Limits...
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.

Show MeSH

Related in: MedlinePlus

The pSET152 derived integrative plasmids used to introduce the (A) codA or (B) codA::upp cassette into R. equi RE1, expressed under control of the aphII kanamycin resistance cassette promoter (PaphII). The apramycin resistance cassette (aac(3)IV), the Streptomyces PhiC31 integrase gene (int) and the RP4 origin of transfer (oriT) are also indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2602762&req=5

Figure 2: The pSET152 derived integrative plasmids used to introduce the (A) codA or (B) codA::upp cassette into R. equi RE1, expressed under control of the aphII kanamycin resistance cassette promoter (PaphII). The apramycin resistance cassette (aac(3)IV), the Streptomyces PhiC31 integrase gene (int) and the RP4 origin of transfer (oriT) are also indicated.

Mentions: The aphII promoter region was amplified from pRESQ (37) using PCR primers Pkan-F and Pkan-E5-R (Table 1). The obtained PCR product of 367 bp was blunt-ligated into EcoRV digested pBluescript(II)KS (Stratagene), resulting in pBs-Pkan. A SalI/NotI restriction released a 431 bp fragment comprising the aphII promoter which was then cloned into SalI/NotI digested pORF-codA::upp (InvivoGen, San Diego, USA), yielding plasmid pORF-Pkan-codAupp. The Pkan-codA::upp cassette was subsequently isolated from pORF-Pkan-codAupp as a 2.4 kb SmaI/NheI fragment and ligated into EcoRV/XbaI digested pSET152 resulting in plasmid pSET-Pkan-codAupp (Figure 2). The Pkan-codA cassette (1733 bp) was amplified from pSET-Pkan-codAupp using primers Pkan-F and codA-R2 (Table 1) and ligated into EcoRV digested pSET152, resulting in pSET-Pkan-codA (Figure 2). Suicide plasmid pSelAct (Figure 3) was constructed by ligating a 2.4 kb Klenow-treated EcoRI/NheI fragment of pORF-Pkan-codAupp into SspI digested pBs-Apra-ori dephosphoryated with alkaline phosphatase. Plasmid pBs-Apra-ori was constructed from pBluescript(II)KS in which the bla cassette was removed with BspHI, followed by Klenow treatment and replaced by an apramycin-oriT cassette obtained as a 1.3 kb XbaI fragment (Klenow treated) from pIJ773 (13).Figure 1.


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)

The pSET152 derived integrative plasmids used to introduce the (A) codA or (B) codA::upp cassette into R. equi RE1, expressed under control of the aphII kanamycin resistance cassette promoter (PaphII). The apramycin resistance cassette (aac(3)IV), the Streptomyces PhiC31 integrase gene (int) and the RP4 origin of transfer (oriT) are also indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: The pSET152 derived integrative plasmids used to introduce the (A) codA or (B) codA::upp cassette into R. equi RE1, expressed under control of the aphII kanamycin resistance cassette promoter (PaphII). The apramycin resistance cassette (aac(3)IV), the Streptomyces PhiC31 integrase gene (int) and the RP4 origin of transfer (oriT) are also indicated.
Mentions: The aphII promoter region was amplified from pRESQ (37) using PCR primers Pkan-F and Pkan-E5-R (Table 1). The obtained PCR product of 367 bp was blunt-ligated into EcoRV digested pBluescript(II)KS (Stratagene), resulting in pBs-Pkan. A SalI/NotI restriction released a 431 bp fragment comprising the aphII promoter which was then cloned into SalI/NotI digested pORF-codA::upp (InvivoGen, San Diego, USA), yielding plasmid pORF-Pkan-codAupp. The Pkan-codA::upp cassette was subsequently isolated from pORF-Pkan-codAupp as a 2.4 kb SmaI/NheI fragment and ligated into EcoRV/XbaI digested pSET152 resulting in plasmid pSET-Pkan-codAupp (Figure 2). The Pkan-codA cassette (1733 bp) was amplified from pSET-Pkan-codAupp using primers Pkan-F and codA-R2 (Table 1) and ligated into EcoRV digested pSET152, resulting in pSET-Pkan-codA (Figure 2). Suicide plasmid pSelAct (Figure 3) was constructed by ligating a 2.4 kb Klenow-treated EcoRI/NheI fragment of pORF-Pkan-codAupp into SspI digested pBs-Apra-ori dephosphoryated with alkaline phosphatase. Plasmid pBs-Apra-ori was constructed from pBluescript(II)KS in which the bla cassette was removed with BspHI, followed by Klenow treatment and replaced by an apramycin-oriT cassette obtained as a 1.3 kb XbaI fragment (Klenow treated) from pIJ773 (13).Figure 1.

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