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Appl Environ Microbiol. 2006 Jul;72(7):4717-25.

Generating tetracycline-inducible auxotrophy in Escherichia coli and Salmonella enterica serovar Typhimurium by using an insertion element and a hyperactive transposase.

Author information

1
Lehrstuhl für Mikrobiologie, Friedrich Alexander Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany.

Abstract

We report the construction and application of a novel insertion element for transposase-mediated mutagenesis in gram-negative bacteria. Besides Km(r) as a selectable marker, the insertion element InsTet(G-)1 carries the anhydrotetracycline (atc)-regulated outward-directed PA promoter so that atc-dependent conditional gene knockouts or knockdowns are generated. The complex formed between the purified hyperactive transposase and InsTet(G-)1 was electroporated into Escherichia coli or Salmonella enterica serovar Typhimurium, and mutant pools were collected. We used E. coli strains with either TetR or the reverse variant revTetR(r2), while only TetR was employed in Salmonella. Screening of the InsTet(G-)1 insertion mutant pools revealed 15 atc-regulatable auxotrophic mutants for E. coli and 4 atc-regulatable auxotrophic mutants for Salmonella. We have also screened one Salmonella mutant pool in murine macrophage-like J774-A.1 cells using ampicillin enrichment. Two mutants with the InsTet(G-)1 insertion in the gene pyrE or argA survived this procedure, indicating a reduced intracellular growth rate in J774-A.1 cells. The nature of the mutants and the modes of their regulation are discussed.

PMID:
16820464
PMCID:
PMC1489314
DOI:
10.1128/AEM.00492-06
[Indexed for MEDLINE]
Free PMC Article

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