RNA-seqanalysis of deinococcus wild-type and mazEF mutant strains after MMC treatment
ABSTRACT: RNA-seq was used to analysis of mazEF toxin-antitoxin system mediated post-transcriptional inhibition through its endoribonuclease activity. DNA damage agents ,such as MMC, activates the mazEF system leading to cells growth arrest. In the present study, results reveal that a larger number of genes involved in amino acid and carbohydrate metabolism, ion transport and transcription was down-regulated in the wild-type compared with the mazEF-dr mutant. Overall design: Total RNAs were extrated from wild-type deinococcus radiodurans and mazEF mutant strain with or without MMC treatment.
Project description:Transcriptional profiling of Deinococcus radiodurans comparing control untreated wild type cells with wild type cells treated with 100 µM CdCl2. Two-condition experiment, wild type vs. 100uM CdCl2. Biological replicates: 4 time courses (0.25, 0.5, 1, 2h), dye-swapped, independently grown and harvested. Two replicate per array.
Project description:Transcriptional profiling of Deinococcus radiodurans comparing control untreated wild type cells with wild type cells treated with 100 µM CdCl2. Overall design: Two-condition experiment, wild type vs. 100uM CdCl2. Biological replicates: 4 time courses (0.25, 0.5, 1, 2h), dye-swapped, independently grown and harvested. Two replicate per array.
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. Overall design: Using Illumina HiSeq 2000, RNA-Seq was performed to explore the transcriptome in non-irradiated and irradiated Deinococcus deserti strains lacking gene irrE or ddrOc.
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.
Project description:The previously uncharacterized proteins HigB (unannotated) and HigA (PA4674) of Pseudomonas aeruginosa PA14 were found to form a type II TA system in which antitoxin HigA masks the RNase activity of toxin HigB through direct binding. To determine the physiological role of HigB/HigA in P. aeruginosa, a whole-transcriptome experiment was performed for the higA antitoxin deletion mutant of the PA14 strain compared to the wild-type PA14 strain. The rationale was that for the strain that lacks the antitoxin, the effect of the toxin could be discerned due to enhanced activity of the toxin. Furthermore, toxin HigB reduces production of the virulence factors pyochelin, pyocyanin, swarming, and biofilm formation. Overall design: The higA mutant of Pseudomonas aeruginosa PA14 compared to the P. aeruginosa PA14 wild-type