Published in

American Society for Microbiology, Applied and Environmental Microbiology, 7(80), p. 2299-2306, 2014

DOI: 10.1128/aem.00084-14

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Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus

Journal article published in 2014 by Axel Thiel, Grégoire Michoud, Yann Moalic, Didier Flament, Mohamed Jebbar ORCID
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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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 (Delta 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 (Delta pyrF) or UBOCC-3260 (Delta pyrF Delta 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.