Moonlighting and Mimicry: Identification and Functional Characterization of a SAM-dependent Methyltransferase Responsible for Methylation of EF-Tu in Pseudomonas Aeruginosa

Abstract

Phosphorylcholine (ChoP) is a charged molecule that, in humans, is the major recognition component of platelet activating factor (PAF) by its cognate receptor, platelet activating factor receptor (PAFR). ChoP-like molecules have been detected on the surfaces of numerous respiratory pathogens where they confer persistence related phenotypes mediated through an increase in adhesion. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen which is often associated with nosocomial pneumonia. Previously, in P. aeruginosa a ChoP-like modification was detected using ChoP-specific antibodies on surface exposed elongation factor-Tu (EF-Tu), a normally cytoplasmic protein involved in protein chain elongation. Here we identify a gene, eftM, which is required for the modification of EF-Tu. To our surprise, mass spectrometry analysis of modified EF-Tu revealed the modification was not ChoP but rather trimethyl-lysine at amino acid residue 5. This trimethyl-lysine acts as a structural mimic of ChoP and confers similar phenotypes to P. aeruginosa as ChoP does to other respiratory pathogens. Bioinformatic analysis of EftM showed amino acid sequence similarity to Sadenosylmethionine (SAM)-dependent methyltransferases. We confirmed biochemically that EftM is able to bind SAM and used it to directly methylate lysine 5 of EF-Tu both in vivo and in vitro. Mass spectrometry analysis of products from these reactions confirmed that EF-Tu is trimethylated at lysine 5. In vivo, P. aeruginosa methylates EF-Tu only at temperatures closer to ambient, 25°C, and not at body temperature, 37°C. We show that this temperature-dependent methylation phenotype is not due to differences in transcription of eftM. The methyltransferase activity of the laboratory P. aeruginosa strain PAO1 EftM is slightly higher at 25°C compared to 37°C, while the rate of