Today was the last day of ISME and a great conclusion to the conference. Today’s theme seemed to surround microbial methods and my favorite talks were split between 2 subterranauts, Karen Lloyd and Roland Hatzenpichler. Karen spoke in the archaea themed session about exploring single cell genomes and exploring novel enzyme function in the uncultured MCG archaea group and Roland presented in the single cell session about a new technique to visualize newly synthesized proteins in situ.
The MCG archaeal group is widespread across the marine subsurface sediment and, during Karen’s post-doc at Aarhaus, they were able to obtain a single cell genome of a member of this elusive group. Looking into the genome, Karen found that many of the predicted ORFs did not annotate well. As she was unhappy with the quality of the functional assignments, she began thinking about ways that one could determine function from organisms that are poorly related to the culture-centric databases. Now, many microbiologis have met these cross roads where a genome is assembled yet a large proportion of the predicted open reading frames (orfs) lack strong functional assignments. However, what makes Karen’s story unique, is that she began collaborating with a group interested in crystalizing her low-identity proteins. Through this collaboration, they were able to generate a crystal structure of a protein annotated as a peptidase or ester hydrolase.
When they compared this crystal structure to the structure of best culture match in the BLAST database, they found that the MCG protein structure was somewhat similar to a known peptidase but contained some distinguishing features. Most exciting was the fact that the active site of the MCG peptidase and its closest relative were different. With this information, they decided to test the activity of their novel protein and determine whether or not the changes in the protein structure led to a change function. Doing this they saw that, although both proteins were peptidases, they had different activities towards different amino acids. In particular, they found that their achaeal peptidase has a broad peptidolytic activity with a preference for amino terminal cysteine but not cysteine. This observation is particularly interesting since terminal cysteine will be converted to cysteine in the presence in oxygen and, therefore, indicative of a peptidase adapted to anoxic sediments.
For the other days at ISME, I have mentioned my favorite bioinformatic tools of the day; however, Roland’s work is just too cool to skip over! He attacks the question of how to distinguish and target the active members of the microbial community through a really cool technique that I would describe as a sort of FISH for proteins. What happens is Roland incubates a culture with a compound called AHA which looks like L-methionine with the exception that AHA contains an azide moiety instead of S-CH3. Similar to radiotracer experiments, the active organisms will incorporate AHA into their proteins and, using some chemistry, cells that contain AHA will fluoresce under fluourescent microscopy — meaning that if you were to able to inoculate a microbial community with AHA you could go as far as cell sorting the most active members for downstream single cell sequencing! For more details on the experimental procedure, Roland’s work was recently published in Environmental Microbiology and definitely worth a look.