Position-Dependent Activation and Repression by As(III) in an Engineered BetI Transcriptional Repressor

Abstract

Arsenite [As(III)]-responsive transcriptional regulation is widely employed by microorganisms for detoxification and homeostasis. Engineering As(III)-responsive behavior into transcription factors that are intrinsically metal-insensitive remains challenging. Here, we introduced a third cysteine residue at different positions in the engineered BetIL79C/Q81C scaffold and evaluated the resulting whole-cell sfGFP reporter responses. The magnitude and direction of the As(III)-dependent reporter change varied with the position of the introduced cysteine. Under combined As(III) and choline inputs, selected variants exhibited operational OR-like or As(III)-inhibited reporter-output patterns over defined concentration ranges. To explore possible structural interpretations for these reporter phenotypes, we performed molecular dynamics simulations using predefined dithiol- and trithiol-coordination models. Simulations of these models yielded distinct fluctuation patterns in the DNA-binding domain, providing testable structural hypotheses for the observed reporter phenotypes. Because neither As(III) binding to the engineered cysteine sites nor As(III)-dependent changes in BetI–DNA binding were directly measured, the reporter assays and simulations do not establish the underlying molecular mechanism. Nevertheless, our experiments show that cysteine placement can tune the direction of As(III)-dependent whole-cell sfGFP output in an engineered BetI regulatory system and thereby expand the design space for multi-input transcriptional devices.