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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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Survival and proliferation of R. equi strains in the human monocyte cell line U937. Macrophage cell suspensions were infected with wild type virulent strain R. equi RE1 (filled diamond), mutant strain R. equi RE1ΔsupAB (filled square) and non-virulent (control) strain R. equi 103− (filled triangle). Following a 1-h incubation to allow phagocytosis, cells were washed and treated with gentamycin to kill remaining extra-cellular bacteria. The numbers of intracellular bacteria were determined by plate counts following macrophage lysis. The data represent the averages for two independent experiments. Plate counts were carried out in duplicate.
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Figure 7: Survival and proliferation of R. equi strains in the human monocyte cell line U937. Macrophage cell suspensions were infected with wild type virulent strain R. equi RE1 (filled diamond), mutant strain R. equi RE1ΔsupAB (filled square) and non-virulent (control) strain R. equi 103− (filled triangle). Following a 1-h incubation to allow phagocytosis, cells were washed and treated with gentamycin to kill remaining extra-cellular bacteria. The numbers of intracellular bacteria were determined by plate counts following macrophage lysis. The data represent the averages for two independent experiments. Plate counts were carried out in duplicate.

Mentions: Intracellular survival and proliferation of the R. equi RE1ΔsupAB mutant in the human monocyte cell line U937 was compared to those of wild type strain RE1 (Figure 7). The avirulent, plasmid free strain R. equi 103− (43) was included as a negative control for macrophage survival (Figure 7). The results revealed that the RE1ΔsupAB mutant is able to survive and proliferate in macrophages comparable to the wild-type parent strain RE1. By contrast, the avirulent strain 103− failed to proliferate, resulting in reduced numbers of intracellular bacteria in time (Figure 7). These results indicate that cholesterol metabolism is not essential for macrophage survival of R. equi RE1 and suggest that cholesterol metabolism is not important for virulence of R. equi RE1 in vivo. These observations are consistent with the finding that ChoE is not important in the virulence of R. equi (28).Figure 7.


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)

Survival and proliferation of R. equi strains in the human monocyte cell line U937. Macrophage cell suspensions were infected with wild type virulent strain R. equi RE1 (filled diamond), mutant strain R. equi RE1ΔsupAB (filled square) and non-virulent (control) strain R. equi 103− (filled triangle). Following a 1-h incubation to allow phagocytosis, cells were washed and treated with gentamycin to kill remaining extra-cellular bacteria. The numbers of intracellular bacteria were determined by plate counts following macrophage lysis. The data represent the averages for two independent experiments. Plate counts were carried out in duplicate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Survival and proliferation of R. equi strains in the human monocyte cell line U937. Macrophage cell suspensions were infected with wild type virulent strain R. equi RE1 (filled diamond), mutant strain R. equi RE1ΔsupAB (filled square) and non-virulent (control) strain R. equi 103− (filled triangle). Following a 1-h incubation to allow phagocytosis, cells were washed and treated with gentamycin to kill remaining extra-cellular bacteria. The numbers of intracellular bacteria were determined by plate counts following macrophage lysis. The data represent the averages for two independent experiments. Plate counts were carried out in duplicate.
Mentions: Intracellular survival and proliferation of the R. equi RE1ΔsupAB mutant in the human monocyte cell line U937 was compared to those of wild type strain RE1 (Figure 7). The avirulent, plasmid free strain R. equi 103− (43) was included as a negative control for macrophage survival (Figure 7). The results revealed that the RE1ΔsupAB mutant is able to survive and proliferate in macrophages comparable to the wild-type parent strain RE1. By contrast, the avirulent strain 103− failed to proliferate, resulting in reduced numbers of intracellular bacteria in time (Figure 7). These results indicate that cholesterol metabolism is not essential for macrophage survival of R. equi RE1 and suggest that cholesterol metabolism is not important for virulence of R. equi RE1 in vivo. These observations are consistent with the finding that ChoE is not important in the virulence of R. equi (28).Figure 7.

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