title: Construction of a tetracycline inducible expression vector and characterization of its use in Vibrio cholerae.
abstract: We report the construction of a tetracycline inducible expression vector that allows regulated gene expression in the enteric pathogen Vibrio cholerae. The expression vector, named pXB300, contains the tetracycline regulatory elements from Tn10, a multiple cloning site downstream of the tetA promoter and operator sequences, a ColE1 origin of replication, a β-lactamase resistance gene for positive selection, and the hok/sok addiction system for selection in the absence of antibiotic. The function of the tetracycline expression system was demonstrated by cloning lacZ under control of the tetA promoter and quantifying β-galactosidase expression in Escherichia coli and V. cholerae. The utility for pXB300 was documented by complementation of V. cholerae virulence mutants during growth under virulence inducing conditions. The results showed that pXB300 allowed high-level expression of recombinant genes with linear induction in response to the exogenous concentration of the inducer anhydrotetracycline. We further show that pXB300 was reliably maintained in V. cholerae during growth in the absence of antibiotic selection.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
authors.forename: X Renee
authors.initials: XR
authors.lastname: Bina
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
authors.forename: Eileen A
authors.initials: EA
authors.lastname: Wong
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Bina
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA. Electronic address: JBina@pitt.edu.
authors.forename: James E
authors.initials: JE
authors.lastname: Bina
mesh_terms: D017920:Antiporters / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D002772:Cholera Toxin / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000187:drug effects* / Q000235:genetics; D009154:Mutation; D010957:Plasmids / Q000187:drug effects / Q000235:genetics; D011401:Promoter Regions, Genetic; D013752:Tetracycline / Q000494:pharmacology*; D013754:Tetracyclines / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics; D014734:Vibrio cholerae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D013754:Tetracyclines; D014157:Transcription Factors; D037521:Virulence Factors; C079051:tetA protein, Bacteria; C499324:toxR protein, Vibrio cholerae; C016229:4-epianhydrotetracycline; D002772:Cholera Toxin; D013752:Tetracycline
keywords: Anhydrotetracycline; Expression vector; ToxR regulon; Vibrio cholerae