Sulfolobus species have been developed as excellent model organisms to address fundamental questions of archaeal biology. Interesting patterns of natural variation among Sulfolobus islandicus strains have been identified through genome sequencing. Experimentally testing hypotheses about the biological causes and consequences of this natural variation requires genetic tools that apply to a diversity of strains. Previously, a genetic transformation system for S. islandicus was reported, in which overexpression of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene on the shuttle vector pSSR allowed the selection of transformants resistant to high concentrations of the thermostable antibiotic simvastatin. Here, we developed a novel gene knockout system based on simvastatin resistance. With this system, we created via homologous recombination an in-frame, markerless deletion of the intact S. islandicus M.16.4 pyrEF genes encoding orotidine-59-monophosphate pyrophosphorylase (OPRTase) and orotidine-59-monophosphate decarboxylase (OMPdecase), and a disruption of the lacS gene encoding β-galactosidase. Phenotypic analyses of the mutants revealed that the pyrEF deletion mutant lost the ability to synthesize uracil, and the lacS deletion mutants exhibited a white colour after X-Gal staining, demonstrating that the β-galactosidase function was inactivated. Our data demonstrate efficient tools to generate gene knockouts in a broad range of wild-type Sulfolobus strains.
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