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Major Soluble Proteome Changes in Deinococcus Deserti Over The Major soluble proteome changes in Deinococcus deserti over the earliest stages following gamma-ray irradiation Alain Dedieu, Elodie Sahinovic, Philippe Guerin, Laurence Blanchard, Sylvain Fochesato, Bruno Meunier, Arjan de Groot, J. Armengaud To cite this version: Alain Dedieu, Elodie Sahinovic, Philippe Guerin, Laurence Blanchard, Sylvain Fochesato, et al.. Major soluble proteome changes in Deinococcus deserti over the earliest stages following gamma-ray irradia- tion. Proteome Science, BioMed Central, 2013, 11 (1), pp.3. 10.1186/1477-5956-11-3. hal-02011060 HAL Id: hal-02011060 https://hal.archives-ouvertes.fr/hal-02011060 Submitted on 29 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Dedieu et al. Proteome Science 2013, 11:3 http://www.proteomesci.com/content/11/1/3 RESEARCH Open Access Major soluble proteome changes in Deinococcus deserti over the earliest stages following gamma-ray irradiation Alain Dedieu1*, Elodie Sahinovic1, Philippe Guérin1, Laurence Blanchard2,3,4, Sylvain Fochesato2,3,4, Bruno Meunier5, Arjan de Groot2,3,4 and Jean Armengaud1 Abstract Background: 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. Results: 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. Conclusions: 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. Keyword: Proteome, Post-translational modification, Irradiation, Early response, Reference 2D map, Kinetics, Hierarchical clustering Introduction damaging conditions, D. deserti and other Deinococcaceae Deinococcus deserti belongs to the Deinococcaceae,a [2,3] exhibit an extraordinary ability to withstand ioni group of extremely radiotolerant bacteria. D. deserti zing radiation. Chromosomes with numerous radiation- VCD115 has been isolated from upper sand layers of the or desiccation-induced double-strand breaks can be Sahara desert [1]. In this environment, cells are exposed repaired in a few hours in D. deserti [4]. To learn about to very changing life conditions with cycles of high tem- the specificities of this bacterium compared with other peratures, high UV irradiation, desiccating conditions, Deinococcaceae, its entire genome sequence has been low temperatures and rehydrating conditions. Probably determined [4]. It consists of a 2.8-Mb chromosome and as a consequence of adaptation to such harsh, DNA- three large plasmids called P1 (324 kb), P2 (314 kb), and P3 (396 kb). The comparative analysis showed some inter- esting differences between D. deserti and other sequenced * Correspondence: [email protected] Deinococcus species. For example, D. deserti possesses 1Laboratoire de Biochimie des Systèmes Perturbés, CEA Marcoule, DSV, iBEB, SBTN, LBSP, BAGNOLS-SUR-CEZE F-30207, France supplementary genes involved in DNA repair, such as Full list of author information is available at the end of the article © 2013 Dedieu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dedieu et al. Proteome Science 2013, 11:3 Page 2 of 14 http://www.proteomesci.com/content/11/1/3 three recA that code for two different RecA proteins, up-regulated after exposure to low dose of γ radiation whereas it lacks homologs of several radiation-induced in a PprI-dependent manner in D. radiodurans [29]. genes in Deinococcus radiodurans (e.g., ddrP encoding a PprI, also called IrrE, is a Deinococcus-specific tran- putative DNA ligase). scriptional factor essential for radiotolerance [30-32]. The extreme radiotolerance of Deinococcaceae was the Moreover, phosphorylation of two proteins (DR_A0283 object of intense investigations during the last years which encodes a serine protease and DR_1343, a using D. radiodurans as model. In cells subjected to ir- glyceraldehyde-3-phosphate dehydrogenase) was shown to radiation, DNA recombinase RecA was the first protein be dependent upon the presence of PprI. More recently, a that was found strongly induced [5]. RecA is essential 2D-proteome reference map has been reported for for radiotolerance [6] and for the fidelity of DNA repair Deinococcus geothermalis grown in standard conditions and genome stability in D. radiodurans [7]. The molecu- [33], as well as a shotgun analysis of its major membrane lar mechanisms underlying DNA repair were also exam- components [34]. ined by transcriptomics [8-10] leading to the description Comparisons of the genome sequences of various of a repertoire of genes responding to acute gamma ir- Deinococcaceae shed new light on their potential arsenal radiation, including genes involved in DNA replication, related to DNA repair and radiotolerance [4,35,36]. repair and recombination, cell wall metabolism, cellular Convinced that comparison of the proteome dynamics transport and many with uncharacterized functions. An- of several species will also be fruitful, we performed a other transcriptional study has shown that 72 genes were time course analysis of the proteome of D. deserti after up-regulated three fold or higher in D. radiodurans fol- exposure to 3 kGy γ-radiations. The proteome from lowing gamma irradiation [11]. In this study, besides cells harvested at different time lapses after irradiation genes with already known assigned function linked to were analyzed with a 2-D gel approach. A hierarchical DNA repair (gyrA, gyrB, uvrA, uvrB, ruvB and recA), five clustering analysis pointed at accumulated proteins at novel genes with unknown or hypothetical assigned the earliest stages after irradiation. Among these are functions were highlighted: ddrA, ddrB, ddrC, ddrD and several important DNA repair proteins. Interestingly, pprA (ddr, DNA damage response; ppr, pleiotropic pro- post-translational modifications of GyrA were detected. tein promoting DNA repair). Genetic analyses demon- We also observed the up-regulation of novel proteins strated a role of these five genes in radiotolerance [11], such as the conserved Deide_20140 acetyltransferase, and further studies have reported that DdrA, DdrB and Deide_19260 response regulator, Deide_21840 PilT pro- PprA are involved in DNA repair [12-19]. Radiotoler- tein, as well as specific D. deserti proteins such as the ance of D. radiodurans probably results from a com- Deide_02842 restriction enzyme. The physiological sig- bination of different highly coordinated physiological nificance of these novel results is discussed. pathways [20]. Besides enzymatic pathways such as DNA repair, passive contributions to radiotolerance have been Results described such as the nucleoid structure where DNA Time course analysis of D. deserti cells after drastic molecules are highly condensed, which would limit dif- irradiation fusion of DNA fragments [21], and the high intracellular We established a reference proteome map for D. deserti Mn2+/Fe2+ ratio that limits protein oxidation [20,22-24]. cells harvested at the exponential phase (Additional file 1: Today, the survival kit components of Deinococcaceae Figure S1 & Additional file 2: Table S1). We observed are far to be exhaustive and some possible factors re- that several stress response and DNA repair proteins are main controversial [25]. already found in standard condition, for example two To further analyze these mechanisms of radioresis- desiccation tolerance-associated proteins (Deide_07540, tance, proteomic approaches are promising as both mass Deide_08510) and RecAC (Deide_19450). We investigated
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