Sulfolobus metallicus is a thermoacidophilic crenarchaeon used in high-temperature bioleaching processes that is able to grow under stressing conditions such as high concentrations of heavy metals. Nevertheless, the genetic and biochemical mechanisms responsible for heavy metal resistance in S. metallicus remain uncharacterized. Proteomic analysis of S. metallicus cells exposed to 100？mM Cu revealed that 18 out of 30 upregulated proteins are related to the production and conversion of energy, amino acids biosynthesis, and stress responses. Ten of these last proteins were also up-regulated in S. metallicus treated in the presence of 1？mM Cd suggesting that at least in part, a common general response to these two heavy metals. The S. metallicus genome contained two complete cop gene clusters, each encoding a metallochaperone (CopM), a Cu-exporting ATPase (CopA), and a transcriptional regulator (CopT). Transcriptional expression analysis revealed that copM and copA from each cop gene cluster were cotranscribed and their transcript levels increased when S. metallicus was grown either in the presence of Cu or using chalcopyrite (CuFeS2) as oxidizable substrate. This study shows for the first time the presence of a duplicated version of the cop gene cluster in Archaea and characterizes some of the Cu and Cd resistance determinants in a thermophilic archaeon employed for industrial biomining. 1. Introduction Bioleaching is the biological conversion of an insoluble metal compound into a water soluble form [1, 2]. Microbe-based processes have clear economic advantages in the extraction of metals from many low-grade deposits [3], and these metal-extraction processes are usually more environmentally friendly than physical-chemical processes [3–5]. Some ores are refractory to mesophilic leaching and temperatures preferably as high as 75–85°C are required [6, 7]. At high temperatures, biomining consortia are dominated by thermoacidophilic Archaea from the genus Sulfolobus, Acidianus, and Metallosphaera [8]. Metals play an integral role in the life process of microorganisms, but at high levels both essential and nonessential metals can damage cell membranes, alter enzyme specificity, disrupt cellular functions, and damage the structure of DNA [9, 10]. Acid-leaching solutions are characterized by high metal concentrations that are toxic to most life, and as might be expected, microorganisms that grow in mineral-rich environments are, in most cases, remarkably tolerant to a wide range of metal ions [3, 11] and should possess robust metal resistance mechanisms [11–15].
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