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author:("dediu, Alain")
1.  Major soluble proteome changes in Deinococcus deserti over the earliest stages following gamma-ray irradiation 
Proteome Science  2013;11:3.
Deinococcus deserti VCD115 has been isolated from Sahara surface sand. This radiotolerant bacterium represents an experimental model of choice to understand adaptation to harsh conditions encountered in hot arid deserts. We analysed the soluble proteome dynamics in this environmentally relevant model after exposure to 3 kGy gamma radiation, a non-lethal dose that generates massive DNA damages. For this, cells were harvested at different time lapses after irradiation and their soluble proteome contents have been analysed by 2-DE and mass spectrometry.
In the first stage of the time course we observed accumulation of DNA damage response protein DdrB (that shows the highest fold change ~11), SSB, and two different RecA proteins (RecAP and RecAC). Induction of DNA repair protein PprA, DNA damage response protein DdrD and the two gyrase subunits (GyrA and GyrB) was also detected. A response regulator of the SarP family, a type II site-specific deoxyribonuclease and a putative N-acetyltransferase are three new proteins found to be induced. In a more delayed stage, we observed accumulation of several proteins related to central metabolism and protein turn-over, as well as helicase UvrD and novel forms of both gyrase subunits differing in terms of isoelectric point and molecular weight.
Post-translational modifications of GyrA (N-terminal methionine removal and acetylation) have been evidenced and their significance discussed. We found that the Deide_02842 restriction enzyme, which is specifically found in D. deserti, is a new potential member of the radiation/desiccation response regulon, highlighting the specificities of D. deserti compared to the D. radiodurans model.
PMCID: PMC3564903  PMID: 23320389
Proteome; Post-translational modification; Irradiation; Early response; Reference 2D map; Kinetics; Hierarchical clustering
2.  Proteomics-based Refinement of Deinococcus deserti Genome Annotation Reveals an Unwonted Use of Non-canonical Translation Initiation Codons* 
Deinococcaceae are a family of extremely radiation-tolerant bacteria that are currently subjected to numerous studies aimed at understanding the molecular mechanisms for such radiotolerance. To achieve a comprehensive and accurate annotation of the Deinococcus deserti genome, we performed an N terminus-oriented characterization of its proteome. For this, we used a labeling reagent, N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide, to selectively derivatize protein N termini. The large scale identification of N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide-modified N-terminal-most peptides by shotgun liquid chromatography-tandem mass spectrometry analysis led to the validation of 278 and the correction of 73 translation initiation codons in the D. deserti genome. In addition, four new genes were detected, three located on the main chromosome and one on plasmid P3. We also analyzed signal peptide cleavages on a genome-wide scale. Based on comparative proteogenomics analysis, we propose a set of 137 corrections to improve Deinococcus radiodurans and Deinococcus geothermalis gene annotations. Some of these corrections affect important genes involved in DNA repair mechanisms such as polA, ligA, and ddrB. Surprisingly, experimental evidences were obtained indicating that DnaA (the protein involved in the DNA replication initiation process) and RpsL (the S12 ribosomal conserved protein) translation is initiated in Deinococcaceae from non-canonical codons (ATC and CTG, respectively). Such use may be the basis of specific regulation mechanisms affecting replication and translation. We also report the use of non-conventional translation initiation codons for two other genes: Deide_03051 and infC. Whether such use of non-canonical translation initiation codons is much more frequent than for other previously reported bacterial phyla or restricted to Deinococcaceae remains to be investigated. Our results demonstrate that predicting translation initiation codons is still difficult for some bacteria and that proteomics-based refinement of genome annotations may be helpful in such cases.
PMCID: PMC2830850  PMID: 19875382
3.  Alliance of Proteomics and Genomics to Unravel the Specificities of Sahara Bacterium Deinococcus deserti 
PLoS Genetics  2009;5(3):e1000434.
To better understand adaptation to harsh conditions encountered in hot arid deserts, we report the first complete genome sequence and proteome analysis of a bacterium, Deinococcus deserti VCD115, isolated from Sahara surface sand. Its genome consists of a 2.8-Mb chromosome and three large plasmids of 324 kb, 314 kb, and 396 kb. Accurate primary genome annotation of its 3,455 genes was guided by extensive proteome shotgun analysis. From the large corpus of MS/MS spectra recorded, 1,348 proteins were uncovered and semiquantified by spectral counting. Among the highly detected proteins are several orphans and Deinococcus-specific proteins of unknown function. The alliance of proteomics and genomics high-throughput techniques allowed identification of 15 unpredicted genes and, surprisingly, reversal of incorrectly predicted orientation of 11 genes. Reversal of orientation of two Deinococcus-specific radiation-induced genes, ddrC and ddrH, and identification in D. deserti of supplementary genes involved in manganese import extend our knowledge of the radiotolerance toolbox of Deinococcaceae. Additional genes involved in nutrient import and in DNA repair (i.e., two extra recA, three translesion DNA polymerases, a photolyase) were also identified and found to be expressed under standard growth conditions, and, for these DNA repair genes, after exposure of the cells to UV. The supplementary nutrient import and DNA repair genes are likely important for survival and adaptation of D. deserti to its nutrient-poor, dry, and UV-exposed extreme environment.
Author Summary
D. deserti belongs to the Deinococcaceae, a family of bacteria characterized by an exceptional ability to withstand the lethal effects of DNA-damaging agents, including ionizing radiation, UV light, and desiccation. It was isolated from Sahara surface sands, an extreme and nutrient-poor environment, regularly exposed to intense UV radiation, cycles of extreme temperatures, and desiccation. The evolution of organisms that are able to survive acute irradiation doses of 15,000 Gy is difficult to explain given the apparent absence of highly radioactive habitats on Earth over geologic time. Thus, it seems more likely that the natural selection pressure for the evolution of radiation-resistant bacteria was chronic exposure to nonradioactive forms of DNA damage, in particular those promoted by desiccation. Here, we report the first complete genome sequence of a bacterium, D. deserti VCD115, isolated from hot, arid desert surface sand. Accurate genome annotation of its 3,455 genes was guided by extensive proteome analysis in which 1,348 proteins were uncovered after growth in standard conditions. Supplementary genes involved in manganese import, in nutrient import, and in DNA repair were identified and are likely important for survival and adaptation of D. deserti to its hostile environment.
PMCID: PMC2669436  PMID: 19370165
4.  THUMP from archaeal tRNA:m22G10 methyltransferase, a genuine autonomously folding domain 
Nucleic Acids Research  2006;34(9):2483-2494.
The tRNA:m22G10 methyltransferase of Pyrococus abyssi (PAB1283, a member of COG1041) catalyzes the N2,N2-dimethylation of guanosine at position 10 in tRNA. Boundaries of its THUMP (THioUridine synthases, RNA Methyltransferases and Pseudo-uridine synthases)—containing N-terminal domain [1–152] and C-terminal catalytic domain [157–329] were assessed by trypsin limited proteolysis. An inter-domain flexible region of at least six residues was revealed. The N-terminal domain was then produced as a standalone protein (THUMPα) and further characterized. This autonomously folded unit exhibits very low affinity for tRNA. Using protein fold-recognition (FR) methods, we identified the similarity between THUMPα and a putative RNA-recognition module observed in the crystal structure of another THUMP-containing protein (ThiI thiolase of Bacillus anthracis). A comparative model of THUMPα structure was generated, which fulfills experimentally defined restraints, i.e. chemical modification of surface exposed residues assessed by mass spectrometry, and identification of an intramolecular disulfide bridge. A model of the whole PAB1283 enzyme docked onto its tRNAAsp substrate suggests that the THUMP module specifically takes support on the co-axially stacked helices of T-arm and acceptor stem of tRNA and, together with the catalytic domain, screw-clamp structured tRNA. We propose that this mode of interactions may be common to other THUMP-containing enzymes that specifically modify nucleotides in the 3D-core of tRNA.
PMCID: PMC1459410  PMID: 16687654
5.  Deciphering structure and topology of conserved COG2042 orphan proteins 
The cluster of orthologous group COG2042 has members in all sequenced Eukaryota as well as in many Archaea. The cellular function of these proteins of ancient origin remains unknown. PSI-BLAST analysis does not indicate a possible link with even remotely-related proteins that have been functionally or structurally characterized. As a prototype among COG2042 orthologs, SSO0551 protein from the hyperthermophilic archaeon Sulfolobus solfataricus was purified to homogeneity for biophysical characterization.
The untagged protein is thermostable and behaves as a monomeric protein in gel filtration experiment. Several mass spectrometry-based strategies were combined to obtain a set of low resolution structural information. Kinetic data from limited proteolysis with various endoproteases are concordant in pointing out that region Glu73-Arg78 is hyper-sensitive, and thus accessible and flexible. Lysine labeling with NHS-biotin and cross-linking with DTSSP revealed that the 35 amino acid RLI motif at the N terminus is solvent exposed. Cross-links between Lys10-Lys14 and Lys23-Lys25 indicate that these residues are spatially close and in adequate conformation to be cross-linked. These experimental data have been used to rank multiple three-dimensional models generated by a de novo procedure.
Our data indicate that COG2042 proteins may share a novel fold. Combining biophysical, mass-spectrometry data and molecular model is a useful strategy to obtain structural information and to help in prioritizing targets in structural genomics programs.
PMCID: PMC549553  PMID: 15701177

Results 1-5 (5)