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.

Carotenoids are isoprenoid lipids found across the tree of life with important implications in oxidative stress adaptations, photosynthetic metabolisms, as well as in membrane dynamics. The canonical view is that C40 carotenoids are synthesized from phytoene and C30 carotenoids from diapophytoene. Squalene is mostly associated with the biosynthesis of polycyclic triterpenes, although there have been suggestions that it could also be involved in the biosynthesis of C30 carotenoids. However, demonstration of the existence of this pathway in nature is lacking. Here, we demonstrate that C30 carotenoids are synthesized from squalene in the Planctomycetes bacteria and that this squalene route to C30 carotenoids is the most widespread in prokaryotes. Using the evolutionary history of carotenoid and squalene amino oxidases, we propose an evolutionary scenario to explain the origin and diversification of the different carotenoid and squalene-related pathways. We show that carotenoid biosynthetic pathways have been constantly transferred and neofunctionalized during prokaryotic evolution. One possible origin of the squalene pathway connects it with the one of C40 carotenoid synthesis of Cyanobacteria. The widespread occurrence of the squalene route to C30 carotenoids in Bacteria increases the functional repertoire of squalene, establishing it as a general hub of carotenoids and polycyclic triterpenes synthesis.

Rhizobia are a group of bacteria that increase soil nitrogen content through symbiosis with legume plants. The soil and symbiotic host are potentially stressful environments, and the soil will likely become even more stressful as the climate changes. Many rhizobia within the Bradyrhizobium clade, like Bradyrhizobium diazoefficiens, possess the genetic capacity to synthesize hopanoids, steroid-like lipids similar in structure and function to cholesterol. Hopanoids are known to protect against stresses relevant to the niche of B. diazoefficiens. Paradoxically, mutants unable to synthesize the extended class of hopanoids participate in symbioses with success similar to that of the wild type, despite being delayed in root nodule initiation. Here, we show that in B. diazoefficiens, the growth defects of extended-hopanoid-deficient mutants can be at least partially compensated for by the physicochemical environment, specifically, by optimal osmotic and divalent cation concentrations. Through biophysical measurements of lipid packing and membrane permeability, we show that extended hopanoids confer robustness to environmental variability. These results help explain the discrepancy between previous in-culture and in planta results and indicate that hopanoids may provide a greater fitness advantage to rhizobia in the variable soil environment than the more controlled environments within root nodules. To improve the legume-rhizobium symbiosis through either bioengineering or strain selection, it will be important to consider the full life cycle of rhizobia, from soil to symbiosis. IMPORTANCE 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. Efforts to use rhizobia as biofertilizers have been effective; however, long-term integration of rhizobia into the soil community has been less successful. This work represents a small step toward improving the legume-rhizobium symbiosis by identifying a cellular component-hopanoid lipids-that confers robustness to environmental stresses rhizobia are likely to encounter in soil microenvironments as sporadic desiccation and flooding events become more common.

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.

Omics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N2-fixing pure cultures with propionate as the carbon source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-fixing pure culture. Nitrogenase, SuF (Fe-Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis.

Hopanoid lipids, bacteriohopanols and bacteriohopanepolyols, are membrane components exclusive to bacteria. Together with their diagenetic derivatives, they are commonly used as biomarkers for specific bacterial groups or biogeochemical processes in the geologic record. However, the sources of hopanoids to marine and freshwater environments remain inadequately constrained. Recent marker gene studies suggest a widespread potential for hopanoid biosynthesis in marine bacterioplankton, including nitrifying (i.e., ammonia- and nitrite-oxidizing) bacteria. To explore their hopanoid biosynthetic capacities, we studied the distribution of hopanoid biosynthetic genes in the genomes of cultivated and uncultivated ammonia-oxidizing (AOB), nitrite-oxidizing (NOB), and complete ammonia-oxidizing (comammox) bacteria, finding that biosynthesis of diverse hopanoids is common among seven of the nine presently cultivated clades of nitrifying bacteria. Hopanoid biosynthesis genes are also conserved among the diverse lineages of bacterial nitrifiers detected in environmental metagenomes. We selected seven representative NOB isolated from marine, freshwater, and engineered environments for phenotypic characterization. All tested NOB produced diverse types of hopanoids, with some NOB producing primarily diploptene and others producing primarily bacteriohopanepolyols. Relative and absolute abundances of hopanoids were distinct among the cultures and dependent on growth conditions, such as oxygen and nitrite limitation. Several novel nitrogen-containing bacteriohopanepolyols were tentatively identified, of which the so called BHP-743.6 was present in all NOB. Distinct carbon isotopic signatures of biomass, hopanoids, and fatty acids in four tested NOB suggest operation of the reverse tricarboxylic acid cycle in Nitrospira spp. and Nitrospina gracilis and of the Calvin-Benson-Bassham cycle for carbon fixation in Nitrobacter vulgaris and Nitrococcus mobilis. We suggest that the contribution of hopanoids by NOB to environmental samples could be estimated by their carbon isotopic compositions. The ubiquity of nitrifying bacteria in the ocean today and the antiquity of this metabolic process suggest the potential for significant contributions to the geologic record of hopanoids.

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.