Construction of a tetracycline inducible expression vector and characterization of its use in Vibrio cholerae

• A tetracycline-regulated expression vector was constructed for Vibrio cholerae.
• The vector was based on the Tn10 tetracycline regulatory elements.
• Heterologous gene expression was titratable with anhydrotetracycline.
• The expression vector encoded a multiple cloning site and plasmid addiction system.
• The expression vector represents a new tool for Vibrio cholerae genetic analysis.

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.