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Chembiochem. 2019 Jun 3;20(11):1430-1437. doi: 10.1002/cbic.201800741. Epub 2019 Apr 5.

Identification and Quantification of (t)RNA Modifications in Pseudomonas aeruginosa by Liquid Chromatography-Tandem Mass Spectrometry.

Author information

1
TWINCORE, Centre for Experimental and Clinical Infection Research GmbH, Institute for Molecular Bacteriology, Feodor-Lynen-Strasse 7, 30625, Hannover, Germany.
2
Helmholtz Centre for Infection Research, Department of Chemical Biology, Inhoffenstrasse 7, 38124, Braunschweig, Germany.
3
Centre of Biomolecular Drug Research (BMWZ), Institute of Organic Chemistry, Leibniz Universität, Schneiderberg 38, 30167, Hannover, Germany.
4
Hannover Medical School, Research Core Unit Metabolomics, Carl-Neuberg-Strasse 1, 30625, Hannover, Germany.
5
Helmholtz Centre for Infection Research, Department of Molecular Bacteriology, Inhoffenstrasse 7, 38124, Braunschweig, Germany.

Abstract

Transfer RNA (tRNA) modifications impact the structure and function of tRNAs, thus affecting the efficiency and fidelity of translation. In the opportunistic pathogen Pseudomonas aeruginosa translational regulation plays an important but less defined role in adaptation to changing environments. In this study, we have explored tRNA modifications in P. aeruginosa through LC-MS/MS approaches. Neutral loss scanning (NLS) demonstrated the potential to identify previously unknown modifications, whereas multiple reaction monitoring (MRM) was able to detect modifications with high specificity and sensitivity. In this study, the MRM-based external calibration method allowed for quantification of the four canonical and 32 modified ribonucleosides, out of which 21 tRNA modifications were quantified in the total tRNA pool of P. aeruginosa PA14. We also purified the single tRNA isoacceptors tRNA-ArgUCU, tRNA-LeuCAA, and tRNA-TrpCCA and determined their specific modification patterns, both qualitatively and quantitatively. Deeper insights into the nature and dynamics of tRNA modifications in P. aeruginosa should pave the way for further studies on post-transcriptional gene regulation as a relatively unexplored molecular mechanism of controlling bacterial pathogenicity and mode of growth.

KEYWORDS:

Pseudomonas aeruginosa; gene expression; mass spectrometry; tRNA; tRNA modifications

PMID:
30644616
DOI:
10.1002/cbic.201800741

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