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A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality.

van der Geize R, de Jong W, Hessels GI, Grommen AW, Jacobs AA, Dijkhuizen L - Nucleic Acids Res. (2008)

Bottom Line: Phenotypic analysis of the generated DeltasupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands. r.van.der.geize@rug.nl

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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Growth curves in mineral medium supplemented with cholesterol (0.5 g/l) of R. equi RE1 wild-type (filled diamond), RE1 ΔsupAB mutant strain (filled square), RE1 ΔsupAB + pSET-supAB complemented strain (filled triangle), and RE1 ΔsupAB + pSET152 control strain (x). Protein content (mg/l) of the culture was used as a measure for biomass formation. The data represent the averages for two independent experiments.
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Figure 6: Growth curves in mineral medium supplemented with cholesterol (0.5 g/l) of R. equi RE1 wild-type (filled diamond), RE1 ΔsupAB mutant strain (filled square), RE1 ΔsupAB + pSET-supAB complemented strain (filled triangle), and RE1 ΔsupAB + pSET152 control strain (x). Protein content (mg/l) of the culture was used as a measure for biomass formation. The data represent the averages for two independent experiments.

Mentions: R. equi RE1 wild type and the RE1ΔsupAB mutant strain were grown in MM-Ac liquid medium and used to inoculate MM-cholesterol liquid medium. The RE1ΔsupAB mutant was completely blocked in growth on cholesterol as sole carbon and energy source (Figure 6). Growth on acetate or the steroid substrate 4-androstene-3,17-dione (AD) was unaffected and comparable to the wild type strain (data not shown). This indicated that supAB are essential for cholesterol catabolism, probably acting as the permease subunits of the cholesterol ABC transporter. These results are fully consistent with the phenotype of the supAB mutant of strain RHA1 (6).Figure 6.


A novel method to generate unmarked gene deletions in the intracellular pathogen Rhodococcus equi using 5-fluorocytosine conditional lethality.

van der Geize R, de Jong W, Hessels GI, Grommen AW, Jacobs AA, Dijkhuizen L - Nucleic Acids Res. (2008)

Growth curves in mineral medium supplemented with cholesterol (0.5 g/l) of R. equi RE1 wild-type (filled diamond), RE1 ΔsupAB mutant strain (filled square), RE1 ΔsupAB + pSET-supAB complemented strain (filled triangle), and RE1 ΔsupAB + pSET152 control strain (x). Protein content (mg/l) of the culture was used as a measure for biomass formation. The data represent the averages for two independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602762&req=5

Figure 6: Growth curves in mineral medium supplemented with cholesterol (0.5 g/l) of R. equi RE1 wild-type (filled diamond), RE1 ΔsupAB mutant strain (filled square), RE1 ΔsupAB + pSET-supAB complemented strain (filled triangle), and RE1 ΔsupAB + pSET152 control strain (x). Protein content (mg/l) of the culture was used as a measure for biomass formation. The data represent the averages for two independent experiments.
Mentions: R. equi RE1 wild type and the RE1ΔsupAB mutant strain were grown in MM-Ac liquid medium and used to inoculate MM-cholesterol liquid medium. The RE1ΔsupAB mutant was completely blocked in growth on cholesterol as sole carbon and energy source (Figure 6). Growth on acetate or the steroid substrate 4-androstene-3,17-dione (AD) was unaffected and comparable to the wild type strain (data not shown). This indicated that supAB are essential for cholesterol catabolism, probably acting as the permease subunits of the cholesterol ABC transporter. These results are fully consistent with the phenotype of the supAB mutant of strain RHA1 (6).Figure 6.

Bottom Line: Phenotypic analysis of the generated DeltasupAB mutant confirmed that supAB are essential for growth of R. equi on cholesterol.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands. r.van.der.geize@rug.nl

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.

Show MeSH
Related in: MedlinePlus