Nondestructive Chemical Sensing within Bulk Soil Using 1000 Biosensors Per Gram of Matrix

Abstract

Gene expression can be monitored in hard-to-image environmental materials using gas-reporting biosensors, but these outputs have only been applied in autoclaved matrices that are hydrated with rich medium. To better understand the compatibility of indicator gas reporting with environmental samples, we evaluated how matrix hydration affects the gas signal of an engineered microbe added to a sieved soil. A gas-reporting microbe presented a gas signal in a forest soil (Alfisol) when hydrated to an environmentally relevant osmotic pressure. When the gas signal was concentrated prior to analysis, a biosensor titer of 103 cells/gram of soil produced a significant signal when soil was supplemented with halides. A signal was also observed without halide amendment, but a higher cell titer (106 cells/gram of soil) was required. A sugar-regulated gas biosensor was able to report with a similar level of sensitivity when added to an unsterilized soil matrix, illustrating how gas concentration enables biosensing within a soil containing environmental microbes. These results establish conditions where engineered microbes can report on gene expression in living environmental matrices with decreased perturbation of the soil environment compared to previously reported approaches, using biosensor titers that are orders of magnitude lower than the number of cells typically observed in a gram of soil.

Using Methyl Bromide for Interspecies Cell–Cell Signaling and As a Reporter in a Model Soil Consortium

“…MSCs of varying complexity have been identified that can be stored and revived for soil studies. , These consortia represent an opportunity to advance soil biosensors by testing the compatibility of native soil microbes with indicator gas reporters. − To investigate whether these reporters are compatible with MSC studies, we first evaluated whether individual MSC members present a gas signal that can be detected using GC-MS analysis. These measurements focused on five microbes from a chitin-degrading consortium of microbes, including Dyadobacter fermentans (Df), Ensifer adhaerens (Ea), Rhodococcus sp003130705 (Rh), Streptomyces sp001905665 (St), and Variovorax beijingensis (Vb).…”

“…To investigate if soil affects the Vb gas signal, we evaluated the accumulation of CH 3 Br in the headspace of a soil containing different titers of Vb-mht (10 7 or 10 8 CFU) held at different hydration levels (32 or 64% field capacity). For these experiments, we used a Texas Alfisol, which we previously characterized . Soils were hydrated using MIDV1 containing 100 mM NaBr because this medium was designed to have an osmotic pressure closer to the range found in natural soil conditions .…”

“…To overcome this challenge, we show that one member of this MSC can be programmed to generate a methyl halide signal that greatly exceeds the background methyl halide generated by other MSC members, and we demonstrate the utility of this approach by characterizing an MSC promoter in both a liquid and a soil environment. For the soil measurements, we hydrated previously dried soils using liquid cultures containing the MSC . We find that this synthetic signal can be read out directly using gas chromatography–mass spectrometry (GC-MS) or indirectly using a Methylorubrum extorquens CM4 biosensor, a soil-derived microbe that is distinct from the MSC, which expresses a fluorescent protein in response to methyl halide sensing.…”

“…For the soil measurements, we hydrated previously dried soils using liquid cultures containing the MSC. 20 We find that this synthetic signal can be read out directly using gas chromatography−mass spectrometry (GC-MS) or indirectly using a Methylorubrum extorquens CM4 biosensor, 22 a soilderived microbe that is distinct from the MSC, which expresses a fluorescent protein in response to methyl halide sensing. Furthermore, using soil habitats that range in size from 1 to 80 g of soil, we show that methyl halide cell−cell signaling can be achieved in soils hydrated across a range of levels observed in the environment.…”

Genetic Circuit Design in Rhizobacteria

“…Non-optical reporters have been used to build genetically encoded sensors for soil bacteria processes. The Silberg Lab developed a soil-functional reporter system using sensor circuits that biosynthesized indicator gaseous compounds, methyl bromide and ethylene [163,[209][210][211]. In their initial circuits, a ratiometric gas signal was generated by inducerregulated methyl halide transferase (MHT) expression/ methyl bromide production and constitutive ethylene forming enzyme (EFE) expression/ethylene production.…”