Using protein-stable isotope probing to link taxa and metabolic function in photic microbial communities
EMSL Project ID
60003
Abstract
An important goal in microbial community ecology is linking metabolic functions with the taxa performing them. To this end, we have exploited well-studied Yellowstone hot spring phototrophic microbial mat communities, whose relative taxonomic simplicity and high-biomass make them ideal for using advanced molecular approaches to understand principles of microbial community ecology. One type of mat includes cyanobacteria, whose oxygenic photosynthesis causes diel cycling of oxygen; another type of mat contains only anoxygenic phototrophic taxa and is perpetually anoxic. Furthermore, these mat communities are natural bioreactors for light harvesting and sequestration of dissolved inorganic carbon (DIC), from which to learn how nature has maximized efficient light utilization and DIC uptake. Metagenomic analyses have revealed the predominant taxa inhabiting these mats and metatranscriptomic and meta-metabolomic analyses have provided insights into their metabolisms. This proposal addresses two hypotheses that relate to the role that two poorly understood processes play in DIC sequestration. First, we hypothesize that the anoxygenic phototroph Roseiflexus, an abundant member of the cyanobacterial mat, undergoes a photomixotrophic metabolism during mid day (under maximum light conditions), in which DIC and organic metabolites are combined. Second, we hypothesize that nonphototrophic, anaerobic, acetogenic bacteria convert DIC to acetic acid during dark anoxic periods in the cyanobacterial mat (and also in light periods in perpetually anoxic mats produced only by anoxygenic phototrophs). To test these hypotheses we developed an approach based on stable-isotope probing (SIP), which inovolves uptake and conversion of 13C-labeled substrates into proteins of taxa inhabiting the mats. This causes shifts the mass spectra of peptides derived from such proteins, and these shifts can be detected in metaproteomic analyses we call Protein-SIP. The peptide sequences can then be used to identify which proteins of which taxa have incorporated the 13C-labeled substrate. To date, we have applied Protein-SIP to demonstrate that 13C-bicarbonate incorporation by Roseiflexus occurs during mid-day. Work proposed herein addressing the first hypothesis focuses on using Protein-SIP to track the incorporation of 13C-labeled organic compounds we know from metametabolomic analyses are produced and consumed in the mat at different times of day. SIP labeling of mat samples has been done with 13C-acetate, -propionate, -glycolate and -lactate. Work proposed herein addressing the second hypothesis focuses on using Protein-SIP to test whether acetogenic community members incorporate 13C-bicarbonate in darkness (or in light and dark in the mat that is perpetually anoxic). SIP labeling of mat samples with 13C-bicarbonate incubated in these mats under these conditions has been done. What remains is to conduct Protein-SIP analyses to associate the incorporated 13C with peptides and proteins of different taxa. We propose to analyze seven samples using EMSL’s Orbitrap Mass Spectrometer, resulting in the generation of data on labeled peptides of each taxonomic group that can be used to evaluate these hypotheses.
Project Details
Project type
Limited Scope
Start Date
2021-10-31
End Date
2022-04-22
Status
Closed
Released Data Link
Team
Principal Investigator
Co-Investigator(s)