(640c) Development of a Transcriptional Hydrogen Sulfide Biosensor to Function in the Anaerobic Gut Microenvironment

Inflammatory bowels disease (IBD) is a gastrointestinal condition (GI) that currently afflicts more than 1% of adults, and is becoming more prevalent. Hydrogen sulfide (H₂S), a gaseous compound produced by both host epithelial cells and the gut microbiota, has long been associated with IBD, but its effects are disputed and the mechanisms underlying its role in disease unclear. Some studies suggest therapeutic, restorative effects of H₂S in low levels, while others imply that higher levels are toxic, leading to disruption of the mucus barrier and promotion of an inflammatory cascade. Unraveling the dose-dependent effects of H₂S is technically challenging due to its high volatility and reactivity, making it difficult to accurately dose and measure in experimental settings. Development of a genetically encoded biosensor for H₂S would overcome the challenges of traditional chemical assays, enabling non-invasive spatiotemporally resolved measurements.

Here we report the development of a transcriptional H₂S biosensor in Escherichia coli, specifically engineered to function under both anaerobic conditions (normal gut physiology) and under aerobic conditions (characteristic of inflammation). The sensor is based on the H₂S-sensing transcriptional architecture of Rhodobacter capsulatus, a photolithoautotrophic microbe that can use sulfide as an electron donor for photosynthesis. Here H₂S is oxidized by sulfide:quinone oxidoreductase (Sqr) to form a persulfide, which forms an intramolecular tetrasulfide bond with the repressor SqrR, leading to dissociation from its cognate promoter and transcription of the target gene. We reconstituted the system in E. coli, individually validating and optimizing each component to maximize sensor dynamic and operating range. Optimization of the SqrR-controlled promoter and 5’-untranslated region through screening of upstream elements and RBS engineering led to a 5-fold increase in fluorescence reporter output. Improving functional SqrR expression through use of the SUMO tag provided tight repression of the promoter. Heterologous expression of the R. capsulatus Sqr resulted in rapid aerobic conversion of H₂S to glutathione persulfide, as determined by LC-HRMS. These combined efforts resulted in a H₂S sensor with an operational range under aerobic conditions from 50 µM to 1 mM, with a 15-fold dynamic range.

Adapting the sensor for anaerobic conditions required additional engineering of the electron transport chain. Specifically, while the primary quinone used by E. coli under aerobic conditions is ubiquinone, under anaerobic conditions this is replaced by menaquinone, the donor for fumarate and nitrate reductase. However, we found the R. capsulatus Sqr was incapable of using menaquinone. Testing an uncharacterized homolog from Wolinella succinogenes – an anaerobe known to couple sulfide oxidation to fumarate reduction which we therefore hypothesized may encode a menaquinone-dependent Sqr – resulted in robust H₂S conversion to persulfide in E. coli with either nitrate or fumarate as terminal electron acceptors. Integration of the W. succinogenes Sqr into the sensor architecture resulted in a sensor capable of detecting up to 750 µM H₂S under anaerobic conditions. This work provides a genetically encoded H₂S biosensor that could be used for non-invasive detection of elevated sulfide levels in animal models, and which may have eventual utility as an early diagnostic. More broadly, it also provides a roadmap for the construction and optimization of biosensors that require enzymatic conversion of the target molecule for detection.