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Applied and Environmental Microbiology, July 2006, p. 4717-4725, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00492-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase

Martin Köstner, Britta Schmidt, Ralph Bertram, and Wolfgang Hillen*

Lehrstuhl für Mikrobiologie, Friedrich Alexander Universität Erlangen-Nürnberg, D-91058 Erlangen, Germany

Received 1 March 2006/ Accepted 21 March 2006

We report the construction and application of a novel insertion element for transposase-mediated mutagenesis in gram-negative bacteria. Besides Kmr as a selectable marker, the insertion element InsTetG–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 InsTetG–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 revTetRr2, while only TetR was employed in Salmonella. Screening of the InsTetG–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 InsTetG–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.

* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie, Friedrich Alexander Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany. Phone: 49-9131-85-28081. Fax: 49-9131-85-28082. E-mail: whillen{at}biologie.uni-erlangen.de.

Applied and Environmental Microbiology, July 2006, p. 4717-4725, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.00492-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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