Open AccessGenerating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase
Author(s) -
Martin Köstner,
B. Schmidt,
Ralph Bertram,
Wolfgang Hillen
Publication year - 2006
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.00492-06
Subject(s) - tetr , salmonella enterica , escherichia coli , transposase , auxotrophy , mutant , biology , salmonella , transposable element , mutagenesis , enterobacteriaceae , microbiology and biotechnology , aroa , transposon mutagenesis , insertion , genetics , gene , bacteria , gene expression , repressor
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 intoEscherichia coli orSalmonella enterica serovar Typhimurium, and mutant pools were collected. We usedE. coli strains with either TetR or the reverse variant revTetRr2 , while only TetR was employed inSalmonella . Screening of the InsTetG− 1 insertion mutant pools revealed 15 atc-regulatable auxotrophic mutants forE. coli and 4 atc-regulatable auxotrophic mutants forSalmonella . We have also screened oneSalmonella mutant pool in murine macrophage-like J774-A.1 cells using ampicillin enrichment. Two mutants with the InsTetG− 1 insertion in the genepyrE orargA 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.