Unraveling the small proteome of the plant symbiont Sinorhizobium meliloti by ribosome profiling and proteogenomics.

Autor: Hadjeras L; Institute of Molecular Infection Biology, University of Würzburg, 97080 Würzburg, Germany., Heiniger B; Molecular Ecology, Agroscope and SIB Swiss Institute of Bioinformatics, 8046 Zurich, Switzerland., Maaß S; Institute of Microbiology, University of Greifswald, 17489 Greifswald, Germany., Scheuer R; Institute of Microbiology and Molecular Biology, University of Giessen, 35392 Giessen, Germany., Gelhausen R; Bioinformatics Group, Department of Computer Science, University of Freiburg, 79110 Freiburg, Germany., Azarderakhsh S; Institute of Microbiology and Molecular Biology, University of Giessen, 35392 Giessen, Germany., Barth-Weber S; Institute of Microbiology and Molecular Biology, University of Giessen, 35392 Giessen, Germany., Backofen R; Bioinformatics Group, Department of Computer Science, University of Freiburg, 79110 Freiburg, Germany., Becher D; Institute of Microbiology, University of Greifswald, 17489 Greifswald, Germany., Ahrens CH; Molecular Ecology, Agroscope and SIB Swiss Institute of Bioinformatics, 8046 Zurich, Switzerland., Sharma CM; Institute of Molecular Infection Biology, University of Würzburg, 97080 Würzburg, Germany., Evguenieva-Hackenberg E; Institute of Microbiology and Molecular Biology, University of Giessen, 35392 Giessen, Germany.
Jazyk: angličtina
Zdroj: MicroLife [Microlife] 2023 Mar 10; Vol. 4, pp. uqad012. Date of Electronic Publication: 2023 Mar 10 (Print Publication: 2023).
DOI: 10.1093/femsml/uqad012
Abstrakt: The soil-dwelling plant symbiont Sinorhizobium meliloti is a major model organism of Alphaproteobacteria. Despite numerous detailed OMICS studies, information about small open reading frame (sORF)-encoded proteins (SEPs) is largely missing, because sORFs are poorly annotated and SEPs are hard to detect experimentally. However, given that SEPs can fulfill important functions, identification of translated sORFs is critical for analyzing their roles in bacterial physiology. Ribosome profiling (Ribo-seq) can detect translated sORFs with high sensitivity, but is not yet routinely applied to bacteria because it must be adapted for each species. Here, we established a Ribo-seq procedure for S. meliloti 2011 based on RNase I digestion and detected translation for 60% of the annotated coding sequences during growth in minimal medium. Using ORF prediction tools based on Ribo-seq data, subsequent filtering, and manual curation, the translation of 37 non-annotated sORFs with ≤ 70 amino acids was predicted with confidence. The Ribo-seq data were supplemented by mass spectrometry (MS) analyses from three sample preparation approaches and two integrated proteogenomic search database (iPtgxDB) types. Searches against standard and 20-fold smaller Ribo-seq data-informed custom iPtgxDBs confirmed 47 annotated SEPs and identified 11 additional novel SEPs. Epitope tagging and Western blot analysis confirmed the translation of 15 out of 20 SEPs selected from the translatome map. Overall, by combining MS and Ribo-seq approaches, the small proteome of S. meliloti was substantially expanded by 48 novel SEPs. Several of them are part of predicted operons and/or are conserved from Rhizobiaceae to Bacteria, suggesting important physiological functions.
Competing Interests: The authors declare that they have no conflicts of interest.
(© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje