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...

Summary Single‐stranded DNA binding proteins (SSBs) have been identified in all three domains of life. Here, we report the identification of a novel crenarchaeal SSB protein that is distinctly different from its euryar‐chaeal counterparts. Rather than comprising four DNA‐binding domains and a zinc‐finger motif within a single polypeptide of 645 amino acids, as for Methanococcus jannaschii, the Sulfolobus solfataricus SSB protein (SsoSSB) has a single DNA‐binding domain in a polypeptide of just 148 amino acids with a eubacterial‐like acidic C‐terminus. SsoSSB protein was purified to homogeneity and found to form tetramers in solution, suggesting a quaternary structure analogous to that of E. coli SSB protein, despite possessing DNA‐binding domains more similar to those of eukaryotic Replication Protein A (RPA). We demonstrate distributive binding of SsoSSB to ssDNA at high temperature with an apparent site size of approximately five nucleotides (nt) per monomer. Additionally, the protein is functional both in vitro and in vivo, stimulating RecA protein‐mediated DNA strand‐exchange and rescuing the ssb‐1 lethal mutation of E. coli respectively. We dis‐cuss possible evolutionary relationships amongst the various members of the SSB/RPA family.

DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of three Sulfolobus solfataricus strains to UV-C irradiation, which often results in double-strand break formation. We examined S. solfataricus strain P2 obtained from two different sources and S. solfataricus strain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for doublestrand break repair genes were also distinct for each strain, indicating that these Sulfolobus solfataricus strains have differential responses to UV-C-induced DNA double-strand break damage.Cells have evolved molecular mechanisms to meet the challenge of maintaining genomic integrity by rapidly responding to environmental stresses that can damage proteins and DNA. One of the most common forms of damage is caused by UV light (UV) exposure. High-energy short-wavelength UV-C light is absorbed directly by DNA and induces both cyclobutane pyrimidine dimers between adjacent thymidine or cytosine residues as well as pyrimidine-pyrimidone photoproducts between adjacent pyrimidine residues. Mechanisms for repair of these lesions appear to be present in all organisms and are thought to occur through either light-independent nucleotide excision repair (NER) or light-dependent photoreactivation using photolyases (for reviews, see references 33 and 34). UV-C irradiation also causes the production of reactive oxygen species, which can result in DNA double-strand breaks (DSBs) (6, 44). Our primary understanding for repair of DSBs has come from studies focused primarily on bacteria and eukaryotes. In Escherichia coli, these breaks are repaired through the action of the RecA protein, which assists in recombinational repair of single-strand regions produced through replication fork arrest at UV lesions and in DSB repair by extended synthesis-dependent strand annealing (SDSA) and homologous recombination (9, 19). Eukaryotes employ nonhomologous end joining as well as DSB repair by SDSA and homologous recombination mechanisms to repair these breaks (for recent reviews, see ref...

Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene (malA) encoding the major soluble α-glucosidase (maltase) and flanking sequences fromSulfolobus solfataricus were cloned and characterized. This is the first report of an α-glucosidase gene from the archaeal domain. malA is 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5′ side by an unusual 1-kb intergenic region. Northern blot analysis of themalA region identified transcripts for malA and an upstream open reading frame located 5′ to the 1-kb intergenic region. The malA transcription start site was located by primer extension analysis to a guanine residue 8 bp 5′ of themalA start codon. Gel mobility shift analysis of themalA promoter region suggests that sequences 3′ to position −33, including a consensus archaeal TATA box, play an essential role in malA expression. malA homologs were detected by Southern blot analysis in other S. solfataricus strains and in Sulfolobus shibatae, while no homologs were evident in Sulfolobus acidocaldarius, lending further support to the proposed revision of the genus Sulfolobus. Phylogenetic analyses indicate that the closest S. solfataricusα-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from Escherichia colirevealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for the S. solfataricus α-glucosidase in carbohydrate metabolism.

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