Biochemical Investigation of Interactions Between Sagebrush Chemicals and Gene Regulatory Elements in the Sage Grouse Microbiome

Digestive microbial communities exist at the interface of chemically defended plants and herbivores that eat them. Riboswitches are a common RNA element found in microbes that regulate the expression of downstream genes by directly binding molecular ligands. However, the existence of microbial riboswitches that respond to chemicals ingested by herbivore hosts has not been confirmed, and it is difficult to directly observe these interactions. We, therefore, set out to investigate riboswitches in the digestive microbiota of Greater Sage-Grouse ( Centrocercus urophasianus ), which specializes in chemically defended sagebrush ( Artemisia spp. ), using a combination of bioinformatics and biochemical approaches. By searching metagenomic sequence data from the sage-grouse cecum, we identified numerous examples of several classes of riboswitches. We chose several specific sequence variants to synthesize and study in the lab. We synthesized the riboswitches using in vitro transcription and tested their ability to bind non-volatile sagebrush chemical extracts using a biochemical probing approach (in-line probing). The approach demonstrates a pathway toward confirming a mechanism for how the digestive microbiota of herbivores detect and respond to the chemically diverse diets of their hosts.