title: Proteomics-based refinement of Deinococcus deserti genome annotation reveals an unwonted use of non-canonical translation initiation codons.
abstract: Deinococcaceae are a family of extremely radiation-tolerant bacteria that are currently subjected to numerous studies aimed at understanding the molecular mechanisms for such radiotolerance. To achieve a comprehensive and accurate annotation of the Deinococcus deserti genome, we performed an N terminus-oriented characterization of its proteome. For this, we used a labeling reagent, N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide, to selectively derivatize protein N termini. The large scale identification of N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide-modified N-terminal-most peptides by shotgun liquid chromatography-tandem mass spectrometry analysis led to the validation of 278 and the correction of 73 translation initiation codons in the D. deserti genome. In addition, four new genes were detected, three located on the main chromosome and one on plasmid P3. We also analyzed signal peptide cleavages on a genome-wide scale. Based on comparative proteogenomics analysis, we propose a set of 137 corrections to improve Deinococcus radiodurans and Deinococcus geothermalis gene annotations. Some of these corrections affect important genes involved in DNA repair mechanisms such as polA, ligA, and ddrB. Surprisingly, experimental evidences were obtained indicating that DnaA (the protein involved in the DNA replication initiation process) and RpsL (the S12 ribosomal conserved protein) translation is initiated in Deinococcaceae from non-canonical codons (ATC and CTG, respectively). Such use may be the basis of specific regulation mechanisms affecting replication and translation. We also report the use of non-conventional translation initiation codons for two other genes: Deide_03051 and infC. Whether such use of non-canonical translation initiation codons is much more frequent than for other previously reported bacterial phyla or restricted to Deinococcaceae remains to be investigated. Our results demonstrate that predicting translation initiation codons is still difficult for some bacteria and that proteomics-based refinement of genome annotations may be helpful in such cases.
authors.affiliation: Laboratoire de Biochimie des Systèmes Perturbés, Service de Biochimie et Toxicologie Nucléaire, Institut de Biologie Environnementale et Biotechnologie (iBEB), Direction des Sciences du Vivant (DSV), Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), F-30207 Bagnols-sur-Cèze, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Baudet
authors.forename: Philippe
authors.initials: P
authors.lastname: Ortet
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Gaillard
authors.forename: Bernard
authors.initials: B
authors.lastname: Fernandez
authors.forename: Philippe
authors.initials: P
authors.lastname: Guérin
authors.forename: Christine
authors.initials: C
authors.lastname: Enjalbal
authors.forename: Gilles
authors.initials: G
authors.lastname: Subra
authors.forename: Arjan
authors.initials: A
authors.lastname: de Groot
authors.forename: Mohamed
authors.initials: M
authors.lastname: Barakat
authors.forename: Alain
authors.initials: A
authors.lastname: Dedieu
authors.forename: Jean
authors.initials: J
authors.lastname: Armengaud
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D018387:Codon, Initiator / Q000235:genetics*; D030562:Databases, Protein; D034301:Deinococcus / Q000235:genetics*; D005798:Genes, Bacterial; D016680:Genome, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics*; D021382:Protein Sorting Signals; D040901:Proteomics / Q000379:methods*; D017386:Sequence Homology, Amino Acid; D013194:Staining and Labeling
chemical_list: D001426:Bacterial Proteins; D018387:Codon, Initiator; D009943:Organophosphorus Compounds; D010455:Peptides; D021382:Protein Sorting Signals; C405845:tris(trimethoxyphenyl)phosphonium acetyl