b CRISPR (clustered regularly interspaced short palindromic repeats) elements and cas (CRISPR-associated) genes are widespread in Bacteria and Archaea. The CRISPR/Cas system operates as a defense mechanism against mobile genetic elements (i.e., viruses or plasmids). Here, we investigate seven CRISPR loci in the genome of the crenarchaeon Thermoproteus tenax that include spacers with significant similarity not only to archaeal viruses but also to T. tenax genes. The analysis of CRISPR RNA (crRNA) transcription reveals transcripts of a length between 50 and 130 nucleotides, demonstrating the processing of larger crRNA precursors. The organization of identified cas genes resembles CRISPR/Cas subtype I-A, and the core cas genes are shown to be arranged on two polycistronic transcripts: cascis (cas4, cas1/2, and csa1) and cascade (csa5, cas7, cas5a, cas3, cas3=, and cas8a2). Changes in the environmental parameters such as UV-light exposure or high ionic strength modulate cas gene transcription. Two reconstitution protocols were established for the production of two discrete multipartite Cas protein complexes that correspond to their operonic gene arrangement. These data provide insights into the specialized mechanisms of an archaeal CRISPR/ Cas system and allow selective functional analyses of Cas protein complexes in the future.
The Crenarchaeon Sulfolobus acidocaldarius has been described to synthesize trehalose via the maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) pathway and the trehalose glycosyltransferring synthase (TreT) pathway has been predicted. Deletion mutant analysis of single and double deletion strains of ΔtreY and ΔtreT in S. acidocaldarius revealed that next to these two pathways a third, novel trehalose biosynthesis pathway is operative in vivo: the trehalose-6-phosphate (T6P) synthase/T6P phosphatase (TPS/TPP) pathway. In contrast to known TPS proteins, which belong to the GT20 family, the S. acidocaldarius TPS belongs to the GT4 family establishing a new function within this group of enzymes. This novel GT4-like TPS turned out to be mainly present in the Sulfolobales. The ΔtreY/ΔtreT/Δtps triple mutant of S. acidocaldarius lacking the ability to synthesize trehalose showed no altered phenotype under standard conditions or heat stress, but was unable to grow under salt stress. Accordingly, in the wild type strain a significant increase of intracellular trehalose formation was observed under salt stress. Quantitative real-time PCR showed a salt stress mediated induction of all three trehalose synthesizing pathways. This demonstrates in Archaea that trehalose plays an essential role for growth under high salt conditions. Importance The metabolism and function of trehalose as compatible solute was not well understood in Archaea. This combined genetic and enzymatic approach at the interface of microbiology, physiology and microbial ecology gives important insights into survival under stress, adaptation to extreme environments, and the role of compatible solutes in Archaea. Here we unravelled the complexity of trehalose metabolism and present a comprehensive study on trehalose function in stress response in S. acidocaldarius. This sheds light on the general microbiology and the fascinating metabolic repertoire of Archaea involving many novel biocatalysts such as glycosyltransferases with great potential in biotechnology.
The role of the disaccharide trehalose, its biosynthesis pathways and their regulation in Archaea are still ambiguous. In Thermoproteus tenax a fused trehalose-6-phosphate synthase/phosphatase (TPSP), consisting of an N-terminal trehalose-6-phosphate synthase (TPS) and a C-terminal trehalose-6-phosphate phosphatase (TPP) domain, was identified. The tpsp gene is organized in an operon with a putative glycosyltransferase (GT) and a putative mechanosensitive channel (MSC). The T. tenax TPSP exhibits high phosphatase activity, but requires activation by the co-expressed GT for bifunctional synthase-phosphatase activity. The GT mediated activation of TPS activity relies on the fusion of both, TPS and TPP domain, in the TPSP enzyme. Activation is mediated by complex-formation in vivo as indicated by yeast two-hybrid and crude extract analysis. In combination with first evidence for MSC activity the results suggest a sophisticated stress response involving TPSP, GT and MSC in T. tenax and probably in other Thermoproteales species. The monophyletic prokaryotic TPSP proteins likely originated via a single fusion event in the Bacteroidetes with subsequent horizontal gene transfers to other Bacteria and Archaea. Furthermore, evidence for the origin of eukaryotic TPSP fusions via HGT from prokaryotes and therefore a monophyletic origin of eukaryotic and prokaryotic fused TPSPs is presented. This is the first report of a prokaryotic, archaeal trehalose synthase complex exhibiting a much more simple composition than the eukaryotic complex described in yeast. Thus, complex formation and a complex-associated regulatory potential might represent a more general feature of trehalose synthesizing proteins.
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