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Genes 2018, 9(2), 77; https://doi.org/10.3390/genes9020077

Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus

1
Université de Brest (UBO, UBL), Institut Universitaire Européen de la Mer (IUEM)–UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes (LM2E), Rue Dumont d’Urville, F-29280 Plouzané, France
2
CNRS, IUEM–UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes (LM2E), Rue Dumont d’Urville, F-29280 Plouzané, France
3
Ifremer, UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes (LM2E), Technopôle Pointe du diable, F-29280 Plouzané, France
*
Author to whom correspondence should be addressed.
Received: 8 January 2018 / Revised: 30 January 2018 / Accepted: 2 February 2018 / Published: 7 February 2018
(This article belongs to the Special Issue Genetics and Genomics of Extremophiles)
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Abstract

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 before and after transformation. Wild-Type (WT) T. barophilus is less sensitive to 6-MP than WT T. kodakarensis, and an increase of cell resistance was achieved after deletion of the T. barophilus TERMP_00517 gene homologous to T. kodakarensis TK0664. Results confirmed the natural resistance of T. barophilus to 6-MP and show that TK0664 can confer sensitivity. This new counterselection system vastly improves genetic manipulations in T. barophilus MP, with a strong decrease in false positives to <15%. Using this genetic tool, we have started to investigate the functions of several genes involved in genomic maintenance (e.g., polB and rnhB). View Full-Text
Keywords: archaea; piezophiles; hyperthermophiles; genetics; gene deletion; deep sea; hydrothermal vents archaea; piezophiles; hyperthermophiles; genetics; gene deletion; deep sea; hydrothermal vents
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Birien, T.; Thiel, A.; Henneke, G.; Flament, D.; Moalic, Y.; Jebbar, M. Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus. Genes 2018, 9, 77.

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