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An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.

Choi KH, Schweizer HP - BMC Microbiol. (2005)

Bottom Line: Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker.The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.With appropriate modifications, the method should be applicable to other bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.

ABSTRACT

Background: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.

Results: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.

Conclusion: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.

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PCR analysis of marked and unmarked P. aeruginosa PA1520 deletion strains. Colony PCR was performed on either wild-type PAO1 and its PA1520 mutant derivatives, either containing a marked (PA1520::FRT-Gm-FRT) or unmarked (PA1520::FRT) PA1520 deletion. The sizes of the expected PCR fragments are indicated. Note that the short deletion removes 41 bp of the PA1520 coding sequence, corresponding to codons 131 to 145, but replaces these sequences with a 85 bp FRT scar. Lane M contained Hi-Lo molecular size markers of the indicated sizes from Minnesota Molecular.
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Figure 5: PCR analysis of marked and unmarked P. aeruginosa PA1520 deletion strains. Colony PCR was performed on either wild-type PAO1 and its PA1520 mutant derivatives, either containing a marked (PA1520::FRT-Gm-FRT) or unmarked (PA1520::FRT) PA1520 deletion. The sizes of the expected PCR fragments are indicated. Note that the short deletion removes 41 bp of the PA1520 coding sequence, corresponding to codons 131 to 145, but replaces these sequences with a 85 bp FRT scar. Lane M contained Hi-Lo molecular size markers of the indicated sizes from Minnesota Molecular.

Mentions: To expedite gene replacement, an improved electroporation method [17] was used for transfer of the suicide plasmid pEX18ApGW-Gene::Gm into P. aeruginosa instead of the traditionally employed conjugation method. Although after transfer into P. aeruginosa double cross-over events can occur frequently (>50%), they can also be rare (<1–5%). In the latter instance, merodiploids formed via integration of the suicide plasmid by a single cross-over event (Fig. 4). The merodiploid state was then resolved via sucrose selection in the presence of gentamycin, resulting in deletion of the wild type gene. For generation of unmarked deletion mutants, the Gmr marker was subsequently removed by deletion of a 967 bp fragment using Flp recombinase. The presence of the desired deletion in either the marked or unmarked PA1520 mutant was verified by colony PCR utilizing the gene-specific up and down primers (Fig. 5).


An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.

Choi KH, Schweizer HP - BMC Microbiol. (2005)

PCR analysis of marked and unmarked P. aeruginosa PA1520 deletion strains. Colony PCR was performed on either wild-type PAO1 and its PA1520 mutant derivatives, either containing a marked (PA1520::FRT-Gm-FRT) or unmarked (PA1520::FRT) PA1520 deletion. The sizes of the expected PCR fragments are indicated. Note that the short deletion removes 41 bp of the PA1520 coding sequence, corresponding to codons 131 to 145, but replaces these sequences with a 85 bp FRT scar. Lane M contained Hi-Lo molecular size markers of the indicated sizes from Minnesota Molecular.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1173109&req=5

Figure 5: PCR analysis of marked and unmarked P. aeruginosa PA1520 deletion strains. Colony PCR was performed on either wild-type PAO1 and its PA1520 mutant derivatives, either containing a marked (PA1520::FRT-Gm-FRT) or unmarked (PA1520::FRT) PA1520 deletion. The sizes of the expected PCR fragments are indicated. Note that the short deletion removes 41 bp of the PA1520 coding sequence, corresponding to codons 131 to 145, but replaces these sequences with a 85 bp FRT scar. Lane M contained Hi-Lo molecular size markers of the indicated sizes from Minnesota Molecular.
Mentions: To expedite gene replacement, an improved electroporation method [17] was used for transfer of the suicide plasmid pEX18ApGW-Gene::Gm into P. aeruginosa instead of the traditionally employed conjugation method. Although after transfer into P. aeruginosa double cross-over events can occur frequently (>50%), they can also be rare (<1–5%). In the latter instance, merodiploids formed via integration of the suicide plasmid by a single cross-over event (Fig. 4). The merodiploid state was then resolved via sucrose selection in the presence of gentamycin, resulting in deletion of the wild type gene. For generation of unmarked deletion mutants, the Gmr marker was subsequently removed by deletion of a 967 bp fragment using Flp recombinase. The presence of the desired deletion in either the marked or unmarked PA1520 mutant was verified by colony PCR utilizing the gene-specific up and down primers (Fig. 5).

Bottom Line: Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker.The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.With appropriate modifications, the method should be applicable to other bacteria.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.

ABSTRACT

Background: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process.

Results: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family.

Conclusion: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.

Show MeSH
Related in: MedlinePlus