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Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation.

ABSTRACT: Deinococcus radiodurans R1 (DEIRA) is a bacterium best known for its extreme resistance to the lethal effects of ionizing radiation, but the molecular mechanisms underlying this phenotype remain poorly understood. To define the repertoire of DEIRA genes responding to acute irradiation (15 kGy), transcriptome dynamics were examined in cells representing early, middle, and late phases of recovery by using DNA microarrays covering approximately 94% of its predicted genes. At least at one time point during DEIRA recovery, 832 genes (28% of the genome) were induced and 451 genes (15%) were repressed 2-fold or more. The expression patterns of the majority of the induced genes resemble the previously characterized expression profile of recA after irradiation. DEIRA recA, which is central to genomic restoration after irradiation, is substantially up-regulated on DNA damage (early phase) and down-regulated before the onset of exponential growth (late phase). Many other genes were expressed later in recovery, displaying a growth-related pattern of induction. Genes induced in the early phase of recovery included those involved in DNA replication, repair, and recombination, cell wall metabolism, cellular transport, and many encoding uncharacterized proteins. Collectively, the microarray data suggest that DEIRA cells efficiently coordinate their recovery by a complex network, within which both DNA repair and metabolic functions play critical roles. Components of this network include a predicted distinct ATP-dependent DNA ligase and metabolic pathway switching that could prevent additional genomic damage elicited by metabolism-induced free radicals.

SUBMITTER: Liu Y

PROVIDER: S-EPMC153069 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature

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Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation.

Liu Yongqing Y Zhou Jizhong J Omelchenko Marina V MV Beliaev Alex S AS Venkateswaran Amudhan A Stair Julia J Wu Liyou L Thompson Dorothea K DK Xu Dong D Rogozin Igor B IB Gaidamakova Elena K EK Zhai Min M Makarova Kira S KS Koonin Eugene V EV Daly Michael J MJ

Proceedings of the National Academy of Sciences of the United States of America 20030321 7

Deinococcus radiodurans R1 (DEIRA) is a bacterium best known for its extreme resistance to the lethal effects of ionizing radiation, but the molecular mechanisms underlying this phenotype remain poorly understood. To define the repertoire of DEIRA genes responding to acute irradiation (15 kGy), transcriptome dynamics were examined in cells representing early, middle, and late phases of recovery by using DNA microarrays covering approximately 94% of its predicted genes. At least at one time point ...[more]

PMID: 12651953

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Project description:5'- and 3'-end healing are key steps in nucleic acid break repair in which 5'-OH and 3'-PO4 or 2',3'-cyclic-PO4 ends are converted to 5'-PO4 and 3'-OH termini suitable for sealing by polynucleotide ligases. Here, we characterize Deinococcus radiodurans HD-Pnk as a bifunctional end-healing enzyme composed of N-terminal HD (histidine-aspartate) phosphoesterase and C-terminal P-loop polynucleotide kinase (Pnk) domains. HD-Pnk phosphorylates 5'-OH DNA in the presence of ATP and magnesium. HD-Pnk has 3'-phosphatase and 2',3'-cyclic-phosphodiesterase activity in the presence of transition metals, optimally cobalt or copper, and catalyzes copper-dependent hydrolysis of p-nitrophenylphosphate. HD-Pnk is encoded by the LIG-PARG-HD-Pnk three-gene operon, which includes polynucleotide ligase and poly(ADP-ribose) glycohydrolase genes. We show that whereas HD-Pnk is inessential for Deinococcus growth, its absence sensitizes by 80-fold bacteria to killing by 9 kGy of ionizing radiation (IR). HD-Pnk protein is depleted during early stages of post-IR recovery and then replenished at 15 h, after reassembly of the genome from shattered fragments. ?HD-Pnk mutant cells are competent for genome reassembly, as gauged by pulsed-field gel electrophoresis. Our findings suggest a role for HD-Pnk in repairing residual single-strand gaps or nicks in the reassembled genome. HD-Pnk-Ala mutations that ablate kinase or phosphoesterase activity sensitize Deinococcus to killing by mitomycin C.IMPORTANCE End healing is a process whereby nucleic acid breaks with "dirty" 3'-PO4 or 2',3'-cyclic-PO4 and 5'-OH ends are converted to 3'-OH and 5'-PO4 termini that are amenable to downstream repair reactions. Deinococcus radiodurans is resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlights Deinococcus HD-Pnk as a bifunctional 3'- and 5'-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.

Project description:The extremophile Deinococcus radiodurans maintains a highly organized and condensed nucleoid as its default state, possibly contributing to its high tolerance to ionizing radiation (IR). Previous studies of the D. radiodurans nucleoid were limited by reliance on manual image annotation and qualitative metrics. Here, we introduce a high-throughput approach to quantify the geometric properties of cells and nucleoids using confocal microscopy, digital reconstructions of cells, and computational modeling. We utilize this novel approach to investigate the dynamic process of nucleoid condensation in response to IR stress. Our quantitative analysis reveals that at the population level, exposure to IR induced nucleoid compaction and decreased the size of D. radiodurans cells. Morphological analysis and clustering identified six distinct sub-populations across all tested experimental conditions. Results indicate that exposure to IR induced fractional redistributions of cells across sub-populations to exhibit morphologies associated with greater nucleoid condensation and decreased the abundance of sub-populations associated with cell division. Nucleoid-associated proteins (NAPs) may link nucleoid compaction and stress tolerance, but their roles in regulating compaction in D. radiodurans are unknown. Imaging of genomic mutants of known and suspected NAPs that contribute to nucleoid condensation found that deletion of nucleic acid-binding proteins, not previously described as NAPs, can remodel the nucleoid by driving condensation or decondensation in the absence of stress and that IR increased the abundance of these morphological states. Thus, our integrated analysis introduces a new methodology for studying environmental influences on bacterial nucleoids and provides an opportunity to further investigate potential regulators of nucleoid condensation.IMPORTANCEDeinococcus radiodurans, an extremophile known for its stress tolerance, constitutively maintains a highly condensed nucleoid. Qualitative studies have described nucleoid behavior under a variety of conditions. However, a lack of quantitative data regarding nucleoid organization and dynamics has limited our understanding of the regulatory mechanisms controlling nucleoid organization in D. radiodurans. Here, we introduce a quantitative approach that enables high-throughput quantitative measurements of subcellular spatial characteristics in bacterial cells. Applying this to wild-type or single-protein-deficient populations of D. radiodurans subjected to ionizing radiation, we identified significant stress-responsive changes in cell shape, nucleoid organization, and morphology. These findings highlight this methodology's adaptability and capacity for quantitatively analyzing the cellular response to stressors for screening cellular proteins involved in bacterial nucleoid organization.

Dataset Information

Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation.

Publications

Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation.

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