Archaea represent a considerable fraction of the prokaryotic world in marine and terrestrial ecosystems, indicating that organisms from this domain might have a large impact on global energy cycles. However, many novel archaeal lineages that have been detected by molecular phylogenetic approaches have remained elusive because no laboratory-cultivated strains are available. Environmental genomic analyses have recently provided clues about the potential metabolic strategies of several of the uncultivated and abundant archaeal species, including non-thermophilic terrestrial and marine crenarchaeota and methanotrophic euryarchaeota. These initial studies of natural archaeal populations also revealed an unexpected degree of genomic variation that indicates considerable heterogeneity among archaeal strains. Here, we review genomic studies of uncultivated archaea within a framework of the phylogenetic diversity and ecological distribution of this domain.

SummarySulfolobus solfataricus has developed into an important model organism for molecular and biochemical studies of hyperthermophilic archaea. Although a number of in vitro systems have been established for the organism, efficient tools for genetic manipulations have not yet been available for any hyperthermophile. In this work, we have developed a stable and selectable shuttle vector based on the virus SSV1 of Sulfolobus shibatae . We have introduced pUC18 for propagation in Escherichia coli and the genes pyrEF coding for orotidine-5 ¢ ¢ ¢ ¢ -monophosphate pyrophosphorylase and orotidine-5 ¢ ¢ ¢ ¢ -monophosphate decarboxylase of Sulfolobus solfataricus as selectable marker to complement pyrimidine auxotrophic mutants. Furthermore, the beta-galactosidase gene ( lacS ) was introduced into this vector as a reporter under the control of the strong and heat-inducible promoter of the Sulfolobus chaperonin (thermosome). After transformation of a S. solfataricus pyrEF/lacS double mutant, the vector was found to reside as a single-copy vector, stably integrated into the host chromosome via the site-specific recombination system of SSV1. Specific beta-galactosidase activities in transformants were found to be fourfold higher than in wild-type S. solfataricus cells, and increased to more than 10-fold after heat shock. Greatly increased levels of lacS mRNA were detected in Northern analyses, demonstrating that this reporter gene system is suitable for the study of regulated promoters in Sulfolobus and that the vector can also be used for the high-level expression of genes from hyperthermophilic archaea.

The homologous and heterologous expression of genes is a prerequisite for most biochemical studies of protein function. A vast variety of systems have been developed for protein production in members of the Bacteria and Eukarya, using numerous combinations of vector and promoter systems. Members of the Archaea, the third domain of life, are much less amenable to genetic manipulation. Transformation tools for the production of recombinant proteins exist for only a few species (for a review, see reference 2). A shuttle vector has been described for the expression of bacterial and methanococcal genes in Methanococcus maripaludis (12). Heterologous expression of bacterial and eukaryotic genes as well as of homologous genes has been achieved in the genetically most accessible archaea, the mesophilic, salt-dependent Halobacterium spp. (14,17,33). However, no such system has existed so far for thermophilic or hyperthermophilic archaea. Mesophilic hosts, in particular Escherichia coli, have been used to produce thermostable proteins for biochemical characterization and crystallographic studies (e.g., see references 22, 23, 29, and 34). However, a considerable number of proteins of hyperthermophiles fold into their native state only under natural conditions of high temperature or in the presence of their native cofactors. Furthermore, the production of recombinant and tagged proteins in native thermophilic hosts allows the identification of associated factors or even larger protein complexes.The crenarchaeote Sulfolobus solfataricus has developed into an important model organism for molecular and biochemical studies of hyperthermophilic archaea. It grows optimally at 80°C and pH 3 under aerobic and heterotrophic conditions. Since many studies on the transcription, translation, and replication of this extremophile have been performed in vitro (4,5,10,40), it is highly desirable to develop genetic tools for in vivo studies and for high-level production of proteins in this organism. Initial transformation systems and selectable markers have been established for Sulfolobus solfataricus in some laboratories (3,6,7,9,16,41). We have recently developed a reporter gene system based on the virus SSV1 as well as the selectable marker genes pyrEF for the complementation of uracil auxotrophic mutants. The latter genes allow stabilization of the propagation of the vector by growing transformants under selective conditions (16). Moreover, strong, heat-inducible expression of the reporter gene lacS, coding for ␤-galactosidase, was demonstrated when it was placed under the control of the promoter of the major heat shock chaperonin gene tf55␣ (16).In this study, we have used and improved this vector system for the heterologous and homologous expression of genes in S. solfataricus. We have constructed a set of entry vectors and introduced a transcriptional terminator and a second inducible promoter. Our system allows for the high-level production of functional and tagged cytoplasmic and membrane-associated

Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Lubelska, J. M., Jonuscheit, M., Schleper, C., Albers, S-V., & Driessen, A. J. M. (2006). Regulation of expression of the arabinose and glucose transporter genes in the thermophilic archaeon Sulfolobus solfataricus. Extremophiles, 10(5), 383-391. DOI: 10.1007/s00792-006-0510-7 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Abstract Sugar uptake in Sulfolobus solfataricus, a thermoacidophilic archaeon, occurs through high-affinity binding of protein-dependent ABC transporters. We have investigated the expression patterns of two sugar transport operons, that is, the glucose and arabinose transporters. Analysis of the araS promoter activity, and the mRNA and protein levels in S. solfataricus cells grown on different carbon sources showed that expression of the arabinose transporter gene cluster is highly regulated and dependent on the presence of arabinose in the medium. Glucose in the growth medium repressed the expression of the arabinose transport genes. By means of primer extension, the transcriptional start site for the arabinose operon was mapped. Interestingly, expression of the arabinose transporter is down-regulated by addition of a selective set of amino acids to the medium. Expression of the glucose transporter genes appeared constitutive. These data confirm the earlier observation of a catabolite repression-like system in S. solfataricus.