Presentation Information
[3FMBS-12]Bidirectional functional switching of proteins via arsenic–cysteine interactions for microbial arsenic sensing and synthetic control
○Shigeko Kawai-Noma1 (1. Chiba University (Japan))
Keywords:
arsenite,aggregation,whole cell sensor,protein engineering
Background: Arsenic is a ubiquitous and highly toxic metalloid, and its detection remains an important challenge. While ICP-MS offers high sensitivity, it requires expensive instrumentation. Microbial biosensors based on arsenic-responsive regulators such as ArsR provide cost-effective alternatives, but most rely on ON-type transcriptional outputs, limiting functional diversity. Arsenic selectively binds to cysteine residues, inducing structural changes and aggregation. We hypothesized that this interaction can be used to control protein function. Here, we developed two complementary systems: (i) an OFF-type transcription factor sensor based on aggregation, and (ii) an ON-type enzyme activation system based on protein assembly.
Methods: For the OFF-type sensor, we utilized LuxR, a cysteine-rich transcriptional activator that aggregates upon arsenic binding, resulting in loss of activity. To enhance sensitivity, a chimeric protein composed of LuxR and a super-repressor ArsR mutant (ArsR_C34Y) was constructed to suppress the arsenic efflux pump ArsB. The construct was expressed in Escherichia coli, and GFP fluorescence was used as a readout. Intracellular arsenic levels were quantified by ICP-MS. For the ON-type system, cysteine residues were introduced into chloramphenicol acetyltransferase (CAT) to enable arsenic-mediated intermolecular assembly. Variants with increased activity in the presence of arsenic were selected and combined with a transcriptional regulation system.
Results: The LuxR-based system showed an OFF-type response, where arsenic-induced aggregation reduced fluorescence output. The ArsR_C34Y-LuxR construct improved sensitivity, showing maximal response at 1 ng/mL arsenic. ICP-MS analysis revealed that intracellular arsenic reached a saturation level (~25–30 ag/cell), and the engineered strain achieved this threshold at lower external concentrations due to suppressed efflux. Sensor output correlated with intracellular arsenic levels, indicating that accumulation governs performance. In contrast, engineered CAT variants exhibited increased enzymatic activity upon arsenic addition, consistent with assembly via cysteine coordination. Integration with transcriptional regulation suggested potential for arsenic-responsive logic circuits.
Discussion: These results demonstrate that arsenic–cysteine interactions enable bidirectional control of protein function. Sensitivity enhancement in the LuxR system arises from modulation of intracellular arsenic rather than protein design alone. The CAT system reveals a distinct activation mechanism via metal-induced assembly. Together, these findings highlight aggregation as a functional, rather than purely toxic, phenomenon.
Conclusion: We established OFF- and ON-type protein switching systems based on arsenic–cysteine interactions, providing a versatile framework for biosensing and synthetic biology applications.
Methods: For the OFF-type sensor, we utilized LuxR, a cysteine-rich transcriptional activator that aggregates upon arsenic binding, resulting in loss of activity. To enhance sensitivity, a chimeric protein composed of LuxR and a super-repressor ArsR mutant (ArsR_C34Y) was constructed to suppress the arsenic efflux pump ArsB. The construct was expressed in Escherichia coli, and GFP fluorescence was used as a readout. Intracellular arsenic levels were quantified by ICP-MS. For the ON-type system, cysteine residues were introduced into chloramphenicol acetyltransferase (CAT) to enable arsenic-mediated intermolecular assembly. Variants with increased activity in the presence of arsenic were selected and combined with a transcriptional regulation system.
Results: The LuxR-based system showed an OFF-type response, where arsenic-induced aggregation reduced fluorescence output. The ArsR_C34Y-LuxR construct improved sensitivity, showing maximal response at 1 ng/mL arsenic. ICP-MS analysis revealed that intracellular arsenic reached a saturation level (~25–30 ag/cell), and the engineered strain achieved this threshold at lower external concentrations due to suppressed efflux. Sensor output correlated with intracellular arsenic levels, indicating that accumulation governs performance. In contrast, engineered CAT variants exhibited increased enzymatic activity upon arsenic addition, consistent with assembly via cysteine coordination. Integration with transcriptional regulation suggested potential for arsenic-responsive logic circuits.
Discussion: These results demonstrate that arsenic–cysteine interactions enable bidirectional control of protein function. Sensitivity enhancement in the LuxR system arises from modulation of intracellular arsenic rather than protein design alone. The CAT system reveals a distinct activation mechanism via metal-induced assembly. Together, these findings highlight aggregation as a functional, rather than purely toxic, phenomenon.
Conclusion: We established OFF- and ON-type protein switching systems based on arsenic–cysteine interactions, providing a versatile framework for biosensing and synthetic biology applications.
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