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

Abstract

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 (C35) 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.

Hopanoids Confer Robustness to Physicochemical Variability in the Niche of the Plant Symbiont Bradyrhizobium diazoefficiens

“…The hpnH mutant manifests growth defects at high osmolarity and is unable to grow under low pH or microaerobic conditionsall conditions thought to characterize the root nodule. While the hpnH mutant exhibits defects in planta, especially in root nodule initiation, the nitrogen fixation rate in symbiosis with the tropical legume Aeschynomene afraspera when normalized for nodule dry weight is not significantly different between the hpnH mutant and WT and the majority of hpnH-infected nodules grow at rates comparable to the WT (30). Given the growth defects of the hpnH mutant observed in vitro in the presence of environmental stresses expected within the root nodule, these results were surprising.…”

“…Removing the ability to synthesize C 35 or "extended" hopanoids (hpnH), however, was achieved, and has a large effect on the fitness of B. diazoefficiens in vitro (25,30). The hpnH mutant manifests growth defects at high osmolarity and is unable to grow under low pH or microaerobic conditionsall conditions thought to characterize the root nodule.…”

The role of hopanoids in fortifying rhizobia against a changing climate

“…BTAi1 ∆ shc and B. diazoefficiens ∆ hpnH ) or the addition of VLCFAs on LA ( B . ORS278 ∆ lpxXL ) (Silipo et al ., 2014; Kulkarni et al ., 2015; Busset et al ., 2017; Belin et al ., 2019); the specific loss of HoLA has not yet been examined because the enzyme that conjugates hopanoids to lipid A remains unknown. All of these HoLA deficient strains were sensitive to stresses affecting the OM, including hyperosmolarity, in free‐living, rich‐media cultures.…”

“…Mutant analysis has been used to approach this question with respect to hyperosmotic stress, yet comes with important caveats: (i) deletion of any biosynthetic step can lead to accumulation of precursors or induce lipidome remodelling to compensate (Neubauer et al, 2015) and (ii) membrane protein function is tuned to its native lipid environment, so removing a membrane component likely results in broad reduction of membrane protein function (Amin and Hazelbauer, 2012;Rice et al, 2014). In these analysis, HoLA deficient mutants have been created by eliminating either hopanoids (B. BTAi1 Δshc and B. diazoefficiens ΔhpnH) or the addition of VLCFAs on LA (B. ORS278 ΔlpxXL) (Silipo et al, 2014;Kulkarni et al, 2015;Busset et al, 2017;Belin et al, 2019); the specific loss of HoLA has not yet been examined because the enzyme that conjugates hopanoids to lipid A remains unknown. All of these HoLA deficient strains were sensitive to stresses affecting the OM, including hyperosmolarity, in free-living, rich-media cultures.…”

Revisiting the precursors of the most abundant natural products on Earth: A look back at 30+ years of bacteriohopanepolyol (BHP) research and ahead to new frontiers

“…Such data could reveal the nitrogen species used by/the metabolism of the hitherto unknown source organisms (potentially aided by δ 2 H analysis; Wijker et al, 2019), which in turn should help constrain the bacterial producers (both in soils and in situ in marine OMZs). Given that many BHPs in soils accumulate in the rhizosphere and are produced by N2-fixing plant symbionts (e.g., Ricci et al, 2014;Kulkarni et al, 2015;Belin et al, 2019;Tookmanian et al, 2021), nucleoside BHPs may be synthesized by N2-fixing bacteria, rather than by heterotrophic bacteria. Fixation of atmospheric N2 and nitrate/ammonia utilization should be directly distinguishable in nucleoside δ 15 N values, especially in settings where soil N is artificially 15 Nenriched due to the use of fertilizers or bacterial denitrification (e.g., Hobbie and Ouimette, 2009;Denk et al, 2017).…”