Extended hopanoid loss reduces bacterial motility and surface attachment and leads to heterogeneity in root nodule growth kinetics in a Bradyrhizobium-Aeschynomene symbiosis

Hopanoids are steroid-like bacterial lipids that enhance membrane rigidity and promote bacterial growth under diverse stresses. Hopanoid biosynthesis genes are conserved in nitrogen-fixing plant symbionts, and we previously found that the extended (C 35 ) class of hopanoids in Bradyrhizobium diazoefficiens are required for efficient symbiotic nitrogen fixation in the tropical legume host Aeschynomene afraspera . Here we demonstrate that the nitrogen fixation defect conferred by extended loss can fully be explained by a reduction in root nodule sizes rather than per-bacteroid nitrogen fixation levels. Using a single-nodule tracking approach to track A. afraspera nodule development, we provide a quantitative model of root nodule development in this host, uncovering both the baseline growth parameters for wild-type nodules and a surprising heterogeneity of extended hopanoid mutant developmental phenotypes. These phenotypes include a delay in root nodule initiation and presence of a subpopulation of nodules with slow growth rates and low final volumes, which are correlated with reduced motility and surface attachment in vitro and lower bacteroid densities in planta , respectively. This work provides a quantitative reference point for understanding the phenotypic diversity of ineffective symbionts in A. afraspera and identifies specific developmental stages affected by extended hopanoid loss for future mechanistic work.