Sulfolobus species have become the model organisms for studying the unique biology of the crenarchaeal division of the archaeal domain. In particular, Sulfolobus islandicus provides a powerful opportunity to explore natural variation via experimental functional genomics. To support these efforts, we further expanded genetic tools for S. islandicus by developing a stringent positive selection for agmatine prototrophs in strains in which the argD gene, encoding arginine decarboxylase, has been deleted. Strains with deletions in argD were shown to be auxotrophic for agmatine even in nutrient-rich medium, but growth could be restored by either supplementation of exogenous agmatine or reintroduction of a functional copy of the argD gene from S. solfataricus P2 into the Δ argD host. Using this stringent selection, a robust targeted gene knockout system was established via an improved next generation of the MID ( m arker i nsertion and unmarked target gene d eletion) method. Application of this novel system was validated by targeted knockout of the upsEF genes involved in UV-inducible cell aggregation formation.

The S-layer is considered to be the sole component of the cell wall in Sulfolobales , a taxonomic group within the Crenarchaeota whose cellular features have been suggested to have a close relationship to the last archaea-eukaryote common ancestor. In this study, we genetically dissect how the two previously characterized S-layer genes as well as a newly identified S-layer-associated protein-encoding gene contribute to the S-layer architecture in Sulfolobus . We provide genetic evidence for the first time showing that the slaA gene is a key cell morphology determinant and may play a role in Sulfolobus cell division or/and cell fusion.

Extremophiles are group of microorganisms that possess ability to tolerate and live under the extremes of physico-chemical, geological and nutritional conditions. Such microorganisms are evolutionary relics and have evolved adaptation strategies at cellular, biochemical and molecular levels. They produce enzymes that are capable to maintain stability and function under the multitudes of extremities. These organisms also produce variety of other molecules and metabolites, such as extremolytes and surface-active compounds to protect against extremes of salinity, pH, pressure, temperatures and solar radiation. Investigations on these microorganisms can further open new avenues and opportunity for research and biotechnological applications in the areas of waste water treatment, bio-plastics, biofuel, cosmetics, agriculture, food and pharmaceuticals. Further, extremophiles have potential roles to play in bioremediation, astrobiology and biorefinery.

Sulfolobus islandicus is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the archaeal domain. As the tree of life grows, identifying the cellular functions of genes within this lineage will have significant impacts on our understanding of the evolution of the l ast a rchaeal e ukaryote c ommon a ncestor (LEACA) and the differentiation of archaea from eukaryotes during the evolution of the modern-day cell. To increase our understanding of this key archaeal organism, we report a novel high-throughput method for targeted gene inactivation in S. islandicus through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD ⁺ transformants showed that S. islandicus can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native argD marker replacement was as few as 10 bp, whereas argD marker replacement was frequently observed when increasing the size of homology to 30 to 50 bp. The microhomology-mediated gene inactivation system developed here will greatly facilitate isolation of S. islandicus gene deletion strains, making generation of a collection of genome-wide targeted mutants feasible and providing a tool to investigate homologous recombination in this organism. IMPORTANCE Current procedures for the construction of deletion mutants of S. islandicus are still tedious and time-consuming. We developed a novel procedure based on microhomology-mediated HR, allowing for rapid and efficient removal for genetic regions as large as 50 kb. Our work will greatly facilitate functional genomic studies in this promising model organism. Additionally, we developed a quantitative genetic assay to measure HR properties in S. islandicus , providing evidence that the ability to incorporate short, mismatched donor DNA into the genome through HR was probably a common trait for members of the Sulfolobus genus that are recombinogenic.

Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 ( apt ). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus . IMPORTANCE Currently, the pyrEF /5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF /5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt /6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF -deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains.