Uncovering the genetics of RIF-1, a sphingolipid responsible for rosette colony formation in choanoflagellates

University of Hawaii Center for Microbial Oceanography Research and Education, Alegado lab, Summer 2015

Choanoflagellates exist in both colonial and unicellular forms and are each composed of a flagellum, microvilli, and a cell body. Algoriphagus machipongonensis triggers rosette colony formation in choanoflagellates through the release of Rosette Inducing Factor-1 (RIF-1) via outer membrane vesicles. RIF-1 shows a similar structure to the essential eukaryotic sphingolipid, important in cell recognition, lipid rafts, signal, and cell proliferation. However, it is still unknown as to which genes encode for steps in the RIF-1 biochemical pathway. Transposon mutagenesis was used to create a library of Algoriphagus mutants which would be co-cultured with choanoflagellates to determine if they still trigger rosette colony formation. The Algoriphagus mutants that no longer triggered colony formation were isolated and cultured to re-verify their inability to produce colonies and ability to grow with WT biomass production to ensure that major pathways were not disrupted. After re-verification, the DNA from the mutants was isolated and saved for sequencing. Using those sequences, we can isolate the locations of genes essential to RIF-1 production. In total, 20,000 strains were created for the transposon library, and 86 strains were isolated for their genomic DNA. Additionally, electron microscopy was used to image and characterize WT and mutant Algoriphagus strains using uranyl acetate negative staining.