Recent Contributions of Proteomics to Our Understanding of Reversible Nε-Lysine Acylation in Bacteria

Abstract

Post-translational modifications (PTMs) have been extensively studied in both eukaryotes and prokaryotes. Lysine acetylation, originally thought to be a rare occurrence in bacteria, is now recognized as a prevalent and important PTM in more than 50 species. This expansion in interest in bacterial PTMs became possible with the advancement of mass spectrometry technology and improved reagents such as acyl-modification specific antibodies. In this Review, we discuss how mass spectrometry-based proteomic studies of lysine acetylation and other acyl modifications have contributed to our understanding of bacterial physiology, focusing on recently published studies from 2018 to 2023. We begin with a discussion of approaches used to study bacterial PTMs. Next, we discuss newly characterized acylomes, including acetylomes, succinylomes, and malonylomes, in different bacterial species. In addition, we examine proteomic contributions to our understanding of bacterial virulence and biofilm formation. Finally, we discuss the contributions of mass spectrometry to our understanding of the mechanisms of acetylation, both enzymatic and nonenzymatic. We end with a discussion of the current state of the field and possible future research avenues to explore.

Keywords: KAT; KDAC; PTM; acetyl; acetylation; acetylome; post-translational modification; succinylation; succinylome.

Figure 1

Bottom-up, middle-down, and top-down MS strategies. For the bottom-up approach, proteins are digested into small peptides (0.8–3 kDa), most often using the enzyme trypsin. Each peptide will have an arginine or lysine at the C-terminus. For middle-down analysis, proteins are partially digested using Glu-C or Asp-N, which yields longer peptides (3–9 kDa). Glu-C peptides will have a glutamate at the C-terminus. The top-down approach does not use digestion and analyzes intact proteins. No matter which approach is used, the peptides or proteins are analyzed by MS and bioinformatics, depending on the specific conditions required for each approach.

Figure 2

General MS workflow for PTM discovery. Bacterial cells are grown under desired conditions, and cells are lysed. Following extraction, proteins are digested into smaller fragments using an MS-compatible enzyme. PTMs are enriched using specific antiacyl antibodies, which are then further subjected to separation, often by liquid chromatography. Peptides are analyzed by MS techniques, and bioinformatic platforms are used for the identification and quantification of PTMs.

Figure 3

Summary of unique and common acetylated and succinylated sites and proteins in various bacteria. Venn diagrams illustrating the overlapping acetyl and succinyl sites in (A) S. coelicolor, adapted from ref (76) under the Creative Commons Attribution (CC BY 4.0) license. (B) VISA, adapted from ref (77) under the CC BY 4.0 license. (C) E. tarda, adapted from ref (78) under the CC BY 4.0 license. (D) A. hydrophila, adapted from ref (79) under the CC BY 4.0 license. (E) D. radiodurans, adapted from ref (62) under the CC BY 4.0 license. (F) V. alginolyticus, adapted from ref (80) under the CC BY 4.0 license. (G) P. gingivalis, modified with permission from ref (81). John Wiley & Sons, copyright 2020.

Figure 4

Biofilm phenotype of deacetylation mimic mutants of YmcA (RicA) and GtaB. B. subtilis variants with the noted acetylation sites mutated to the deacetylation mimic arginine. The top row shows pellicle formation at the air–liquid interface, and the bottom row shows the biofilm colony morphology on solid media. (A) The ymcAK64R mutant had a severe defect in biofilm formation, as evidenced by less wrinkling on the pellicle and a smaller sized colony, which suggests that acetylation of YmcA at K64 is regulatory and required for this process. (B) The gtaBK89R mutant is also defective in biofilm formation. These mutants had featureless pellicles and a larger colony size with less pronounced wrinkles. This suggests that the acetylation of K89 is required for proper GtaB function. Modified from ref (90) under the Creative Commons Attribution (CC BY 4.0) license.

Figure 5

Acyl analysis of two virulence factors of P. aeruginosa. The virulence factors CbpD (A) and LasB (B) with their identified PTMs are noted. The possible acyl modifications of lysine residues include acetylation, butyrylation, crotonylation, malonylation, propionylation, and succinylation. Also noted are the lysine modifications of mono-, di-, and trimethylation and serine, threonine, and tyrosine phosphorylation sites. Reprinted from ref (96). Copyright 2019, American Chemical Society.

Figure 6

Bacterial mechanisms of acetylation and deacetylation. (1) Acetylation is carried out by lysine acetyltransferases (KATs), which catalyze the transfer of the acetyl group from Ac-CoA to the target lysine residue. Alternatively, lysines can be nonenzymatically acetylated, predominantly by using Ac-P as the donor. (2) These reactions are reversible by the action of the lysine deacetylases (KDACs), which in bacteria are mostly NAD⁺-dependent sirtuins. Reproduced from ref (11) under the Creative Commons Attribution (CC BY 4.0) license.