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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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Introduction of E. coli codA::upp cassette, encoding CD and UPRT, confers 5-FC sensitivity to R. equi strain RE1. Panels show wild type strain RE1, recombinant strain RE1 containing pSET152, recombinant strain RE1 containing plasmid pSET-codA::upp streaked on mineral acetate agar (MM-Ac) containing (A) no addition, (B) 5-FC (100 μg/ml) or (C) 5-FU (50 μg/ml) and on (D) LB agar containing 5-FC (100 μg/ml).
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Figure 1: Introduction of E. coli codA::upp cassette, encoding CD and UPRT, confers 5-FC sensitivity to R. equi strain RE1. Panels show wild type strain RE1, recombinant strain RE1 containing pSET152, recombinant strain RE1 containing plasmid pSET-codA::upp streaked on mineral acetate agar (MM-Ac) containing (A) no addition, (B) 5-FC (100 μg/ml) or (C) 5-FU (50 μg/ml) and on (D) LB agar containing 5-FC (100 μg/ml).

Mentions: An obvious prerequisite for the applicability of a 5-FC based conditionally lethal positive selection system is natural resistance of R. equi strain RE1 towards 5-FC. To test this, R. equi RE1 was streaked onto acetate mineral (MM-Ac) agar plates supplemented with 5-FC or 5-FU and incubated for 3 days at 30°C. Examination of the plates revealed that R. equi was resistant to high concentrations of 5-FC (100 μg/ml), but highly sensitive to a lower concentration of 5-FU (50 μg/ml), indicating the feasibility of developing a 5-FC based positive selection system for R. equi (Figure 1). Next, we examined whether expression of the E. coli genes codA and upp in R. equi would confer sensitivity to 5-FC and thus could act as a suicide marker. The integrative E. coli-Streptomyces shuttle vector pSET152 provided a stable and convenient vehicle to introduce codA or a functional codA::upp fusion into the R. equi genome (40,41). The aphII promoter region of the kanamycin resistance cassette (Pkan) was used to drive expression of codA or codA::upp, respectively. The two resulting integrative plasmids, pSET-Pkan-codA and pSET-Pkan-codAupp (Figure 2), were mobilized separately by electroporation to R. equi RE1. Transformants harboring either pSET-Pkan-codA or pSET-Pkan-codAupp were streaked onto MM-Ac agar plates (Figure 1A), MM-Ac agar plates supplemented with 100 μg/ml 5-FC (Figure 1B), or MM-Ac agar plates supplemented with 50 μg/ml 5-FU (Figure 1C) and incubated for 3 days at 30°C. The presence of the codA::upp cassette rendered R. equi sensitive to 5-FC, whereas expression of codA alone did not result in 5-FC sensitivity (Figure 1B). Growth of the R. equi strain carrying the codA::upp cassette under nonselective conditions was similar to that of wild type (Figure 1A), indicating that expression is conditionally lethal and not detrimental to cells when 5-FC is omitted from the medium. Importantly, no 5-FC selection was observed when complex agar media (LB) was used (Figure 1D). This may be due to repression of the pyrimidine salvage pathway affecting 5-FC uptake.


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)

Introduction of E. coli codA::upp cassette, encoding CD and UPRT, confers 5-FC sensitivity to R. equi strain RE1. Panels show wild type strain RE1, recombinant strain RE1 containing pSET152, recombinant strain RE1 containing plasmid pSET-codA::upp streaked on mineral acetate agar (MM-Ac) containing (A) no addition, (B) 5-FC (100 μg/ml) or (C) 5-FU (50 μg/ml) and on (D) LB agar containing 5-FC (100 μg/ml).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Introduction of E. coli codA::upp cassette, encoding CD and UPRT, confers 5-FC sensitivity to R. equi strain RE1. Panels show wild type strain RE1, recombinant strain RE1 containing pSET152, recombinant strain RE1 containing plasmid pSET-codA::upp streaked on mineral acetate agar (MM-Ac) containing (A) no addition, (B) 5-FC (100 μg/ml) or (C) 5-FU (50 μg/ml) and on (D) LB agar containing 5-FC (100 μg/ml).
Mentions: An obvious prerequisite for the applicability of a 5-FC based conditionally lethal positive selection system is natural resistance of R. equi strain RE1 towards 5-FC. To test this, R. equi RE1 was streaked onto acetate mineral (MM-Ac) agar plates supplemented with 5-FC or 5-FU and incubated for 3 days at 30°C. Examination of the plates revealed that R. equi was resistant to high concentrations of 5-FC (100 μg/ml), but highly sensitive to a lower concentration of 5-FU (50 μg/ml), indicating the feasibility of developing a 5-FC based positive selection system for R. equi (Figure 1). Next, we examined whether expression of the E. coli genes codA and upp in R. equi would confer sensitivity to 5-FC and thus could act as a suicide marker. The integrative E. coli-Streptomyces shuttle vector pSET152 provided a stable and convenient vehicle to introduce codA or a functional codA::upp fusion into the R. equi genome (40,41). The aphII promoter region of the kanamycin resistance cassette (Pkan) was used to drive expression of codA or codA::upp, respectively. The two resulting integrative plasmids, pSET-Pkan-codA and pSET-Pkan-codAupp (Figure 2), were mobilized separately by electroporation to R. equi RE1. Transformants harboring either pSET-Pkan-codA or pSET-Pkan-codAupp were streaked onto MM-Ac agar plates (Figure 1A), MM-Ac agar plates supplemented with 100 μg/ml 5-FC (Figure 1B), or MM-Ac agar plates supplemented with 50 μg/ml 5-FU (Figure 1C) and incubated for 3 days at 30°C. The presence of the codA::upp cassette rendered R. equi sensitive to 5-FC, whereas expression of codA alone did not result in 5-FC sensitivity (Figure 1B). Growth of the R. equi strain carrying the codA::upp cassette under nonselective conditions was similar to that of wild type (Figure 1A), indicating that expression is conditionally lethal and not detrimental to cells when 5-FC is omitted from the medium. Importantly, no 5-FC selection was observed when complex agar media (LB) was used (Figure 1D). This may be due to repression of the pyrimidine salvage pathway affecting 5-FC uptake.

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