Project description:Radiation-resistant Deinococcus radiodurans is an extreme microorganism capable of withstanding high levels of ionizing radiation and chemical mutagens. It possesses remarkable DNA repair capabilities and serves as a model organism for studying stress resistance mechanisms. However, our understanding of the relationship between the biological characteristics of this species and its chromosomal 3D structure remains limited. In this study, we employed chromosome conformation capture and sequencing (3C-seq) technology to determine the 3D genome structure of D. radiodurans and to further investigate the changes of chromosome conformation induced by ultraviolet irradiation. We observed that the overall chromatin folding structure of the cells became much looser after UV irradiation, with smaller chromosomal interaction domains (CIDs) merging to form larger CIDs. Integrating transcriptomic data analysis, we found that the majority of upregulated differentially expressed genes were significantly enriched near specific CID boundaries. Additionally, we comprehensively elucidated that Dr_ebfC as a nuclear-associated protein, serves as a global regulatory factor in gene expression processes and may modulate transcriptional regulation by altering chromosomal structure, thereby influencing the physiological state of the bacterium. Overall, our study provides insights into the chromosomal conformational changes of D. radiodurans under different conditions, offering valuable resources for further understanding the molecular mechanisms underlying its extreme resistance.
Project description:Background: The International Space Station is an orbiting laboratory for microbial research in space, where microorganisms can be exposed to multiple extremes. Dehydrated cell clusters of extremophilic bacterium Deinococcus radiodurans have survived 3-year exposure outside the International Space Station in frames of the Tanpopo mission. We investigated the robust molecular machinery of Deinococcus radiodurans involved in its recovery after long-term space travel. Methods: The space-exposed and ground control cells of Deinococcus radiodurans were recovered in a complex medium for 5 and 15 h and investigated using integrative –omics techniques combined with electron microscopy tools. Consolidative transcriptomic, proteomic, and metabolomic analyses were performed to investigate molecular kinetics of cell recovery after 3-year exposure to low Earth orbit. Results: Ultrastructure analysis showed that Deinococcus radiodurans cells remained intact after low Earth orbit exposure for 3 years. Multiscale molecular analysis revealed significant alterations in response to long-term space travel. Key adaptations included upregulated DNA repair genes,stress response regulators, and oxidative stress scavenging enzymes. Proteins associated with transmembrane processes, cell division, and stress defence were also upregulated. Metabolomic analysis showed that only a few amino acids, sugars, and specific metabolites were more abundant after low Earth orbit exposure, suggesting energy conservation for molecular repair and regulation.Primordial stress molecule spermidine is also involved in cells recovery, helping combat the stress factors after space travel. Conclusion: Comparative –omics profile of extracted mRNA, proteins and metabolites allowed us to propose a multiscale dynamic molecular response of Deinococcus radiodurans after 3 years of space exposure. The kinetic profile with 2 timepoints during post-exposure analysis enabled the identification of foreground molecular targets employed by this microorganism in recovery after a space journey. Altogether, a multi-omics approach towards space-exposed cells revealed a strong focus on repair mechanisms, stress defence, and the utilization of external resources during the initial recovery phase. These findings expand our understanding of the molecular mechanisms employed by extremophiles to survive in space, providing implications for astrobiology and future space exploration.
Project description:Abstract Background The extraordinarily resistant bacterium Deinococcus radiodurans withstands harsh environmental conditions present in outer space. Deinococcus radiodurans was exposed for one year outside the International Space Station within Tanpopo orbital mission to investigate microbial survival and space travel. In addition, a ground-based simulation experiment with conditions, mirroring those from Low Earth orbit, was performed. Methods We monitored Deinococcus radiodurans cells during early stage of recovery after Low Earth orbit exposure using electron microscopy tools. Furthermore, proteomic, transcriptomic and metabolomic analyses were performed to identify molecular mechanisms responsible for the survival of Deinococcus radiodurans in Low Earth orbit. Results D. radiodurans cells exposed to low Earth orbit conditions do not exhibit any morphological damage. However, an accumulation of numerous outer-membrane associated vesicles was observed. On levels of proteins and transcripts, a multifaceted response was detected to alleviate cell stress. The UvrABC endonuclease excision repair mechanism was triggered to cope with DNA damage. Defense against reactive oxygen species is mirrored by the increased abundance of catalases and is accompanied by the increased abundance of putrescine which works as scavenging molecule. In addition, several proteins and mRNAs, responsible for regulatory and transporting functions showed increased abundances. The decrease in primary metabolites indicate alternations in the energy status, which is needed to repair damaged molecules. Conclusion Low Earth orbit induced molecular rearrangements trigger multiple components of metabolic stress response and regulatory networks in exposed microbial cells. Presented results show that the non-sporulating bacterium Deinococcus radiodurans survived long-term Low Earth orbit exposure if wavelength below 200 nm are not present, which mirrors the UV spectrum of Mars, where CO2 effectively provides a shield below 190 nm. These results should be considered in the context of planetary protection concerns and the development of new sterilization techniques for future space missions.
Project description:This study tracks the proteome during recovery from 10 kGy acute ionizing radiation (IR) in Deinococcus radiodurans R1 (WT). After 1 hour of recovery post-IR exposure, we observed 37 proteins significantly differentially expressed, including several within the Radiation and Dessication Response (RDR) pathway. Additionally, we also explored the regulatory network of a sRNA named PprS (previously Dsr2) in Deinococcus radiodurans by comparing the proteome of a sRNA knockdown strain (PprSKD, which demonstrates a ~2-fold decrease in PprS expression) to WT D. radiodurans during unirradiated conditions at late-exponential phase. Comparison between these two strains demonstrated decreased levels of one of PprS's targets, PprM, in the PprSKD strain which validated the activation mechanism we propose for PprS on pprM.
Project description:Transcriptional profiling of Deinococcus radiodurans comparing control untreated wild type cells with wild type cells treated with 100 µM CdCl2.
Project description:This study explores the regulatory network of a sRNA named PprS (previously Dsr2) in Deinococcus radiodurans R1. We compared the transcriptomes of two strains with and without exposure to 10 kGy acute ionizing radiation (IR): D. radiodurans R1 (wild-type) and a PprS knockdown mutant (PprSKD) that expresses ~2-fold less PprS compared to WT levels. Analysis of this RNAseq dataset demonstrated significant differential expression of several transcripts in both WT (34) and PprSKD (61) strains during recovery from IR. However, we did not observe any significantly differentially expressed transcripts between the two strains during recovery from IR. 31 transcripts were signficantly differentially expressed when comparing the two strains under unirradiated conditions. To determine the regulatory network that this sRNA, PprS could regulate we performed a MAPS (MS2-affinity purification followed by RNAsequencing) experiment in which a MS2-binding domain tagged PprS was expressed from a constutive promoter from a plasmid (pRADgro) in D. radiodurans R1. A control plasmid (containing only the MS2-binding domain) was also expressed and differential enrichment was compared to this control. MAPS suggested ~130 potential interacting transcripts with PprS and we were able to validate the binding and stabilization of one specific transcript, pprM, for PprS.
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.