Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational β-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which β-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ΔearP, or from a ΔrmlC::acc1 strain deficient in dTDP-l-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-l-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in Δefp, ΔearP, and ΔrmlC::acc1 strains. ΔrmlC::acc1, ΔearP, and Δefp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival.
Rajkovic, A., Erickson, S., Witzky, A., Branson, O.E., Seo, J., Gafken, P.R., Frietas, M.A., Whitelegge, J.P., Faull, K.F., Navarre, W., Darwin, A.J. and Ibba, M. (2015) Cyclic rhamnosylated elongation factor P establishes antibiotic resistance in Pseudomonas aeruginosa. mBio 6 e00823-15. https://doi.org/10.1128/mBio.00823-15
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