title: Characterization of unique modification of flagellar rod protein FlgG by Campylobacter jejuni lipid A phosphoethanolamine transferase, linking bacterial locomotion and antimicrobial peptide resistance.
abstract: Gram-negative bacteria assemble complex surface structures that interface with the surrounding environment and are involved in pathogenesis. Recent work in Campylobacter jejuni identified a gene encoding a novel phosphoethanolamine (pEtN) transferase Cj0256, renamed EptC, that serves a dual role in modifying the flagellar rod protein, FlgG, and the lipid A domain of C. jejuni lipooligosaccharide with a pEtN residue. In this work, we characterize the unique post-translational pEtN modification of FlgG using collision-induced and electron transfer dissociation mass spectrometry, as well as a genetic approach using site-directed mutagenesis to determine the site of modification. Specifically, we show that FlgG is modified with pEtN at a single site (Thr(75)) by EptC and demonstrate enzyme specificity by showing that EptC is unable to modify other amino acids (e.g. serine and tyrosine). Using Campylobacter strains expressing site-directed FlgG mutants, we also show that defects in motility arise directly from the loss of pEtN modification of FlgG. Interestingly, alignments of FlgG from most epsilon proteobacteria reveal a conserved site of modification. Characterization of EptC and its enzymatic targets expands on the increasingly important field of prokaryotic post-translational modification of bacterial surface structures and the unidentified role they may play in pathogenesis.
authors.affiliation: Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Cullen
authors.forename: James A
authors.initials: JA
authors.lastname: Madsen
authors.forename: Petko L
authors.initials: PL
authors.lastname: Ivanov
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
authors.forename: M Stephen
authors.initials: MS
authors.lastname: Trent
mesh_terms: D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000502:physiology*; D004982:Ethanolaminephosphotransferase / Q000235:genetics / Q000378:metabolism*; D005407:Flagella / Q000235:genetics / Q000378:metabolism*; D008050:Lipid A / Q000235:genetics / Q000378:metabolism*; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*
chemical_list: D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D008050:Lipid A; D008074:Lipoproteins; D004982:Ethanolaminephosphotransferase