@article{Hadjeras_Heiniger_Maass-Unrav_the_small-2023,
author = {Hadjeras, Lydia and Heiniger, Benjamin and Maaß, Sandra 
          and Scheuer, Robina and Gelhausen, Rick and Azarderakhsh, 
          Saina and Barth-Weber, Susanne and Backofen, Rolf and 
          Becher, Dörte and Ahrens, Christian H. and Sharma, Cynthia 
          M. and Evguenieva-Hackenberg, Elena},
title = {Unraveling the small proteome of the plant symbiont 
         {Sinorhizobium} meliloti by ribosome profiling and 
         proteogenomics},
journal = {Microlife},
year = {2023},
doi = {10.1093/femsml/uqad012},
volume = {4},
user = {backofen},
pmid = {37223733},
pages = {uqad012},
number = {},
issn = {2633-6693},
abstract = {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.}
}