Project description:Thermococcus gammatolerans, the most radioresistant archaeon known to date, is an anaerobic and hyperthermophilic sulfur-reducing organism living in deep-sea hydrothermal vents. Knowledge of mechanisms underlying archaeal metal tolerance in such metal-rich ecosystem is still poorly documented. We showed that T. gammatolerans exhibits high resistance to cadmium (Cd), cobalt (Co) and zinc (Zn), a weaker tolerance to nickel (Ni), copper (Cu) and arsenate (AsO4) and that cells exposed to 1mM Cd exhibit a cellular Cd concentration of 67µM. A time-dependent transcriptomic analysis using microarrays was performed at a non-toxic (100μM) and a toxic (1mM) Cd dose. The reliability of microarray data was strengthened by real time RT-PCR validations. Altogether, 114 Cd responsive genes were revealed and a substantial subset of genes is related to metal homeostasis, drug detoxification, re-oxidization of cofactors and ATP production. This first genome-wide expression profiling study of archaeal cells challenged with Cd showed that T. gammatolerans withstands induced stress through pathways observed in both prokaryotes and eukaryotes but also through new and original strategies. T. gammatolerans cells challenged with 1mM Cd basically promote: 1) the induction of several transporter/permease encoding genes, probably to detoxify the cell; 2) the upregulation of Fe transporters encoding genes to likely compensate Cd damages in iron-containing proteins; 3) the induction of membrane-bound hydrogenase (Mbh) and membrane-bound hydrogenlyase (Mhy2) subunits encoding genes involved in recycling reduced cofactors and/or in proton translocation for energy production. By contrast to other organisms, redox homeostasis genes appear constitutively expressed and only a few genes encoding DNA repair proteins are regulated. We compared the expression of 27 Cd responsive genes in other stress conditions (Zn, Ni, heat shock, γ-rays), and showed that the Cd transcriptional pattern is comparable to other metal stress transcriptional responses (Cd, Zn, Ni) but not to a general stress response.

Project description:Thermococcus gammatolerans, the most radioresistant archaeon known to date, is an anaerobic and hyperthermophilic sulfur-reducing organism living in deep-sea hydrothermal vents. Knowledge of mechanisms underlying archaeal metal tolerance in such metal-rich ecosystem is still poorly documented. We showed that T. gammatolerans exhibits high resistance to cadmium (Cd), cobalt (Co) and zinc (Zn), a weaker tolerance to nickel (Ni), copper (Cu) and arsenate (AsO4) and that cells exposed to 1mM Cd exhibit a cellular Cd concentration of 66M-BM-5M. A time-dependent transcriptomic analysis using microarrays was performed at a non-toxic (100M-NM-<M) and a toxic (1mM) Cd dose. The reliability of microarray data was strengthened by real time RT-PCR validations. Altogether, 114 Cd responsive genes were revealed and a substantial subset of genes is related to metal homeostasis, drug detoxification, re-oxidization of cofactors and ATP production. This first genome-wide expression profiling study of archaeal cells challenged with Cd showed that T. gammatolerans withstands induced stress through pathways observed in both prokaryotes and eukaryotes but also through new and original strategies. T. gammatolerans cells challenged with 1mM Cd basically promote: 1) the induction of several transporter/permease encoding genes, probably to detoxify the cell; 2) the upregulation of Fe transporters encoding genes to likely compensate Cd damages in iron-containing proteins; 3) the induction of membrane-bound hydrogenase (Mbh) and membrane-bound hydrogenlyase (Mhy2) subunits encoding genes involved in recycling reduced cofactors and/or in proton translocation for energy production. By contrast to other organisms, redox homeostasis genes appear constitutively expressed and only a few genes encoding DNA repair proteins are regulated. We compared the expression of 27 Cd responsive genes in other stress conditions (Zn, Ni, heat shock, M-NM-3-rays), and showed that the Cd transcriptional pattern is comparable to other metal stress transcriptional responses (Cd, Zn, Ni) but not to a general stress response. Kinetics of gene expression changes induced by Cd were performed at two different concentrations (0.1mM and 1mM) and at 3 time points (30, 120 and 270 min). A 0.1mM Cd concentration did not affect the growth rate, whereas 1mM Cd induced a transitory growth arrest for 270min. Late exponentially growing cells (7x107 cells/mL) were exposed to Cd and were collected after 30min, 120 min and after 270 min. For each time point, four slides containing the 2157 oligonucleotide gene probes printed in duplicate were hybridized. The experiment was repeated twice leading to eight data sets per time point (four per biological replicate). Microarray analyses monitored 161 transcriptional changes (M-bM-^NM-^_Fold Change (FC)M-bM-^NM-^\M-bM-^IM-%2 and p-Value M-bM-^IM-$ 0.01) in response to Cd exposure corresponding to 114 unique genes i.e. 5,3% of T. gammatolerans gene content, most of them being upregulated. While about 25% of the upregulated genes exhibited a FC>3 with a maximum of almost 10 for one encoding a conserved hypothetical protein (tg0885, 1mM Cd at 120min), the large majority of the up- and down-regulated genes exhibited a 2 to 3-fold transcriptional change as already described in many archaeal transcriptomic studies.

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Thermococcus gammatolerans

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