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| Birien, Tiphaine; Thiel, Axel; Henneke, Ghislaine; Flament, Didier; Moalic, Yann; Jebbar, Mohamed. |
| A gene disruption system for Thermococcus barophilus was developed using simvastatin (HMG-CoA reductase encoding gene) for positive selection and 5-Fluoroorotic acid (5-FOA), a pyrF gene for negative selection. Multiple gene mutants were constructed with this system, which offers the possibility of complementation in trans, but produces many false positives (<80%). To significantly reduce the rate of false positives, we used another counterselective marker, 6-methylpurine (6-MP), a toxic analog of adenine developed in Thermococcus kodakarensis, consistently correlated with the TK0664 gene (encoding a hypoxanthine-guanine phosphoribosyl-transferase). We thus replaced pyrF by TK0664 on our suicide vector and tested T. barophilus strain sensitivity to 6-MP... |
| Tipo: Text |
Palavras-chave: Archaea; Piezophiles; Hyperthermophiles; Genetics; Gene deletion; Deep sea; Hydrothermal vents. |
| Ano: 2018 |
URL: https://archimer.ifremer.fr/doc/00425/53679/54523.pdf |
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| Thiel, Axel; Michoud, Gregoire; Moalic, Yann; Flament, Didier; Jebbar, Mohamed. |
| 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... |
| Tipo: Text |
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| Ano: 2014 |
URL: http://archimer.ifremer.fr/doc/00187/29818/28263.pdf |
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| Cario, Anaïs; Jebbar, Mohamed; Thiel, Axel; Kervarec, Nelly; Oger, Phil M.. |
| The accumulation of mannosyl-glycerate (MG), the salinity stress response osmolyte of Thermococcales, was investigated as a function of hydrostatic pressure in Thermococcus barophilus strain MP, a hyperthermophilic, piezophilic archaeon isolated from the Snake Pit site (MAR), which grows optimally at 40 MPa. Strain MP accumulated MG primarily in response to salinity stress, but in contrast to other Thermococcales, MG was also accumulated in response to thermal stress. MG accumulation peaked for combined stresses. The accumulation of MG was drastically increased under sub-optimal hydrostatic pressure conditions, demonstrating that low pressure is perceived as a stress in this piezophile, and that the proteome of T. barophilus is low-pressure sensitive. MG... |
| Tipo: Text |
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| Ano: 2016 |
URL: http://archimer.ifremer.fr/doc/00345/45570/45178.pdf |
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| Cario, Anais; Mizgier, Alex; Thiel, Axel; Jebbar, Mohamed; Oger, Phil M.. |
| Most Thermococcales accumulate di-myo-inositol-phosphate (DIP) as an organic solute as a response to heat stress. We have studied the accumulation of this osmolyte in the high-hydrostatic pressure adapted hyperthermophile Thermococcus barophilus. We found no accumulation of DIP under any of the stress conditions tested, although this archaeon harbors the 3 DIP synthesis genes. Lack of synthesis is due to the lack of expression of TERMP_01135 coding for the second step of DIP synthesis. In contrast to other species, the T. barophilus synthesis operon is interrupted by a four gene locus, in reverse orientation. Restoring an operon like structure at the DIP locus restored DIP synthesis, but did not have an impact on growth characteristics, suggesting that... |
| Tipo: Text |
Palavras-chave: High hydrostatic pressure; Heat stress; Di-myo-inositol-phosphate; Thermococcus barophilus. |
| Ano: 2015 |
URL: http://archimer.ifremer.fr/doc/00270/38113/37216.pdf |
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