Ratiometric Gas Reporting: A Nondisruptive Approach To Monitor Gene Expression in Soils

Abstract

Fluorescent proteins are ubiquitous tools that are used to monitor the dynamic functions of natural and synthetic genetic circuits. However, these visual reporters can only be used in transparent settings, a limitation that complicates nondisruptive measurements of gene expression within many matrices, such as soils and sediments. We describe a new ratiometric gas reporting method for nondisruptively monitoring gene expression within hard-to-image environmental matrices. With this approach, CH is continuously synthesized by ethylene forming enzyme to provide information on viable cell number, and CHBr is conditionally synthesized by placing a methyl halide transferase gene under the control of a conditional promoter. We show that ratiometric gas reporting enables the creation of Escherichia coli biosensors that report on acylhomoserine lactone (AHL) autoinducers used for quorum sensing by Gram-negative bacteria. Using these biosensors, we find that an agricultural soil decreases the bioavailable concentration of a long-chain AHL up to 100-fold. We also demonstrate that these biosensors can be used in soil to nondisruptively monitor AHLs synthesized by Rhizobium leguminosarum and degraded by Bacillus thuringiensis. Finally, we show that this new reporting approach can be used in Shewanella oneidensis, a bacterium that lives in sediments.

Soil organic matter attenuates the efficacy of flavonoid-based plant-microbe communication

“…Similarly, soil geochemistry and its interaction with chemical signals could provide opportunities for plants to manipulate interactions with their plant neighbors and affect their competition with proximal plants (28). The observed OC-flavonoid quenching observed here might be used by plants under selection to foster OC deposition.…”

“…3, B and E). To measure quercetin availability, we engineered a biosensor that uses the transcriptional repressor QdoR (18) to regulate the production of an indicator gas (28). When QdoR binds quercetin (Fig.…”

“…When QdoR binds quercetin (Fig. 3, C and F), it switches on the production of an enzyme that synthesizes methyl halide as an indicator gas (28).…”

A split methyl halide transferase AND gate that reports by synthesizing an indicator gas

“…The sMHT fragments characterized herein presented enhanced CH3Br synthesis rates when the fragments were fused to different pairs of interacting proteins, indicating that the sMHT-113 functions as a PCA 15 . Because sMHT generates an indicator gas as an output, this new PCA will enable detection of protein-protein interactions under hard-to-image conditions (e.g., in marine sediments, soils, and wastewater) 23,26 where other PCAs cannot be used due to their generation of a visual signal [6][7][8][9][10][11][12][13][14]28 . Additionally, the sMHT AND gate described herein will be a useful synthetic biology tool for environmental studies that probe micron-scale spatial heterogeneity in hard-to-image matrices.…”

“…This gas diffuses out of matrices and can be quantified in the headspace using a gas chromatograph (GC) mass spectrometer (MS) 25 . MHTs have been used as a reporter to monitor how soil conditions influence conjugation 23 , the bioavailability of acylhomoserine lactones that are used for microbial quorum sensing 26 , and the impact of dissolved organic matter on the bioavailability of flavonoids that mediate plant-microbe communication 27 . Although these studies have provided evidence that MHTs are useful for studying how environmental matrices alter the bioavailability of signals that mediate cell-cell communication, gas…”