Identifying Biosynthetic Gene Clusters Involved in Antagonistic Activity in Pseudomonads

Danielle Weber

Biology

Melinda Grosser

Since the discovery of antibiotics, improper usage has led to the evolution and emergence of antibiotic resistant microorganisms. ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) in particular are among the most deadly due to their unassailable ability to resist antibiotics. To discover new ESKAPE antibiotics and help combat the antibiotic resistance crisis, a molecular strategy was constructed by the educational networks Tiny Earth (in which UNC Asheville is a member) and Small World Initiative. As part of Tiny Earth, 29 soil isolates (SI) were characterized using the 16S rRNA gene and assayed for antibiotic production by Dr. Melinda Grosser’s spring 2022 microbiology class (BIOL 339). Three of these isolates (all pseudomonads that exhibited antagonistic activity) as well as two previously isolated pseudomonads (SS 400 and RGRF B10) from Dr. Amanda Wolfe were tested for their amenability to undergo transposon mutagenesis using transposon pBAM1, which was inserted into genomes using biparental mating and conjugation with E. coli S17-λpir. This transposon mutagenesis would then allow for identification of a biosynthetic gene cluster involved in antibiotic compound production using replica plate screening. Of the five isolates tested (SS 400, RGRF B10, SI JK, SI JC, and SI RS), SI JK was found to be the most efficient in conjugation and screening. One mutant with loss of antagonistic activity against Bacillus subtilis was identified in SI JK. This mutant will go through subsequent whole-genome sequencing to identify a biosynthetic gene locus likely involved in its inhibitory activity.

Jan-9-2024