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
Summary
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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