ArticleNucleic acids research2008
A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality.
Article in Nucleic acids research, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 30 papers.
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Who cites it
30 citing papers in PubMed, 64 citations in OpenAlex.
- Protective immune response against Rhodococcus equi: An innate immunity-focused review.Equine veterinary journal · 2025Review
- Identification of an atypical replicative genetic element inFrontiers in microbiology · 2025Article
- A modular toolkit for environmentalApplied and environmental microbiology · 2024Article
- Gene expression profile analysis and target gene discovery of Mycobacterium tuberculosis biofilm.Applied microbiology and biotechnology · 2021Article
- Approaches to genetic tool development for rapid domestication of non-model microorganisms.Biotechnology for biofuels · 2021Review
- Article
- PhytopathogenicFrontiers in microbiology · 2020Article
- Mycoredoxins Are Required for Redox Homeostasis and Intracellular Survival in the Actinobacterial PathogenAntioxidants (Basel, Switzerland) · 2019Article
- Review
- Identification of a VapA virulence factor functional homolog in Rhodococcus equi isolates housing the pVAPB plasmid.PloS one · 2018Article
- Genome-based exploration of the specialized metabolic capacities of the genus Rhodococcus.BMC genomics · 2017Article
- Discovery and characterization of an FApplied microbiology and biotechnology · 2017Article
- Genome-Wide Transcriptome Profiling of Mycobacterium smegmatis MC² 155 Cultivated in Minimal Media Supplemented with Cholesterol, Androstenedione or Glycerol.International journal of molecular sciences · 2016Article
- Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27.Applied and environmental microbiology · 2016Article
- Transcriptome reprogramming by plasmid-encoded transcriptional regulators is required for host niche adaption of a macrophage pathogen.Infection and immunity · 2015Article
- An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.Infection and immunity · 2015Article
- VirS, an OmpR/PhoB subfamily response regulator, is required for activation of vapA gene expression in Rhodococcus equi.BMC microbiology · 2014Article
- Deletion of the gene encoding the reductase component of 3-ketosteroid 9α-hydroxylase in Rhodococcus equi USA-18 disrupts sterol catabolism, leading to the accumulation of 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid and 1,4-androstadiene-3,17-dione.Microbial cell factories · 2014Article
- IcgA is a virulence factor of Rhodococcus equi that modulates intracellular growth.Infection and immunity · 2014Article
- Article
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Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 DeltasupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol. Macrophage survival assays revealed that the DeltasupAB 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.
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