Transcriptional Regulation of Carbon Utilization in the Hyperthermophilic Archaeon Sulfolobus solfataricus

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Blum, Paul MCB-9974453Small subunit (16S) ribosomal RNA sequence comparisons have identified a unique lineage or grouping of prokaryotic organisms called Archaea. They are as distantly related to Bacteria as they are to Eukaryotes. Archaea generally are subdivided into two major groups called the Euryarchaea and the Crenarchaea; the aerobic hyperthermophile, Sulfolobus solfataricus, is an intensively studied member of the Crenarchaea. Hyperthermophiles are present in both prokaryotic groups and are distinguished by their deeply rooted (slowly evolving) 16S rRNA. Archaea are distinct from Eukaryotes yet there is remarkable conservation between their general (basal) transcription components including RNA polymerase, promoters and promoter binding proteins. Whether this evolutionary overlap includes regulatory (accessory) transcription components is not known. This project focuses on the question of whether the biochemistry of gene expression in Archaea employs mechanisms like those in Eukaryotes or in Bacteria, or instead, altogether new strategies.In previous studies a Catabolite Repression (CR) gene regulatory system was discovered in S. solfataricus. CR controls expression of at least three genes all encoding glucosyl hydrolases which are used for growth on polysaccharides. The affected genes include an a-glucosidase (malA), an a-amylase (amyA), and a b-glycosidase (lacS). These genes are coordinately regulated at the transcriptional level in response to the quality of the carbon source provided for growth. Carbon quality is measured by how readily it can be assimilated into the TCA cycle. As carbon source quality decreases, expression of these genes is increased (CR is relieved). Because these genes are physically unlinked, coordinated expression indicates they are controlled by a trans-acting regulatory factor. The existence of such a factor is further supported by the isolation of unlinked (extragenic) mutations which pleiotropically reduce CR gene transcription. This project will test the following hypotheses: 1) CR alters gene expression through action at cis-acting promoter sequences; 2) Changes in CR gene expression require the action of a trans-acting factor; and 3) Differential regulation of CR-gene expression is promoter specific. The experimental approach will entail the exchange of promoter regions between CR-dependent and independent genes and the construction of promoter hybrids between CR-dependent genes. These constructs will be fused to a reporter gene and analyzed in vivo by enzyme assays, western blot and northern blot. Promoters will be analyzed in vitro by primer extension of transcription products and by gel retardation assays. The anticipated results will provide insight into basic aspects of archaeal hyperthermophilic metabolism and the evolutionary origin of gene expression.