RESUMEN
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems are widely distributed among bacteria and archaea. In this study, we demonstrate the successful utilization of the type I-D CRISPR-Cas system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Given its extreme growth conditions characterized by a temperature of 75°C and pH 3, an uracil auxotrophic selection system was previously established, providing a basis for our investigations. We developed a novel plasmid specifically designed for genome editing, which incorporates a mini-CRISPR array that can be induced using xylose, resulting in targeted DNA cleavage. Additionally, we integrated a gene encoding the ß-galactosidase of Saccharolobus solfataricus into the plasmid, enabling blue-white screening and facilitating the mutant screening process. Through the introduction of donor DNA containing genomic modifications into the plasmid, we successfully generated deletion mutants and point mutations in the genome of S. acidocaldarius. Exploiting the PAM (protospacer adjacent motif) dependence of type I systems, we experimentally confirmed the functionality of three different PAMs (CCA, GTA, and TCA) through a self-targeting assessment assay and the gene deletion of upsE. Our findings elucidate the application of the endogenous Type I-D CRISPR-Cas system for genetic engineering in S. acidocaldarius, thus expanding its genetic toolbox.
RESUMEN
The thermophilic archaeon Sulfolobus acidocaldarius can use different carbon sources for growth, including the pentoses D-xylose and L-arabinose. In this study, we identified the activator XylR (saci_2116) responsible for the transcriptional regulation of the pentose transporter and pentose metabolizing genes in S. acidocaldarius. A xylR deletion mutant showed growth retardation on D-xylose/L-arabinose containing media and the lack of transcription of the respective ABC transporter. In contrast to so far used promoters for expression in S. acidocaldarius, the xylR responsive promoters have a very low background activity. Finally, two XylR dependent promoters next to the long-established maltose inducible promotor were used to construct a high-throughput expression vector system for S. acidocaldarius to efficiently clone and express proteins in S. acidocaldarius.
RESUMEN
BACKGROUND: Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. METHODS: We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. RESULTS: Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. CONCLUSIONS: Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. GENERAL SIGNIFICANCE: This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research.