HC 4: Linking identity and function

Functional genes as markers

Instead of looking only at broad identification genes (like 16S rRNA), researchers can target functional genes that encode specific traits or processes.

• nifH → nitrogen fixation

• amoA → ammonia oxidation (nitrification)

• antibiotic resistance genes → resistance traits

Functional gene markers help answer: “What can this microbial community do?” rather than only: “Who is there?”

SIP

• PLFA + labeled substrate (13C)

• analyses df=ifferent microbial groups as peaks

The idea is simple: you feed a microbial community a labelled compound, and only the microbes that actively metabolise it will incorporate the isotope into their cells. You then detect the “labelled” microbes using different molecular or biochemical approaches.

SIP: DNA/RNA vs PLFA

• Heavy DNA/RNA = active users of the substrate

• Labelled PLFAs indicate active microbial biomass using the substrate (Only living, active cells can incorporate the isotope into membrane lipids)

Single-cell approaches

• MAR-FISH

• NanoSIMS

• RAMAN Micro/Spectroscopy

MAR-FISH (/STAR-FISH)

You have two different steps:

1.fluorescent in situ hybridization (FISH);

• you have a probe that’s specific for specific taxa; sticks to the taxa where the probe has affinity for.

2.looking at autoradiogram/ at radioactive substrate

• you add radioactive glucose

• every place where you see a spot, is where the substrate has been taken up.

Then you overlay the FISH and the radioactive results:

• you can see which organisms are the taxa of interest AND are being active in taking up the substate.

• With different fluorescent labels you cat target different taxa, and you can determine which ones are taking up substrate and which ones aren’t.

NanoSIMS

You zap a cell, all the chemicals get scattered and land on detectors, so heavy and light atoms land on different places

• NanoSIMS tells you where specific elements or isotopes are located at a very small scale, allowing researchers to link microbial identity with actual activity

• useful for studying microbial interactions, nutrient cycling, and host–microbe relationships.

SIP + NanoSIMS

a method that tracks isotope uptake to directly see which individual microbial cells are actively metabolising a substrate, at extremely high spatial resolution.

Raman spectroscopy

shining a laser on a single cell and measuring how the light is scattered.

• Most light bounces off unchanged, but a small part interacts with the molecules inside the cell (like proteins, lipids, and DNA) and changes energy
  »» creates a unique spectral pattern (biochemical fingerprint)

• this is compared to a reference database of known cells
  » Based on how similar the patterns are, the cell can be classified as most likely belonging to a certain species/functional group

Raman-SIP

combines Raman spectroscopy with Stable Isotope Probing (SIP) to link microbial identity with activity at the single-cell level

• If they actively metabolise it, the isotope is incorporated into their biomass, which causes a detectable shift in their Raman spectrum —> identify which individual cells are actively using a specific substrate (without cultivation or DNA extraction)

“shows who is active and what they are consuming at single-cell resolution”