Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus.
Genes (Basel)
; 9(2)2018 Feb 07.
Article
em En
| MEDLINE
| ID: mdl-29414865
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. barophilusTERMP_00517 gene homologous to T. kodakarensisTK0664. 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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Coleções:
01-internacional
Base de dados:
MEDLINE
Idioma:
En
Revista:
Genes (Basel)
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
França