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Unlabelled: 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., Importance: Because 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 C35 hopanoids 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., (Copyright © 2015 Kulkarni et al.)

Lipopolysaccharides (LPSs) are major components of the outer membrane of Gram-negative bacteria and are essential for their growth and survival. They act as a structural barrier and play an important role in the interaction with eukaryotic hosts. Here we demonstrate that a photosynthetic Bradyrhizobium strain, symbiont of Aeschynomene legumes, synthesizes a unique LPS bearing a hopanoid covalently attached to lipid A. Biophysical analyses of reconstituted liposomes indicate that this hopanoid-lipid A structure reinforces the stability and rigidity of the outer membrane. In addition, the bacterium produces other hopanoid molecules not linked to LPS. A hopanoid-deficient strain, lacking a squalene hopene cyclase, displays increased sensitivity to stressful conditions and reduced ability to survive intracellularly in the host plant. This unusual combination of hopanoid and LPS molecules may represent an adaptation to optimize bacterial survival in both free-living and symbiotic states.

Lipid molecules preserved in sedimentary rocks facilitate the reconstruction of events that have shaped the evolution of the Earth's biosphere. A key limitation for the interpretation of many of these molecular fossils is that their biological roles are still poorly understood. Here, we use Rhodopseudomonas palustris TIE-1 to identify factors that induce biosynthesis of 2-methyl hopanoids (2-MeBHPs), progenitors of 2-methyl hopanes, one of the most abundant biomarkers in the rock record. This is the first dissection of the regulation of hpnP, the gene encoding the C-2 hopanoid methylase, at the molecular level. We demonstrate that EcfG, the general stress response factor of alphaproteobacteria, regulates expression of hpnP under a variety of challenges, including high temperature, pH stress, and presence of nonionic osmolytes. Although higher hpnP transcription levels did not always result in higher amounts of total methylated hopanoids, the fraction of a particular kind of hopanoid, 2-methyl bacteriohopanetetrol, was consistently higher in the presence of most stressors in the wild type, but not in the ΔecfG mutant, supporting a beneficial role for 2-MeBHPs in stress tolerance. The ΔhpnP mutant, however, did not exhibit a growth defect under the stress conditions tested except in acidic medium. This indicates that the inability to make 2-MeBHPs under most of these conditions can readily be compensated. Although stress is necessary to regulate 2-MeBHP production, the specific conditions under which 2-MeBHP biosynthesis is essential remain to be determined.