Reverse Genetics to Investigate the Roles of Two Genes in the Biosynthesis and Export of Nonribosomal Peptide Natural Products

Student: Lydia Arnold ’22
Research Mentors: Yitao Dai and Alessandra Eustaquio (University of Illinois at Chicago Department of Pharmaceutical Sciences and Center for Biolecular Sciences: College of Pharmacy)

A specific genus of bacteria is found to colonize on healthy marine sponges. These bacteria produce a natural product that allows the bacteria to survive on the sponge more easily. We studied the production of this natural product and its benefit to the bacteria.

Microbial secondary metabolites (natural products) play important roles in organismal behavior and in microbe-host interactions. Genomes of Pseudovibrio marine bacteria isolated from healthy marine sponges contain a nonribosomal peptide synthetase, polyketide synthase gene cluster shown to encode the biosynthesis of pseudovibriamides which were shown to affect swarming motility and biofilm formation. The goal of this project was to use reverse genetics methods for Pseudovibrio brasiliensis strain Ab134 to study two flanking genes, open reading frame (ORF) 1 and 2. Based on sequence similarity, we hypothesized ORF1 encodes a major facilitator superfamily transporter potentially involved in pseudovibriamide export, and ORF2, a GCN5-related N-acetyltransferase (GNAT) potentially involved in pseudovibriamide propionylation. Gene deletion mutants for ORF2 were obtained via homologous recombination and confirmed by Polymerase Chain Reaction. Comparative metabolite analysis of ORF2 mutant and wild-type strains using matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry suggest that ORF2 is not involved in the production of pseudovibriamides. Moreover, the ORF2 mutant’s swarming motility was comparable to the wild-type strain. Deletion of ORF1 is still ongoing. Once mutants are obtained, we will perform comparative swarming assays and metabolite analysis of supernatant and cell pellet extracts to test the hypothesis that ORF1 is a pseudovibriamide transporter.