Project description:Deinococcus 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.
Project description:The extreme radiation resistance of Deinococcus bacteria requires the radiation-stimulated cleavage of protein DdrO by a specific metalloprotease called IrrE. DdrO is the repressor of a predicted radiation/desiccation response (RDR) regulon, composed of radiation-induced genes having a conserved DNA motif (RDRM) in their promoter regions. Here, we showed that addition of zinc ions to purified apo-IrrE, and short exposure of Deinococcus cells to zinc ions, resulted in cleavage of DdrO in vitro and in vivo, respectively. Binding of IrrE to RDRM-containing DNA or interaction of IrrE with DNA-bound DdrO was not observed. The data are in line with IrrE being a zinc peptidase, and indicate that increased zinc availability, caused by oxidative stress, triggers the in vivo cleavage of DdrO unbound to DNA. Transcriptomics and proteomics of Deinococcus deserti confirmed the IrrE-dependent regulation of predicted RDR regulon genes and also revealed additional members of this regulon. Comparative analysis showed that the RDR regulon is largely well conserved in Deinococcus species, but also showed diversity in the regulon composition. Notably, several RDR genes with an important role in radiation resistance in Deinococcus radiodurans, for example pprA, are not conserved in some other radiation-resistant Deinococcus species.
Project description:Nucleoids have been purified from Deinococcus deserti and Deinococcus radiodurans cells subjected to irradiation and short recovery (six replicates for each strain). The nucleoids have been analyzed by shotgun proteomics as described in Toueille M et al (2012) J Proteomics. Comparative proteomics pointed at specific proteins recruited at the nucleoids during the recovery stage after DNA damages produced by the irradiation. Data processing and bioinformatics: Peak lists were generated with the MASCOT DAEMON software (version 2.2.2) from Matrix Science using the extract_msn.exe data import filter (ThermoFisher) from the Xcalibur FT package (version 2.0.7) from ThermoFisher. Data import filter options were set at: 400 (minimum mass), 5000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1000 (threshold). Using the MASCOT search engine (version 2.2.04) from Matrix Science, we searched all MS/MS spectra against in-house polypeptide sequence databases. For Deinococcus radiodurans MS/MS assignments, the database contained i) the sequence of all currently annotated proteins coded by D. radiodurans BAA-816 genome (2629 proteins from chromosome 1 (NC_001263), 368 proteins from chromosome 2 (NC_001264), 130 proteins from plasmid MP1 (NC_000958), and 35 proteins from plasmid CP1 (NC_000958)), ii) 28 manual protein sequence curations from D. radiodurans R1, and iii) 121 additional ORF sequences predicted by the CONSORF consensus prediction system.This database thus comprises 3,311 polypeptide sequences, totaling 1,006,757 amino acids. For Deinococcus deserti MS/MS assignments, the database contained the sequence of all annotated proteins coded by D. deserti VCD115 chromosome and plasmids. This database comprises 3,455 polypeptide sequences, totaling 1,083,334 amino acids. Searches for tryptic peptides were performed with the following parameters: full-trypsin specificity, a mass tolerance of 10 ppm on the parent ion and 0.5 Da on the MS/MS, static modifications of carboxyamidomethylated Cys (+57.0215), and dynamic modifications of oxidized Met (+15.9949). The maximum number of missed cleavages was set at 1. All peptide matches with a peptide score below a stringent P value of 0.001 were filtered by the IRMa 1.18.1 parser. A protein was considered validated when at least two different peptides were detected in the same experiment. False-positives rate for protein identification was estimated using the appropriate decoy database as below 0.1% with these parameters.