Response to excess copper in the hyperthermophile Sulfolobus solfataricus strain 98/2
Introduction
Copper is a transition metal and an important trace element because of the essential role it plays in a range of biological processes. In contrast, the occurrence of copper levels beyond the physiological range causes serious damage to all molecular components. Studies on yeast have led to the proposition that virtually no free copper ions are present in the cell under normal conditions [1]. The response of cells to copper excess/deficiency is accomplished through the interplay of copper-binding proteins, copper-responsive regulators, transporters for the efflux and uptake of copper, and copper-requiring enzymes. Genetic determinants of copper homeostasis have been described for several bacterial species [2], [3], [4], [5], [6], [7]. In particular, the various components of the Enterococcus irae and Escherichia coli copper-homeostasis systems, their regulation, and interactions have been thoroughly studied [2], [8]. Many sequenced archaeal genomes encode homologs of Cu (I) and Cu (II) transporting ATPases [9]. However, investigations of the response in archaea to changes of copper levels are still limited. Structural studies of individual functional domains of the Cu (I)-transporting ATPase CopA in Archeoglobus fulgidus have provided useful insights into its activities and functions [10], [11], [12], [13], [14]. A. fulgidus also possesses a Cu(II)-transporting ATPase, CopB, that has been biochemically characterized [15]. The transcriptional analysis of a cop locus responsible for survival in the presence of copper has been reported in the extreme acidophilic archaeon “Ferroplasma acidarmanus” strain Fer1, where cotranscription of genes encoding the copper-binding protein CopZ and the putative copper-transporting ATPase CopB was shown to increase in response to Cu (II) [16]. An interesting mechanism for copper detoxification has been described in Sulfolobus metallicus, which is based on sequestration by organic phosphate, possibly followed by active efflux of the metal-phosphate complex [17]. The Sulfolobus solfataricus genome encodes a cop locus, which includes the three open reading frames (ORFs) Sso2651, Sso2651, and Sso10823, encoding the CopA ATPase, a copper-responsive regulator, and a putative copper-binding protein, respectively [18]. Cotranscription of Sso2651 and Sso10823 has been reported to specifically increase in the presence of copper, while the copper-responsive regulator binds sequences surrounding the putative copA promoter in S. solfataricus strain P2 [19]. In this study, the response of S. solfataricus to copper has been further investigated in the strain 98/2. The selection of the genetically tractable strain 98/2 [20] will expand the scope of analyses aimed to the elucidation of archaeal interactions with copper. To gain better insights into the Sulfolobus response to copper levels, the transcription of the three genes of the copRTA operon has been examined under different conditions and in a time course experiment, and the changes in the amount of copper associated with the cells have been monitored over time. Based on the data obtained, a preliminary model for the maintenance of copper homeostasis in Sulfolbus is proposed.
Section snippets
Materials and methods
Growth conditions.Sulfolobus solfataricus strains 98/2 or P2 (DSM 1617) were cultured at 80 °C in a defined standard medium (SM) as described in [20], the medium was supplemented with 0.2% sucrose as the carbon and energy source. Batch cultures were inoculated to obtain a density corresponding to an OD540 of about 0.025, with aliquots withdrawn from mid-log phase cultures. Growth was monitored at a wavelength of 540 nm on a Beckman DU-520 spectrophotometer (Beckman Coulter, USA). All the
Physiological response to copper
To establish the optimal concentration of copper to be used in this study, cells were exposed to CuCl2 at concentrations ranging from 0 mM to 2.5 mM. The MIC, defined as the lowest concentration that completely inhibits cell growth immediately after exposure, was determined to be 1.5 mM (Fig. 1A). In response to copper concentrations that were equal or greater than 1.5-mM CuCl2, a lag phase of variable duration was observed. The duration of the lag phase was directly proportional to the metal
Conclusions
When S. solfataricus cells are challenged with copper, their rate of growth slows down, and this decrease is proportional to the concentration of copper tested. Although immediately after copper challenge a MIC of 1.5 mM copper has been determined, cells become resistant to environs containing up to 2.5 mM of CuCl2 after prolonged exposure. An increased level of the transcripts of genes directly involved in copper detoxification, namely copA and copT, which are co-transcribed, is observed after
Acknowledgments
This research was supported by Hatch Project NJ01136 and a SEBS Pre-Tenure Award to EB, a GAANN fellowship to AV, and NIH Bridge Award GM58389 to MC.
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Present address: Department of Molecular Biology and Biochemistry, Rutgers University, USA.
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