Single-celled organisms called bacterioplankton spend their lives drifting in open ocean, visible to the naked eye only en masse. But don't be fooled by their slight size: These minuscule critters play a hefty role in the carbon cycle. Heterotrophic microbes, by some estimates, process half of the organic carbon in the ocean fixed by phytoplankton and other autotrophs through photosynthesis.
But different communities of free-floating microbes use resources differently, and microbiologists have long sought robust tools to help identify how individual taxa interact with carbon. Think of it as spying on a sprawling dinner party without actually seeing anyone eat: How can you identify who ate what? [image: phytoplankton - the foundation of the ocean food chain]
Previous experimental methods aimed at answering this question have used and combine a variety of innovative tools. Some use metagenomics and gene expression to look at DNA and RNA in an entire microbial community; others use FISH (fluorescence in situ hybridization) to tag single cells as they metabolize carbon. Another looks at how RNA molecules incorporate carbon isotopies.
A new technique introduced by a collaboration of researchers from Oregon State University, Lawrence Livermore National Lab, and Oak Ridge National Lab uses proteomics – the large-scale analysis of proteins produced by an organism – to identify who ate what. In a study published today in mSystems, Samuel Bryson and his collaborators report on the initial test of a new technique they're calling proteomic stable isotope probing, or SIP. The work was funded by the Gordon and Betty Moore Foundation Marine Microbiology Initiative.
Previous techniques have used carbon isotopes to chart the metabolic responses of the organisms, Bryson says, but proteomic SIP has an advantage – it allows the direct measurement of how microbes take in resources and synthesize proteins.
He and his team experimented on samples from two North Pacific locations – one from the Oregon Coast and another from California's Monterey Bay. To each sample they added dissolved free amino acids (DFAA) containing the isotope Carbon-13. DFAAs are an important source of organic carbon in the ocean and originate from phytoplankton bloom die-offs, lysing cells, and other events.
After 15 hours, and then again after 32 hours, the researchers used a proteomics approach developed by researchers at Oak Ridge National Laboratory, in Tennessee, to identify how quickly the amino acids were being consumed and how the bacterial proteins changed in response. In particular, says Bryson, “we wanted to see if taxa continue to accumulate the substrate [the DFAAs] over time,”
Since certain peptides are indicative of particular bacterial communities, a proteomics approach can identify which taxa take up the carbon, and how much. The researchers found high levels of proteins enriched with Carbon-13, for example, in Rhodobacterales bacteria – but not in Flavobacteriales or SAR11 communities.
This initial test run of the technology suggests the method offers a detailed look at the metabolic responses of these bacteria to carbon. Ryan Mueller, senior author on the paper, says their technique provides direct evidence of information at three levels: “How much is being used, by whom, and how they're using it to produce new proteins,” he says.
Bryson notes that their proteomics approach is part of a wider trend in microbiology to call on various “-omics” data to better understand how a community of bacterioplankton respond to their environment. “We're figuring out how to use all that data,” he says. He says the proteomic-SIP model isn't limited to DFAAs, and he and his team have already begun running experiments looking at how microorganisms take up other substrates like glucose or lipids.
“This is one of the first steps at linking the process to the specific organisms and how they're responding,” says Mueller.
-- Stephen Ornes