24. An extension of Tiny Earth: Characterization of Strain S5F11

An extension of Tiny Earth: Characterization of Strain S5F11

Eleanor Behling, Justin Campbell, Grace O’Hare, Katlyn Wendel, Kathleen Wolf, and Dr. Hans Wildschutte
Bowling Green State University

Tiny Earth (TE) is a program that crowdsources antibiotic discovery through exposing students to microbiology research. The TE program begins with the collection of a soil sample and the isolation of strains from the environmental sample. Strains were identified as Pseudomonas by 16S rRNA gene sequencing and BLAST analysis. An antagonistic assay was performed using all isolates in an effort to identify if any strains could inhibit the growth of another. Several strains did show inhibition, suggesting the production of an antibiotic compound. The traditional TE program relies on biochemical techniques to isolate the active secondary metabolite. However, Biochemical analyses are complex and difficult to undertake with an undergraduate research group; as such, no novel antibiotics have been discovered through TE. Instead, TE at Bowling Green State University’s uses a different approach involving transposon mutagenesis to disrupt the production of an antibiotic substance. Mutant strains that have lost the ability to inhibit the growth of another are subject to characterization in a second semester of TE. In our class, the arbitrary PCR product from the mutant was aligned to the whole genome sequence to identify the transposon-disrupted gene. Here we identified a biosynthetic gene cluster in S5F11 that was able to inhibit five and eleven multi-drug resistant Pseudomonas aeruginosa and Burkholderia pathogens respectively. Using the sequenced genome, we were able to perform an average nucleotide analysis and predict antibiotic resistance genes. This bioinformatic workflow has the potential to facilitate antibiotic discovery through TE.