Chemist Pieter Dorrestein’s laboratory group has been developing mass spectrometry methods to look at what molecules are produced by microbes interacting in a petri dish. But recently, the group jumped out of the dish and into the dirt—to analyze a soil-dwelling lichen and discover how the molecules made by a complex microbial community spatially map to that structure.
Collaborating with an international team of researchers and drawing on a global database of metabolomic data, his team generated the first images of an intact microbial community’s molecular architecture, published this week in mSystems.
“There are microbial communities around us everywhere,” says Dorrestein, a professor at the University of California, San Diego school of pharmacy. “There is not a single example of a community where we understand the molecules that drive and shape the community.”
The team chose the Peltigera hymenina lichen, collected from British Columbia, to test how well their mass spectrometry (MS) tools could tease apart the chemistry associated with a multi-microbial community. Indeed, a metagenomic analysis of the sample revealed the lichen held 342 phyla, including 74 representing viruses, 236 bacteria, 8 eukaryotes and 6 archaea.
Which begs the question: is it even appropriate to think of a lichen as an individual species? “I don’t have a good answer for you! This question is better left to taxonomists,” says Dorrestein. “But it is clearly built out of microbes and that is why we picked it.” He also notes that this is the first observation of so many bacteriophages and viruses residing within a lichen. “Right now, I don’t have a guess as to what these viruses are doing. But they probably play a bigger role than previously estimated in shaping microbial communities.”
Dorrestein’s group wanted to dig deeper into how microbial communities are sculpted by the chemicals being produced by their members. Knowing who is in the community through genomics is the first step, but finding out what products they are making is a key next step.
“It’s still really important to take an inventory,” he says. “If Darwin didn’t collect an inventory of finches, he wouldn’t have developed his theory [of evolution]. We need to understand all the components that make up a particular organism.”
To that end, the group took an inventory of the molecules present in the lichen and then looked at the spatial arrangement of certain molecules. First, the team annotated all of the identified genes from the lichen and found that 13% of them, or 75,076 genes, were classified as enzymes that produce secondary metabolites. Dorrestein explains that as much as 20% of many microbes’ genetic makeup is dedicated to producing just a handful of small molecules in response to living communally.
Next, the team scraped samples from 110 spots on the postage stamp-sized piece of lichen, extracted the metabolites, and analyzed them by chromatography coupled to MS. They ran the same analysis on metabolites extracted from about 70 microbes isolated from the lichen and grown up in laboratory cultures. Comparing the MS spectra generated from both sets of samples told the team which metabolites in the lichen were produced by which of the individual bacteria, cyanobacteria, or fungi grown in the lab.
To identify as many molecules as possible, the team relied on the crowd-sourced Global Natural Products Social (GNPS) molecular networking infrastructure. Built by an international effort earlier this year, GNPS holds MS data from 17,000 users in 122 countries. “It is a place where you can analyze untargeted metabolomics data,” explains Dorrestein. As people annotate their public MS spectra, or ‘molecular barcodes’, matches with other researchers’ data are sent by email. “In that way, your data continues to be alive.”
Using those public data sets, the team identified the likely producer for about 40% of the lichen’s molecules and provided all the annotations currently possible with public reference data. For a subset molecules made by fungal and cyanobacteria members of the lichen, the team wanted to map where on the lichen’s three-dimensional layers those molecules reside. The team took a tiny sliver of a cross-section of the lichen sample and subjected it to imaging mass spec (IMS).
Sitting on a moving microscope stage, the cross-section was swept under a laser, which ionized and sent molecule fragments from each particular spot on the sample flying into the IMS to be analyzed. Because the stage ‘remembers’ each exact spot on the sample that generates each data point, the team could use false colors to build a picture of the molecules detected from the lichen cross-section.
Overlaying the different molecular profiles, reveals where each type of molecule nestles within the lichen’s layers. It shows that potential defense molecules made by fungal members appear in different layers and that chlorophyll made by the cyanobacteria members, not surprisingly heavily coats the uppermost, sun-facing layer.
Of course, Dorrestein says, this initial molecular blueprint for how a lichen is built through chemistry raises more questions than answers. “It was very interesting that there is less and less chlorophyll made as you move down through the layers. Are those older layers completely functional?” he wonders. But the study shows that ultimately researchers can use MS methods to draw the more intricate picture of the key energy sources and protections needed to assemble a lichen, or any other microbial collective for that matter.
Image: The distribution of fungal molecules PF1140, asperphenamate, and alantolactone (left) and cyanobacterial molecules (chlorophyll and heterocyst glycolipid) (right) and a representative member of the molecular family of compounds with spectral similarity to lupeol is shown. The complete overlap of cyanobacterial chlorophyll pigment (green) and heterocyst glycolipid (red) results in cyanobacteria appearing yellow.