Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Genes Année : 2018

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

Résumé

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).
Fichier principal
Vignette du fichier
genes-09-00077.pdf (1.99 Mo) Télécharger le fichier
Origine : Publication financée par une institution

Dates et versions

hal-04202052 , version 1 (15-04-2024)

Identifiants

Citer

Tiphaine Birien, Axel Thiel, Ghislaine Henneke, Didier Flament, Yann Moalic, et al.. Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus. Genes, 2018, 9 (2), pp.77. ⟨10.3390/genes9020077⟩. ⟨hal-04202052⟩
28 Consultations
0 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More