Genetic Manipulations of the Hyperthermophilic Piezophilic Archaeon Thermococcus barophilus

Type Article
Date 2014-04
Language English
Author(s) Thiel Axel1, 2, 3, Michoud Gregoire1, 2, 3, Moalic Yann1, 2, 3, Flament DidierORCID1, 2, 3, Jebbar Mohamed1, 2, 3
Affiliation(s) 1 : Univ Bretagne Occidentale, UMR 6197, Lab Microbiol Environm Extremes, Inst Univ Europeen Mer, Plouzane, France.
2 : CNRS, UMR 6197, Lab Microbiol Environm Extremes, Inst Univ Europeen Mer, Plouzane, France.
3 : IFREMER, UMR 6197, Lab Microbiol Environm Extremes, Plouzane, France.
Source Applied And Environmental Microbiology (0099-2240) (Amer Soc Microbiology), 2014-04 , Vol. 80 , N. 7 , P. 2299-2306
DOI 10.1128/AEM.00084-14
WOS© Times Cited 22
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.
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