Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase

Joshua P Owings,Emily G Kuiper,Samantha M Prezioso,Jeffrey Meisner,John J Varga,Natalia Zelinskaya,Eric B Dammer,Duc M Duong,Nicholas T Seyfried,Sebastián Albertí,Graeme L Conn,Joanna B Goldberg

doi:10.1074/jbc.m115.706853

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.

Highlights

Protein post-translational modification adds an additional level of complexity that can influence protein function as well as change the protein charge and tertiary structure
In Escherichia coli, for example, elongation factor-thermo-unstable (EF-Tu) lysine 57 is monomethylated during logarithmic growth phase [10], but as cells transition to stationary phase, there is a gradual conversion of monomethyl lysine to dimethyl lysine [11]
EftM Is a SAM-dependent Methyltransferase—We have previously demonstrated that the laboratory P. aeruginosa strain PAO1 shows EF-Tu methylation at 25 °C but not at 37 °C (Fig. 1)

Summary

Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase*

We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. EftM activity is associated with increased adhesion to and invasion of respiratory epithelial cells and is more prominent at 22–25 °C than at 37 °C [14] These observations and the fact that modified EF-Tu can be recognized by antibodies to phosphorylcholine lead to the speculation that trimethylation of EF-Tu functions like phosphorylcholine-modified surface-associated molecules of other respiratory pathogens. Analyses of EftM stability reveal that the protein undergoes an irreversible structural reorganization at 37 °C, resulting in loss of methyltransferase activity Together, these data suggest that the in vivo temperature-dependent methylation and associated effect on adhesion may be explained by direct thermoregulation of EftM, resulting in structural instability at human body temperature (37 °C)

Experimental Procedures

Results

Discussion