show Abstracthide AbstractDeinococcus deserti is a desiccation- and radiation-tolerant desert bacterium. Differential RNA sequencing was performed to explore the specificities of its transcriptome. Strikingly, for 1174 (60%) mRNAs the transcription start site was found exactly at (916 cases, 47%) or very close to the translation initiation codon AUG or GUG. Such proportion of leaderless mRNAs, which may resemble ancestral mRNAs, is unprecedented for a bacterial species. Proteomics showed that leaderless mRNAs are efficiently translated in D. deserti. Interestingly, we also found 173 additional transcripts with a 5'-AUG or 5'-GUG that would make them competent for ribosome binding and translation into novel small polypeptides. Fourteen of these are predicted to be leader peptides involved in transcription attenuation. Another 30 correlated with new gene predictions and/or showed conservation with annotated and non-annotated genes in other Deinococcus species, and five of these novel polypeptides were indeed detected by mass spectrometry. The data also allowed re-annotation of the start codon position of 257 genes, including several DNA repair genes. Moreover, several novel highly radiation-induced genes were found and their potential roles are discussed. Based on our RNA sequencing and proteogenomics data, we propose that translation of many of the novel leaderless transcripts, which may have resulted from single nucleotide changes and maintained by selective pressure, provides a new explanation for the generation of a cellular pool of small peptides important for protection of proteins against oxidation and thus for radiation/desiccation tolerance and adaptation to harsh environmental conditions. Overall design: Using Illumina HiSeq 2000, RNA-Seq was performed to explore the transcriptome, including transcription start sites identification, in non-irradiated and irradiated Deinococcus deserti.