Grant: Research Tools and Instruments - Category 1 (<$150,000)

My research program seeks to discover and characterize enzymes (protein catalysts) that produce the vital algal metabolite dimethylsulfoniopropionate (DMSP). DMSP is important to 1) marine algae for cellular protection; 2) marine bacteria preferring DMSP as a sulfur source; 3) supplying half the sulfur in the global biogeochemical sulfur cycle; and 4) climate regulation since DMSP-derived aerosols form clouds. I exploit 3 complementary strategies (comparative genomics, bacterial functional complementation assays, and enzyme purification) to discover genes encoding DMSP metabolism enzymes. Comparative genomics and bacterial functional complementation assays identify DMSP gene candidates that we express in bacterial hosts as recombinant enzymes which then need purification. The native algal DMSP enzymes can be purified to enable protein sequencing tools which reveal the genes encoding these DMSP enzymes. These strategies all *depend* on 1) separation of nucleic acids, 2) purified, high-quality native and recombinant enzymes, 3) derivatization and separation of enzyme products and substrates in harsh chemicals to confirm and study DMSP enzyme function. I am applying for funds for a refrigerated microcentrifuge with an aerosol-tight, chemical-resistant rotor that is immediately needed to replace a 23 yr old inoperable and unrepairable unit. This centrifuge will be used to purify nucleic acids, DMSP metabolites, help study the native DMSP enzymes from algal extracts or recombinant enzymes from transgenic bacterial extracts. Purified DMSP enzymes will be characterized with respect to their biochemical structure, regulation, kinetic behaviour, and identity. DMSP gene identification will advance understanding of how widespread DMSP production is in marine algae and can enable studies predicting the response of algal DMSP production to future oceanic conditions. These DMSP enzymes may also be introduced into crop plants to confer DMSP's cellular protection on them, leading to stress-resistant crops. The requested centrifuge will see substantial use by my postdoctoral (2), MSc (2) and undergraduate (15+) students, and technicians (2), giving excellent training for the next generation of scientists in Canada.