Abstract

The Pseudomonas aeruginosa methyltransferase EftM trimethylates elongation factor-Tu (EF-Tu) on lysine 5 to form a post-translational modification important for initial bacterial adherence to host epithelial cells. EftM methyltransferase activity is directly temperature regulated. The protein stability of EftM is tuned with a melting temperature (Tm) around 37 °C such that the enzyme is stable and active at 25 °C, but is completely inactivated by protein unfolding at higher temperatures. This leads to higher observable levels of EF-Tu trimethylation at the lower temperature. Here we report an additional layer of thermoregulation resulting in lower eftM mRNA transcript level at 37 °C compared to 25 °C and show that this regulation occurs at the level of transcription initiation. To begin to define the impact of this system on P. aeruginosa physiology, we demonstrate that EF-Tu is the only observable substrate for EftM. Further, we interrogated the proteome of three different wild-type P. aeruginosa strains, their eftM mutants, and these mutants complemented with eftM and conclude that trimethylation of EF-Tu by EftM does not impact EF-Tu’s canonical function in translation. In addition to furthering our knowledge of this Pseudomonas virulence factor, this study provides an intriguing example of a protein with multiple layers of thermoregulation.

Highlights

  • Www.nature.com/scientificreports are decreased in expression at 37 °C

  • Another example of a P. aeruginosa virulence factor that is down-regulated upon transition to 37 °C is EftM, a S-adenosyl-L-methionine (SAM)-dependent methyltransferase that trimethylates elongation factor-Tu (EF-Tu) on lysine 5 (K5me3)

  • In Escherichia coli, EF-Tu lysine 56 is methylated in response to bacterial energy state with monomethylation being observed during exponential phase growth and dimethylation being observed during nutrient limitation, such as in stationary phase

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Summary

Introduction

Www.nature.com/scientificreports are decreased in expression at 37 °C. One example of this regulatory trend in P. aeruginosa is Piv (protease IV; PA4175). EF-Tu is an elongation factor that delivers charged tRNA to the ribosome during translation and contributes to proofreading of the growing peptide chain[11] This canonical function is essential for bacterial cells[12]; post-translational modifications can alter this function. We utilized whole-cell proteomics to assess the proteome of three different P. aeruginosa strains in a label-free, unbiased manner to expose any effect K5me[3] has on the global Pseudomonas proteomic landscape These analyses reveal that methylation of EF-Tu by EftM has limited impact on the proteome under the conditions examined

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