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

ABSTRACT: 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.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.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.

SUBMITTER: Choi KH

PROVIDER: S-EPMC1173109 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

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

Choi Kyoung-Hee KH Schweizer Herbert P HP

BMC microbiology 20050523

<h4>Background</h4>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.<h4>Results</h4>We describe a method for rapid generation ...[more]

PMID: 15907219

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Project description:BackgroundPseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune compromised patients. As such P. aeruginosa is one of the most well studied organisms in terms of its genetics. However, the construction of gene deletions and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum two weeks. ??METHODS: We have developed a rapid streamlined method to create clean deletion mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting deletion plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and deletion mutants are identified via sacB mediated sucrose counter-selection. ?RESULTS: T?he current method was employed to generate clean gene deletions of the heme assimilation system anti-? factor, hasS and the virulence regulator involving ECF system anti-? and ? factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene deletion was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI deletions, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly deletion mutants were constructed with single base pair precision to generate the respective vreA and vreI deletions, while maintaining the start and stop codon of the respective genes. Overall, this method allows for rapid construction of gene deletions in P. aeruginosa with base pair precision.ConclusionThis method from the construction of the suicide vector to sequence confirmation of the unmarked gene deletion can be performed in one week, without the requirement for expensive proprietary reagents or instruments. The precision of Gibson assembly and the fact the accuracy in generating the desirable construct is 95%, makes this a viable and attractive alternative to previous methods.

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

Publications

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

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