(590c) Fungal Highways Enable Migration and Communication of Engineered Bacteria in Soil

To realize the potential of synthetic biology, genetic circuits need to function in mixtures like soil, outside of rich media and controlled conditions. Soil is of particular interest because it facilitates essential natural chemical cycles, supports a diverse microbiome with immense biosynthetic capacity, and interfaces with civilization through agriculture, the built environment, and military action. However, soil is not nutrient-rich (compared to laboratory media) and is poorly mixed. Thus, if a typical host like E. coli even survives, it cannot penetrate into the soil or maintain burdensome plasmids for long. Yet, study of natural soil microbiomes has revealed that bacterial migration and long-distance chemical signaling occurs, facilitated by filamentous fungal highways.

Here, we report an interkingdom microbial consortium that enables genetic circuit delivery and function in soil. We harnessed the bacterial and chemical transmission properties of fungal highways to deliver a motile engineered bacteria down into soil where it senses a chemical signal and sends that signal to the surface. Several innovations were required. First, we selected an alternative bacterial host – Pseudomonas putida. P. putida is a motile soil bacterium with developed genetic techniques, but few inducible systems. We tested several genetic circuits for function in P. putida, eventually identifying quorum sensing circuits Lux and Las as the most robust, achieving 15-fold induction in soil. We found that function in M9 medium was a good predictor for function in soil. Next, we prototyped several fungal species for growth rate, soil penetration, and compatibility with P. putida, finding that Lyophyllum atratum extends P. putida soil survival and accelerates migration.

Finally, we tested bacterial migration and signal propagation through a soil column, with and without fungi. Bacterial migration in response to a salicylic acid chemoattractant was 15 mm/day whereas no migration was observed in response to water as control. Signal propagated 35 mm and fluorescent protein expression lasted for 48 hours in response to 3OC6-HSL in soil without fungi. With the fungal partner, signal propagated 80 mm and fluorescent protein expression lasted for 120 hours. Interkingdom networks appear key to engineering robust genetic circuit function in soil. Thus, this study builds the foundation for synthetic biology solutions in agriculture, environmental remediation, and buried chemical detection.