ABSTRACT
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.
ABSTRACT
Thermoacidophilic archaea belonging to the order Sulfolobales thrive in extreme biotopes, such as sulfuric hot springs and ore deposits. These microorganisms have been model systems for understanding life in extreme environments, as well as for probing the evolution of both molecular genetic processes and central metabolic pathways. Thermoacidophiles, such as the Sulfolobales, use typical microbial responses to persist in hot acid (e.g. motility, stress response, biofilm formation), albeit with some unusual twists. They also exhibit unique physiological features, including iron and sulfur chemolithoautotrophy, that differentiate them from much of the microbial world. Although first discovered >50 years ago, it was not until recently that genome sequence data and facile genetic tools have been developed for species in the Sulfolobales. These advances have not only opened up ways to further probe novel features of these microbes but also paved the way for their potential biotechnological applications. Discussed here are the nuances of the thermoacidophilic lifestyle of the Sulfolobales, including their evolutionary placement, cell biology, survival strategies, genetic tools, metabolic processes and physiological attributes together with how these characteristics make thermoacidophiles ideal platforms for specialized industrial processes.
