Model Systems: A bridge between environmental science and controlled experimentation.
The power of environmental science is that it tells us what's happening on the planet, in all of its messy complexity. But that complexity is exactly what makes understanding the fundamental basis of environmental processes so challenging. Developing model systems from the environment allows us to isolate specific variables, to begin to understand how mechanisms at a cellular level contribute to (and cause) environmental phenomena.
Methyloprofundus sedimenti
Patricia isolated Methyloprofundus in 2013, and we've been characterizing its behavior ever since. Genus Methyloprofundus lives (occasionally) in the water column, in sediments, and most intriguingly, inside Bathymodiolus gill cells (bacteriocytes). The isolation of this genus allows new areas of investigation into inter-kingdom molecular communication and the evolution of the invertebrate immune system.
Methyloprofundus is a large methanotrophic bacterium that, in culture, is often present in a state of near-division (appearing as a diplococcus.) We've generated high resolution imaging and discovered that it has a proteinaceous surface layer, and multiple storage granule types. These granules appear to serve several functions including partitioning, energy storage, and carbon for transition from starvation to active growth.
Methyloprofundus is a large methanotrophic bacterium that, in culture, is often present in a state of near-division (appearing as a diplococcus.) We've generated high resolution imaging and discovered that it has a proteinaceous surface layer, and multiple storage granule types. These granules appear to serve several functions including partitioning, energy storage, and carbon for transition from starvation to active growth.
M. sedimenti encodes a specific cellular program to persist for (at least) months of carbon starvation, and to compete rapidly and effectively during methane flux. This cellular program includes down-regulating most genes during starvation, but up-regulating methane monooxygenase transcripts. That last part is surprising and requires us to re-think environmental transcriptomics.
Membranes are preserved during early starvation as a potential energy source, and provide localization for newly-synthesized pMMO in the event of methane flux. During methane flux, nascent methane monooxygenase transcripts are translated - within minutes, These newly synthesized monooxygenases metabolize methane to produce methanol - which is assimilated internally but also excreted to the environment. Lipid saturations remodulate and cells begin to divide 24 hours after methane has become available. These data are forming our understanding of the cellular program Methyloprofundus uses to survive the 'feast or famine' conditions that typify the natural world.
Membranes are preserved during early starvation as a potential energy source, and provide localization for newly-synthesized pMMO in the event of methane flux. During methane flux, nascent methane monooxygenase transcripts are translated - within minutes, These newly synthesized monooxygenases metabolize methane to produce methanol - which is assimilated internally but also excreted to the environment. Lipid saturations remodulate and cells begin to divide 24 hours after methane has become available. These data are forming our understanding of the cellular program Methyloprofundus uses to survive the 'feast or famine' conditions that typify the natural world.
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We also investigated the nature of the surface layer protein by performing extraction of the exterior proteins (while minimizing cell lysis). This 'extracellular protein prep' was then subjected to proteomics, and the protein responsible for the studded surface layer was identified.
The predicted surface layer protein of M. sedimenti is only loosely related at the primary sequence level to known proteins. Structural predictions of this protein (left, below) suggest that it closely mimics secretory component of IgA1 (center, below). This result is ...tantalizingly... so cool - because it suggests that Methyloprofundus gains access to Bathymodiolus bacteriocytes through associations at the mucosal boundary of bacteriocytes. This is an area we'd love to explore in depth.
Using cryoelectron tomography in collaboration with Drs.Grant Jensen and Elitza Tocheva, we are able to image a 'tilt series' of high resolution, near native slices through a M. sedimenti cell. This movie allows us to visualize the spatial relationships between membranes, storage granules, ribosomes, and additional cellular features.
Current and future directions.
* We're interested in investigating the potential role of the surface layer protein in bacteria:mussel interactions (molecular dialog molecules).
* We're also examining lipid changes in bacterial membranes in greater depth. These modulations appear crucial to survival during starvation and crucial to function during recovery.
* ...and we're detailing the cellular response of this bacterium during starvation and during recovery from starvation: Unpublished data suggests that a transient carbon fixation event occurs as cells are entering dormancy. Additionally, recovery from starvation involves methanol secretion to the environment - What does M. sedimenti gain from this loss of carbon?
* Long term: We'd love to develop primary bacteriocyte cultures from Bathymodiolus to image bacterial uptake - and investigate the role of the mussel cytoskeleton in this process.
* We're also examining lipid changes in bacterial membranes in greater depth. These modulations appear crucial to survival during starvation and crucial to function during recovery.
* ...and we're detailing the cellular response of this bacterium during starvation and during recovery from starvation: Unpublished data suggests that a transient carbon fixation event occurs as cells are entering dormancy. Additionally, recovery from starvation involves methanol secretion to the environment - What does M. sedimenti gain from this loss of carbon?
* Long term: We'd love to develop primary bacteriocyte cultures from Bathymodiolus to image bacterial uptake - and investigate the role of the mussel cytoskeleton in this process.
Candidatus Sphingobium alkanivorans
Historically, genus Sphingobium has been associated with a variety of pollutants including trichloroethylene and polycyclic aromatic hydrocarbons. The Porter Ranch gas leak revealed that Sphingobium can also remediate alkane gases. The genes that allow this are plasmid based, much like the genes that allow other members of Sphingobium to remediate larger hydrocarbon pollutants. We are in the initial stages of learning about this new cultivar.
Having S. alkanivorans in hand has allowed us to sequence the genome and identify potential biochemical pathways. S. alkanivorans encodes a pMMO related enzymes as well as soluble MMO machinery (sMMO). Thus, two pathways of alkane oxidation are encoded by the bacterium. These genes are carried exclusively on a megaplasmid, a strategy that has been demonstrated previously in other Sphingobium species. The idea of metabolic flexibility between species - the 'meta organism' - is one that we love thinking about, in light of these sorts of findings.
Identifying the genes for alkane oxidation led immediately to experiments, asking if S. alkanivorans can oxidize methane. In pure culture, S. alkanivorans does not oxidize methane, but does oxidize ethane. Additionally, genes for alkane oxidation (pmo and smo) are not expressed in the absence of gas, or in the presence of only methane. However, these genes are dramatically upregulated in the presence of ethane (expression data is not shown.)
Given the substantial bloom of S. alkanivorans during the Aliso Canyon gas leak (see related information under 'Porter Ranch'), we continue to investigate whether methane oxidation may be possible, facilitated by S. alkanivorans living in complex communities or under specific nutritional conditions. Early results are mixed!
Current and future directions.
* Measure gene expression of pmo and smo in the presence of mixed gases.
* Assess the effects of methanol and formaldehyde on the growth of S. alkanivorans. Perhaps this organism metabolizes methane but requires a partner to detoxify a downstream product.
* Evaluate relatedness of this isolate to the many Sphingomonadaceae in the Aliso Canyon region. What can we learn about the evolutionary strategies used by Sphinomonads?
* Ultimately, we hope to develop this or another organism from the Porter Ranch leak site as a bioremediation tool to help lower the carbon footprint from the fossil fuel industry.
* Assess the effects of methanol and formaldehyde on the growth of S. alkanivorans. Perhaps this organism metabolizes methane but requires a partner to detoxify a downstream product.
* Evaluate relatedness of this isolate to the many Sphingomonadaceae in the Aliso Canyon region. What can we learn about the evolutionary strategies used by Sphinomonads?
* Ultimately, we hope to develop this or another organism from the Porter Ranch leak site as a bioremediation tool to help lower the carbon footprint from the fossil fuel industry.