title: Evidence for the recent origin of a bacterial protein-coding, overlapping orphan gene by evolutionary overprinting.
abstract: BACKGROUND
  Gene duplication is believed to be the classical way to form novel genes, but overprinting may be an important alternative. Overprinting allows entirely novel proteins to evolve de novo, i.e., formerly non-coding open reading frames within functional genes become expressed. Only three cases have been described for Escherichia coli. Here, a fourth example is presented.
  
  
  RESULTS
  RNA sequencing revealed an open reading frame weakly transcribed in cow dung, coding for 101 residues and embedded completely in the -2 reading frame of citC in enterohemorrhagic E. coli. This gene is designated novel overlapping gene, nog1. The promoter region fused to gfp exhibits specific activities and 5' rapid amplification of cDNA ends indicated the transcriptional start 40-bp upstream of the start codon. nog1 was strand-specifically arrested in translation by a nonsense mutation silent in citC. This Nog1-mutant showed a phenotype in competitive growth against wild type in the presence of MgCl2. Small differences in metabolite concentrations were also found. Bioinformatic analyses propose Nog1 to be inner membrane-bound and to possess at least one membrane-spanning domain. A phylogenetic analysis suggests that the orphan gene nog1 arose by overprinting after Escherichia/Shigella separated from the other γ-proteobacteria.
  
  
  CONCLUSIONS
  Since nog1 is of recent origin, non-essential, short, weakly expressed and only marginally involved in E. coli's central metabolism, we propose that this gene is in an initial stage of evolution. While we present specific experimental evidence for the existence of a fourth overlapping gene in enterohemorrhagic E. coli, we believe that this may be an initial finding only and overlapping genes in bacteria may be more common than is currently assumed by microbiologists.
authors.affiliation: Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85350, Freising, Germany. Lea.Fellner@dlr.de.
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, Constance, Germany. simon@dbvis.inf.uni-konstanz.de.
authors.forename: Svenja
authors.initials: S
authors.lastname: Simon
authors.affiliation: Lehrstuhl für Ernährungsphysiologie, Wissenschaftszentrum Weihenstephan, Technische Universität München, Gregor-Mendel-Straße 2, D-85354, Freising, Germany. christian.scherling@klinkner.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Scherling
authors.affiliation: Research Unit Analytical BioGeoChemistry, Deutsches Forschungszentrum für Gesundheit und Umwelt GmbH, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85754, Neuherberg, Germany. michael.witting@helmholtz-muenchen.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Witting
authors.affiliation: Institute of Communications Engineering, Universität Ulm, Albert-Einstein-Allee 43, 89081, Ulm, Germany. steffen.schober.de@ieee.org.|Present address: Blue Yonder GmbH, Ohiostraße 8, Karlsruhe, Germany. steffen.schober.de@ieee.org.
authors.forename: Steffen
authors.initials: S
authors.lastname: Schober
authors.affiliation: Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85350, Freising, Germany. Christine.Polte@chemie.uni-hamburg.de.|Present address: Institut für Biochemie und Molekularbiologie, Universität Hamburg, Martin-Luther-King Platz 6, 20146, Hamburg, Germany. Christine.Polte@chemie.uni-hamburg.de.
authors.forename: Christine
authors.initials: C
authors.lastname: Polte
authors.affiliation: Research Unit Analytical BioGeoChemistry, Deutsches Forschungszentrum für Gesundheit und Umwelt GmbH, Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85754, Neuherberg, Germany. schmitt-kopplin@helmholtz-muenchen.de.
authors.forename: Philippe
authors.initials: P
authors.lastname: Schmitt-Kopplin
authors.affiliation: Lehrstuhl für Datenanalyse und Visualisierung, Fachbereich Informatik und Informationswissenschaft, Universität Konstanz, Box 78, 78457, Constance, Germany. keim@uni-konstanz.de.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Keim
authors.affiliation: Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85350, Freising, Germany. siegfried.scherer@wzw.tum.de.
authors.forename: Siegfried
authors.initials: S
authors.lastname: Scherer
authors.affiliation: Lehrstuhl für Mikrobielle Ökologie, Wissenschaftszentrum Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, 85350, Freising, Germany. neuhaus@wzw.tum.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Neuhaus
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D018387:Codon, Initiator; D019295:Computational Biology; D054308:Enteropathogenic Escherichia coli / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D019143:Evolution, Molecular; D005243:Feces / Q000382:microbiology; D005807:Genes, Overlapping; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D009876:Operon; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D012760:Shigella / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D018387:Codon, Initiator