A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae

The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements P(tac) and P(BAD) to versions that have an additional theophylline-responsive riboswitch (P(tac)-riboswitch and P(BAD)-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with P(tac) > P(BAD) > P(BAD)-riboswitch > P(tac)-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for P(tac) > P(tac)-riboswitch > P(BAD); while the newly developed P(BAD)-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible P(BAD)-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of V. cholerae and could be adapted for use in other species.