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Rapid and highly efficient method for scarless mutagenesis within the Salmonella enterica chromosome.

Blank K, Hensel M, Gerlach RG - PLoS ONE (2011)

Bottom Line: Here we demonstrate, for the first time, the combination of the Red recombinase system with I-SceI endonuclease-based selection of successful recombinants after electroporation with short synthetic oligonucleotides.We show the generation of scarless gene knockouts as well as site-directed mutagenesis using the Salmonella virulence-associated two component signaling system PhoPQ.The presented approach is very versatile for generating in-frame deletions, point mutations or insertions within bacterial chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Junior Research Group 3, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.

ABSTRACT
Direct manipulation of bacterial chromosomes by recombination-based techniques has become increasingly important for both cognitive and applied research. Here we demonstrate, for the first time, the combination of the Red recombinase system with I-SceI endonuclease-based selection of successful recombinants after electroporation with short synthetic oligonucleotides. We show the generation of scarless gene knockouts as well as site-directed mutagenesis using the Salmonella virulence-associated two component signaling system PhoPQ. The presented approach is very versatile for generating in-frame deletions, point mutations or insertions within bacterial chromosomes.

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Related in: MedlinePlus

Quantification of intracellular survival within RAW264.7 macrophage-like cells using gentamicin protection assay.The intracellular replication rates of the mutant strains WRG6 (ΔphoQ) and WRG23 (phoQ-T48I) were compared to S. Typhimurium WT and the previously characterized mutants P2D6 (ssaV::mTn5) and CS022 (pho-24). After infection with a MOI of 5 non-internalized bacteria were killed by gentamicin treatment and the intracellular bacteria were quantified 2 h and 16 h after infection by plating of cell lysates. Intracellular replication rates are expressed by the quotient of 16 h colony forming units (CFU) and the 2 h CFU. One representative of three independent experiments done in triplicates is shown. Statistical analysis by Student's t-test was done by comparison with the WT or by comparing individual strains as depicted: *** P<0.001.
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pone-0015763-g004: Quantification of intracellular survival within RAW264.7 macrophage-like cells using gentamicin protection assay.The intracellular replication rates of the mutant strains WRG6 (ΔphoQ) and WRG23 (phoQ-T48I) were compared to S. Typhimurium WT and the previously characterized mutants P2D6 (ssaV::mTn5) and CS022 (pho-24). After infection with a MOI of 5 non-internalized bacteria were killed by gentamicin treatment and the intracellular bacteria were quantified 2 h and 16 h after infection by plating of cell lysates. Intracellular replication rates are expressed by the quotient of 16 h colony forming units (CFU) and the 2 h CFU. One representative of three independent experiments done in triplicates is shown. Statistical analysis by Student's t-test was done by comparison with the WT or by comparing individual strains as depicted: *** P<0.001.

Mentions: For further phenotypic characterization of the obtained mutants, we used RAW264.7 mouse macrophage-like cells in an infection model and gentamicin protection assays as described [22] to quantify intracellular survival. Using a multiplicity of infection (MOI) of 5, constitutive active PhoQ (WRG23) as well as a phoQ deletion (WRG6) were attenuated comparable to the ssaV-deficient strain P2D6 which cannot translocate SPI-2 effectors [23] or the previously described T48I-harboring strain CS022 (Fig. 4) [18]. For complementation WRG6 and WRG23 were transformed with the low-copy plasmid pWRG103 containing PphoP-phoPQ. With introduction of pWRG103 intracellular replication rates of WRG6 as well as WRG23 could be restored to WT levels (Fig. 4).


Rapid and highly efficient method for scarless mutagenesis within the Salmonella enterica chromosome.

Blank K, Hensel M, Gerlach RG - PLoS ONE (2011)

Quantification of intracellular survival within RAW264.7 macrophage-like cells using gentamicin protection assay.The intracellular replication rates of the mutant strains WRG6 (ΔphoQ) and WRG23 (phoQ-T48I) were compared to S. Typhimurium WT and the previously characterized mutants P2D6 (ssaV::mTn5) and CS022 (pho-24). After infection with a MOI of 5 non-internalized bacteria were killed by gentamicin treatment and the intracellular bacteria were quantified 2 h and 16 h after infection by plating of cell lysates. Intracellular replication rates are expressed by the quotient of 16 h colony forming units (CFU) and the 2 h CFU. One representative of three independent experiments done in triplicates is shown. Statistical analysis by Student's t-test was done by comparison with the WT or by comparing individual strains as depicted: *** P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3021506&req=5

pone-0015763-g004: Quantification of intracellular survival within RAW264.7 macrophage-like cells using gentamicin protection assay.The intracellular replication rates of the mutant strains WRG6 (ΔphoQ) and WRG23 (phoQ-T48I) were compared to S. Typhimurium WT and the previously characterized mutants P2D6 (ssaV::mTn5) and CS022 (pho-24). After infection with a MOI of 5 non-internalized bacteria were killed by gentamicin treatment and the intracellular bacteria were quantified 2 h and 16 h after infection by plating of cell lysates. Intracellular replication rates are expressed by the quotient of 16 h colony forming units (CFU) and the 2 h CFU. One representative of three independent experiments done in triplicates is shown. Statistical analysis by Student's t-test was done by comparison with the WT or by comparing individual strains as depicted: *** P<0.001.
Mentions: For further phenotypic characterization of the obtained mutants, we used RAW264.7 mouse macrophage-like cells in an infection model and gentamicin protection assays as described [22] to quantify intracellular survival. Using a multiplicity of infection (MOI) of 5, constitutive active PhoQ (WRG23) as well as a phoQ deletion (WRG6) were attenuated comparable to the ssaV-deficient strain P2D6 which cannot translocate SPI-2 effectors [23] or the previously described T48I-harboring strain CS022 (Fig. 4) [18]. For complementation WRG6 and WRG23 were transformed with the low-copy plasmid pWRG103 containing PphoP-phoPQ. With introduction of pWRG103 intracellular replication rates of WRG6 as well as WRG23 could be restored to WT levels (Fig. 4).

Bottom Line: Here we demonstrate, for the first time, the combination of the Red recombinase system with I-SceI endonuclease-based selection of successful recombinants after electroporation with short synthetic oligonucleotides.We show the generation of scarless gene knockouts as well as site-directed mutagenesis using the Salmonella virulence-associated two component signaling system PhoPQ.The presented approach is very versatile for generating in-frame deletions, point mutations or insertions within bacterial chromosomes.

View Article: PubMed Central - PubMed

Affiliation: Junior Research Group 3, Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany.

ABSTRACT
Direct manipulation of bacterial chromosomes by recombination-based techniques has become increasingly important for both cognitive and applied research. Here we demonstrate, for the first time, the combination of the Red recombinase system with I-SceI endonuclease-based selection of successful recombinants after electroporation with short synthetic oligonucleotides. We show the generation of scarless gene knockouts as well as site-directed mutagenesis using the Salmonella virulence-associated two component signaling system PhoPQ. The presented approach is very versatile for generating in-frame deletions, point mutations or insertions within bacterial chromosomes.

Show MeSH
Related in: MedlinePlus