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

Abstract

Rhizobia, such as B. diazoefficiens , play an important role in the nitrogen cycle by making nitrogen gas bioavailable through symbiosis with legume plants. As climate change threatens soil health, this symbiosis has received increased attention as a more sustainable source of soil nitrogen than the energy-intensive Haber-Bosch process.

Hopanoid lipids promote soybean-Bradyrhizobiumsymbiosis

“…We also found that high doses of cumate inhibited bacterial growth (Fig. S1) , and therefore chose 25 µM cumate for all following assays, consistent with previous studies (24).…”

“…To characterize hopanoid functions in B. diazoefficiens , a cumate-inducible shc mutant strain Pcu- shc ::Δ shc , abbreviated as Pcu- shc hereafter, was constructed as previously published (24). Briefly, an exogenous copy of shc was placed after a cumate-inducible promoter (Pcu) and integrated into the genome, after which the endogenous shc was deleted ( Fig.…”

“…This validation typically requires lipid extraction and quantification by mass spectrometry, a technically challenging approach that is less widely available than methods for tracking protein depletion. Here we provide a thorough validation of an inducible total hopanoids mutant strain, previously generated by colleagues through a clever genetic approach to bypass the difficulty of selecting for a simple shc knockout (24). We demonstrate that the strain is suitable for cumate dose-dependent manipulation of not only shc expression but also membrane hopanoid content in a pure culture setting.…”

“…In the context of temperature stress, the Δ hpnH mutant of B. diazoefficiens also cannot grow at 37 °C (3) suggesting extended hopanoids are the primary contributors to this phenotype in the Pcu- shc strain. This Δ hpnH strain has more mild growth inhibition under osmotic stress (24) than Pcu- shc +E, and the 2-methylated hopanoid mutant Δ hpnP has no osmotic stress phenotype (3), indicating that the short hopanoid class is the most relevant to osmotic stress survival. Overall, the stress sensitivity phenotypes of Pcu- shc +E under our culturing conditions are more severe than previously published mutants.…”

“…As we’ve demonstrated, the inducible mutant strategy used to develop the Pcu- shc strain (24) is an extremely useful workaround for generating mutants of B. diazoefficiens genes that are essential or otherwise difficult to delete cleanly. However, it has limited use in planta , as we could not rescue shc depletion phenotypes in the host.…”