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

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 BetI_L79C/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.