Giacalone David, Schutt Emilly, McRose Darcy L
Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Appl Environ Microbiol. 2025 Jan 31;91(1):e0219424. doi: 10.1128/aem.02194-24. Epub 2024 Dec 17.
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.
土壤微生物群落对植物健康和养分获取至关重要。越来越清楚的是，微生物之间以及微生物与植物之间的许多相互作用都受小型生物活性分子或“次生代谢产物”的调控，这些分子有助于交流、竞争和养分吸收。然而，次生代谢产物生物地理学——谁在何处产生何种次生代谢产物以及原因——尚处于起步阶段。此外，次生代谢产物生物合成基因在标准实验室条件下通常处于沉默或低表达状态，这使得研究这些小分子极其困难。为了开始应对这两个挑战，我们聚焦于氧化还原活性代谢产物（RAMs），这是一类特定的小分子，并利用了最近的研究发现，即许多RAMs有助于获取磷，且它们的产生常常受到这种大量营养素胁迫的刺激。我们开发了一种筛选产生RAMs细菌的方法，该方法利用磷限制来刺激代谢产物生物合成，并使用比色法（亚铁嗪）铁还原测定来鉴定氧化还原活性。我们从美国各地（亚利桑那州圣丽塔实验场、堪萨斯州康扎草原生物站和马萨诸塞州哈佛森林）采集的草以及商业番茄植株中分离出557种根际细菌，并对它们进行RAMs产生的筛选。我们鉴定出了128种土壤分离株，它们分属于变形菌门、放线菌门、厚壁菌门和拟杆菌门的至少19个属，这些菌株在磷胁迫下产生RAMs。我们的工作表明，在磷胁迫下RAMs的产生在各种土壤细菌中很常见，并提供了一种快速筛选这些小分子的方法。
通过分泌次生代谢产物，植物根部的细菌可以抵御疾病并帮助获取必需养分。然而，合成次生代谢产物的基因在标准实验室条件下通常不活跃或低表达。这一事实使得研究这些小分子变得困难，并阻碍了发现可能在农业和生物医学环境中起关键作用的新型小分子。在此，我们聚焦于氧化还原活性代谢产物（RAMs），这是一类次生代谢产物，可帮助细菌溶解磷，且在磷有限时经常产生。我们开发了一种筛选方法，通过结合比色铁还原测定与磷限制来刺激生物合成，从而快速鉴定产生RAMs的细菌。该筛选方法表明，产生RAMs的细菌在土壤中的普遍程度远超以往认知，并且这种方法可用于鉴定RAMs产生菌。
