title: Translatomics combined with transcriptomics and proteomics reveals novel functional, recently evolved orphan genes in Escherichia coli O157:H7 (EHEC).
abstract: BACKGROUND
  Genomes of E. coli, including that of the human pathogen Escherichia coli O157:H7 (EHEC) EDL933, still harbor undetected protein-coding genes which, apparently, have escaped annotation due to their small size and non-essential function. To find such genes, global gene expression of EHEC EDL933 was examined, using strand-specific RNAseq (transcriptome), ribosomal footprinting (translatome) and mass spectrometry (proteome).
  
  
  RESULTS
  Using the above methods, 72 short, non-annotated protein-coding genes were detected. All of these showed signals in the ribosomal footprinting assay indicating mRNA translation. Seven were verified by mass spectrometry. Fifty-seven genes are annotated in other enterobacteriaceae, mainly as hypothetical genes; the remaining 15 genes constitute novel discoveries. In addition, protein structure and function were predicted computationally and compared between EHEC-encoded proteins and 100-times randomly shuffled proteins. Based on this comparison, 61 of the 72 novel proteins exhibit predicted structural and functional features similar to those of annotated proteins. Many of the novel genes show differential transcription when grown under eleven diverse growth conditions suggesting environmental regulation. Three genes were found to confer a phenotype in previous studies, e.g., decreased cattle colonization.
  
  
  CONCLUSIONS
  These findings demonstrate that ribosomal footprinting can be used to detect novel protein coding genes, contributing to the growing body of evidence that hypothetical genes are not annotation artifacts and opening an additional way to study their functionality. All 72 genes are taxonomically restricted and, therefore, appear to have evolved relatively recently de novo.
authors.affiliation: Lehrstuhl für Mikrobielle Ökologie, Zentralinstitut für Ernährungs- und Lebensmittelforschung, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany. neuhaus@wzw.tum.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Neuhaus
authors.affiliation: Lehrstuhl für Mikrobielle Ökologie, Zentralinstitut für Ernährungs- und Lebensmittelforschung, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany. r.landstorfer@gmx.de.
authors.forename: Richard
authors.initials: R
authors.lastname: Landstorfer
authors.affiliation: Lehrstuhl für Mikrobielle Ökologie, Zentralinstitut für Ernährungs- und Lebensmittelforschung, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany. fellnerlea@hotmail.com.
authors.forename: Lea
authors.initials: L
authors.lastname: Fellner
authors.affiliation: Lehrstuhl für Datenanalyse und Visualisierung, Fachbereich Informatik und Informationswissenschaft, Universität Konstanz, Box 78, 78457, Konstanz, Germany. simon@dbvis.inf.uni-konstanz.de.
authors.forename: Svenja
authors.initials: S
authors.lastname: Simon
authors.affiliation: Department of Informatics - Bioinformatics & TUM-IAS, Technische Universität München, Boltzmannstraße 3, 85748, Garching, Germany. andrea.schafferhans@rostlab.org.
authors.forename: Andrea
authors.initials: A
authors.lastname: Schafferhans
authors.affiliation: Department of Informatics - Bioinformatics & TUM-IAS, Technische Universität München, Boltzmannstraße 3, 85748, Garching, Germany. goldberg@rostlab.org.
authors.forename: Tatyana
authors.initials: T
authors.lastname: Goldberg
authors.affiliation: Chair of Proteomics and Bioanalytics, Wissenschaftszentrum Weihenstephan, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany. h4r4ld.marx@googlemail.com.
authors.forename: Harald
authors.initials: H
authors.lastname: Marx
authors.affiliation: Institute of Cell Biophysics, Russian Academy of Sciences, Moscow Region, 142290, Pushchino, Russia. ozoline@rambler.ru.
authors.forename: Olga N
authors.initials: ON
authors.lastname: Ozoline
authors.affiliation: Department of Informatics - Bioinformatics & TUM-IAS, Technische Universität München, Boltzmannstraße 3, 85748, Garching, Germany. rost@rostlab.org.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Rost
authors.affiliation: Chair of Proteomics and Bioanalytics, Wissenschaftszentrum Weihenstephan, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354, Freising, Germany. kuster@wzw.tum.de.|Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS), Technische Universität München, Gregor-Mendel-Str. 4, 85354, Freising, Germany. kuster@wzw.tum.de.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kuster
authors.affiliation: Lehrstuhl für Datenanalyse und Visualisierung, Fachbereich Informatik und Informationswissenschaft, Universität Konstanz, Box 78, 78457, Konstanz, Germany. keim@informatik.uni-konstanz.de.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Keim
authors.affiliation: Lehrstuhl für Mikrobielle Ökologie, Zentralinstitut für Ernährungs- und Lebensmittelforschung, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85354, Freising, Germany. siegfried.scherer@wzw.tum.de.
authors.forename: Siegfried
authors.initials: S
authors.lastname: Scherer
mesh_terms: D000818:Animals; D002417:Cattle; D019295:Computational Biology; D019453:Escherichia coli O157 / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D013058:Mass Spectrometry; D010641:Phenotype; D020543:Proteome / Q000235:genetics*; D012329:RNA, Bacterial / Q000235:genetics; D017423:Sequence Analysis, RNA; D059467:Transcriptome
chemical_list: D029968:Escherichia coli Proteins; D020543:Proteome; D012329:RNA, Bacterial