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

Type Article
Date 2018-02
Language English
Author(s) Birien Tiphaine1, 2, 3, Thiel Axel1, 2, 3, Henneke GhislaineORCID1, 2, 3, Flament DidierORCID1, 2, 3, Moalic Yann1, 2, 3, Jebbar MohamedORCID1, 2, 3
Affiliation(s) 1 : Univ Brest UBO UBL, IUEM, UMR 6197, Lab Microbiol Environm Extremes LM2E, Rue Dumont Urville, F-29280 Plouzane, France.
2 : CNRS, IUEM, UMR 6197, Lab Microbiol Environm Extremes LM2E, Rue Dumont Urville, F-29280 Plouzane, France.
3 : IFREMER, UMR 6197, Lab Microbiol Environm Extremes LM2E, Technopole Pointe Diable, F-29280 Plouzane, France.
Source Genes (2073-4425) (Mdpi), 2018-02 , Vol. 9 , N. 2 , P. 77 (13p.)
DOI 10.3390/genes9020077
WOS© Times Cited 11
Keyword(s) archaea, piezophiles, hyperthermophiles, genetics, gene deletion, deep sea, hydrothermal vents

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).

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