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Mercury resistance mediated by mercuric reductase (MerA) is widespread among bacteria and operates under the control of MerR. MerR represents a unique class of transcription factors that exert both positive and negative regulation on gene expression. Archaea and bacteria are prokaryotes, yet little is known about the biological role of mercury in archaea or whether a resistance mechanism occurs in these organisms. The archaeon Sulfolobus solfataricus was sensitive to mercuric chloride, and low-level adaptive resistance could be induced by metal preconditioning. Protein phylogenetic analysis of open reading frames SSO2689 and SSO2688 clarified their identity as orthologs of MerA and MerR. Northern analysis established that merA transcription responded to mercury challenge, since mRNA levels were transiently induced and, when normalized to 7S RNA, approximated values for other highly expressed transcripts. Primer extension analysis of merA mRNA predicted a noncanonical TATA box with nonstandard transcription start site spacing. The functional roles of merA and merR were clarified further by gene disruption. The merA mutant exhibited mercury sensitivity relative to wild type and was defective in elemental mercury volatilization, while the merR mutant was mercury resistant. Northern analysis of the merR mutant revealed merA transcription was constitutive and that transcript abundance was at maximum levels. These findings constitute the first report of an archaeal heavy metal resistance system; however, unlike bacteria the level of resistance is much lower. The archaeal system employs a divergent MerR protein that acts only as a negative transcriptional regulator of merA expression.The element mercury is a toxic heavy metal that occurs naturally in several forms, including elemental (Hg 0 ), ionized (inorganic salts Hg 2ϩ and Hg ϩ ), organic (typically alkylated), or sulfidic (cinnabar). Mercury use is widespread, particularly in the production of gold, vaccines, antimicrobials, amalgams, and electronics. Mercuric chloride (HgCl 2 ) is most often used in experimental studies because it is soluble and poisonous. Mercury is a redox-active transition metal in both biotic and abiotic environments. In vivo, mercury plays a critical role in modulating cellular redox status by depleting antioxidant pools (16). Both ionic and organic mercury form covalent bonds with sulfur atoms in cysteine residues of target proteins.Bacteria respond to mercury exposure using several strategies. While mechanisms involving tolerance occur (33, 34, 58), enzymatic reduction of mercuric ion to elemental mercurycatalyzed by-products of the mer operon is the only resistance mechanism that has been described (reviewed in references 4, 30, 31, and 50). The mer operon (merTPCAD) encodes a group of proteins involved in the detection, transport, and reduction of mercury. The NADPH-dependent enzyme, mercuric reductase (MerA), transfers two electrons to mercuric ion, Hg 2ϩ , reducing it to elemental mercury Hg 0 . Elemental mercury is volatile and is re...

A role for DnaK, the major E. coli Hsp70, in chaperoning de novo protein folding has remained elusive. Here we show that under nonstress conditions DnaK transiently associates with a wide variety of nascent and newly synthesized polypeptides, with a preference for chains larger than 30 kDa. Deletion of the nonessential gene encoding trigger factor, a ribosome-associated chaperone, results in a doubling of the fraction of nascent polypeptides interacting with DnaK. Combined deletion of the trigger factor and DnaK genes is lethal under normal growth conditions. These findings indicate important, partially overlapping functions of DnaK and trigger factor in de novo protein folding and explain why the loss of either chaperone can be tolerated by E. coli.

An ␣-amylase was purified from culture supernatants of Sulfolobus solfataricus 98/2 during growth on starch as the sole carbon and energy source. The enzyme is a homodimer with a subunit mass of 120 kDa. It catalyzes the hydrolysis of starch, dextrin, and ␣-cyclodextrin with similar efficiencies. Addition of exogenous glucose represses production of ␣-amylase, demonstrating that a classical glucose effect is operative in this organism. Synthesis of [35 S]-␣-amylase protein is also subject to the glucose effect. ␣-Amylase is constitutively produced at low levels but can be induced further by starch addition. The absolute levels of ␣-amylase detected in culture supernatants varied greatly with the type of sole carbon source used to support growth. Aspartate was identified as the most repressing sole carbon source for ␣-amylase production, while glutamate was the most derepressing. The pattern of regulation of ␣-amylase production seen in this organism indicates that a catabolite repression-like system is present in a member of the archaea.Catabolite repression is a paradigm for studies concerned with global and specific gene control mechanisms (22). In prokaryotes, catabolite repression together with transient repression and inducer exclusion make up what has been termed the glucose effect or repression of catabolic enzyme synthesis by glucose (23). However, for eukaryotes, the term catabolite repression is more generally used as a pseudonym for the glucose effect. In fact, evidence for transient repression, inducer exclusion, and requisite aspects of catabolite repression, including the ability to grow most rapidly on preferred carbon sources, is not well demonstrated (for reviews, see references 29 and 31). Catabolite repression in prokaryotes and eukaryotes has received wide attention, but the existence of an analogous process in the archaea has not been addressed. One hallmark of this process in gram-negative bacteria consists of the global mode of gene regulation of catabolite-repressible genes mediated by the small molecule cyclic AMP (cAMP) (8). Although the role of cAMP in some prokaryotes is well accepted, it has been eliminated as an effector in the corresponding catabolite response in the gram-positive bacterium Bacillus subtilis (for a review, see reference 14). In eukaryotes, including the budding yeast Saccharomyces cerevisiae, cAMP plays a crucial but indirect role in mediating the glucose effect. Interestingly, cAMP has been reported in a range of archaea (21).Sulfolobus solfataricus is an extremely thermophilic organism which inhabits acidic hot springs. S. solfataricus is a member of the archaea and has been assigned to a subdivision termed the crenarchaeota by rRNA gene (rDNA) sequence analysis (32). It is capable of diverse modes of metabolism at temperatures ranging between 70 and 90ЊC. It can grow either lithoautotrophically, oxidizing sulfur (4, 15), or chemoheterotrophically on starch or other sugars as sole carbon and energy sources (7,11). Recent studies also suggest that hot springs cont...

Despite their taxonomic description, not all members of the order Sulfolobales are capable of oxidizing reduced sulfur species, which, in addition to iron oxidation, is a desirable trait of biomining microorganisms. However, the complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ϳ2,300 open reading frames [ORFs]) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins involved (directly or indirectly) with bioleaching. As expected, the M. sedula genome contains genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation, and a putative tetrathionate hydrolase, implicated in sulfur oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, was also identified. These iron-and sulfur-oxidizing components are missing from genomes of nonleaching members of the Sulfolobales, such as Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole-genome transcriptional response analysis showed that 88 ORFs were up-regulated twofold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included genes for components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized.Biomining exploits acidophilic microorganisms to recover valuable metals (i.e., Cu and Au) from ores ( 52, 56, 57, 59) in biohydrometallurgical processes conducted at temperatures ranging from ambient ( 44, 71 ) to 80°C (17). Higher-temperature operations involve consortia of extremely thermoacidophilic archaea from the genera Sulfolobus, Acidianus, and Metallosphaera (49,50). For example, Sulfolobus metallicus, certain Acidianus species ( 12, 47 ), and Metallosphaera sedula (29, 33) all can mobilize metals from metal sulfides. However, not all members of these genera are metal bioleachers. In fact, the three Sulfolobus species with completed genome sequences (S. solfataricus, S. acidocaldarius, and S. tokodaii) are apparently unable to effect metal sulfide oxidation. Since genome sequences are not yet available for metal-bioleaching extreme thermoacidophiles, genetic features characteristic of this physiological capability remain to be seen.