1887

Abstract

species have been developed as excellent model organisms to address fundamental questions of archaeal biology. Interesting patterns of natural variation among 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 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 M.16.4 genes encoding orotidine-5′-monophosphate pyrophosphorylase (OPRTase) and orotidine-5′-monophosphate decarboxylase (OMPdecase), and a disruption of the gene encoding β-galactosidase. Phenotypic analyses of the mutants revealed that the deletion mutant lost the ability to synthesize uracil, and the 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 strains.

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2012-06-01
2020-08-12
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