A Novel OxyR Sensor and Regulator of Hydrogen Peroxide Stress with One Cysteine Residue in Deinococcus radiodurans

In bacteria, OxyR is a peroxide sensor and transcription regulator, which can sense the presence of reactive oxygen species and induce antioxidant system. When the cells are exposed to H2O2, OxyR protein is activated via the formation of a disulfide bond between the two conserved cysteine residues (C199 and C208). In Deinococcus radiodurans, a previously unreported special characteristic of DrOxyR (DR0615) is found with only one conserved cysteine. dr0615 gene mutant is hypersensitive to H2O2, but only a little to ionizing radiation. Site-directed mutagenesis and subsequent in vivo functional analyses revealed that the conserved cysteine (C210) is necessary for sensing H2O2, but its mutation did not alter the binding characteristics of OxyR on DNA. Under oxidant stress, DrOxyR is oxidized to sulfenic acid form, which can be reduced by reducing reagents. In addition, quantitative real-time PCR and global transcription profile results showed that OxyR is not only a transcriptional activator (e.g., katE, drb0125), but also a transcriptional repressor (e.g., dps, mntH). Because OxyR regulates Mn and Fe ion transporter genes, Mn/Fe ion ratio is changed in dr0615 mutant, suggesting that the genes involved in Mn/Fe ion homeostasis, and the genes involved in antioxidant mechanism are highly cooperative under extremely oxidant stress. In conclusion, these findings expand the OxyR family, which could be divided into two classes: typical 2-Cys OxyR and 1-Cys OxyR

Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis

Discovery of a splicing regulator required for cell cycle progression.

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms

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Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organism's dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical

TgRRM1 is associated with U4/U6.U5 subcomplex of the spliceosome.

<p>(A) TgRRM1 was immunoprecipitated with anti-myc antibody from the whole cell lysates (shown here) or nuclear extracts (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305.s004" target="_blank">Dataset S4</a>) of the transgenic clone 12-109C6 expressing wt-TgRRM1^myc. The original mutant strain 12-109C6 was used as a negative control. Eluates from experimental (wt-TgRRM1^myc) and control immunoprecipitations were resolved on SDS-PAGE and stained with Commassie Blue. Digestion of the gel slices and mass-spectrometry analysis of protein peptides is described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#s4" target="_blank">Material and Methods</a>. Proteins identified in the series of gel slices marked by braces (}) are numbered here and in the splicing diagram below. These numbers also correspond to the protein list in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen-1003305-t001" target="_blank">Table 1</a>. Stars indicate host cell protein contamination. TGME49_119920 (“a”) was a single protein identified in the negative control pull-down. The highly abundant ROP1 protein (TGME49_109590, “b”) was also found in these experiments and is a common contaminant in <i>Toxoplasma</i> co-IP/proteomic analyses. Molecular mass standards are indicated between the gel lanes. (B) Schematic diagrams spliceosome assembly comprising the stepwise incorporation of U1 (green), U2 (blue), U4/U6 (yellow) and U5 (orange) snRNP complexes to intronic sequences of pre-mRNA. Core proteins in each snRNP complex are shown in the color boxes on the right of the scheme. Splicing factors co-precipitated with TgRRM1 belonged to U4/U6 or U5 snRNPs and are denoted by the black boxes in each column. Numbers on the right of the boxes correspond to the protein number in the gel slices in (A) and to the proteins listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen-1003305-t001" target="_blank">Table 1</a>. A complete list of identified proteins is included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305.s004" target="_blank">Dataset S4</a>.</p

Expression of TgRRM1 is cell cycle–dependent.

<p>(A) A genetically rescued mutant 12-109C6 clone expressing wt-TgRRM1^myc under control of the native TgRRM1 promoter was evaluated for cell cycle expression. Parasites were grown for 24 h at 34°C and then processed for IFA by co-staining with anti-myc (green = TgRRM1^myc protein), anti-IMC1 (red) and DAPI (blue) as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen-1003305-g001" target="_blank">Figure 1</a>. Four image panels (G1 to C phases) show the basic cell cycle profile of wt-TgRRM1^myc expression. Magnification bar (2 µm) is shown. Note that intravacuolar parasites were tightly synchronized allowing the cell cycle position of each vacuole (defined on the left) to be assigned based on known characteristics <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Gubbels1" target="_blank">[6]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Striepen1" target="_blank">[7]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Hu1" target="_blank">[49]</a>. Peak expression of wt-TgRRM1^myc was observed in the G1 panel, while the protein was nearly undetectable in parasites undergoing cytokinesis (M/C and C panels) demonstrating this factor is tightly cell cycle regulated. A fifth image panel (bottom) is included that pinpoints wt-TgRRM1^myc expression with respect to the G1 and S transition; co-staining in the panel is red = anti-myc, green = anti-centrin1, and blue = DAPI. The marker guide panel included here is an inverse image of the merged blue (DAPI) and green (centrin1) images to highlight the centrosome content marked by adjacent red dots. Note, parasites in these two separate vacuoles have single nuclei with no internal daughters, which places their cell cycle position on either side of the G1/S boundary based on single versus double centrosomes (G1 versus S phase, respectively). Strong wt-TgRRM1^myc expression was detected in the vacuole where parasites possessed a single centrosome (vacuole of 4 in G1), whereas wt-TgRRM1^myc was downregulated in S phase parasites associated with recently duplicated centrosomes (vacuole of 2). (B) Cyclical profile of TgRRM1 mRNA spanning nearly two tachyzoite division cycles also shows G1 phase peak expression. The graph is based on expression values obtained from our <i>Toxoplasma</i> cell cycle transcriptome microarray dataset <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Behnke1" target="_blank">[13]</a>. (C) Immunostaining of <i>Plasmodium falciparum</i> merozoites shows distinct cell cycle distribution of PfRRM1 in the nucleus of the ring stage parasites. While the protein is detected in discrete nuclear bodies in ring stages (see Inset), it appears diffused in the nuclei and cytoplasm of trophozoites and schizont stages and barely detectable by IFA. (R- ring; T- trophozoite; S- schizont; Hoechst- nucleic acid stain). (D) A time course immunoblot analysis of <i>P. falciparum</i> ring (R) (8–16 hours post-invasion), trophozoites (T) (24–32 hours post-invasion), and schizont (S) (36–44 hours post-invasion) stages shows constitutive overall expression of PfRRM1 throughout the intraerythrocytic cycle. Anti-Histone H3 antibody was used as a loading control. (E) The graph represents the percentile value of PF13_0318 mRNA measured in the synchronized population of <i>P. falciparum</i> 3D7 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003305#pgen.1003305-Bartfai1" target="_blank">[50]</a>.</p