Specific Hopanoid Classes Differentially Affect Free-Living and Symbiotic States of Bradyrhizobium diazoefficiens

Abstract

A 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 bacterium Bradyrhizobium diazoefficiens and the legume Aeschynomene afraspera requires 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 C35 hopanoids or the accumulation of the C30 hopanoid diploptene. Our results also show that C35 hopanoids are necessary for symbiosis only with the host Aeschynomene afraspera but not with soybean. This difference is likely related to the presence of cysteine-rich antimicrobial peptides in Aeschynomene nodules 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.

Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency

“…For example, the putative T2SS determined S‐layer could together with the LPS and the hopanoid lipids constitute a multilayered envelope which provides sufficient strength to the bacteria to withstand the NCR peptides or other stresses in the symbiotic nodule cells. Removing the individual components of this envelope could have an imperceptible or moderate effect on the bacteroids (Silipo et al ., ; Kulkarni et al ., ; Busset et al ., ; this work) but, possibly, removing multiple parts simultaneously would be much more deleterious for the bacteroids. Redundancy on the other hand can be achieved by homologous genes or through a functional complementation by an unrelated pathway.…”

Hopanoids play a role in stress tolerance and nutrient storage in the cyanobacterium Nostoc punctiforme

“…Interestingly, N. punctiforme S hpnP mutant did not show any growth defects at extreme temperatures, but did display a depressed growth rate at ambient temperature. Experiments with ∆hpnP mutants of R. palustris (Kulkarni et al., ) and Bradyrhizobium diazoefficiens (Kulkarni et al., ) have not shown growth rate defects at any temperature. Taken together, these data suggest that while 2‐methylhopanoids do not act as temperature‐dependent membrane rigidfiers per se , their absence may cause a subtle species‐specific phenotype that warrants further investigation.…”

“…A particular subclass of hopanoids, 2‐methylhopanoids, were once thought to be biomarkers of cyanobacteria and oxygenic photosynthesis (Summons, Jahnke, Hope, & Logan, ), yet this conclusion has been abandoned in the last decade due to insights gained from subsequent molecular and cellular biological studies (reviewed in Newman et al., ; Rashby, Sessions, Summons, & Newman, ; Welander, Coleman, Sessions, Summons, & Newman, ). Although some evidence indicates hopanes and 2‐methylhopanes may be general stress markers (Kulkarni, Wu, & Newman, ; Kulkarni et al., ; Welander et al., , ), much remains to be learned before we can reach a more nuanced interpretation, including whether or how broadly the stress phenotypes are spread among the various hopanoid‐producing phyla.…”

“…A major constraint on our interpretative confidence arises from the fact that while hopanoids are made by phylogenetically diverse bacteria, to date, studies of hopanoid function have mainly been performed in Proteobacteria. Such work has shown that hopanoids assist in rigidifying cell membranes, aid in stress tolerance, and are important in facilitating plant–microbe symbioses (Kulkarni et al., , ; Ricci et al., ; Sáenz et al., ; Schmerk, Bernards, & Valvano, ; Silipo et al., ; Welander et al., ; Wu, Bialecka‐Fornal, & Newman, ). Although today Alphaproteobacteria appear to be the dominant producers of hopanoids in most environments (Pearson & Rusch, ; Ricci et al., ), cyanobacteria have long been known to contribute to hopanoid deposition in certain locales, such as Yellowstone National Park (Jahnke et al., ).…”

“…Their presence in these different membrane compartments raised the question of whether hopanoids might play multiple physiological roles, including in the context of symbiosis. Symbiotic niches correlate with the presence of hopanoid biosynthetic genes in modern environments (Ricci et al., ), and recent studies of two different Alphaproteobacteria linked hopanoid production to symbiotic fitness (Kulkarni et al., ; Silipo et al., ). However, the mechanisms whereby hopanoids contribute to these effects are unclear, and whether this linkage extends to cyanobacteria or other phyla is unknown.…”