Monitoring Microbial Genetic Circuits in Soils Using Orthogonal Enzymes That Synthesize Volatile Metabolites

The tools of synthetic biology have the potential to significantly improve our understanding of the roles that microbes play in soil development, water quality, crop yields, and greenhouse gas production. However, synthetic biology has not yet seen widespread applications within many environmental matrices because it is hard to monitor the dynamics of genetic circuit outputs using visual reporters within soils. To develop strategies that enable the non-disruptive detection of microbial gene expression within organisms that are situated in hard-to-image environmental matrices, we have been investigating whether natural and engineered enzymes can be used as reporters of microbial gene expression. For these studies, we have been exploring whether enzymes that produce small volatile metabolites can be used individually or in parallel to report on gene expression. For our initial efforts, we evaluated whether horizontal gene transfer can be monitored within a soil over a range of environmentally-relevant hydration conditions by coupling expression of a methyl halide transferase to the transfer of a conjugative plasmid. We found that that the methyl halide released from a soil matrix displays a strong linear correlation with the number of transconjugant bacteria that form in soils. We have also engineered a split methyl halide transferase whose fragments must associate to form an active enzyme, and we are using this split protein to monitor conjugation between two microbes within the context of a mixed microbial community. To study the activities of conditional promoters in soils, we have been using ethylene forming enzyme and methyl halide transferases to develop a ratiometric gas reporting strategy. With this ratiometric reporting approach, one enzyme is constitutively produced to provide information on cell number and metabolic activity, while the second enzyme is coupled to a conditional promoter. We have found that the ratio of the two volatile metabolites synthesized by these enzymes can be used to report on the concentration of diffusible signals used for microbe-microbe and microbe-plant communication within soils. Ongoing efforts are focused on using these gas-reporting strategies to study the effects of matrix composition on cell-cell signaling and horizontal gene transfer. Our results demonstrate how volatile gas production can be used to monitor microbial processes within bulk soils that are not compatible with visual reporters. This approach for monitoring gene expression is expected to have applications in studying natural and engineered genetic circuits a wide range of microbes and matrices.