The phospho-ferrozine assay: a tool to study bacterial redox-active metabolites produced at the plant root.

Soil microbial communities are pivotal to plant health and nutrient acquisition. It is becoming increasingly clear that many interactions, both among and between microbes and plants, are governed by small bioactive molecules or "secondary metabolites" that can aid in communication, competition, and nutrient uptake. Yet, secondary metabolite biogeography - who makes what, where, and why-is in its infancy. Further, secondary metabolite biosynthesis genes are often silent or weakly expressed under standard laboratory conditions, making it incredibly difficult to study these small molecules. To begin to address these dual challenges, we focused on redox-active metabolites (RAMs), a specific class of small molecules, and took advantage of recent findings that many RAMs aid in acquiring phosphorus and that their production is frequently stimulated by stress for this macronutrient. We developed a screen for RAM-producing bacteria that leverages phosphorus limitation to stimulate metabolite biosynthesis and uses a colorimetric (ferrozine) iron-reduction assay to identify redox activity. We isolated 557 root-associated bacteria from grasses collected at sites across the United States (Santa Rita Experimental Range [AZ], Konza Prairie Biological Station [KS], and Harvard Forest [MA]) and from commercial tomato plants and screened them for RAM production. We identified 128 soil isolates of at least 19 genera across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes that produced RAMs under phosphorus stress. Our work reveals that the production of RAMs under phosphorus stress is common across diverse soil bacteria and provides an approach to screen for these small molecules rapidly.IMPORTANCEBy secreting secondary metabolites, bacteria at the plant root can defend against diseases and help acquire essential nutrients. However, the genes that synthesize secondary metabolites are typically inactive or are weakly expressed under standard laboratory conditions. This fact makes it difficult to study these small molecules and hinders the discovery of novel small molecules that may play crucial roles in agricultural and biomedical settings. Here, we focus on redox-active metabolites (RAMs), a class of secondary metabolites that can help bacteria solubilize phosphorus and are often produced when phosphorus is limited. We developed a screen that rapidly identifies RAM-producing bacteria by utilizing a colorimetric iron-reduction assay in combination with phosphorus limitation to stimulate biosynthesis. The screen reveals that RAM-producing bacteria are far more prevalent in soil than previously appreciated and that this approach can be used to identify RAM producers.