Nucleic Acids Research Advance Access originally published online on November 4, 2008
Nucleic Acids Research 2008 36(22):e151; doi:10.1093/nar/gkn811
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2008, Vol. 36, No. 22 e151
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality
1Groningen Biomolecular Sciences and Biotechnology Institute (GBB), Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren and 2Intervet Schering-Plough Animal Health, Bacteriological R&D, Wim de Körverstraat 35, Postbus 31, 5830 AA Boxmeer, The Netherlands
*To whom correspondence should be addressed. Tel: +31 50 363 2257; Fax: +31 50 363 2254; Email: r.van.der.geize{at}rug.nl
Received September 16, 2008. Accepted October 10, 2008.
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 
supAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the 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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.