Characterization of N-terminal protein modifications in Pseudomonas aeruginosa PA14

Even though protein initiator methionine excision (NME) and N-terminal acetylation (NTA) have been relatively well investigated in eukaryotic proteomes, few studies were dedicated to these modifications in bacteria up to now. In this work, we investigated, for the first time, the N-terminal proteome of the bacterium Pseudomonas aeruginosa PA14 by studying the NME and NTA processes using proteomic approaches. For NME, most of proteins had their initiator Met cleaved (63%) and the nature of the penultimate residue seems to be essential for this cleavage. Concerning NTA, two methods were applied (protein fractionation and peptide enrichment). This allowed us to identify 117 Nα-acetylated proteins, among them 113 have not yet been described as modified in bacteria. Most often, the non-acetylated form was over-represented compared to the acetylated form, arguing that this latter was a minor part of the total abundance of a given protein. Furthermore, some proteins with acetylated initiator methionine were observed. The present work significantly enlarges the number of N-terminally modified proteins in bacteria and confirms that these modifications are a general and fundamental process, not only restricted to eukaryotes.

Protein modifications in prokaryotes have been detected more recently than in eukaryotes. Methionine cleavage and N-terminal acetylation are two common protein N-terminal modifications. Despite their importance in bacterial processes, they are less investigated. The characterization of N-terminal acetylation in bacteria is a challenge because no antibody exists and it is a less frequent modification than in eukaryotes. We used proteomic approaches (enrichment, fractionation, nanoLC-MS/MS, and bioinformatic analyses) to investigate the N-terminal methionine excision and to profile the N-terminal acetylome of P. aeruginosa strain PA14. From our results, around 60% of the proteins had their iMet cleaved. In total, 117 proteins were identified constituting the largest dataset in prokaryotes. Among them, proteins kept their initiator methionine and were acetylated. These results may facilitate the design of experiments to better understand the role of acetylation at the protein N-terminus of P. aeruginosa PA14.