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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 (?pyrF) 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 (?pyrF) or UBOCC-3260 (?pyrF ?hisB), 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.

SUBMITTER: Thiel A 

PROVIDER: S-EPMC3993132 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Genetic manipulations of the hyperthermophilic piezophilic archaeon Thermococcus barophilus.

Thiel Axel A   Michoud Grégoire G   Moalic Yann Y   Flament Didier D   Jebbar Mohamed M  

Applied and environmental microbiology 20140131 7


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 poin  ...[more]

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