@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, Dorte 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},
  volume =	 {4},
  number =	 {},
  pages =	 {uqad012},
  user =	 {backofen},
  pmid =	 {37223733},
  doi = 	 {10.1093/femsml/uqad012},
  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.}
}