Enhancement of Sensitivity in Aggregation-Based Whole-Cell Arsenite Sensor Utilizing Arsenic Metabolism Regulation

Arsenite [As(III)] is a toxic substance widely present on Earth, and the development of low-cost and simple microbial-based As(III) sensors has been attracting attention. Recently, we discovered that the protein LuxR, which contains multiple cysteine residues with high affinity for As(III), forms an insoluble structure upon binding to As(III) and exhibits OFF-switching properties as a quorum sensing transcriptional activator. Based on this property, the LuxR sensor operates on a new principle distinct from conventional whole-cell As(III) sensors; however, its sensitivity remains a challenge. In this study, we aimed to improve the sensitivity of the whole-cell OFF-type As(III) sensor by increasing the frequency of intracellular interactions between the sensor protein and As(III). We utilized the super-repressor properties of ArsR, a transcriptional repressor of the As(III)-metabolizing <i>ars</i> operon, achieved by replacing C34 in its As(III)-binding domain with Y. By linking ArsR_C34Y with the OFF-type As(III) sensor protein LuxR, we constructed a single plasmid to create a portable ArsR_C34Y-LuxR sensor protein. By suppressing the expression of ArsB, an As(III) efflux transporter encoded in the <i>ars</i> operon, using ArsR_C34Y, we successfully enhanced the sensitivity of the OFF-type As(III) response.