Genetic manipulations of the hyperthermophilic piezophilic archaeon Thermococcus barophilus. - Université de Bretagne Occidentale Access content directly
Journal Articles Applied and Environmental Microbiology Year : 2014

Genetic manipulations of the hyperthermophilic piezophilic archaeon Thermococcus barophilus.

Abstract

In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (ΔpyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophilus was inserted into the chromosome of UBOCC-3256 (ΔpyrF) or UBOCC-3260 (ΔpyrF ΔhisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.
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Dates and versions

hal-01019712 , version 1 (07-07-2014)

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Axel Thiel, Grégoire Michoud, Yann Moalic, Didier Flament, Mohamed Jebbar. Genetic manipulations of the hyperthermophilic piezophilic archaeon Thermococcus barophilus.. Applied and Environmental Microbiology, 2014, 80 (7), pp.2299-306. ⟨10.1128/AEM.00084-14⟩. ⟨hal-01019712⟩
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