In trace amounts, copper is essential for the function of key enzymes in prokaryotes and eukaryotes. Organisms have developed sophisticated mechanisms to control the cytosolic level of the metal, manage its toxicity and survive in copper-rich environments. Here we show that the Sulfolobus CopR represents a novel class of copper-responsive regulators, unique to the archaeal domain. Furthermore, by disruption of the ORF Sso2652 (copR) of the Sulfolobus solfataricus genome, we demonstrate that the gene encodes a transcriptional activator of the copper-transporting ATPase CopA gene and co-transcribed copT, encoding a putative copper-binding protein. Disruption resulted in a loss of copper tolerance in two copR-knockout mutants, while metals such as zinc, cadmium and chromium did not affect their growth. Copper sensitivity in the mutant was linked to insufficient levels of expression of CopA and CopT. The findings were further supported by time-course inductively coupled plasma optical emission spectrometry measurements, whereby continued accumulation of copper in the S. solfataricus mutant was observed. In contrast, copper accumulation in the wild-type stabilized after reaching approximately 6 pg (µg total protein)–1. Complementation of the disrupted mutant with a wild-type copy of the copR gene restored the wild-type phenotype with respect to the physiological and transcriptional response to copper. These observations, taken together, lead us to propose that CopR is an activator of copT and copA transcription, and the member of a novel class of copper-responsive regulators.

Copper is an essential micronutrient, but toxic in excess. Sulfolobus solfataricus cells have the ability to adapt to fluctuations of copper levels in their external environment. To better understand the molecular mechanism behind the organismal response to copper, the expression of the cluster of genes copRTA, which encodes the copper-responsive transcriptional regulator CopR, the copper-binding protein CopT, and CopA, has been investigated and the whole operon has been shown to be cotranscribed at low levels from the copR promoter under all conditions, whereas increased transcription from the copTA promoter occurs in the presence of excess copper. Furthermore, the expression of the copper-transporting ATPase CopA over a 27-hour interval has been monitored by quantitative real-time RT-PCR and compared to the pattern of cellular copper accumulation, as determined in a parallel analysis by Inductively Coupled Plasma Optical Emission spectrometry (ICP-OES). The results provide the basis for a model of the molecular mechanisms of copper homeostasis in Sulfolobus, which relies on copper efflux and sequestration.

Geobacillus kaustophilus strain A1 was previously isolated from a geothermal environment for its ability to grow in the presence of high arsenate levels. In this study, the molecular mechanisms of arsenate resistance of the strain were investigated. As(V) was reduced to As(III), as shown by HPLC analysis. Consistent with the observation that the micro-organism is not capable of anaerobic growth, no respiratory arsenate reductases were identified. Using specific PCR primers based on the genome sequence of G. kaustophilus HTA426, three unlinked genes encoding detoxifying arsenate reductases were detected in strain A1. These genes were designated arsC1, arsC2 and arsC3. While arsC3 is a monocistronic locus, sequencing of the regions flanking arsC1 and arsC2 revealed the presence of additional genes encoding a putative arsenite transporter and an ArsR-like regulator upstream of each arsenate reductase, indicating the presence of sequences with putative roles in As(V) reduction, As(III) export and arsenic-responsive regulation. RT-PCR demonstrated that both sets of genes were co-transcribed. Furthermore, arsC1 and arsC2, monitored by quantitative real-time RT-PCR, were upregulated in response to As(V), while arsC3 was constitutively expressed at a low level. A mechanism for regulation of As(V) detoxification by Geobacillus that is both consistent with our findings and relevant to the biogeochemical cycle of arsenic and its mobility in the environment is proposed. INTRODUCTIONMicro-organisms have an important impact on the biogeochemical transformations of arsenic, and their activities affect the mobility and toxicity of this element. Elevated amounts of arsenic can occur, especially in geothermal environments (Stauffer & Thompson, 1984). Thus, thermophilic micro-organisms such as Geobacillus species that thrive in geothermal soils and sediments are of particular interest for studying the mechanisms for the detoxification of arsenic compounds.Genes for arsenic detoxification were first discovered and characterized in Gram-negative bacteria (Chen et al., 1986;Rosen, 1999). These genes are often plasmid-encoded and are widespread in prokaryotes (Bruhn et al., 1996;Ji & Silver, 1992b;Oden et al., 1994;Rosenstein et al., 1992). In thoroughly studied systems, the ars operon has been reported to contain the five genes arsRDABC, as in Escherichia coli (Cai & DuBow, 1996;Rosen et al., 1992), or at least the three genes arsRBC, as in Staphylococcus aureus (Silver et al., 1993). ArsA and ArsB are components of an arsenite-transporting ATPase, where ArsA is the ATPase and ArsB is the transmembrane component of the complex. In micro-organisms in which ArsA is absent, ArsB acts as a single-component transporter. Both ArsA and ArsB may confer resistance to arsenite and antimonite (Rosen, 1999). In its dimeric form, ArsR is an arsenite-responsive repressor that binds to the ars promoter (Xu & Rosen, 1997). ArsC is the arsenate reductase that converts arsenate to arsenite, conferring resistance to arsenate (Martin et al., 2001 Here w...

Mesophilic glucuronidases are the most widely used reporters of gene expression in plants, but unsuitable as reporters in (hyper-)thermophiles due their insufficient thermal stability. Here we present the native 66.8 kDa thermostable β-glucuronidase of Sulfolobus solfataricus. The enzyme activity is characterized in a wide temperature range ideal for, but not limited to, in vivo genetic study of hyperthermophiles. As a proof of concept, we demonstrate its use as a reporter of gene expression in Sulfolobus, by monitoring a promoter fusion created with the β-glucuronidase coding gene gusB and a copper-responsive promoter.