Video: MMB reacting to a magnetic field.
Visualizing the activity of sediment microbes. DAPI, blue. FISH, green. BONCAT, magenta. Bar, 10 micron.
Biogeochemical function and in situ activity of yet uncultured archaea in hot springs
Development of new substrate analog probing approaches for singe cell resolved activity tracing is performed in collaboration with JGI and EMSL via a FICUS project. Our work on developing Next-generation physiology approaches for visualizing activity of marine microbes at nm to cm scale is funded by the Marine Microbiology Initiative of the Gordon and Betty Moore Foundation and the NSF and is performed in collaboration with Erik Grumstrup and Stephan Warnat, Valerie Copie and Brian Bothner (MSU), Peter Girguis (Harvard), Jeff Marlow (Boston U), and Mark Ellisman (UCSD). The acquisition of a Raman microscope was spearheaded by Roland and funded by the NSF MRI program and the M.J. Murdock Charitable Trust.
Research on MMB is funded by the NASAExobiology program.
Next-generation physiology approaches to probe in situ function of uncultured microbes
Video: Five Sisters hot springs group.
Our hot spring research is supported by a FICUS project with the JGI and EMSL, an NSF EPSCoR project, the NASA Exobiology program, and a NASA Early Career Fellowship to Roland. Nick's work at the JGI is supported by an Office of Science Graduate Student Research (SCGSR) award by the Department of Energy. Research is performed in collaboration with Emiley Eloe-Fadrosh and Tanja Woyke (JGI), Luke McKay, Tim McDermott, Bill Inskeep, Robin Gerlach, Brent Peyton, and others at MSU's Thermal Biology Institute.
If the physiology of uncultured cells is to be determined, approaches that target the single cell level are essential in order to link the identity of a cell to its specific function. Ideally, these approaches are not destructive to the cell so that it can be analyzed using complementary downstream applications, for example targeted cultivation or genome sequencing. Our lab has recently introduced the term Next-generation physiology approaches to refer to such techniques. Our lab is pioneering the development and application of a new approach to studying anabolically active, but uncultured cells. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of synthetic amino acids that exploit the substrate promiscuity of the translational machinery. After incorporation into new proteins, these amino acids can be fluorescently detected via azide-alkyne click chemistry, a highly selective and biocompatible labeling reaction. When used in conjunction with rRNA-targeted fluorescence in situ hybridization (FISH), BONCAT allows the identity of a cell and its in situ activity to be linked. BONCAT is intrinsically high throughput and, when combined with cell-sorting devices enables cells to be separated from samples based on their anabolic activity and to study the response of microbes to substrate amendment. In contrast to isotope labeling approaches, which require highly specialized instruments, BONCAT-FISH and BONCAT-FACS use standard microscopes and flow cytometers that are readily available to microbiology labs. We are currently expanding our work to other classes of biomolecules (DNA, lipids, peptidoglycan) and are developing new approaches to target cellular in situ activity on single cell level.
In collaboration with the labs of Andreas Teske (Univ. of North Carolina, Chapel Hill), Brett Baker (Univ. of Texas at Austin), Samantha Joye (U Georgia), Virginia Edgcomb (WHOI), and Karthik Anantharaman (U Wisconsin) we study the genomics and ecophysiology of uncultured bacteria and archaea in cold and hydrothermal sediments in the Guaymas and Pescadero deep-sea basins in the Gulfs of California and Baja, respectively. Specifically, we seek to determine the identities, in situ activities, and niche-determining factors of cells involved in the degradation of high molecular weight carbon (HMW) compounds of photosynthetic origin, detrital protein and lipids, and HMW hydrocarbons, as well as deep-sea methanogenic lineages. We also seek to determine how pressure, temperature, and other physicochemical parameters affect microbial in situ activity. Techniques used in this line of research include lab mesocosms that replicate in situ conditions (e.g., temperature, pressure), BONCAT-FACS, SIP-Raman, and metatranscriptomics.
Investigated by: Viola Krukenberg. New postdoc starting in spring 2021. We are also looking for a new graduate student to join this project in fall 2021.
Investigated by: New postdoc starting early summer 2021
Our research is funded by Moore–Simons Project on the Origin of the Eukaryotic Cell. MSU press release
Best of" video, research cruise AT42-05 that sampled Guaymas basin sediments. Credit: WHOI and R. Hatzenpichler.
Our work on Guaymas and Pescadero basin microbiology and biogeochemistry is supported by the Systems and Synthetic Biology, Biological Oceanography, and Chemical Oceanography programs of the NSF. Previous research on microbes in coastal sediments was supported by the Gordon and Betty Moore Foundation.
Understanding the origins of multicellularity and the organization of complex life is a critical endeavor in biology. Past studies on the transition from single cells to multicellular entities mostly focused on volvocine green algae and early radiating animal taxa as experimental systems. While multicellular bacteria exist, their organization appears comparatively simple, and multicellularity seems to occur only as an adaptation to changing environmental conditions or as a single step in a complex life cycle.
A group of delta-Proteobacteria termed multicellular magnetotactic bacteria (MMB) constitutes the only known exception to this view. MMB are mono-species consortia typically 3-15 μm in size. They are comprised of 10-60 cells arranged in symmetry around a central acellular compartment. Each cell is multiply flagellated and contains magnetic crystals, called magnetosomes, which are used to guide the consortia along the geomagnetic field lines. The life cycle of MMB has no known unicellular stage. Division occurs by separation of a MMB into two apparently identical daughter consortia, while disaggregated cells rapidly lose viability. These characteristics render MMB the only identified bacteria with an obligate multicellular lifestyle and make them a prime subject for the study of the early evolution of advanced life, the cellular and molecular organization of multicellular entities, and the extent of division of metabolic labor and cellular differentiation in bacteria.
Some of the currently most pressing questions are: do individual cells within MMB consortia exhibit cell differentiation and division of labor? Are the consortia clonal and, if not, what are the evolutionary consequences? Which genetic factors do MMB share with other multicellular microbes and which ones set them apart from their unicellular relatives? What is the diversity and ecophysiology of MMB? What are the factors controlling their ecology and the fine interplay of aero-, chemo-, magneto-, and phototaxis?
Investigated by: George Schaible
In addition to bioorthogonal labeling, we use stable isotope probing in combination with single cell visualization instruments to study substrate uptake and in situ activity. Specifically, we use SIP-FISH-Raman (via a Horiba Confocal Raman microspectroscope that was brought to MSU by Roland in 2019) to study the biochemistry and in situ biogeochemical function of uncultured cells. In collaboration with other researchers at MSU, we also are developing new Raman-activated cell sorting technology. In addition, we collaborate with the Environmental Molecular Sciences Laboratory to use their nano-scale secondary ion mass spectrometry (nanoSIMS) instrument.
We are currently looking for a highly motivated and experienced postdoctoral researcher to join a research project funded by the Simons Foundation's Origin of the Eukaryotic Cell Initiative. In collaboration with the labs of Brett Baker (UT Austin), Mark Ellisman (UCSD), and Thijs Ettema (Wageningen University), we seek to obtain a comprehensive genetic catalog of Asgard archaea diversity, determine their (eco)physiology, and characterize their cellular ultrastructure. To achieve this, we will employ an array of “omics”, physiological, and microscopic approaches. Determining the identity of archaea most closely related to eukaryotes, their physiological interactions, and cellular structure will transform our understanding of eukaryogenesis.
Our research activities focus on microbial ecophysiology: the study of the physiology of microorganisms with respect to their habitat. We are interested in how the activity of the “uncultured majority” – the large number of microbes that evades cultivation under laboratory conditions – impacts humans and the environment on a micron to global scale. We are convinced that only by gaining an understanding of microbes directly in their habitats researchers will be able to elucidate the mechanisms of microbial interactions with the biotic and abiotic world. To accomplish these goals, we apply an integrative approach that bridges the two extremes of the microbial scale bar: the individual cell and the whole community.
Very broadly, the research questions our lab addresses are:
(1) who is doing what (linking phylogenetic identity and physiological function),
(2) what are the abiotic and biotic factors controlling microbial in situ activity,
(3) how does this activity affect the environment and ultimately humans,
(4) what are the limits to metabolism in terms of energy, space, and time, and
(5) how can we discover novel structures and functions within uncultured microbial lineages?
Our approach to these problems is inherently multi-disciplinary and multi-scaled. In order to address previously unrecognized physiologies and cellular interactions of uncultured microbes, we employ a unique combination of metagenomics (as hypotheses generator), high-through-put metabolic screening via bioorthogonal labeling (to identify geochemical and biotic parameters driving ecology), and single cell resolved stable isotope probing via Raman and nanoSIMS (to identify specific growth-sustaining substrates). Because these approaches target the whole microbiome as well as the individual cell we do not depend on samples enriched in a target population, as is often necessitated in ecological studies. Our main study sites are sediments from a variety of geothermal, deep-sea, and coastal habitats.
If you are interested in learning more about Next-generation physiology approaches, please read our recent review. Next-generation physiology methods are either targeted at intrinsic characteristics of the cell (label-free methods) or employ substrate analog probing or stable isotope probing to study the in situ phenotype of cells. For a protocol on how to perform BONCAT-experiments see our book chapter. For talking about or teaching about BONCAT, free to use our slides.
Investigated by: Will Christian & Zack Jay, in close collaboration with Qian Wang, Tim McDermott, and Brian Bother (all MSU)
We study the ecological niches, biogeochemical roles, in situ activities, and biotechnological potential of lineages of (hyper)thermophilic uncultured archaea in geothermal features of Yellowstone National park. We currently focus our efforts on the lineages Aig-, Bathy-, Kor-, Verstraete-, and Thaumarchaeota and are interested in expanding our view towards novel archaeal groups. For this, we recently performed a microbiological and geochemical survey of >100 features in five regions of Yellowstone National Park (YNP, see map to the right). Amongst other excited results, we have identified YNP to be a yet unrecognized hot spot for anaerobic methane cycling.
We achieve our goal of linking new archaeal taxonomies with in situ function by a combination of metagenome sequencing of environmental DNA and testing genome-derived hypotheses by single cell targeted physiology approaches, including stable isotope probing and bioorthogonal labeling. The latter two approaches distinguish our lab from many other labs, who do not put their genomic hypotheses to an experimental test.