In-Place Microscopy and the spatial organization of metabolic activity

One of environmental microbiology's "grand challenges" is to faithfully connect findings at widely disparate spatial scales. From the planetary to the molecular scales, microbes have impact across 17 orders of magnitude, and as results are extended from one domain to another, errors propagate while emergent properties and cryptic cycles may be obscured. Through a project funded by the Moore Foundation's Marine Microbiology Initiative, the primary focus of my current work is developing novel approaches to measure metabolic activity in-place while maintaining microscale spatial organization. By incubating environmental samples with non-canonical amino acids, embedding in resin, and performing electron microscopy, micro-CT, and multiplexed FISH, we can see how organism-organism and organism-substrate relationships influence metabolic activity. Ultimately, by integrating "slices" of sample material, it will be possible to directly observe microbial communities from the organism scale through to the ecosystem scale. These techniques will be developed first in estuary sediments and ultimately applied to deep-sea and subsurface samples, exposing critical microbial communities in new detail.

Relevant Publications: Investigations on-going


Revealing & Characterizing novel biological methane sinks in the Deep Sea

At marine methane seeps, an estimated one-fifth of the planet’s methane streams out of the Earth’s crust and into seafloor sediments, representing a critical control point for an important greenhouse gas. As a metabolic by-product of the anaerobic oxidation of methane (AOM), authigenic carbonate rock precipitates, frequently leading to extensive pavements and mounds. Along the Costa Rica margin, for example, seep-associated mounds were observed approximately every four kilometers, highlighting the pervasiveness of carbonate substrate within methane-linked geochemical systems.

This potentially voluminous habitat had not been previously examined as an active microbial habitat. Through a coordinated field campaign at Hydrate Ridge, Oregon, we recovered and quantified endolithic microbial constituents, which were identified by fluorescence in situ hybridization (FISH). Data demonstrated that aggregates were more abundant and larger within carbonate structures than the surrounding sediment, and that aggregate architecture was more diffusely packed. Multiple lines of evidence pointed to substantial metabolic activity of these rock-hosted (endolithic) communities: radiotracer experiments using 14-C methane revealed a full catabolic oxidation to bicarbonate, while nanoSIMS analysis probing 15-N incorporation demonstrated methane-dependent anabolic growth. Surprisingly, similar analysis of sediments and carbonates located at sites believed to be dormant also showed measurable quantities of methane oxidation. This sustained potential for activity indicates that biomarkers within ancient seep structures are likely time-integrated reflections of a range of physicochemical environments, weighted toward particularly productive or recent regimes, and tempered by remineralization, succession and competition among microbial communities. Archaeal and bacterial phylogenetic analyses were conducted using 16S rRNA gene clone libraries, allowing the interrogation of two distinct variables on microbial diversity: physical substrate type (sediment, nodule, carbonate rock) and methane seepage activity (active, low-activity, off-seep). A range of statistical analyses demonstrated that the archaeal community is shaped primarily by seepage activity, while bacterial communities depend more strongly on substrate type. The detection of active endolithic methanotrophic communities - at actively seeping locations and beyond - has important implications for carbon cycling and methane storage. When the vast reservoir of seafloor and sub-seafloor carbonate rock is taken into account, endolithic AOM likely represents a significant methane sink on a global scale, whose presence and continued functionality play key roles in climate regulation.

The observation of active anaerobic methanotrophs within carbonate rocks signified a novel type of endolithic life. In particular, the distinct relationship between metabolism and physical substrate was unaccounted for by the traditional subdivisions of endolithic organisms (euendoliths, cryptoendoliths, and chasmoendoliths). We proposed the term "Autoendolith" to describe organisms - typified by endolithic anaerobic methanotrophs - whose metabolic activity directly or indirectly causes rock formation, and continue to remain active following lithification. Whereas the three previously named categories degrade rock or passively inhabit its cavities, seeking environmental stability or energy sources from the rock setting, autoendoliths construct the structures in which they reside, actively shaping their habitat and seemingly complicating future habitation prospects through pore space filling. Autoendoliths signify intriguing model systems for metabolism - environment feedbacks and offer a new functional category of geobiological process.

Relevant publications:

Marlow, J., Borrelli, C., Jungbluth, S., Hoffman, C., Marlow, J., and P. Girguis, (2017), Telepresence is a Potentially Transformative Tool for Field Science, Proceedings of the National Academy of Sciences. [Link]

Loyd, S., Sample, J., Tripati, R., Defliese, W., Brooks, K., Hovland, M., Torres, M., Marlow, J., Hancock, L., Martin, R., Lyons, T., and A. Tripati, (2016), Methane seep carbonates yield clumped isotope signatures out of equilibrium with formation temperatures, Nature Communications. [Link]

Case, D., Pasulka, A., Marlow, J., Grupe, B., Levin, L., and V. Orphan, (2015), Methane Seep Carbonates Host Distinct, Diverse, and Dynamic Microbial Assemblages, mBio.

Marlow, J., Peckmann, J., and V. Orphan, (2015), Autoendoliths: A Distinct Class of Rock-Hosted Microbial Life, Geobiology. [Link]

Marlow, J., (2015), Methane Fuels Rock-Hosted Ecosystem in the Deep Sea, Microbe Magazine. [Link]

Marlow, J., Steele, J., Case, D., Levin, L., and V. Orphan, (2014), Microbial Abundance and Diversity Patterns Associated with Sediments and Carbonates from the Methane Seep Environments of Hydrate Ridge, OR, Frontiers in Aquatic Microbiology. [Link]

Marlow, J., Steele, J., Thurber, A., Ziebis, W., Levin, L., and V. Orphan, (2014), Carbonate Hosted Methanotrophy: An Unrecognized Methane Sink in the Deep Sea, Nature Communications. [Link]

Thurber, A., Levin, L., Orphan, V., and J. Marlow, (2012), Archaea in metazoan diets: implications for food webs and biogeochemical cycling, ISME Journal. [Link]

Assessing Culture-Independent Metabolic Activity with Stable Isotope Approaches

The vast majority of microbial life on Earth has yet to be cultured, creating challenges for microbiologists seeking genetic or functional details. However, culture-independent work is also an opportunity, exposing inter-organism relationships and providing a more faithful representation of environmental systems. In this context, I have embraced and developed stable isotope probing (SIP) approaches for studying both catabolic and anabolic activity in complex microbial systems.

Metaproteomics is a uniquely valuable tool that provides a functional understanding of microbial systems, moving beyond metabolic potential afforded by nucleotide-based study toward metabolic reality based on enzyme presence. Incorporating SIP with a generalizable growth indicator (such as 15N-ammonium) represents an important entry in the analysis of metabolic activity in low-energy microbial systems, due to its spatially broad, yet functionally- and phylogenetically-specific search space. The procedure is able to identify particular metabolic pathways or enzyme-mediated responses that can be integrated across constituents of a particular lineage, accessing a segment of the low-activity biosphere that would go undetected by other methods due to low levels of anabolism by individual organisms. Through a partnership with the Mass Spectrometry Group at Oak Ridge National Lab, we optimized experimental and computational procedures for the analysis of proteomic data from methane seep sediment, which represents one of the most complicated microbiological communities to date that has been sampled for metaproteomic analysis. These efforts have recovered previously unseen proteins in the reverse methanogenesis pathway and revealed intriguing patterns of nitrogen cycling and protein synthesis. Twenty-eight previously unreported post-translational modification events of McrA were detected, indicating dynamic enzymatic machinery and offering an additional dimension of functional diversity beyond gene-dictated sequence. Community-wide, GroEL and ATPase proteins were the most abundantly detected protein types during the experiment, while RNA polymerase associated with putative sulfur-oxidizing Epsilonproteobacteria and multiple lineages of aerobic Gammaproteobacteria were more abundant in the pre-incubation protein fraction. Twenty-six proteins of unknown function were consistently detected, suggesting that they play important roles in methane seep ecosystems - these proteins represent first-line targets for further investigation of the methanotrophic system.

To more easily measure rates of methane oxidation, we developed a novel method using singly-deuterated methane, CH3D, as a metabolic substrate. Methane activation generates water-exchangeable hydrogen atoms, and the quantification of D/H ratios in the aqueous medium offers a measure of metabolic activity. To compare this method with the better-established 14C radiotracer approach, parallel experiments were conducted using seep sediments and carbonates, as well as aerobic methanotrophic cultures (both Type I and II). Because these two approaches measure the flow of different elements of the methane molecule, the resulting signals are not equivalent, but the scaling factors are substrate-agnostic and remarkably consistent within distinct metabolic modes (e.g., oxic, micro-oxic, and anoxic conditions). Given this consistency and the approach's logistical ease and analytical precision, deuterated methane represents a valuable tool in the arsenal of methanotrophic empiricism.  

Relevant publications:

Marlow, J., and R. Hatzenpichler, (2017), Assessing Metabolic Activity at Methane Seeps: A Testing Ground for Slow-Growing Environmental Systems. In: Kallmeyer, J. (Ed.), Life in Extreme Environments: Life at Vents and Seeps, Berlin: De Gruyter. [Link]

Marlow, J., Steele, J., Ziebis, W., Scheller, S., Case, D., Reynard, L., and V. Orphan, (2017), Monodeuterated Methane: An Isotopic Probe to Measure Biological Methane Metabolism Rates, mSphere. [Link]

Marlow, J., Skennerton, C., Li, Z., Chourey, K., Hettich, R., Pan, C., and V. Orphan, (2016), Proteomic Stable Isotope Probing Reveals Biosynthesis Dynamics of Slow-Growing Methane-Based Microbial Communities, Frontiers in Microbiology. [Link]

Sivan, O., Antler, G., Turchyn, A, Marlow, J., and V. Orphan, (2014), Iron Oxides Stimulate Sulfate Driven Anaerobic Methane Oxidation in Seeps, Proceedings of the National Academy of Sciences. [Link]

Activity-based controls on Methane Metabolism and Molecular Machinery

In support of a 2016-2017 ARPA-E project aimed at developing infrastructure-compatible fuels from methane feedstock, we explored the details of methane-based metabolisms using uncultured seafloor methane seep communities as inspiration. One key objective was to express a methane-activating metabolic pathway in a fast-growing, genetically tractable organism; once incorporated into central metabolism, methane-derived carbon can relatively easily be used to produce targeted products such as plastics or liquid fuels. When expressing a methanogenic version of the key enzyme methyl coenzyme M reductase (Mcr) in E. coli, we observed a distinct profile of post-translational modifications, indicating that protein sequence alone does not determine the final form and activity of the enzyme. Through affinity-based protein pull-down experiments, we subsequently identified several putative methyltransferases that may be critical in generating these modifications.

Related work incorporated anaerobic methanotrophic, sulfide-producing organisms into an industrially relevant platform by developing a sediment-free community. We identified optimized salinity and temperature settings, and demonstrated enhanced metabolic rates on a carbon cloth scaffold. High throughput sequencing revealed a streamlined community composition, and fluorescence in situ hybridization microscopy showed a novel spatial arrangement of AOM consortia partners, potentially due to the substrate's electrical conductivity. These findings are integral to the first-stage of a proposed two-component system to produce biofuel from methane, through sulfide production. 

Relevant publications:

Marlow, J.*, Kumar, A.*, Enalls, B., Reynard, L., Tuross, N., Stephanopoulos, G., and P. Girguis, Biotechnology & Bioengineering (2018), Harnessing a Mixed Microbial Community in Support of a Methane-Fueled, Sediment-Free System for Utilization of Distributed Sources of Natural Gas. [Link]

Energetic Perspectives on Methanotrophic Limits, "Extremophilic" Life, and Astrobiology

Understanding the energetic limits of microbial metabolism offers an important constraint when probing the boundaries of habitable environments, boundaries that dictate the potential extent of biogeochemical processes. By developing a reaction transport model for subsurface AOM, we have provided thermodynamic support for the metabolism on ancient Mars and constrained its potential relevance in terrestrial systems. In a martian context, geologically promising provinces of reactant provisioning (i.e., sulfate minerals and evidence of methane-generating serpentinization) were highlighted, representing promising sites for biomarker investigations.

An energy-centric view of metabolism and biological limitations also prompted a re-consideration of the term "extremophile." For such a term to be broadly meaningful, it must refer to an objective measure of extremeness—an advantageous capability enacted in response to a common challenge. One such challenge, something that all living organisms must face, is the acquisition of chemical energy to drive cellular reactions. Perhaps the ways in which organisms handle this task could separate the truly industrious from the merely viable. This framework represents fertile ground for continued empirical and theoretical work, determining energetic balance sheets to identify and pursue energetic extremophiles.

Relevant publications:

Marlow, J., and J. Amend, (2015), The Real Extremophiles: Life Through an Energetics Lens, The Scientist. [Link]

Marlow, J., LaRowe, D., Ehlmann, B., Amend, J., and V. Orphan, (2014), The Potential for Biologically Catalyzed Anaerobic Methane Oxidation on Ancient Mars, Astrobiology. [Link]

Marlow, J., Martins, Z., and M. Sephton, (2011), Organic host analogues and the search for life on Mars, International Journal of Astrobiology. [Link]

Marlow, J., Martins, Z., and M. Sephton, (2008), Mars on Earth: Streamlining the Search for the Perfect Mars Analog Soil, Astronomy and Geophysics. [Link]

Microbial Colonization and Succession on Recently-Erupted Basaltic Rocks: An analog for life detection on mars

Extrusive volcanic activity constantly creates new basaltic crust, and while this newly formed rock substrate ultimately hosts complex microbial communities, the time frame of colonization and the resulting successional dynamics are unknown. In order to investigate this process, we aim to collect recently-erupted material from the Marum Crater lava lake on Ambrym Island, Vanuatu. Samples across a range of ages - from seconds to decades old - will be analyzed for microbial abundance and diversity analyses in order to determine the temporal colonization limit of life.

The no- / low-biomass Marum Crater samples are ideal test samples for the SHERLOC instrument, which is slated to fly on NASA's Mars 2020 rover mission. The instrument provides co-localized mineralogical identification and aromatic molecule detection utilizing deep UV and Raman spectroscopy, respectively. Mapping the recently produced volcanic rocks may reveal mineralogical associations of early colonization and continued community development.

Relevant publications: Investigations on-going

Lev, E., Ruprecht, P., Oppenheimer, C., Peters, N., Patrick, M., Hernandez, P., Spampinato, L., and J. Marlow, (In Review), A Global Synthesis of Lava Lake Dynamics.