Genetic manipulations of the hyperthermophilic piezophilic archaeon Thermococcus barophilus.
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.
Université de Bretagne Occidentale, UMR 6197-Laboratoire de Microbiologie des Environnements Extrêmes, Institut Universitaire Européen de la Mer, Plouzané, France.
Axel
A
Thiel
Grégoire
G
Michoud
Yann
Y
Moalic
Didier
D
Flament
Mohamed
M
Jebbar
D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D012641:Selection, Genetic; D019712:Thermococcus / Q000235:genetics*; D014170:Transformation, Genetic