Ancient putative microbial structures that appear in the rock record commonly serve as evidence of early life on Earth, but the details of their formation remain unclear. The study of modern microbial mat structures can help inform the properties of their ancient counterparts, but modern mineralizing mat systems with morphological similarity to ancient structures are rare. Here, we characterize partially lithified microbial mats containing cm-scale dendrolitic coniform structures from a geothermal pool (“Cone Pool”) at Little Hot Creek, California, that if fully lithified, would resemble ancient dendrolitic structures known from the rock record. Light and electron microscopy revealed that the cm-scale ‘dendrolitic cones’ were comprised of intertwined microbial filaments and grains of calcium carbonate. The degree of mineralization (carbonate content) increased with depth in the dendrolitic cones. Sequencing of 16S rRNA gene libraries revealed that the dendrolitic cone tips were enriched in OTUs most closely related to the genera Phormidium, Leptolyngbya, and Leptospira, whereas mats at the base and adjacent to the dendrolitic cones were enriched in Synechococcus. We hypothesize that the consumption of nutrients during autotrophic and heterotrophic growth may promote movement of microbes along diffusive nutrient gradients, and thus microbialite growth. Hour-glass shaped filamentous structures present in the dendrolitic cones may have formed around photosynthetically-produced oxygen bubbles—suggesting that mineralization occurs rapidly and on timescales of the lifetime of a bubble. The dendrolitic-conical structures in Cone Pool constitute a modern analog of incipient microbialite formation by filamentous microbiota that are morphologically distinct from any structure described previously. Thus, we provide a new model system to address how microbial mats may be preserved over geological timescales.
| URL | https://www.bco-dmo.org/dataset/654179 |
|---|---|
| Created | August 16, 2016 |
| Modified | August 10, 2017 |
| State | Preliminary and in progress |
| Brief Description | 40 16S amplicon libraries collected from sediments of the Adelie Basin |
Samples were collected at 66.413S, 140.425E from depths 0.05 – 97 meters below sea floor.
Samples for molecular biological analysis were collected shipboard from 10 cm sections of the intact core using sterile cut-end 5 mL syringes. Samples were immediately frozen at –80 degrees C and maintained at this temperature during transport and storage to the home laboratory. Genomic DNA was extracted from sediment samples using a bead beating/phenol chloroform protocol. Amplicons of the 16S rRNA gene were prepared for 454 pyrosequencing using a PCR-touchdown annealing temperature strategy from Don et al. (1991) with procedure modifications and modified universal primers 515f and 927r of Osburn et al. (2011). Forward primers included the 454 Life Science A adaptors and a sample specific 8 nt barcode. The reverse primer included the 454 Life Science B adaptors. Amplicon concentrations for each sample were quantified using a 2100 Bioanalyzer (Agilent Technologies, Colorado Springs, CO, USA), pooled (22 ng DNA/sample) and concentrated using a Savant DNA 12 Speed Vac Concentrator (Thermo Scientific, Waltham, MA, USA). The pooled DNA was gel purified using the Montage DNA Gel Extraction Kit (Millipore, Bellerica, MA, USA), and sequenced on a Roche 454 FLX titanium platform at Engencore, University of South Carolina (now Selah Genomics).
Pyrosequencing reads were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME) Pipeline. Reads between 200 and 500 base pairs with a quality score of 27 or above were denoised using the QIIME denoiser for titanium runs, and clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using UCLUST ( Edgar, 2010 ). Taxonomy of OTUs was assigned by BLAST against the Silva SSU NR Reference database, release 102 within QIIME. Chimeras were identified and removed using QIIME’s ChimeraSlayer Wrapper.
Sequences were deposited into the MG-RAST database under accession numbers 4624791.3–4634830.3.
| Name | Affiliation | Contact |
|---|---|---|
| Stephanie A. Carr | Colorado School of Mines (CSM) | ✓ |
| Beth N. Orcutt | Colorado School of Mines (CSM) | ✓ |
| John R. Spear | Bigelow Laboratory for Ocean Sciences | |
| Shannon Rauch | Colorado School of Mines (CSM) | |
| Shannon Rauch | Colorado School of Mines (CSM) | |
| Shannon Rauch | Woods Hole Oceanographic Institution (WHOI BCO-DMO) |
| Project Title | Functional potential of the uncultivated Candidate Phylum OP9 from deep Antarctic marine sediments using single cell genome techniques |
|---|---|
| Acronym | Adelie Basin Atribacteria |
| URL | https://www.bco-dmo.org/project/626786 |
| Created | November 20, 2015 |
| Modified | November 20, 2015 |
Bacteria belonging to the newly classified candidate phylum “Atribacteria” (formerly referred to as “OP9” and “JS1”) are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica’s Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.
This project was funded by a C-DEBI Postdoctoral Fellowship
| Name | Affiliation | Role |
|---|---|---|
| Stephanie A. Carr | Colorado School of Mines (CSM) | Principal Investigator |
| Beth N. Orcutt | Bigelow Laboratory for Ocean Sciences | Co-Principal Investigator |
| John R. Spear | Colorado School of Mines (CSM) | Co-Principal Investigator |
| URL | https://www.bco-dmo.org/dataset/654189 |
|---|---|
| Created | August 16, 2016 |
| Modified | August 22, 2016 |
| State | Preliminary and in progress |
| Brief Description | This is the manually curated genome of the SAG SCGC AD-561-N23 |
Samples for molecular biological analysis were collected shipboard from 10 cm sections of the intact core using sterile cut-end 5 mL syringes. Samples were immediately frozen at –80 degrees C and maintained at this temperature during transport and storage to the home laboratory. Sorting of individual cells from unfixed frozen sediment from 97.41 mbsf was attempted for single cell genomics using the Single Cell Genomics Center (SCGC) at the Bigelow Laboratory for Ocean Sciences. While the sediment was not preserved with recommended fixatives to prevent cell lysis during storage, and although the samples had gone through at least one round of freeze-thaw for bulk DNA extraction which may have introduced cell lysis, the high relative abundance of target microorganisms in the sample suggested this approach might still be successful in recovering intact cells. Approximately 0.5 g of frozen sediment was diluted in 1 mL of filter-sterilized seawater and vortexed for 30 s to liberate cells from the sediment matrix, modifying methods developed previously. Sediment was then separated from cells by gentle centrifugation at 2000 rpm for 30 s The cell suspension was treated with SYTO-9 DNA stain and sorted into two 384-well plates using SCGC’s s tandard pipeline. Both sorted plates were subjected to physical lysis treatments (five freeze- thaw cycles), and the second plate also experienced an alkaline lysis treatment. DNA amplification by multiple displacement amplification.
The initial assembly of Atribacteria bacterium SCGC AD- 561-N23 is publically available within the IMG system (taxon ID 2588254308) and the sequence for the 16S rRNA gene is available within the IMG system and Supplementary Materials. A detailed assembly procedure (QC.finalReport.pdf ) can be downloaded from: http://genome.jgi.doe.gov/CandivSCAD561N23/CandivSCAD561N23.download.html.
Briefly, single-cell amplified genomic (SAG) DNA was sequenced, assembled and annotated at the United States Department of Energy’s Joint Genome Institute (JGI) following their standard pipeline for Illumina HiSeq 2000 platform sequencing. Illumina reads were screened using JGI’s in-house DUK filtering program (Mingkun et al., unpublished). Trimmed reads were assembled using SPAdes (version 3.0.0) with the following parameters (–t 8 –m 40 – –sc – –careful – –12; Bankevich et al., 2012). Once released to Integrated Microbial Genomes (IMG) system, manual screening and removal of potential contaminate sequences according to JGI’s single cell data decontamination protocol (Clingenpeel, 2015). Scaffolds with GC contents that varied from the genome average more than 10% and clustered as a distinct group according to a kmer analysis (IMG, fragment window 5000 bp, fragment step 500 bp, oligomer size 5, minimum variation 10) were identified as potential contaminates and were removed from the de novo assembly (with the exception of scaffolds that contained ribosomal DNA). This screened genome was submitted to the IMG database as GOLD project Gp0087948, titled “Candidate division JS 1 bacterium SCGC AD-560-N23 (manually screened)”. Gene annotations were performed using both IMG and the Rapid Annotation using Subsystem Technology (RAST) platforms (Aziz et al., 2008; Overbeek et al., 2013 ;Markowitz et al., 2014). Discrepancies between annotations were investigated by comparing coding sequences of genes against GenBank non-redundant protein sequence and Swiss- Prot Databases by BLASTP (Altschul et al., 1990). Genome completeness was estimated by comparing the annotated genome sequence against a list of conserved single copy bacterial genes.
This screened genome was submitted to the IMG database as GOLD project Gp0087948 with an IMG taxon ID of 2626541500, and to MG-RAST under accession numbers 4624791.3–4634830.3.
| Name | Affiliation | Contact |
|---|---|---|
| Stephanie A. Carr | Colorado School of Mines (CSM) | ✓ |
| Beth N. Orcutt | Colorado School of Mines (CSM) | ✓ |
| John R. Spear | Bigelow Laboratory for Ocean Sciences | |
| Shannon Rauch | Colorado School of Mines (CSM) | |
| Shannon Rauch | Colorado School of Mines (CSM) | |
| Shannon Rauch | Woods Hole Oceanographic Institution (WHOI BCO-DMO) |
| Project Title | Functional potential of the uncultivated Candidate Phylum OP9 from deep Antarctic marine sediments using single cell genome techniques |
|---|---|
| Acronym | Adelie Basin Atribacteria |
| URL | https://www.bco-dmo.org/project/626786 |
| Created | November 20, 2015 |
| Modified | November 20, 2015 |
Bacteria belonging to the newly classified candidate phylum “Atribacteria” (formerly referred to as “OP9” and “JS1”) are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica’s Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.
This project was funded by a C-DEBI Postdoctoral Fellowship
| Name | Affiliation | Role |
|---|---|---|
| Stephanie A. Carr | Colorado School of Mines (CSM) | Principal Investigator |
| Beth N. Orcutt | Bigelow Laboratory for Ocean Sciences | Co-Principal Investigator |
| John R. Spear | Colorado School of Mines (CSM) | Co-Principal Investigator |
| Project Title | Functional potential of the uncultivated Candidate Phylum OP9 from deep Antarctic marine sediments using single cell genome techniques |
|---|---|
| Acronym | Adelie Basin Atribacteria |
| URL | https://www.bco-dmo.org/project/626786 |
| Created | November 20, 2015 |
| Modified | November 20, 2015 |
Bacteria belonging to the newly classified candidate phylum “Atribacteria” (formerly referred to as “OP9” and “JS1”) are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica’s Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.
This project was funded by a C-DEBI Postdoctoral Fellowship
| Name | Affiliation | Role |
|---|---|---|
| Stephanie A. Carr | Colorado School of Mines (CSM) | Principal Investigator |
| Beth N. Orcutt | Bigelow Laboratory for Ocean Sciences | Co-Principal Investigator |
| John R. Spear | Colorado School of Mines (CSM) | Co-Principal Investigator |
Bacteria belonging to the newly classified candidate phylum “Atribacteria” (formerly referred to as “OP9” and “JS1”) are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica’s Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.
A cruise is scheduled for December 5-23, 2013 to visit the Dorado Outcrop, a new Major Program focus site of C-DEBI, with Geoff Wheat from the University of Alaska, Fairbanks as Chief Scientist. I have been given the opportunity to participate aboard this expedition as a shipboard microbiologist. In collaboration with the microbiology team (lead by Dr. B. Orcutt), I will part take in routine microbial and geochemical analyses, which will contribute to the following primary expedition objectives: To determine the ecological and biogeochemical nature of microbial communities in cool crustal fluids from the Dorado Outcrop, and determine the degree to which the communities and chemistry vary by comparison to ridge axis systems; and to determine the influence of cool crustal fluid circulation on sediment and hard-rock microbial ecology and biogeochemistry. These objectives will be further fulfilled post-expedition by various shore-based scientists, using samples that I will help to collect and preserve onboard. As a shipboard scientist, I am also entitled to, and responsible for, contributing to post-expedition research. My specific research plan is to employ single cell genomic techniques to identify the biogeochemical potential of uncultivable heterotrophic organisms present within aerobic marine sediment of the Dorado Outcrop. Revealing the potential functions and life requirements of uncultured organisms will greatly benefit our understanding of elemental cycling in the marine subsurface biosphere.
Bacteria belonging to the newly classified candidate phylum “Atribacteria” (formerly referred to as “OP9” and “JS1”) are common in anoxic methane-rich sediments. However, the metabolic functions and biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica’s Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 meters below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.
Over the course of this project, Spear and Boyd took several research trips to Yellowstone National Park for field data collection and sample acquisition. In this project, a mineral coupon holding a field deployable, sampling container was developed that could hold several crushed rock types and/or other materials, to establish the growth of biofilms when the coupons were deployed in hot springs for periods of time. We suspended these mineral coupons in several hot springs of varying chemistry, pH and temperature to observe change over time. One objective was to determine basal rates of metabolic transformation of small organic molecules in ecosystems previously found to be supported by heterotrophic metabolism. Co-I Boyd and graduate student Matthew Urschel determined rates of dissolved inorganic carbon (DIC) mineralization / assimilation to evaluate the importance of different carbon substrates that support metabolism. Urschel and Boyd found in 9 of 13 high-temperature hot springs studied (all greater than the upper temperature limit for photosynthesis of 73C), rates of DIC assimilation were greater than those of formate and acetate assimilation, but 2 exhibited rates of formate and acetate assimilation that exceeded those of DIC assimilation. DIC, formate and acetate mineralization and assimilation was positively correlated with spring pH and showed little correlation with spring temperature. Overall, results indicate that dominant chemoautotrophs in high-temperature communities are facultatively autotrophic or mixotrophic and are adapted to fluctuating nutrient availability. This likely allows hot-spring microbial communities (and other similar subsurface biosphere organisms) to take advantage of energy-rich organic substrates when they become available. The results substantiate earlier efforts by PI Spear for the importance of chemolithoautotrophs to sustain high-temperature, non-photosynthetic hot spring communities. However, the rate of carbon assimilation by these organisms suggests that members of these non-photosynthetic communities may be reliant upon, or take advantage of, organic carbon to support their metabolism. Results indicate that subsurface microbiota in hot environments are versatile in their ability to adapt to environmental fluctuation.
