Understanding the assembly of the methane generating enzyme in Methanosarcina acetivorans

Nearly all biologically produced methane comes from Archaea known as methanogens. Whether it is emitted from a cow, natural wetlands, rice paddies or bioreactors, methane in these ecosystems is produced almost entirely from methanogenic Archaea living in oxygen-free niches. The enzyme responsible for methane production is methyl-coenzyme M reductase (Mcr), which contains 6 protein subunits, unique cofactors and a raft of post-translational modifications. In this project I am using Cas9-based genome editing, physiology and structural biology to understand what intermediate states exist in Mcr assembly. Genetic modification of the Mcr operon in the native host has allowed for the purification of Mcr assembly intermediates in large enough quantities for our collaborator Aaron Joiner to generate high resolution models from single-particle cryoEM. Our ongoing work on this project focuses on refining these models and attempting to generate a mechanistic understanding of the Mcr-associated proteins that help with the assembly process.

Physiological and transcriptional response to methane inhibition in a methanogenic archaea

The methane generating enzyme (Mcr) is required for the viability of methanogenic Archaea because all methanogens must make methane to survive. Mcr is also found in the methanotrophic archaea I studied in my PhD, where it is used in the reverse direction to consume methane. In both cases Mcr is one of the most abundant proteins in the cell, and the mRNA encoding it is one of the most prevalent in the transcriptome. This has led to the common assumption that Mcr is rate-limiting in both metabolisms. In this project I have placed the Mcr operon under the control of an inducible promoter allowing for tight control the amount of Mcr that is expressed. We are interested in understanding the aberrant (linear) growth phenotypes we observe when Mcr transcription is inhibited as well as understanding the global transcriptional response to Mcr limitation in Methanosarcina acetivorans. We hope to test whether Mcr abundance is actually limiting methanogenesis under ideal lab conditions, and see whether our induced limitation reveals unknown backup metabolic processes.

Developing the Archaeoglobi into a model system for the study of methane metabolism in Archaea

The past few years have seen tectonic shifts in our understanding of how broadly distributed methane metabolism is in the Archaea. In a 2015 study we found the first metagenomic evidence for the methane generating enzyme (Mcr) outside of the Euryarchaeota—the clade of Archaea previously thought to contain all methane metabolizing archaea (4). Subsequent studies by many groups have validated and extended these observations to an ever-increasing number of uncultivated Archaea. A study I helped with in 2019 found Mcr homologs in an uncultivated member of the hyperthermophilic archaeal class Archaeoglobi (12). A handful of the Archaeoglobi have been isolated in pure culture, including the sulfate reducing Archaeoglobus sp. and the metal and nitrate reducing Ferroglobus and Geoglobus; all of which can use a broad range of complex carbon compounds. My hope for this project is to develop the Archaeoglobi into genetically tractable model organisms to study how methanogenesis and methane oxidation can be gained and lost in Archaea. An added benefit of studying these organisms is that one species contains and extended complex I operon, which is the focus of one of my past research projects.

Evolutionary and Phylogenetic Analysis of Anaerobic Methanotrophic Archaea

This project led by myself and co-first author Connor Skennerton was a multi-institute collaborative analysis of all currently available metagenome-assembled genomes (MAGs) of Anaerobic Methanotrophic Archaea (ANME) (27). This work represents the state-of-the-art in our understanding of how the symbiosis between ANME and their bacterial partners functions to remove this potent greenhouse gas from marine methane seeps. The "parts list" of ANME-specific proteins we highlight here are thought to be crucial for the adaptation of a methanogenic ancestor to a methanotrophic lifestyle. The most exciting of these parts was the acquisition of extracellular electron transport machinery, which allows the ANME to share electrons directly with their syntrophic partner without a diffusible intermediate.

Characterization of an ultrafast swimming phototrophic bacteria

While working as a teaching assistant at the Woods Hole Microbial Diversity course I enriched a new genus of purple sulfur bacteria based on their communal swimming and biofilm forming behavior (21). Nearly all bacteria are small enough and slow enough to live in what is called a low-Reynolds number regime, where substrate supply is determined entirely by diffusive and not advective transport. High speed video microscopy and single particle tracking revealed this new bacteria to be the fastest swimming phototrophic bacteria, with top speeds of just over 800 microns per second. This size and speed puts them in an intermediate regime where our modelling suggests the fluid flow they produce can actually increase their substrate supply beyond what diffusion alone can generate.

Mapping the activity of individual cells in symbiotic methane oxidizing consortia

The anaerobic oxidation of methane is a hugely important process in many marine and terrestrial environments that prevents methane, a strong greenhouse gas, from escaping into the atmosphere. The microorganisms responsible for this are archaea known as ANME that live in symbiotic consortia with sulfate reducing bacteria (SRB). In this project led by myself and co-first author Shawn McGlynn we combined stable isotope incubation experiments coupled to fluorescent in situ hybridization (FISH) and nanometer-scale secondary ion mass spectrometry (nanoSIMS) to map the activity of individual cells in relation to other cells in their structured community (5). These spatially-indexed activity measurements, combined with genomic analysis and modelling, indicate that the ANME-SRB syntrophy is based on the direct transfer of electrons between the symbiotic partners.

Mapping the activity of electroactive microbes at sub-micron resolution

Electroactive microorganisms such as Geobacter sulfurreducens are able to grow using anodes as their terminal electron acceptor. These features have resulted in great interest in using these organisms in bioelectrochemical reactors, where organic matter degradation is linked directly to electricity generation. The current density generated in these systems is an important value that determines the applicability of these technologies, so understanding how cell growth is limited in these communities is of great importance. In this work led by myself and co-first author Fernanda Jiménez Otero we used stable isotope pulses and nanoSIMS imaging to map the activity of G. sulfurreducens at sub-micron resolution within anodic biofilms. We showed that growth largely decreases with distance from the anode surface, suggesting that biofilm conductivity, not carbon substrate supply, was limiting in our reactors (15). We further showed how changes in the applied potential on the anode can effect the pattern of cell activity in the biofilms.

NanoSIMS and modeling shed light on extracellular electron transfer mechanisms

In a series of three papers led by Xiaojia He during his time at the University of Georgia we combined our FISH-nanoSIMS approach to visualize the metabolic activity of single cells in complex microbial communities with Xiaojia's metabolic modeling approaches. Through this series of papers we were able to iterate between experimentation and modeling to produce a detailed understanding of the interplay between physical community structure and overall function of the community. These papers have greatly advanced our understanding of what is possible in the ANME-SRB symbiosis, as well as helped shed light on what limits the current densities that can be achieved by Geobacter biofilms grown on electrodes (18, 13, 17). Further application of the isotope probing and imagine techniques, in conjunction with genetic manipulation and bioelectrochemical analysis, helped explain increased current density in Geobacter mutants led by Fernanda Jiménez Otero (20).

Evolutionary convergence on structural modifications to respiratory Complex I for increased proton pumping

In a comprehensive phylogenomic analysis of respiratory Complex I operons across all prokaryotic genomes I found a repeated pattern of incorporation of an additional proton pumping subunit. Leveraging recent crystal structures of Complex I the acquisition of these pumping subunits co-occurs with amino acid insertions necessary for changing the quaternary structure to incorporate the extra subunit. Remarkably, this genetic and structural reorganization evolved independently in three unrelated phylogenetic groups. These observations help explain a longstanding mystery about carbon fixation in ammonium-oxidizing Nitrospira (10). In a comparative genomic study led by Hank Yu on the newly-discovered chemolithoautotrophic manganese oxidizing bacteria we found that these organisms have iterated on this evolutionary process, bringing in yet another pumping subunit, indicating the ease with which this design principle can be repeatedly utilized for augmenting microbial energy metabolism (24).

Investigations into hyperalkaliphilic life in serpentinizing systems

Serpentinization is a geologic process that can produce marine hydrothermal vents or terrestrial springs that are characterized by very low redox potential and very high pH. The chemical gradients that occur when these fluids mix with more oxidizing waters of the ocean or the oxygenated atmosphere make for a great habitat for extremophilic microorganisms. In a project led by Leah Trutschel in Annie Rowe's lab, I helped with a series of field campaigns to characterize the chemistry and microbiology of Ney Springs, a serpentinizing spring from Northern California with the most extreme eH/pH of any natural waters on Earth (26). Ongoing work on this system is attempting cultivation of some of the dominant organisms in the pH 12 waters, as well as continued monitoring of the microbial community dynamics over time.

Understanding the breadth and significance of nitrogen fixation in marine methane seep ecosystems

During my time in the Orphan lab I worked on three studies all led by Anne Dekas that helped us better understand the role of nitrogen fixation in marine sediments. In this work I helped set up stable isotope probing incubations with 15N-labeled nitrogen gas so that we could trace bulk nitrogen fixation rates with Elemental Analyzer Isotope Ratio Mass Spectrometry, as well as trace nitrogen fixation into individual organisms using our combined FISH-nanoSIMS approach. I also extracted DNA and RNA from our sediment incubations to help understand which organisms in the community contained the genetic potential for nitrogen fixation and who were actively transcribing nitrogenase genes. Most of our work focused on methane seep ecosystems(2,6), but we also compared the nitrogen fixation rates and active organisms between these ecosystems and other marine sediments such as background non-seep sediments and the communities surrounding whalefalls(9).

NanoSIMS investigations of other structured microbial communities

I was lucky enough to be a collaborator on a number of other projects where we could use our FISH-nanoSIMS approach to help other groups answer interesting questions of their structure microbial communities. In a project led by DeAnna Bublitz I helped design experiments and develop a sample preparation strategy to trace isotopically labelled D-alanine into the peptidoglycan layer of Moranella sp. which live inside Tremblaya sp., which in turn live inside of special bacteriocyte cells in Mealybugs (14). This application of phylogenetic imaging and isotope imaging helped us make the case that peptidoglycan production of the innermost bacterial symbiont was dependent on biosynthetic processes that have been exported to the insect genome. In another study led by Julia Schwartzman we used stable isotope probing to understand the nutrient consumption dynamics of vibrio cultures that form bizarre spherical biofilms, where phenotypic differentiation occurs, resulting in an outer layer of immobile cells forming a shell around a rapidly swimming motile inner core (25).

Remote metorship during COVID-19

During the COVID-19 pandemic many summer research programs were forced to be remote, which is particularly hard in the biological sciences where many of the students hope to get laboratory experience. During the summer of 2020 I volunteered as a mentor for the Research Science Institute (RSI) at MIT, a program I participated in as a student in 2006. During that project my summer student and I expanded on the complex I analysis I did in my PhD (10), generating a series of python programs to screen the entire GTDB prokaryote genome database for complex I operons and layer these gene clusters on phylogentic trees to easily visualize gene synteny changes through evolutionary time. In the summer of 2021 I mentored two additional RSI students and two students from NIH's Bridges to Baccalaureate (B2B) Research Training Program run by the Molecular and Cell Biology department at UC Berkeley. Projects included evolution of flu virus spike proteins and the incorporation of the non-standard amino acid pyrrolysine into proteins in methanogenic archaea.

In-person mentorship

In 2018 I served as a summer research mentor for the Amgen Scholars Program. My mentee and I developed protocols for coupling fluorescence in situ hybridization with laser capture microdissection of individual ANME-SRB aggregates from methane seep sediments. Individual captured aggregates were subjected to DNA extraction and PCR for marker genes to link the spatial organization of the community with strain-level information on the partner organisms.
Summer 2022 I am following up with one of my remote B2B students from last summer who wanted to come back and get some laboratory experience. Together we are working on making some of the genome editing contructs and testing transformation protocols in my Archaeoglobus genetics project.

Science outreach at El Cerrito High School

Starting in 2020 the Miller Insitute at UC Berkeley began working with science teachers at El Cerrito High School to develop outreach aimed at inspiring and educating students about careers in science. I participated virtually during pandemic school closures, and then took over organizing these outreach efforts in early 2021, transitioning them to in-person format. In these classroom visits I share some of my favorite science demos and answer questions about scientific careers. For biology I have brought microscopes and a variety of everyday examples of symbiosis and parasatism in lichens and dodder. For the physics classes I bring a cloud chamber for visualizing cosmic rays and ferrofluid for to visualize magnetic field lines.

Awards

• 2020 – 2023 Miller Research Fellowship
• 2017 – 2019 NIH Predoctoral Training Grant in Molecular and Cell Biology
• 2016 Gordon P. and Virginia G. Eaton Graduate Fellowship
• 2015 Caltech CEMI Fellowship
• 2013 Rose Hills Fellowship
• 2010 NCAA Men’s Soccer Captain, First-Team All-Conference, Brine Award of Distinction
• 2009, 2010 Caltech Upperclass Merit Scholarship
• 2009 NCAA Men’s Soccer Captain, Second-Team All-Conference, Brine Award of Distinction
• 2009 Amgen Scholars Program
• 2009 George W. and Bernice E. Green Memorial Prize
• 2008, 2010 Caltech Summer Undergraduate Research Fellowship
• 2008 Richard G. Brewer Prize in Physics

Metrics

As of July 2022
• Publications 26
• Citations 1933
• h-index 14
• i10-index 15

Teaching and Service

• 2022 Co-chair Gordon Research Seminar Microbial One-Carbon Conversion from the Microscale to the Global Scale

• 2010 – 2019 Teaching assistant for Caltech courses: Microbial Ecology, Evolution, Microbial Metabolic Diversity, Bioengineering Bootcamp, The Great Ideas of Biology, Principles of Biology

• 2017 - 2019, 2022 Teaching assistant for International Geobiology Course, Caltech

• 2016 Teaching assistant for Microbial Diversity Course, Marine Biological Laboratory at Woods Hole

• 2016, 2019 Organizer Southern California Geobiology Symposium

• 2017 – 2018 Organizer Caltech Geoclub Seminar Series

• Reviewer for: Nature Microbiology, Science, Frontiers in Microbiology, Environmental Microbiology, Communications Biology, mSystems, Limnology and Oceanography: Methods, Elementa, Biogeosciences, NASA Astrobiology External Reviewer