ABSTRACTA better understanding of how bacteria resist stresses encountered during the progression of plant-microbe symbioses will advance our ability to stimulate plant growth. Here, we show that the symbiotic system comprising the nitrogen-fixing bacteriumBradyrhizobium diazoefficiensand the legumeAeschynomene afrasperarequires hopanoid production for optimal fitness.While methylated (2Me) hopanoids contribute to growth under plant-cell-like microaerobic and acidic conditions in the free-living state, they are dispensable during symbiosis. In contrast, synthesis of extended (C35) hopanoids is required for growth microaerobically and under various stress conditions (high temperature, low pH, high osmolarity, bile salts, oxidative stress, and antimicrobial peptides) in the free-living state and also during symbiosis. These defects might be due to a less rigid membrane resulting from the absence of free or lipidA-bound C35hopanoids or the accumulation of the C30hopanoid diploptene. Our results also show that C35hopanoids are necessary for symbiosis only with the hostAeschynomene afrasperabut not with soybean. This difference is likely related to the presence of cysteine-rich antimicrobial peptides inAeschynomenenodules that induce drastic modification in bacterial morphology and physiology. The study of hopanoid mutants in plant symbionts thus provides an opportunity to gain insight into host-microbe interactions during later stages of symbiotic progression, as well as the microenvironmental conditions for which hopanoids provide a fitness advantage.IMPORTANCEBecause bradyrhizobia provide fixed nitrogen to plants, this work has potential agronomical implications. An understanding of how hopanoids facilitate bacterial survival in soils and plant hosts may aid the engineering of more robust agronomic strains, especially relevant in regions that are becoming warmer and saline due to climate change. Moreover, this work has geobiological relevance: hopanes, molecular fossils of hopanoids, are enriched in ancient sedimentary rocks at discrete intervals in Earth history. This is the first study to uncover roles for 2Me- and C35hopanoids in the context of an ecological niche that captures many of the stressful environmental conditions thought to be important during (2Me)-hopane deposition. Though much remains to be done to determine whether the conditions present within the plant host are shared with niches of relevance to the rock record, our findings represent an important step toward identifying conserved mechanisms whereby hopanoids contribute to fitness.