystal morphologies that directly reflect different formational settings. The X-ray diffraction (XRD) patterns revealed different crystallographic characters of the four distinct barite crystal morphologies; in particular, the samples that precipitated from supersaturated SrSO4 solution exhibited double peaks at 43° 2-Theta, which matched barite with strontium substitution as barite might have incorporated strontium in its structure. Barite crystals that formed in the presence of organics in the initial solution exhibited a double peak at 33° 2-Theta, which was absent in other samples. Confocal Raman microscopy indicated that all of the samples had typical barium sulfate bands, with a few differences in bands between the samples; for example, the 638 cm−1 band showed splitting or a double band between different samples. The samples that precipitated from solution with organics had organic compounds from the experimental solution included in their composition. In both cases, C–H stretches from 2800 cm−1 to 3000 cm−1 were present as well as bands from 1350 cm−1 to 1500 cm−1, which are typical of organic compounds. Based on our experiments, the variation in barite crystal morphologies reflected changes in initial solution chemistry (or environmental settings).

Celestine in a sulfidic spring barite deposit - A potential biomarker?

We have documented the presence of celestine (SrSO4) within sediment accumulating at an artesian sulfidic spring (Zodletone Spring, Oklahoma) dominated by barite (BaSO4) precipitation associated with microbiological activity. The distribution and speciation of Sr in solid phases was determined by synchrotron-based micro-X-ray fluorescence spectroscopy and micro-X-ray diffraction, and particle morphology and texture was determined using electron microscopy. In all the natural sulfidic spring samples and lithified tufa sample, celestine was detected in fine-grained micron-scale Sr-rich phases but not in euhedral, Sr-poor grains. In parallel laboratory-based precipitation experiments, celestine was not observed even when solutions contained high Sr/Ba. Thermodynamic predictions alone do not account for the presence of micron-scale celestine in the sulfidic spring, and they do not account for the differences in Sr presence and distribution in naturally-occurring versus synthetic grains. While the mechanism is unclear, based on this evidence we hypothesize that the combination of bacterial surfaces and microenvironments within the crusts and microbial mats creates a synergistic effect where Sr is preferentially exchanged over Ba between the overlying stream water and the pore water within the mats allowing celestine to precipitate. Ultimately, our results point to an important role of biological activity for preferential Sr uptake. The presence of micron-scale celestine in ancient barite deposits can therefore potentially be used as a biomarker for conditions similar to modern sulfidic springs.

Metatranscriptomic analysis of a high-sulfide aquatic spring reveals insights into sulfur cycling and unexpected aerobic metabolism.

Zodletone spring is a sulfide-rich spring in southwestern Oklahoma characterized by shallow, microoxic, light-exposed spring water overlaying anoxic sediments. Previously, culture-independent 16S rRNA gene based diversity surveys have revealed that Zodletone spring source sediments harbor a highly diverse microbial community, with multiple lineages putatively involved in various sulfur-cycling processes. Here, we conducted a metatranscriptomic survey of microbial populations in Zodletone spring source sediments to characterize the relative prevalence and importance of putative phototrophic, chemolithotrophic, and heterotrophic microorganisms in the sulfur cycle, the identity of lineages actively involved in various sulfur cycling processes, and the interaction between sulfur cycling and other geochemical processes at the spring source. Sediment samples at the spring’s source were taken at three different times within a 24-h period for geochemical analyses and RNA sequencing. In depth mining of datasets for sulfur cycling transcripts revealed major sulfur cycling pathways and taxa involved, including an unexpected potential role of Actinobacteria in sulfide oxidation and thiosulfate transformation. Surprisingly, transcripts coding for the cyanobacterial Photosystem II D1 protein, methane monooxygenase, and terminal cytochrome oxidases were encountered, indicating that genes for oxygen production and aerobic modes of metabolism are actively being transcribed, despite below-detectable levels (<1 µM) of oxygen in source sediment. Results highlight transcripts involved in sulfur, methane, and oxygen cycles, propose that oxygenic photosynthesis could support aerobic methane and sulfide oxidation in anoxic sediments exposed to sunlight, and provide a viewpoint of microbial metabolic lifestyles under conditions similar to those seen during late Archaean and Proterozoic eons.

Response of the rare biosphere to environmental stressors in a highly diverse ecosystem (Zodletone spring, OK, USA)

Within highly diverse ecosystems, the majority of bacterial taxa are present in low abundance as members of the rare biosphere. The rationale for the occurrence and maintenance of the rare biosphere, and the putative ecological role(s) and dynamics of its members within a specific ecosystem is currently debated. We hypothesized that in highly diverse ecosystems, a fraction of the rare biosphere acts as a backup system that readily responds to environmental disturbances. We tested this hypothesis by subjecting sediments from Zodletone spring, a sulfide- and sulfur-rich spring in Southwestern OK, to incremental levels of salinity (1, 2, 3, 4, and 10% NaCl), or temperature (28°, 30°, 32°, and 70 °C), and traced the trajectories of rare members of the community in response to these manipulations using 16S rRNA gene analysis. Our results indicate that multiple rare bacterial taxa are promoted from rare to abundant members of the community following such manipulations and that, in general, the magnitude of such recruitment is directly proportional to the severity of the applied manipulation. Rare members that are phylogenetically distinct from abundant taxa in the original sample (unique rare biosphere) played a more important role in the microbial community response to environmental disturbances, compared to rare members that are phylogenetically similar to abundant taxa in the original sample (non-unique rare biosphere). The results emphasize the dynamic nature of the rare biosphere, and highlight its complexity and non-monolithic nature.

Controls on stable Sr-isotope fractionation in continental barite

Barite precipitation typically occurs when barium rich fluids mix with sulfate rich fluids, however barite found in the modern continental environment suggests that biological activity can play an important role in barite formation by oxidizing sulfur and/or concentrating barium within microenvironments. These activities induce barite precipitation, and carry with them implications for studies of barite genesis. Strontium (Sr) is incorporated into the barite crystal structure during barite formation preserving a radiogenic and stable Sr-isotope signature in barite, providing information about its formation. Here we present Sr-isotope results from three artesian sulfidic springs with ongoing barite precipitation (Zodletone Spring, Oklahoma; Stinking Spring, Utah; and Doughty Springs, Colorado) to explore the controls on stable Sr-isotope fractionation during barite precipitation in a continental setting. Apparent stable Sr-isotope fractionation for all three sites ranged from −0.6‰ to ~0.0‰ similar to previously published calculated values for equilibrium conditions and measured values of synthetic barite. However, clear relationships do not exist between water and barite chemistry in the natural systems, indicating that barite does not precipitate directly from solution, but heterogeneously within diverse microenvironments created by microbial biomass or on sediment surfaces. The dynamic microenvironments in a continental setting influence the apparent stable Sr-isotope fractionation during barite precipitation because of changing saturation conditions, Sr concentration and/or precipitation of different mineral phases (e.g., celestine). In order to better understand the geochemistry of barite deposits, future work is necessary to study the controls on radiogenic and stable Sr-isotope signatures of barite in the context of the temporally and spatially dynamic nature of the continental setting.

Single-cell Genomics: Definingm Microbiology's Dark Matter

Single-cell Genomics: Definingm Microbiology's Dark Matter

Novel High-Rank Phylogenetic Lineages within a Sulfur Spring (Zodletone Spring, Oklahoma), Revealed Using a Combined Pyrosequencing-Sanger Approach

The utilization of high-throughput sequencing technologies in 16S rRNA gene-based diversity surveys has indicated that within most ecosystems, a significant fraction of the community could not be assigned to known microbial phyla. Accurate determination of the phylogenetic affiliation of such sequences is difficult due to the short-read-length output of currently available high-throughput technologies. This fraction could harbor multiple novel phylogenetic lineages that have so far escaped detection. Here we describe our efforts in accurate assessment of the novelty and phylogenetic affiliation of selected unclassified lineages within a pyrosequencing data set generated from source sediments of Zodletone Spring, a sulfide- and sulfur-rich spring in southwestern Oklahoma. Lineage-specific forward primers were designed for 78 putatively novel lineages identified within the pyrosequencing data set, and representative nearly full-length small-subunit (SSU) rRNA gene sequences were obtained by pairing those primers with reverse universal bacterial primers. Of the 78 lineages tested, amplifiable products were obtained for 52, 32 of which had at least one nearly full-length sequence that was representative of the lineage targeted. Analysis of phylogenetic affiliation of the obtained Sanger sequences identified 5 novel candidate phyla and 10 novel candidate classes (within Fibrobacteres, Planctomycetes, and candidate phyla BRC1, GN12, TM6, TM7, LD1, WS2, and GN06) in the data set, in addition to multiple novel orders and families. The discovery of multiple novel phyla within a pilot study of a single ecosystem clearly shows the potential of the approach in identifying novel diversities within the rare biosphere.

Phylogenetic Diversities and Community Structure of Members of the Extremely Halophilic Archaea (Order Halobacteriales) in Multiple Saline Sediment Habitats

We investigated the phylogenetic diversity and community structure of members of the halophilic Archaea (order Halobacteriales) in five distinct sediment habitats that experience various levels of salinity and salinity fluctuations (sediments from Great Salt Plains and Zodletone Spring in Oklahoma, mangrove tree sediments in Puerto Rico, sediment underneath salt heaps in a salt-processing plant, and sediments from the Great Salt Lake northern arm) using Halobacteriales-specific 16S rRNA gene primers. Extremely diverse Halobacteriales communities were encountered in all habitats, with 27 (Zodletone) to 37 (mangrove) different genera identified per sample, out of the currently described 38 Halobacteriales genera. With the exception of Zodletone Spring, where the prevalent geochemical conditions are extremely inhospitable to Halobacteriales survival, habitats with fluctuating salinity levels were more diverse than permanently saline habitats. Sequences affiliated with the recently described genera Halogranum, Halolamina, Haloplanus, Halosarcina, and Halorientalis, in addition to the genera Halorubrum, Haloferax, and Halobacterium, were among the most abundant and ubiquitous genera, suggesting a wide distribution of these poorly studied genera in saline sediments. The Halobacteriales sediment communities analyzed in this study were more diverse than and completely distinct from communities from typical hypersaline water bodies. Finally, sequences unaffiliated with currently described genera represented a small fraction of the total Halobacteriales communities, ranging between 2.5% (Zodletone) to 7.0% (mangrove and Great Salt Lake). However, these novel sequences were characterized by remarkably high levels of alpha and beta diversities, suggesting the presence of an enormous, yet-untapped supply of novel Halobacteriales genera within the rare biosphere of various saline ecosystems.

Insights into chemotaxonomic composition and carbon cycling of phototrophic communities in an artesian sulfur‐rich spring (Zodletone, Oklahoma, USA), a possible analog for ancient microbial mat systems

Zodletone spring in Oklahoma is a unique environment with high concentrations of dissolved-sulfide (10 mm) and short-chain gaseous alkanes, exhibiting characteristics that are reminiscent of conditions that are thought to have existed in Earth's history, in particular the late Archean and early-to-mid Proterozoic. Here, we present a process-oriented investigation of the microbial community in two distinct mat formations at the spring source, (1) the top of the sediment in the source pool and (2) the purple streamers attached to the side walls. We applied a combination of pigment and lipid biomarker analyses, while functional activities were investigated in terms of oxygen production (microsensor analysis) and carbon utilization ((13)C incorporation experiments). Pigment analysis showed cyanobacterial pigments, in addition to pigments from purple sulfur bacteria (PSB), green sulfur bacteria (GSB) and Chloroflexus-like bacteria (CLB). Analysis of intact polar lipids (IPLs) in the source sediment confirmed the presence of phototrophic organisms via diacylglycerol phospholipids and betaine lipids, whereas glyceroldialkylglyceroltetraether additionally indicated the presence of archaea. No archaeal IPLs were found in the purple streamers, which were strongly dominated by betaine lipids. (13)C-bicarbonate- and -acetate-labeling experiments indicated cyanobacteria as predominant phototrophs in the source sediment, carbon was actively fixed by PSB/CLB/GSB in purple streamers by using near infrared light. Despite the presence of cyanobacteria, no oxygen could be detected in the presence of light, suggesting anoxygenic photosynthesis as the major metabolic process at this site. Our investigations furthermore indicated photoheterotrophy as an important process in both habitats. We obtained insights into a syntrophically operating phototrophic community in an ecosystem that bears resemblance to early Earth conditions, where cyanobacteria constitute an important contributor to carbon fixation despite the presence of high sulfide concentrations.

Fine-Scale Bacterial Beta Diversity within a Complex Ecosystem (Zodletone Spring, OK, USA): The Role of the Rare Biosphere

BackgroundThe adaptation of pyrosequencing technologies for use in culture-independent diversity surveys allowed for deeper sampling of ecosystems of interest. One extremely well suited area of interest for pyrosequencing-based diversity surveys that has received surprisingly little attention so far, is examining fine scale (e.g. micrometer to millimeter) beta diversity in complex microbial ecosystems.Methodology/Principal FindingsWe examined the patterns of fine scale Beta diversity in four adjacent sediment samples (1mm apart) from the source of an anaerobic sulfide and sulfur rich spring (Zodletone spring) in southwestern Oklahoma, USA. Using pyrosequencing, a total of 292,130 16S rRNA gene sequences were obtained. The beta diversity patterns within the four datasets were examined using various qualitative and quantitative similarity indices. Low levels of Beta diversity (high similarity indices) were observed between the four samples at the phylum-level. However, at a putative species (OTU0.03) level, higher levels of beta diversity (lower similarity indices) were observed. Further examination of beta diversity patterns within dominant and rare members of the community indicated that at the putative species level, beta diversity is much higher within rare members of the community. Finally, sub-classification of rare members of Zodletone spring community based on patterns of novelty and uniqueness, and further examination of fine scale beta diversity of each of these subgroups indicated that members of the community that are unique, but non novel showed the highest beta diversity within these subgroups of the rare biosphere.Conclusions/SignificanceThe results demonstrate the occurrence of high inter-sample diversity within seemingly identical samples from a complex habitat. We reason that such unexpected diversity should be taken into consideration when exploring gamma diversity of various ecosystems, as well as planning for sequencing-intensive metagenomic surveys of highly complex ecosystems.

Assessment of the Diversity, Abundance, and Ecological Distribution of Members of Candidate Division SR1 Reveals a High Level of Phylogenetic Diversity but Limited Morphotypic Diversity

We used a combination of 16S rRNA gene clone library surveys, quantitative PCR (qPCR) analysis, and fluorescent in situ hybridization to investigate the diversity, abundance, and distribution of members of candidate division SR1 in multiple habitats. Using SR1-specific 16S rRNA gene primers, we identified multiple novel SR1 lineages in four different anaerobic environments: sediments from Zodletone Spring, a sulfide- and sulfur-rich spring in southwestern Oklahoma; inner layers of microbial mats obtained from Sperm Pool, a high-temperature, low-pH pool (55 degrees C, pH 2.5) in Yellowstone National Park; fresh bovine ruminal contents; and anaerobic freshwater pond sediments (Duck Pond) in Norman, Oklahoma. qPCR analysis indicated that SR1 members constitute a small fraction (<0.01%) of the microbial communities in Duck Pond and ruminal samples but constitute a significant fraction (11.6 and 48.7%) of the total number of bacterial 16S rRNA genes in Zodletone Spring and the inner layers of Sperm Pool microbial mat samples, respectively. By using SR1-specific fluorescent probes, filamentous cells were identified as the sole SR1 morphotype in all environments examined, with the exception of Sperm Pool, where a second bacillus morphotype was also identified. Using a full-cycle 16S rRNA approach, we show that each of these two morphotypes corresponds to a specific phylogenetic lineage identified in the Sperm Pool clone library. This work greatly expands the intralineage phylogenetic diversity within candidate division SR1 and provides valuable quantification and visualization tools that could be used for investigating the ecological roles, dynamics, and genomics of this as-yet-uncultured bacterial phylum.

Halosarcina pallida gen. nov., sp. nov., a halophilic archaeon from a low-salt, sulfide-rich spring

A novel halophilic archaeon, strain BZ256T, was isolated from Zodletone Spring, a sulfide- and sulfur-rich spring in south-western Oklahoma, USA. Cells were non-motile, non-flagellated cocci that divided along two axes, resulting in the formation of sarcina-like clusters. Strain BZ256T grew at salt concentrations ranging from 1.3 to 4.3 M NaCl, with optimum growth at approximately 3.4 M, and required at least 1 mM Mg2+ for growth. The pH range for growth was 5.0 to at least 8.5, and the temperature range for growth was 25-45 degrees C. The two diether phospholipids that are typical of members of the order Halobacteriales, namely phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, were present in strain BZ256T, as were two glycolipids chromatographically identical to S-DGD-1 and DGD-1. The 16S rRNA gene sequence of strain BZ256T showed 96.8 % similarity to that of the type strain of Halogeometricum borinquense, the closest recognized species within the order Halobacteriales. The DNA G+C content of strain BZ256T was 65.4 mol%. Microscopic, physiological, biochemical and phylogenetic comparisons between strain BZ256T and recognized genera of extremely halophilic archaea suggest that this strain represents a member of a novel genus and species within the family Halobacteriaceae, for which the name Halosarcina pallida gen. nov., sp. nov. is proposed. The type strain of Halosarcina pallida is BZ256T (=KCTC 4017T =JCM 14848T).

Phylogenetic and metabolic diversity of Planctomycetes from anaerobic, sulfide- and sulfur-rich Zodletone Spring, Oklahoma.

We investigated the phylogenetic diversity and metabolic capabilities of members of the phylum Planctomycetes in the anaerobic, sulfide-saturated sediments of a mesophilic spring (Zodletone Spring) in southwestern Oklahoma. Culture-independent analyses of 16S rRNA gene sequences generated using Planctomycetes-biased primer pairs suggested that an extremely diverse community of Planctomycetes is present at the spring. Although sequences that are phylogenetically affiliated with cultured heterotrophic Planctomycetes were identified, the majority of the sequences belonged to several globally distributed, as-yet-uncultured Planctomycetes lineages. Using complex organic media (aqueous extracts of the spring sediments and rumen fluid), we isolated two novel strains that belonged to the Pirellula-Rhodopirellula-Blastopirellula clade within the Planctomycetes. The two strains had identical 16S rRNA gene sequences, and their closest relatives were isolates from Kiel Fjord (Germany), Keauhou Beach (HI), a marine aquarium, and tissues of marine organisms (Aplysina sp. sponges and postlarvae of the giant tiger prawn Penaeus monodon). The closest recognized cultured relative of strain Zi62 was Blastopirellula marina (93.9% sequence similarity). Detailed characterization of strain Zi62 revealed its ability to reduce elemental sulfur to sulfide under anaerobic conditions, as well as its ability to produce acids from sugars; both characteristics may potentially allow strain Zi62 to survive and grow in the anaerobic, sulfide- and sulfur-rich environment at the spring source. Overall, this work indicates that anaerobic metabolic abilities are widely distributed among all major Planctomycetes lineages and suggests carbohydrate fermentation and sulfur reduction as possible mechanisms employed by heterotrophic Planctomycetes for growth and survival under anaerobic conditions.

Haladaptatus paucihalophilus gen. nov., sp. nov., a halophilic archaeon isolated from a low-salt, sulfide-rich spring

Two novel strains of halophilic archaea, DX253(T) and GY252, were isolated from Zodletone Spring, a low-salt, sulfide- and sulfur-rich spring in south-western Oklahoma, USA. The cells were cocci or coccobacilli and occurred singly or in pairs. The two strains grew in a wide range of salt concentrations (0.8-5.1 M) and required at least 5 mM Mg(2+) for growth. The pH range for growth was 5-7.5 and the temperature range was 25-45 degrees C. In addition to having the capacity to grow at relatively low salt concentrations, cells remained viable in distilled water after prolonged incubation. The two diether phospholipids that are typical of members of the order Halobacteriales, phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, were present. Phosphatidylglycerol sulfate and two unidentified glycolipids were also detected. Each strain had two distinct 16S rRNA gene sequences that were only 89.5-90.8 % similar to sequences from the most closely related cultured and recognized species within the order Halobacteriales. The DNA G+C content of the type strain was found to be 60.5 mol%. The closest relatives were clones and uncharacterized isolates obtained from coastal salt-marsh sediments with salinities equivalent to that of seawater. The physiological, biochemical and phylogenetic differences between strains DX253(T) and GY252 and other previously described genera of extremely halophilic archaea suggest that these novel strains represent a novel species and genus within the family Halobacteriaceae, for which the name Haladaptatus paucihalophilus gen. nov., sp. nov. is proposed. The type strain is DX253(T) (=JCM 13897(T)=DSM 18195(T)=ATCC BAA-1313(T)=KCTC 4006(T)).

Metagenomic Analysis of the Microbial Community at Zodletone Spring (Oklahoma): Insights into the Genome of a Member of the Novel Candidate Division OD1

A metagenomic library was constructed from the anaerobic sediments of a mesophilic sulfur spring. Thirty-five bacterial 16S rRNA gene-containing clones were identified in this library. Analysis of a genomic fragment belonging to candidate division OD1 provided useful insights into the physiology and biochemistry of this novel, yet-uncultured candidate division.