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Frontiers in Microbiology

Comparative Genomics and Proteomic Analysis of Assimilatory Sulfate Reduction Pathways in Anaerobic Methanotrophic Archaea

Authors: Hang Yu, Dwi Susanti, Shawn E. McGlynn, Connor. T. Skennerton, Karuna Chourey, Ramsunder Iyer, Silvan Scheller, Patricia L. Tavormina, Robert L. Hettich, Biswarup Mukhopadhyay, Victoria J. Orphan

Published: December 3, 2018

C-DEBI Contribution Number: 449

Abstract

Sulfate is the predominant electron acceptor for anaerobic oxidation of methane (AOM) in marine sediments. This process is carried out by a syntrophic consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB) through an energy conservation mechanism that is still poorly understood. It was previously hypothesized that ANME alone could couple methane oxidation to dissimilatory sulfate reduction, but a genetic and biochemical basis for this proposal has not been identified. Using comparative genomic and phylogenetic analyses, we found the genetic capacity in ANME and related methanogenic archaea for sulfate reduction, including sulfate adenylyltransferase, APS kinase, APS/PAPS reductase and two different sulfite reductases. Based on characterized homologs and the lack of associated energy conserving complexes, the sulfate reduction pathways in ANME are likely used for assimilation but not dissimilation of sulfate. Environmental metaproteomic analysis confirmed the expression of 6 proteins in the sulfate assimilation pathway of ANME. The highest expressed proteins related to sulfate assimilation were two sulfite reductases, namely assimilatory-type low-molecular-weight sulfite reductase (alSir) and a divergent group of coenzyme F₄₂₀-dependent sulfite reductase (Group II Fsr). In methane seep sediment microcosm experiments, however, sulfite and zero-valent sulfur amendments were inhibitory to ANME-2a/2c while growth in their syntrophic SRB partner was not observed. Combined with our genomic and metaproteomic results, the passage of sulfur species by ANME as metabolic intermediates for their SRB partners is unlikely. Instead, our findings point to a possible niche for ANME to assimilate inorganic sulfur compounds more oxidized than sulfide in anoxic marine environments.

Source: http://dx.doi.org/10.3389/fmicb.2018.02917

Awards > Research Exchange Grants

International Geobiology Course

Awardee: Heidi Aronson (University of Southern California)

Advisor: Jan P. Amend (University of Southern California)

Hosts: Alex L. Sessions (Caltech), Woody Fischer (Caltech), Victoria J. Orphan (Caltech)

Amount: $2,000.00

Award Dates: June 12, 2018 — July 15, 2018

Abstract

With the generous support from the C-DEBI research exchange grant, I had the opportunity to participate in the International Geobiology Course, which was directed by Drs. Alex Session, Victoria Orphan, and Woody Fischer from the California Institute of Technology (in conjuction with the Agouron Institute, Simons Foundation and USC Wrigley Institute). In this course, I traveled with 15 other geobiology graduate students to Mono Lake, Naples Beach, and Santa Paula Creek where we learned how to collect biological and geochemical samples for analysis at Caltech. At Caltech, I learned cutting-edge laboratory techniques including SEM, stable isotope analysis, SIMS, and NanoSIMS. Finally, on Catalina Island, I worked with three other students on a project that investigated the sulfur cycle at Santa Paula Creek, which we will be presenting at the AGU annual meeting. Participating in this course provided not only comprehensive training in geobiology, but also a unique opportunity to network with established scientists and peers that I hope to collaborate with in the future. Since returning from this course, I have a stronger understanding of current interdisciplinary topics and questions in geobiology and the ways in which these ideas are addressed. I look forward to continuing to pursue research in geobiology and collaborating with the scientists I have connected with on this course.

Awards > Research Exchange Grants

Toward a better understanding of the active deep subsurface biosphere through the Geobiology Summer Course

Awardee: Megan. M. Mullis (Texas A&M University, Corpus Christi)

Advisor: Brandi Kiel Reese (Texas A&M University, Corpus Christi)

Hosts: Alex L. Sessions (Caltech), Woody Fischer (Caltech), Victoria J. Orphan (Caltech)

Amount: $2,000.00

Award Dates: June 12, 2018 — July 15, 2018

Abstract

A C-DEBI research exchange was awarded for travel to the International Geobiology Summer Course hosted by California Institute of Technology (in conjuction with the Agouron Institute, Simons Foundation and USC Wrigley Institute). This course offered many unique opportunities including extensive field sampling, lab work, and data analyses. Field sampling occurred at Mono Lake, Little Hot Creek, the Monterey Formation, and Sulfur Mountain. Laboratory procedures included DNA extraction and PCR, CARD-FISH, microeukaryote culturing, nanoSIMS, beamline, SEM, biomarker, isotopes, and petrography analyses. We found that there was potential for microbial communities to be active at low levels in Mono Lake sediments. We also concluded that there were detrital input of albite and orthoclase into Mono sediments that correlated with El Niño and La Niña events. These data were analyzed and presented for the participants, directors, and course administrators on the final day. This experience not only provided me with technical training, but also allowed me to build an extensive network of colleagues in the field of geobiology. This course was relevant to C-DEBI Research Themes 2 (Activities, Communities, and Ecosystems) and 3 (Metabolism, Survival, and Adaptation) because we connected microbial community structure and potential function to geochemical measurements.

Protocols

Examining active cells after incubation with a labeled substrate (eg Carbon-13, Nitrogen-15, Hydrogen-2 or Sulfur-33/34)

Authors: Elizabeth Trembath-Reichert, Yuki Morono, Fumio Inagaki, Victoria J. Orphan

Posted: January 24, 2018

Source: https://dx.doi.org/10.17504/protocols.io.gwubxew

Publications > Journal Article

Proceedings of the National Academy of Sciences

Methyl-compound use and slow growth characterize microbial life in 2-km-deep subseafloor coal and shale beds

Authors: Elizabeth Trembath-Reichert, Yuki Morono, Akira Ijiri, Tatsuhiko Hoshino, Katherine. S. Dawson, Fumio Inagaki, Victoria J. Orphan

Published: October 3, 2017

C-DEBI Contribution Number: 389

Abstract

The past decade of scientific ocean drilling has revealed seemingly ubiquitous, slow-growing microbial life within a range of deep biosphere habitats. Integrated Ocean Drilling Program Expedition 337 expanded these studies by successfully coring Miocene-aged coal beds 2 km below the seafloor hypothesized to be “hot spots” for microbial life. To characterize the activity of coal-associated microorganisms from this site, a series of stable isotope probing (SIP) experiments were conducted using intact pieces of coal and overlying shale incubated at in situ temperatures (45 °C). The 30-month SIP incubations were amended with deuterated water as a passive tracer for growth and different combinations of ¹³C- or ¹⁵N-labeled methanol, methylamine, and ammonium added at low (micromolar) concentrations to investigate methylotrophy in the deep subseafloor biosphere. Although the cell densities were low (50–2,000 cells per cubic centimeter), bulk geochemical measurements and single-cell–targeted nanometer-scale secondary ion mass spectrometry demonstrated active metabolism of methylated substrates by the thermally adapted microbial assemblage, with differing substrate utilization profiles between coal and shale incubations. The conversion of labeled methylamine and methanol was predominantly through heterotrophic processes, with only minor stimulation of methanogenesis. These findings were consistent with in situ and incubation 16S rRNA gene surveys. Microbial growth estimates in the incubations ranged from several months to over 100 y, representing some of the slowest direct measurements of environmental microbial biosynthesis rates. Collectively, these data highlight a small, but viable, deep coal bed biosphere characterized by extremely slow-growing heterotrophs that can utilize a diverse range of carbon and nitrogen substrates.

Source: http://dx.doi.org/10.1073/pnas.1707525114

Publications > Journal Article

mBio

Methane-Fueled Syntrophy through Extracellular Electron Transfer: Uncovering the Genomic Traits Conserved within Diverse Bacterial Partners of Anaerobic Methanotrophic Archaea

Authors: Connor. T. Skennerton, Karuna Chourey, Ramsunder Iyer, Robert L. Hettich, Gene. W. Tyson, Victoria J. Orphan

Editors: Nicole Dubilier

Published: August 1, 2017

C-DEBI Contribution Number: 374

Abstract

The anaerobic oxidation of methane by anaerobic methanotrophic (ANME) archaea in syntrophic partnership with deltaproteobacterial sulfate-reducing bacteria (SRB) is the primary mechanism for methane removal in ocean sediments. The mechanism of their syntrophy has been the subject of much research as traditional intermediate compounds, such as hydrogen and formate, failed to decouple the partners. Recent findings have indicated the potential for extracellular electron transfer from ANME archaea to SRB, though it is unclear how extracellular electrons are integrated into the metabolism of the SRB partner. We used metagenomics to reconstruct eight genomes from the globally distributed SEEP-SRB1 clade of ANME partner bacteria to determine what genomic features are required for syntrophy. The SEEP-SRB1 genomes contain large multiheme cytochromes that were not found in previously described free-living SRB and also lack periplasmic hydrogenases that may prevent an independent lifestyle without an extracellular source of electrons from ANME archaea. Metaproteomics revealed the expression of these cytochromes at in situ methane seep sediments from three sites along the Pacific coast of the United States. Phylogenetic analysis showed that these cytochromes appear to have been horizontally transferred from metal-respiring members of the Deltaproteobacteria such as Geobacter and may allow these syntrophic SRB to accept extracellular electrons in place of other chemical/organic electron donors.

Source: http://dx.doi.org/10.1128/mbio.00530-17

Publications > Journal Article

PeerJ

Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments

Authors: Elizabeth Trembath-Reichert, David Hamilton Case, Victoria J. Orphan

Published: April 18, 2016

C-DEBI Contribution Number: 345

Abstract

Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range of Deltaproteobacteria diversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seep sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. Many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1, Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involving Epsilon-, Delta-, and Gammaproteobacteria in methane seep ecosystems.

Source: http://dx.doi.org/10.7717/peerj.1913

Protocols

Detection of protein-synthesizing microorganisms in the environment via bioorthogonal non-canonical amino acid tagging (BONCAT)

Authors: Roland Hatzenpichler, Victoria J. Orphan

Posted: July 24, 2016

Source: http://dx.doi.org/10.17504/protocols.io.drp55m

Publications > Journal Article

Proceedings of the National Academy of Sciences

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

Authors: Roland Hatzenpichler, Stephanie A. Connon, Danielle Goudeau, Rex R. Malmstrom, Tanja Woyke, Victoria J. Orphan

Published: June 28, 2016

C-DEBI Contribution Number: 330

Abstract

To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.

Source: http://dx.doi.org/10.1073/pnas.1603757113

Publications > Journal Article

Astrobiology

The Potential for Biologically Catalyzed Anaerobic Methane Oxidation on Ancient Mars

Authors: Jeffrey J. Marlow, Douglas E. LaRowe, Bethany L. Ehlmann, Jan P. Amend, Victoria J. Orphan

Published: April 1, 2014

C-DEBI Contribution Number: 174

Abstract

This study examines the potential for the biologically mediated anaerobic oxidation of methane (AOM) coupled to sulfate reduction on ancient Mars. Seven distinct fluids representative of putative martian groundwater were used to calculate Gibbs energy values in the presence of dissolved methane under a range of atmospheric CO₂ partial pressures. In all scenarios, AOM is exergonic, ranging from −31 to −135 kJ/mol CH₄. A reaction transport model was constructed to examine how environmentally relevant parameters such as advection velocity, reactant concentrations, and biomass production rate affect the spatial and temporal dependences of AOM reaction rates. Two geologically supported models for ancient martian AOM are presented: a sulfate-rich groundwater with methane produced from serpentinization by-products, and acid-sulfate fluids with methane from basalt alteration. The simulations presented in this study indicate that AOM could have been a feasible metabolism on ancient Mars, and fossil or isotopic evidence of this metabolic pathway may persist beneath the surface and in surface exposures of eroded ancient terrains.

Source: http://dx.doi.org/10.1089/ast.2013.1078

Awards > Research Exchange Grants

Determination of deep biosphere cell activity and identity utilizing the state of the art low-biomass, single cell techniques developed at JAMSTEC in their class 10,000 clean room

PI: Elizabeth Trembath-Reichert (California Institute of Technology)

Current Placement: Postdoctoral Researcher, Woods Hole Oceanographic Institution

Advisor: Victoria J. Orphan (California Institute of Technology)

Host: Fumio Inagaki (JAMSTEC)

Amount: $1,375.00

Award Dates: March 17, 2015 — April 10, 2015

Abstract

IODP Expedition 337 set the record for deepest marine scientific drilling down to 2.4 kmbsf. This cruise also had the unique opportunity to retrieve deep cores from the Shimokita coal bed system in Japan with the aseptic and anaerobic conditions necessary to look for deep life. Onboard scientists prepared nearly 1,700 microbiology samples shared among five different countries to study life in the deep biosphere. Samples spanned over 1km in sampling depths and include representatives of shale, sandstone, and coal lithologies. Findings from previous IODP and deep mine expeditions suggest the genetic potential for methylotrophy in the deep subsurface, but it has yet to be observed in incubations. A subset of Expedition 337 anoxic incubations were prepared with a range of ¹³C-methyl substrates (methane, methylamine, and methanol) and maintained near in situ temperatures. To observe ¹³C methyl compound metabolism over time, we monitored the δ¹³C of the dissolved inorganic carbon and methane (by-products of methyl compound metabolism) over a period of 1.5 years. Our geochemical evidence suggests that the coal horizon incubated with ¹³C-methylamine showed the highest activity of all methyl incubations. Therefore, there are not only cells in the deeply buried terrigenous coal bed at Shimokita, but a microbial community that can be activated by methylotrophic compounds. Incubations showing the highest geochemical activity were prepared at the JAMSTEC Kochi Core Center for nanoSIMS analysis in March of 2015, and will be analyzed at Caltech in the coming months. This will allow us to observe if cells also incorporated the labeled methyl compounds into their body mass and provide another line of evidence that these substrates were used by the deep coalbed microbial community.

Awards > Postdoctoral Fellowships

Activity-based cell-sorting and enrichment of newly synthesized proteins via amino acid tagging and click chemistry

Awardee: Roland Hatzenpichler (California Institute of Technology)

Current Placement: Assistant Professor, Montana State University, Bozeman, 2016-

Degree: Ph.D. Microbial Ecology, University of Vienna (2011)

Advisor: Victoria J. Orphan (California Institute of Technology)

Amount: $120,000.00

Award Dates: July 17, 2014 — July 13, 2016

Abstract

The very low rates of activity typically observed for subseafloor microbes make it hard to differentiate slow-growing from inactive cells. During his C-DEBI postdoctoral fellowship Roland Hatzenpichler developed novel approaches to detect biosynthetic activity in uncultured cells directly in their habitat. Based on the incorporation of synthetic amino acids into new proteins and their subsequent detection via fluorescence staining (BONCAT, bioorthogonal non-canonical amino acid tagging), protein synthesis active cells are visualized via epifluorescence microscopy. In combination with fluorescence-activated cell-sorting, they can then be separated from complex samples. After adapting BONCAT for environmental applications, Roland used BONCAT to track the in situ translational activity of syntrophic archaeal-bacterial consortia catalyzing the anaerobic oxidation of methane in deep-sea sediments (Hatzenpichler et al., PNAS 2016). By combining sorting of active consortia, whole genome amplification, and 16S rRNA gene sequencing, the identities of hundreds of individual, biosynthetically active cellular partnerships could be resolved. This approach revealed that representatives of all major methane-oxidizing archaeal clades were active under controlled incubation conditions. Unexpectedly, some consortia were anabolically active even in the absence of methane, suggesting energy sources beyond methane. It also led to the discovery of a previously unrecognized interaction of methane-oxidizing archaea with members of the environmentally highly abundant, yet poorly understood phylum Verrucomicrobia. Since November 1^st 2016, Roland is an Assistant Professor in the Department of Chemistry and Biochemistry at Montana State University, Bozeman. His group focuses on the ecophysiology and in situ activity of uncultured archaea in a variety of sediment environments. www.environmental-microbiology.com

Awards > Research Grants

Microbe-mineral interactions in oligotrophic subseafloor habitats

PI: Victoria J. Orphan (California Institute of Technology)

Co-Is: Joshua Steele (California Institute of Technology),

Current Placement: Microbiologist, SCCWRP, 2014-

Anne E. Dekas (California Institute of Technology)

Current Placement: Assistant Professor, Stanford, 2015-

Amount: $49,974.00

Award Dates: May 1, 2011 — April 30, 2013

Abstract

Although the subsurface biosphere is now recognized as an important reservoir of life on our planet, until recently the microbial community beneath open-ocean oligotrophic gyres (making up the majority of the seafloor) has not been studied in detail (D’Hondt et al., 2004, 2009). IODP Expedition 329 has taken a first step at characterizing the microbial community beneath the South Pacific Gyre. This region has low biological surface productivity and therefore very low organic carbon burial rates (10^-8 and 10^-10 moles C cm^-1 yr^-1), deep oxygen penetration (sediments are oxidized to the basement), and low prokaryotic cell counts (10⁶ cells cm^-3 to <10³ cells cm^-3) (D’Hondt et al., 2009; Fischer et al., 2009, IODP Exp. 329 Preliminary Report, 2011). In these sediments, the dominant fraction of organic carbon may be aggregated or adsorbed to minerals (Arnarson & Kiel 2007). Thus the ability to colonize minerals should be an important ecological adaptation, with those microbes that are able to grow on the minerals creating potential “hotspots” of microbial activity within these oligotrophic sediments. Our project aims to determine whether there is stimulated microbial activity associated in long-term incubations with H¹³CO^3- and ¹⁵NO^3-. Specific mineral and clay fractions in the oligotrophic South Pacific Gyre sediment system were targeted using combination of magnetic and density separation and SEM-EDS. The bacterial and archaeal community were examined by CARD-FISH, CARD-FISH-nanoSIMS, and 16S rRNA tag sequencing. Overall results from this C-DEBI grant have shown the viability of magnetic separation and identification of single cells in subsurface sediments as a method for investigating mineral association in microbial communities. We have identified putatively viable cells attached to 7 Fe/Mn-rich minerals, potentially representing an unexplored strategy for low-carbon environments. We also have discovered a higher level of diversity in the paramagnetic (Fe/Mn-rich) mineral-associated bacteria and higher number of Marine Group I archaeal OTUs compared to the diamagnetic fraction in the oligotrophic subsurface sediment from the South Pacific Gyre.

Person: Victoria J. Orphan

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