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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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Effect of deletions of P. aeruginosa GntR homologs on fabA expression. The indicated genes were deleted in strain PAO1 containing a chromosomally integrated fabA'-lacZ fusion (labelled PAO1). The control was PAO1 with a fabAΔ30'-lacZ fusion; this strain harbours the same fabA'-lacZ fusion but contains a deletion of the putative 30 nucleotide activator-binding site in the fabA promoter region. Strains were grown to mid-log phase in LB medium containing 0.05% Brij 58 +/- 0.05% oleic acid and β-galactosidase expression was monitored in triplicate samples. The dotted line marks expression levels observed in the putative activator binding-site mutant and similar levels would be expected in an activator mutant.
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Figure 6: Effect of deletions of P. aeruginosa GntR homologs on fabA expression. The indicated genes were deleted in strain PAO1 containing a chromosomally integrated fabA'-lacZ fusion (labelled PAO1). The control was PAO1 with a fabAΔ30'-lacZ fusion; this strain harbours the same fabA'-lacZ fusion but contains a deletion of the putative 30 nucleotide activator-binding site in the fabA promoter region. Strains were grown to mid-log phase in LB medium containing 0.05% Brij 58 +/- 0.05% oleic acid and β-galactosidase expression was monitored in triplicate samples. The dotted line marks expression levels observed in the putative activator binding-site mutant and similar levels would be expected in an activator mutant.

Mentions: We are interested in characterizing the activator involved in regulation of expression of the fabAB operon, which encodes two essential enzymes of de novo fatty acid biosynthesis [18]. Expression of the fabAB operon is repressed by exogenously added fatty acids, most effectively by oleic acid, and preliminary results from our laboratory indicate that this may be due to relief of activation by a transcriptional regulator which binds to a 30 nucleotide site located in the fabAB promoter region (Choi and Schweizer, unpublished results). This 30 bp region is only found and conserved in Pseudomonas spp. Its deletion dramatically reduces transcription of the fabAB operon and the residual transcriptional activity is no longer fatty acid responsive (Fig. 6). In E. coli, transcriptional activation of the unlinked fabA and fabB genes is achieved by FadR, a regulator belonging to a group of transcriptional regulators with the GntR family signature [19,20]. The PAO1 chromosome encodes more than 30 regulatory proteins with this signature. Of these, 27 have not been assigned function. Using the methodology described above for one of these genes, PA1520, we deleted 25 of the 27 genes (we could not delete the presumably essential PA1285 and PA2299) in less than 4 weeks in strain PAO1 with a chromosomally integrated fabA'-lacZ transcriptional fusion and analyzed β-galactosidase expression in the resulting mutant strains (Fig. 6). In exponential phase cells, the overall pattern of fabA expression was indistinguishable from the parental strain in all 25 mutants, indicating that none of these genes encodes the fabAB activator. We would have expected an expression pattern similar to that observed in the PAO1::fabAΔ30'-lacZ control strain which contains a deletion of the 30 nucleotide putative activator-binding site.


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

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

Effect of deletions of P. aeruginosa GntR homologs on fabA expression. The indicated genes were deleted in strain PAO1 containing a chromosomally integrated fabA'-lacZ fusion (labelled PAO1). The control was PAO1 with a fabAΔ30'-lacZ fusion; this strain harbours the same fabA'-lacZ fusion but contains a deletion of the putative 30 nucleotide activator-binding site in the fabA promoter region. Strains were grown to mid-log phase in LB medium containing 0.05% Brij 58 +/- 0.05% oleic acid and β-galactosidase expression was monitored in triplicate samples. The dotted line marks expression levels observed in the putative activator binding-site mutant and similar levels would be expected in an activator mutant.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1173109&req=5

Figure 6: Effect of deletions of P. aeruginosa GntR homologs on fabA expression. The indicated genes were deleted in strain PAO1 containing a chromosomally integrated fabA'-lacZ fusion (labelled PAO1). The control was PAO1 with a fabAΔ30'-lacZ fusion; this strain harbours the same fabA'-lacZ fusion but contains a deletion of the putative 30 nucleotide activator-binding site in the fabA promoter region. Strains were grown to mid-log phase in LB medium containing 0.05% Brij 58 +/- 0.05% oleic acid and β-galactosidase expression was monitored in triplicate samples. The dotted line marks expression levels observed in the putative activator binding-site mutant and similar levels would be expected in an activator mutant.
Mentions: We are interested in characterizing the activator involved in regulation of expression of the fabAB operon, which encodes two essential enzymes of de novo fatty acid biosynthesis [18]. Expression of the fabAB operon is repressed by exogenously added fatty acids, most effectively by oleic acid, and preliminary results from our laboratory indicate that this may be due to relief of activation by a transcriptional regulator which binds to a 30 nucleotide site located in the fabAB promoter region (Choi and Schweizer, unpublished results). This 30 bp region is only found and conserved in Pseudomonas spp. Its deletion dramatically reduces transcription of the fabAB operon and the residual transcriptional activity is no longer fatty acid responsive (Fig. 6). In E. coli, transcriptional activation of the unlinked fabA and fabB genes is achieved by FadR, a regulator belonging to a group of transcriptional regulators with the GntR family signature [19,20]. The PAO1 chromosome encodes more than 30 regulatory proteins with this signature. Of these, 27 have not been assigned function. Using the methodology described above for one of these genes, PA1520, we deleted 25 of the 27 genes (we could not delete the presumably essential PA1285 and PA2299) in less than 4 weeks in strain PAO1 with a chromosomally integrated fabA'-lacZ transcriptional fusion and analyzed β-galactosidase expression in the resulting mutant strains (Fig. 6). In exponential phase cells, the overall pattern of fabA expression was indistinguishable from the parental strain in all 25 mutants, indicating that none of these genes encodes the fabAB activator. We would have expected an expression pattern similar to that observed in the PAO1::fabAΔ30'-lacZ control strain which contains a deletion of the 30 nucleotide putative activator-binding site.

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