Abstract
Deinococcaceae are a family of extremely radiation tolerant bacteria that is currently subjected to numerous studies aimed at understanding the molecular mechanisms for such radiotolerance. To achieve a comprehensive and accurate annotation of Deinococcus deserti genome, we performed an N-terminal-oriented characterization of its proteome. For this, we used a labeling reagent, N-Tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), to selectively derivatize protein N-termini. The large scale identification of TMPP-modified Nterminal- most peptides by a shotgun liquid chromatography-tandem mass spectrometry analysis led to the validation of 278 and correction of 73 translation initiation codons in D. deserti genome. In addition, four new genes were detected, three located on the main chromosome and one on plasmid P3. 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 report the use of nonconventional translation initiation codons for two other genes: Deide_03051 and infC. We also analyzed signal peptide cleavages at a genome-wide scale. Based on comparative proteogenomic 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. Our results demonstrate that predicting translation initiation codons is still difficult and that proteomic-based refinement of genome annotations is necessary. Whether such use of noncanonical translation initiation codons is much more frequent for other bacterial phyla than previously reported 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.