Academic Journal
A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality
| العنوان: | A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality |
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| المؤلفون: | van der Geize, R., de Jong, W., Hessels, G. I., Grommen, A. W. F., Jacobs, A. A. C., Dijkhuizen, L. |
| المصدر: | van der Geize , R , de Jong , W , Hessels , G I , Grommen , A W F , Jacobs , A A C & Dijkhuizen , L 2008 , ' A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality ' , Nucleic Acids Research , vol. 36 , no. 22 , 151 . https://doi.org/10.1093/nar/gkn811 |
| سنة النشر: | 2008 |
| المجموعة: | University of Groningen research database |
| مصطلحات موضوعية: | COUNTER-SELECTABLE MARKER, SUBTILIS SACB GENE, CYTOSINE DEAMINASE, BACILLUS-SUBTILIS, ESCHERICHIA-COLI, MYCOBACTERIUM-SMEGMATIS, CHOLESTEROL OXIDASE, POSITIVE SELECTION, CLONING VECTORS, STREPTOMYCES |
| الوصف: | A novel method to efficiently generate unmarked in-frame gene deletions in Rhodococcus equi was developed, exploiting the cytotoxic effect of 5-fluorocytosine (5-FC) by the action of cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) enzymes. The opportunistic, intracellular pathogen R. equi is resistant to high concentrations of 5-FC. Introduction of Escherichia coli genes encoding CD and UPRT conferred conditional lethality to R. equi cells incubated with 5-FC. To exemplify the use of the codA::upp cassette as counter-selectable marker, an unmarked in-frame gene deletion mutant of R. equi was constructed. The supA and supB genes, part of a putative cholesterol catabolic gene cluster, were efficiently deleted from the R. equi wild-type genome. Phenotypic analysis of the generated Delta supAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the Delta supAB mutant is able to survive and proliferate in macrophages comparable to wild type. Thus, cholesterol metabolism does not appear to be essential for macrophage survival of R. equi. The CD-UPRT based 5-FC counter-selection may become a useful asset in the generation of unmarked in-frame gene deletions in other actinobacteria as well, as actinobacteria generally appear to be 5-FC resistant and 5-FU sensitive. |
| نوع الوثيقة: | article in journal/newspaper |
| وصف الملف: | application/pdf |
| اللغة: | English |
| Relation: | https://research.rug.nl/en/publications/d40db2b3-4fa6-413f-a8d7-b2c752e8e6fd |
| DOI: | 10.1093/nar/gkn811 |
| الاتاحة: | https://hdl.handle.net/11370/d40db2b3-4fa6-413f-a8d7-b2c752e8e6fd https://research.rug.nl/en/publications/d40db2b3-4fa6-413f-a8d7-b2c752e8e6fd https://doi.org/10.1093/nar/gkn811 https://pure.rug.nl/ws/files/6727362/2008NuclAcidsResvdGeize.pdf |
| Rights: | info:eu-repo/semantics/openAccess |
| رقم الانضمام: | edsbas.AC37FB69 |
| قاعدة البيانات: | BASE |
| DOI: | 10.1093/nar/gkn811 |
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