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Arginine-rhamnosylation as new strategy to activate translation elongation factor P

Nature Chemical Biology volume 11, pages 266–270 (2015)Cite this article

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A Corrigendum to this article was published on 18 March 2015

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Abstract

Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, translation elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-β-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-L-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a translation elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-L-rhamnose–biosynthesizing enzymes ideal targets for antibiotic development.

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Figure 1: Bioinformatic identification of the EarP-arginine type EF-P subfamily.
Figure 2: Phenotypic analysis of S. oneidensis MR-1 earP and efp deletion mutants.
Figure 3: Dependent peptide MS analysis of the S. oneidensis MR-1 EF-P modification.
Figure 4: In vivo analysis of S. oneidensis MR-1 EF-P functionality depending on NDP-deoxyhexose biosyntheses.
Figure 5: EF-P rhamnosylation, mode of action and impact on pathogenicity.

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  • 24 February 2015

    In the version of this article initially published online, the second author's middle initial was inadvertently omitted. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We would like to thank I. Weitl for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft: Center for Integrated Protein Science Munich (CiPSM; Exc114/2 to K.J. and WI3285/4-1 to D.N.W.) and the Max Planck Society (to E.C.K., K.W., L.S.-A. and M.M.). A.L.S. is funded by an AXA Research Fund Postdoctoral Fellowship. The work of S.H. and J.G. was supported by the Helmholtz Association and the Bundesministerium für Bildung und Forschung. J.-M.C. and J.R are funded by the US National Institutes of Health (CA 091901).

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Authors and Affiliations

  1. Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Munich, Germany

    Jürgen Lassak, Maximilian Fürst, Agata L Starosta, Daniel N Wilson & Kirsten Jung

  2. Department of Biology I, Microbiology, Ludwig-Maximilians-Universität München, Martinsried, Germany

    Jürgen Lassak, Maximilian Fürst & Kirsten Jung

  3. Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Martinsried, Germany

    Eva C Keilhauer & Matthias Mann

  4. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

    Kristin Wuichet & Lotte Søgaard-Andersen

  5. Institute for Molecular Bacteriology, Twincore, Centre for Clinical and Experimental Infection Research, a joint venture of the Helmholtz Centre of Infection Research and the Hannover Medical School, Hannover, Germany

    Julia Gödeke & Susanne Häussler

  6. Department for Biochemistry, Gene Center, Ludwig-Maximilians-Universität München, Munich, Germany

    Agata L Starosta & Daniel N Wilson

  7. Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky, USA

    Jhong-Min Chen & Jürgen Rohr

  8. Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany

    Susanne Häussler

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  1. Jürgen Lassak
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Contributions

J.L. and K.J. designed the experiments and wrote the manuscript. J.L., A.L.S. and D.N.W. discovered EarP and arginine-type EF-P using bioinformatics. K.W., J.L. and L.S.-A. performed phylogenetic analyses. J.L. and M.F. generated S. oneidensis and E. coli deletion mutants as well as all of the plasmids used in the study. All phenotypic analysis was performed by J.L. and M.F. J.L. purified proteins for MS analysis, which was performed by E.C.K. and analyzed by E.C.K. and M.M. TDP-L-rhamnose was synthesized by J.-M.C. and J.R. for in vitro glycosylation performed by J.L. Modeling of modified EF-P to the ribosome was done by D.N.W. Phenotypic characterization of P. aeruginosa Δefp and ΔearP mutants was performed by J.G. and analyzed by J.G. and S.H.

Corresponding authors

Correspondence to Jürgen Lassak or Kirsten Jung.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–12 and Supplementary Tables 1–4. (PDF 3661 kb)

Supplementary Data Set 1

Distribution of EF-P, EpmA, EpmB, EpmC, EarP, and RmlD homologs in the representative genome set (XLS 189 kb)

Supplementary Data Set 2

EF-P, EpmA, EpmB, EpmC, EarP, and RmlD sequences identified in the representative genome set. (XLS 141 kb)

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Lassak, J., Keilhauer, E., Fürst, M. et al. Arginine-rhamnosylation as new strategy to activate translation elongation factor P. Nat Chem Biol 11, 266–270 (2015). https://doi.org/10.1038/nchembio.1751

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