FN Archimer Export Format
PT J
TI Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus
BT 
AF BIRIEN, Tiphaine
   THIEL, Axel
   HENNEKE, Ghislaine
   FLAMENT, Didier
   MOALIC, Yann
   JEBBAR, Mohamed
AS 1:1,2,3;2:1,2,3;3:1,2,3;4:1,2,3;5:1,2,3;6:1,2,3;
FF 1:;2:PDG-REM-EEP-LMEE;3:PDG-REM-EEP-LMEE;4:PDG-REM-EEP-LMEE;5:;6:;
C1 Univ Brest UBO UBL, IUEM, UMR 6197, Lab Microbiol Environm Extremes LM2E, Rue Dumont Urville, F-29280 Plouzane, France.
   CNRS, IUEM, UMR 6197, Lab Microbiol Environm Extremes LM2E, Rue Dumont Urville, F-29280 Plouzane, France.
   IFREMER, UMR 6197, Lab Microbiol Environm Extremes LM2E, Technopole Pointe Diable, F-29280 Plouzane, France.
C2 UBO, FRANCE
   CNRS, FRANCE
   IFREMER, FRANCE
SI BREST
SE PDG-REM-EEP-LMEE
UM BEEP-LM2E
IN WOS Ifremer jusqu'en 2018
   DOAJ
   copubli-france
   copubli-univ-france
IF 3.331
TC 13
UR https://archimer.ifremer.fr/doc/00425/53679/54523.pdf
LA English
DT Article
DE ;archaea;piezophiles;hyperthermophiles;genetics;gene deletion;deep sea;hydrothermal vents
AB 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). 
PY 2018
PD FEB
SO Genes
SN 2073-4425
PU Mdpi
VL 9
IS 2
UT 000427518700023
DI 10.3390/genes9020077
ID 53679
ER

EF