The symbioses between leguminous plants and nitrogen-fixing bacteria known as rhizobia are well known for promoting plant growth and sustainably increasing soil nitrogen. Recent evidence indicates that hopanoids, a family of steroid-like lipids, promote Bradyrhizobium symbioses with tropical legumes. To characterize hopanoids in Bradyrhizobium symbiosis with soybean, we validated a recently published cumate-inducible hopanoid mutant of Bradyrhizobium diazoefficiens USDA110, Pcu-shc::∆shc. GC-MS analysis showed that this strain does not produce hopanoids without cumate induction, and under this condition, is impaired in growth in rich medium and under osmotic, temperature, and pH stress. In planta, Pcu-shc::∆shc is an inefficient soybean symbiont with significantly lower rates of nitrogen fixation and low survival within the host tissue. RNA-seq revealed that hopanoid loss reduces the expression of flagellar motility and chemotaxis-related genes, further confirmed by swim plate assays, and enhances the expression of genes related to nitrogen metabolism and protein secretion. These results suggest that hopanoids provide a significant fitness advantage to B. diazoefficiens in legume hosts and provide a foundation for future mechanistic studies of hopanoid function in protein secretion and motility.
A major problem for global sustainability is feeding our exponentially growing human population while available arable land decreases. Harnessing the power of plant-beneficial microbes is a potential solution, including increasing our reliance on the symbioses of leguminous plants and nitrogen-fixing rhizobia. This study examines the role of hopanoid lipids in the symbiosis between Bradyrhizobium diazoefficiens USDA110, an important commercial inoculant strain, and its economically significant host soybean. Our research extends our knowledge of the functions of bacterial lipids in symbiosis to an agricultural context, which may one day help improve the practical applications of plant-beneficial microbes in agriculture.

Komagataeibacter is the dominant taxon and cellulose-producing bacteria in the Kombucha Microbial Community (KMC). This is the first study to isolate the K. oboediens genome from a reactivated space-exposed KMC sample and comprehensively characterize it. The space-exposed genome was compared with the Earth-based reference genome to understand the genome stability of K. oboediens under extraterrestrial conditions during a long time. Our results suggest that the genomes of K. oboediens IMBG180 (ground sample) and K. oboediens IMBG185 (space-exposed) are remarkably similar in topology, genomic islands, transposases, prion-like proteins, and number of plasmids and CRISPR-Cas cassettes. Nonetheless, there was a difference in the length of plasmids and the location of cas genes. A small difference was observed in the number of protein coding genes. Despite these differences, they do not affect any genetic metabolic profile of the cellulose synthesis, nitrogen-fixation, hopanoid lipids biosynthesis, and stress-related pathways. Minor changes are only observed in central carbohydrate and energy metabolism pathways gene numbers or sequence completeness. Altogether, these findings suggest that K. oboediens maintains its genome stability and functionality in KMC exposed to the space environment most probably due to the protective role of the KMC biofilm. Furthermore, due to its unaffected metabolic pathways, this bacterial species may also retain some promising potential for space applications.

Background: Hopanoids modify plasma membrane properties in bacteria and are often compared to sterols that modulate membrane fluidity in eukaryotes. In some microorganisms, they can also allow adaptations to extreme environments. Methods: Hopanoids were identified by liquid chromatography-mass spectrometry in fourteen strains of thermophilic bacteria belonging to five genera, i.e., Alicyclobacillus, Brevibacillus, Geobacillus, Meiothermus, and Thermus. The bacteria were cultivated at temperatures from 42 to 70 °C. Results: Regardless of the source of origin, the strains have the same tendency to adapt the hopanoid content depending on the cultivation temperature. In the case of aminopentol, its content increases; aminotetrol does not show a significant change; and in the case of aminotriol the content decreases by almost a third. The content of bacteriohopanetetrol and bacteriohopanetetrol glycoside decreases with increasing temperature, while in the case of adenosylhopane the opposite trend was found. Conclusions: Changes in hopanoid content can be explained by increased biosynthesis, where adenosylhopane is the first intermediate in the biosynthesis of the hopanoid side chain.

Bacterial lipids are well-preserved in ancient rocks and certain ones have been used as indicators of specific bacterial metabolisms or environmental conditions existing at the time of rock deposition. Here we show that an anaerobic bacterium produces 3-methylhopanoids, pentacyclic lipids previously detected only in aerobic bacteria and widely used as biomarkers for methane-oxidizing bacteria. Both Rhodopila globiformis, a phototrophic purple nonsulfur bacterium isolated from an acidic warm spring in Yellowstone, and a newly isolated Rhodopila species from a geochemically similar spring in Lassen Volcanic National Park (USA), synthesized 3-methylhopanoids and a suite of related hopanoids and contained the genes encoding the necessary biosynthetic enzymes. Our results show that 3-methylhopanoids can be produced under anoxic conditions and challenges the use of 3-methylhopanoids as biomarkers of oxic conditions in ancient rocks and as prima facie evidence that methanotrophic bacteria were active when the rocks were deposited.

Hopanoids and carotenoids are two of the major isoprenoid-derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols. Methylobacterium extorquens contains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate the role of isoprenoid lipids in surface membrane function and cellular fitness. By genetically knocking out hpnE and crtB we disrupted the production of squalene and phytoene in M. extorquens PA1, which are the presumed precursors for hopanoids and carotenoids respectively. Deletion of hpnE revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in pigmentation with a C30 backbone, rather than the previously predicted canonical C40 phytoene-derived pathway. Phylogenetic analysis suggested that M. extorquens may have acquired the C30 pathway through lateral gene transfer from Planctomycetes. Surprisingly, disruption of carotenoid synthesis did not generate any major growth or membrane biophysical phenotypes, but slightly increased sensitivity to oxidative stress. We further demonstrated that hopanoids but not carotenoids are essential for growth at higher temperatures, membrane permeability and tolerance of low divalent cation concentrations. These observations show that hopanoids and carotenoids serve diverse roles in the outer membrane of M. extorquens PA1.

Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353-358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop-17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated anaerobic growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast anaerobic growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.

The 2-methylhopanes (2-MeHops) are molecular fossils of 2-methylbacteriohopanepolyols (2-MeBHPs) and among the oldest biomarkers on Earth. However, these biomarkers\' specific sources are currently unexplained, including whether they reflect an expansion of marine cyanobacteria. Here, we study the occurrence of 2-MeBHPs and the genes involved in their synthesis in modern bacteria and explore the occurrence of 2-MeHops in the geological record. We find that the gene responsible for 2-MeBHP synthesis (hpnP) is widespread in cyano- and ⍺-proteobacteria, but absent or very limited in other classes/phyla of bacteria. This result is consistent with the dominance of 2-MeBHP in cyano- and ⍺-proteobacterial cultures. The review of their geological occurrence indicates that 2-MeHops are found from the Paleoproterozoic onwards, although some Precambrian samples might be biased by drilling contamination. During the Phanerozoic, high 2-MeHops\' relative abundances (index >15%) are associated with climatic and biogeochemical perturbations such as the Permo/Triassic boundary and the Oceanic Anoxic Events. We analyzed the modern habitat of all hpnP-containing bacteria and find that the only one species coming from an undisputed open marine habitat is an ⍺-proteobacterium acting upon the marine nitrogen cycle. Although organisms can change their habitat in response to environmental stress and evolutionary pressure, we speculate that the high sedimentary 2-MeHops\' occurrence observed during the Phanerozoic reflect ⍺-proteobacteria expansion and marine N-cycle perturbations in response to climatic and environmental change.

Bacterial hopanoid lipids are ubiquitous in the geologic record and serve as biomarkers for reconstructing Earth\'s climatic and biogeochemical evolution. Specifically, the abundance of 2-methylhopanoids deposited during Mesozoic ocean anoxic events (OAEs) and other intervals has been interpreted to reflect proliferation of nitrogen-fixing marine cyanobacteria. However, there currently is no conclusive evidence for 2-methylhopanoid production by extant marine cyanobacteria. As an alternative explanation, here we report 2-methylhopanoid production by bacteria of the genus Nitrobacter, cosmopolitan nitrite oxidizers that inhabit nutrient-rich freshwater, brackish, and marine environments. The model organism Nitrobacter vulgaris produced only trace amounts of 2-methylhopanoids when grown in minimal medium or with added methionine, the presumed biosynthetic methyl donor. Supplementation of cultures with cobalamin (vitamin B12) increased nitrite oxidation rates and stimulated a 33-fold increase of 2-methylhopanoid abundance, indicating that the biosynthetic reaction mechanism is cobalamin dependent. Because Nitrobacter spp. cannot synthesize cobalamin, we postulate that they acquire it from organisms inhabiting a shared ecological niche-for example, ammonia-oxidizing archaea. We propose that during nutrient-rich conditions, cobalamin-based mutualism intensifies upper water column nitrification, thus promoting 2-methylhopanoid deposition. In contrast, anoxia underlying oligotrophic surface ocean conditions in restricted basins would prompt shoaling of anaerobic ammonium oxidation, leading to low observed 2-methylhopanoid abundances. The first scenario is consistent with hypotheses of enhanced nutrient loading during OAEs, while the second is consistent with the sedimentary record of Pliocene-Pleistocene Mediterranean sapropel events. We thus hypothesize that nitrogen cycling in the Pliocene-Pleistocene Mediterranean resembled modern, highly stratified basins, whereas no modern analog exists for OAEs.

Cells, from microbes to mammals, adapt their membrane lipid composition in response to environmental changes to maintain optimal properties. Global patterns of lipidome remodeling are poorly understood, particularly in organisms with simple lipid compositions that can provide insight into fundamental principles of membrane adaptation. Using shotgun lipidomics, we examine the simple yet, as we show here, adaptive lipidome of the plant-associated Gram-negative bacterium Methylobacterium extorquens. We observe that minimally 11 lipids account for 90% of total variability, thus constraining the upper limit of variable lipids required for an adaptive living membrane. Through lipid features analysis, we reveal that acyl chain remodeling is not evenly distributed across lipid classes, resulting in headgroup-specific effects of acyl chain variability on membrane properties. Results herein implicate headgroup-specific acyl chain remodeling as a mechanism for fine-tuning the membrane\'s physical state and provide a resource for using M. extorquens to explore the design principles of living membranes.

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 hopanoid loss can be fully explained by a reduction in root nodule sizes rather than per-bacteroid nitrogen-fixation levels. Using a single-nodule tracking approach to quantify 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 the 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.