Measuring bacterial S‐acylation in Pseudomonas putida

Abstract

In eukaryotes, reversible palmitoylation of cysteine residues via a thioester linkage allows dynamic shuttling of proteins between intracellular compartments. Regulation of protein localization by palmitoylation is involved in key cellular processes including cell signaling, ion transport, and neuronal communication. These dynamic cycles of palmitoylation and depalmitoylation are controlled by two classes of cellular enzymes, protein acyl transferases and acyl‐protein thioesterases. While bacterial proteins are S‐acylated and homologues of human acyl protein thioesterase are present in many gram‐negative bacterium, dynamic cycles of S‐acylation have not been observed in prokaryotes. In this study, we utilized azide mimics of palmitic acid to measure the level of protein S‐acylation in Pseudomonas putida. S‐acylated proteins were then detected by click chemistry using either biotin or fluorophore labeled alkynes. Specific S‐acylated proteins were identified in P. putida and their identities are currently being confirmed. Using deletion strains for potential bacterial palmitoylation machinery, we plan to search for proteins that regulate levels of S‐acylation in P. putida. Funding provided by Butler University Holcomb research fund.