Development of Simplified Soil Microbial Consortia and Activity-Based Probes to Characterize the Activity of Chitin Degrading Enzymes

Primary author: Elias Zegeye
Faculty sponsor: Dr. Aaron Wright

Primary college/unit: Voiland College of Engineering and Architecture
Campus: Pacific Northwest National Laboratory (PNNL)

Abstract:

We investigated the succession, selectivity and dynamics of a soil microbiome during 21 weeks of enrichment on chitin and its monomer in a soil matrix and liquid environment. We hypothesized that the initial species richness would influence the tendency for the selected consortia to stabilize and maintain a relatively constant consortia over time. We found that the lower initial richness stabilizes rapidly, and the resulting community composition differed greatly in soil than liquid medium. The reduced and stable consortia found in this study will aid in the discovery of functionally active chitin-degrading microbes using Activity-Based Probes (ABP). Chitin-derived ABPs (i.e. N-acetyl glucosamine and chitotriose) were developed including 1) a moiety that will covalently label an enzyme upon glycosidic bond hydrolysis, and 2) a chemical handle for isolation of labeled enzymes. The ABPs were first tested on a sample harvested from Cellvibrio japonicus, a soil bacterium with well-characterized chitinolytic enzyme machinery, that have grown on different carbon sources. As a result, the ABPs showed the induction and activities of the chitinase enzyme depend on the carbon sources and the time course of bacterial growth. Additionally, the application of this small chemical probe will be extended to identify and measure the activity of chitinase enzymes in simplified soil consortia. Generally, ABPs can be used to broaden our functional understanding of chitin breakdown and to characterize the soil consortia’s metabolic potential for chitin degradation.