TY - JOUR T1 - Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus A1 - Thiel,Axel A1 - Michoud,Gregoire A1 - Moalic,Yann A1 - Flament,Didier A1 - Jebbar,Mohamed AD - Univ Bretagne Occidentale, UMR 6197, Lab Microbiol Environm Extremes, Inst Univ Europeen Mer, Plouzane, France. AD - CNRS, UMR 6197, Lab Microbiol Environm Extremes, Inst Univ Europeen Mer, Plouzane, France. AD - IFREMER, UMR 6197, Lab Microbiol Environm Extremes, Plouzane, France. UR - https://archimer.ifremer.fr/doc/00187/29818/ DO - 10.1128/AEM.00084-14 N2 - 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. Y1 - 2014/04 PB - Amer Soc Microbiology JF - Applied And Environmental Microbiology SN - 0099-2240 VL - 80 IS - 7 SP - 2299 EP - 2306 ID - 29818 ER -