| Registro completo |
| Provedor de dados: |
ArchiMer
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| País: |
France
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| Título: |
Development of an Effective 6-Methylpurine Counterselection Marker for Genetic Manipulation in Thermococcus barophilus
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| Autores: |
Birien, Tiphaine
Thiel, Axel
Henneke, Ghislaine
Flament, Didier
Moalic, Yann
Jebbar, Mohamed
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| Data: |
2018-02
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| Ano: |
2018
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| Palavras-chave: |
Archaea
Piezophiles
Hyperthermophiles
Genetics
Gene deletion
Deep sea
Hydrothermal vents
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| Resumo: |
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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| Tipo: |
Text
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| Idioma: |
Inglês
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| Identificador: |
https://archimer.ifremer.fr/doc/00425/53679/54523.pdf
DOI:10.3390/genes9020077
https://archimer.ifremer.fr/doc/00425/53679/
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| Editor: |
Mdpi
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| Formato: |
application/pdf
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| Fonte: |
Genes (2073-4425) (Mdpi), 2018-02 , Vol. 9 , N. 2 , P. 77 (13p.)
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| Direitos: |
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
info:eu-repo/semantics/openAccess
restricted use
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