Publication in: Spring 2023 Issue

Heterologous Expression of Pseudomonas aeruginosa ATP Synthase to Facilitate Antibiotic Discovery
Vesper Fraunfelter
Faculty Mentor(s):
Ryan Steed
Abstract / Summary:
Multidrug resistance (MDR), in microorganisms is a rising and prevalent threat, necessitating the constant development of new antibiotics, preferably with novel mechanisms of action to avoid existing resistance. Targeting the bioenergetic pathways of bacteria has shown promise in treating drug-resistant bacteria, as demonstrated by the diarylquinoline bedaquiline, which is effective against the ATP synthase of Mycobacterium tuberculosis. Expanding on this strategy, we are screening quinoline analogs to identify effective inhibitors of ATP synthase of Pseudomonas aeruginosa (PA),, an opportunistic pathogen known to be rapidly developing MDR. For this purpose, we constructed the novel pASH20 plasmid to express PA ATP synthase in Escherichia coli. The plasmid, derived from the ampicillin-resistant vector pBR322, contains the PA ATP synthase (atp), operon under the control of the native E. coli atp (unc), promoter, added silent restriction sites to facilitate mutagenesis, and a hexahistidine tag encoded on the N-terminus of the beta subunit to facilitate future purification. Expression of functional ATP synthase was verified by growth of transformant E. coli on succinate minimal medium and in vitro ATP synthesis and hydrolysis activity assayed in inverted membrane vesicles prepared from transformant E. coli.
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