Diversity of hopanoids and squalene-hopene cyclases across a tropical land-sea gradient

Bacterial hopanoids are ubiquitous in Earth surface environments. They hold promise as environmental and ecological biomarkers, if the phylogeny and physiological drivers of hopanoid biosynthesis can be linked with the distribution of hopanoids observed across a breadth of samples. Here we survey the diversity of hopanoid cyclases from a land‐sea gradient across the island of San Salvador, in the easternmost part of the Bahamas. The distribution of lipids was determined for the same sites, for the first time overlaying quantification of bacteriohopanepolyols with sqhC phylogeny. The results are similar to previous reports: environmental sqhCs average < 65% translated amino acid identity to their closest named relatives, and sequences from putative Proteobacteria dominate. Additionally, a new and apparently ubiquitous group of marine hopanoid producers is identified; it has no identifiable close relatives. The greatest diversity of hopanoid lipids occurs in soil, but hopanoids represent a minor fraction of total soil‐derived lipids. Marine samples contain fewer identifiable hopanoids, but they are more abundant as a fraction of the total extractable lipids. In soil, the dominant compounds are 35‐aminobacteriohopane‐32,33,34‐triol and adenosylhopane. In an upper estuarine sample, bacteriohopanetetrol and 32,35‐anhydrobacteriohopanetetrol dominate; while in lower estuarine and open marine samples, the most abundant are bacteriohopanetetrol and bacteriohopaneribonolactone. Cyclitol ethers are trace components in the soil, absent in the estuary, and of moderate abundance in the open marine setting, suggesting a dominant marine source. Conversely, aminotriol and aminotetrol decrease in abundance or disappear completely from land to ocean, while 2‐methyldiplopterol shows the opposite trend. Small quantities of 2‐methylbacteriohopanepolyols are detectable in all samples. The overall hopanoid distributions may correlate to the major phylogenetic families of hopanoid producers or to the environments in which they are found.     

Hopanoids in marine cyanobacteria: probing their phylogenetic distribution and biological role

Cyanobacteria are key players in the global carbon and nitrogen cycles and are thought to have been responsible for the initial rise of atmospheric oxygen during the Neoarchean. There is evidence that a class of membrane lipids known as hopanoids serve as biomarkers for bacteria, including many cyanobacteria, in the environment and in the geologic record. However, the taxonomic distributions and physiological roles of hopanoids in marine cyanobacteria remain unclear. We examined the distribution of bacteriohopanepolyols (BHPs) in a collection of marine cyanobacterial enrichment and pure cultures and investigated the relationship between the cellular abundance of BHPs and nitrogen limitation in Crocosphaera watsonii, a globally significant nitrogen‐fixing cyanobacterium. In pure culture, BHPs were only detected in species capable of nitrogen fixation, implicating hopanoids as potential markers for diazotrophy in the oceans. The enrichment cultures we examined exhibited a higher degree of BHP diversity, demonstrating that there are presently unaccounted for marine bacteria, possibly cyanobacteria, associated with the production of a range of BHP structures. Crocosphaera watsonii exhibited high membrane hopanoid content consistent with the idea that hopanoids have an important effect on the bulk physical properties of the membrane. However, the abundance of BHPs in C. watsonii did not vary considerably when grown under nitrogen‐limiting and nitrogen‐replete conditions, suggesting that the role of hopanoids in this organism is not directly related to the physiology of nitrogen fixation. Alternatively, we propose that high hopanoid content in C. watsonii may serve to reduce membrane permeability to antimicrobial toxins in the environment.     

Novel hopanoid cyclases from the environment

Hopanoids are ubiquitous isoprenoid lipids found in modern biota, in recent sediments and in low-maturity sedimentary rocks. Because these lipids primarily are derived from bacteria, they are used as proxies to help decipher geobiological communities. To date, much of the information about sources of hopanoids has come from surveys of culture collections, an approach that does not address the vast fraction of prokaryotic communities that remains uncharacterized. Here we investigated the phylogeny of hopanoid producers using culture-independent methods. We obtained 79 new sequences of squalene-hopene cyclase genes (sqhC) from marine and lacustrine bacterioplankton and analysed them along with all 31 sqhC fragments available from existing metagenomics libraries. The environmental sqhCs average only 60% translated amino acid identity to their closest relatives in public databases. The data imply that the sources of these important geologic biomarkers remain largely unknown. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analysed here, yet the geologic record contains abundant hopanoids apparently of cyanobacterial origin. The data also suggest that hopanoid biosynthesis is uncommon: < 10% of bacterial species may be capable of producing hopanoids. A better understanding of the contemporary distribution of hopanoid biosynthesis may reveal fundamental insight about the function of these compounds, the organisms in which they are found, and the environmental signals preserved in the sedimentary record.     

Bacteriohopanepolyols across environmental gradients in Lake Vanda,Antarctica

Bacteriohopanepolyols (BHPs) are bacterial membrane lipids that may be used as biological or environmental biomarkers. Previous studies have described the diversity, distribution, and abundance of BHPs in a variety of modern environments. However, the regulation of BHP production in polar settings is not well understood. Benthic microbial mats from ice‐covered lakes of the McMurdo Dry Valleys, Antarctica provide an opportunity to investigate the sources, physiological roles, and preservation of BHPs in high‐latitude environments. Lake Vanda is one of the most stable lakes on Earth, with microbial communities occupying specific niches along environmental gradients. We describe the influence of mat morphology and local environmental conditions on the diversity and distribution of BHPs and their biological sources in benthic microbial mats from Lake Vanda. The abundance and diversity of C‐2 methylated hopanoids (2‐MeBHP) are of particular interest, given that their stable degradation products, 2‐methylhopanes, are among the oldest and most prevalent taxonomically informative biomarkers preserved in sedimentary rocks. Furthermore, the interpretation of sedimentary 2‐methylhopanes is of great interest to the geobiology community. We identify cyanobacteria as the sole source of 2‐MeBHP in benthic microbial mats from Lake Vanda and assess the hypothesis that 2‐MeBHP are regulated in response to a particular environmental variable, namely solar irradiance.     

Environmental controls on bacteriohopanepolyol profiles of benthic microbial mats from Lake Fryxell,Antarctica

Bacteriohopanepolyols (BHPs) are pentacyclic triterpenoid lipids that contribute to the structural integrity and physiology of some bacteria. Because some BHPs originate from specific classes of bacteria, BHPs have potential as taxonomically and environmentally diagnostic biomarkers. For example, a stereoisomer of bacteriohopanetetrol (informally BHT II) has been associated with anaerobic ammonium oxidation (anammox) bacteria and suboxic to anoxic marine environments where anammox is active. As a result, the detection of BHT II in the sedimentary record and fluctuations in the relative abundance of BHT II may inform reconstructions of nitrogen cycling and ocean redox changes through the geological record. However, there are uncertainties concerning the sources of BHT II and whether or not BHT II is produced in abundance in non‐marine environments, both of which are pertinent to interpretations of BHT II signatures in sediments. To address these questions, we investigate the BHP composition of benthic microbial mats from Lake Fryxell, Antarctica. Lake Fryxell is a perennially ice‐covered lake with a sharp oxycline in a density‐stabilized water column. We describe the diversity and abundance of BHPs in benthic microbial mats across a transect from oxic to anoxic conditions. Generally, BHP abundances and diversity vary with the morphologies of microbial mats, which were previously shown to reflect local environmental conditions, such as irradiance and oxygen and sulfide concentrations. BHT II was identified in mats that exist within oxic to anoxic portions of the lake. However, anammox bacteria have yet to be identified in Lake Fryxell. We examine our results in the context of BHPs as biomarkers in modern and ancient environments.     

Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis

Hopanoids are a class of membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The ethanol fermenter Zymomonas mobilis features the highest measured concentration of hopanoids, leading to the hypothesis that these lipids can protect against the solvent toxicity. However, the lack of genetic tools for manipulating hopanoid composition in this bacterium has limited their further functional analysis. Due to the polyploidy (>50 genome copies per cell) of Z. mobilis, we found that disruptions of essential hopanoid biosynthesis (hpn) genes act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrate that both reduced hopanoid content and modified hopanoid polar head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their head group composition, contributes to fitness at low pH. Spectroscopic analysis of bacterial‐derived liposomes showed that hopanoids protect against several ethanol‐driven phase transitions in membrane structure, including lipid interdigitation and bilayer dissolution. We propose that hopanoids act through a combination of hydrophobic and inter‐lipid hydrogen bonding interactions to stabilize bacterial membranes during solvent stress.     

pH Shaping the Composition of sqhC-Containing Bacterial Communities

Sediment and tailings samples were collected from sites with a contrasting physicochemical gradient to investigate microbial squalene-hopene cyclase (sqhC) composition and distribution in terrestrial environments. Acidobacteria (66%), Alphaproteobacteria (96%) and Gammaproteobacteria (55%) were found to dominate sqhC communities, respectively, at the acidic Dajiuhu Peatland, the alkaline Heshang Cave and strongly acidic tailings of Tongling copper mine in China. Statistical analysis confirmed that pH was the important factor impacting the geographical distribution of sqhC at phylum level. sqhC gene abundance is comparable at the three sites. However, the total amount of hopanoids in per gram total organic matter content (TOC) is 1.75 times higher in the acidic peatland than that in the alkaline Cave and it is below the detecting limit in tailings of Tongling copper mine, inferring the potential impact of pH in regulating the hopanoid production.     

Identification and characterization of Rhodopseudomonas palustris TIE-1 hopanoid biosynthesis mutants

Hopanes preserved in both modern and ancient sediments are recognized as the molecular fossils of bacteriohopanepolyols, pentacyclic hopanoid lipids. Based on the phylogenetic distribution of hopanoid production by extant bacteria, hopanes have been used as indicators of specific bacterial groups and/or their metabolisms. However, our ability to interpret them ultimately depends on understanding the physiological roles of hopanoids in modern bacteria. Toward this end, we set out to identify genes required for hopanoid biosynthesis in the anoxygenic phototroph Rhodopseudomonas palustris TIE-1 to enable selective control of hopanoid production. We attempted to delete 17 genes within a putative hopanoid biosynthetic gene cluster to determine their role, if any, in hopanoid biosynthesis. Two genes, hpnH and hpnG, are required to produce both bacteriohopanetetrol and aminobacteriohopanetriol, whereas a third gene, hpnO, is required only for aminobacteriohopanetriol production. None of the genes in this cluster are required to exclusively synthesize bacteriohopanetetrol, indicating that at least one other hopanoid biosynthesis gene is located elsewhere on the chromosome. Physiological studies with the different deletion mutants demonstrated that unmethylated and C(30) hopanoids are sufficient to maintain cytoplasmic but not outer membrane integrity. These results imply that hopanoid modifications, including methylation of the A-ring and the addition of a polar head group, may have biologic functions beyond playing a role in membrane permeability.     

Targeted genomic detection of biosynthetic pathways: anaerobic production of hopanoid biomarkers by a common sedimentary microbe

The lipid biomarker principle requires that preservable molecules (molecular fossils) carry specific taxonomic, metabolic, or environmental information. Historically, an empirical approach was used to link specific taxa with the compounds they produce. The lipids extracted from numerous, but randomly cultured species provided the basis for the interpretation of biomarkers in both modern environments and in the geological record. Now, with the rapid sequencing of hundreds of microbial genomes, a more focused genomic approach can be taken to test phylogenetic patterns and hypotheses about the origins of biomarkers. Candidate organisms can be selected for study on the basis of genes that encode proteins fundamental to the synthesis of biomarker compounds. Hopanoids, a class of pentacyclic triterpenoid lipid biomarkers, provide an illustrative example. For many years, interpretations of biomarker data were made with the assumption that hopanoids are produced only by aerobic organisms. However, the recent discovery of ¹³C‐depleted hopanoids in environments undergoing anaerobic methane oxidation and in enrichment cultures of anammox planctomycetes indicates that some hopanoids are produced anaerobically. To further examine the potential distribution of hopanoid biosynthesis by anaerobes, we searched publicly available genomic databases for the presence of squalene‐hopene cyclase genes in known obligate or facultative anaerobes. Here we present evidence that Geobacter sulfurreducens, Geobacter metallireducens, and Magnetospirillum magnetotacticum, all bacteria common in anoxic environments, have the appropriate genes for hopanoid biosynthesis. We further show that these data accurately predict that G. sulfurreducens does produce a variety of complex hopanoids under strictly anaerobic conditions in pure culture.     

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

Hopanes are abundant in ancient sedimentary rocks at discrete intervals in Earth history, yet interpreting their significance in the geologic record is complicated by our incomplete knowledge of what their progenitors, hopanoids, do in modern cells. To date, few studies have addressed the breadth of diversity of physiological functions of these lipids and whether those functions are conserved across the hopanoid‐producing bacterial phyla. Here, we generated mutants in the filamentous cyanobacterium, Nostoc punctiforme, that are unable to make all hopanoids (shc) or 2‐methylhopanoids (hpnP). While the absence of hopanoids impedes growth of vegetative cells at high temperature, the shc mutant grows faster at low temperature. This finding is consistent with hopanoids acting as membrane rigidifiers, a function shared by other hopanoid‐producing phyla. Apart from impacting fitness under temperature stress, hopanoids are dispensable for vegetative cells under other stress conditions. However, hopanoids are required for stress tolerance in akinetes, a resting survival cell type. While 2‐methylated hopanoids do not appear to contribute to any stress phenotype, total hopanoids and to a lesser extent 2‐methylhopanoids were found to promote the formation of cyanophycin granules in akinetes. Finally, although hopanoids support symbiotic interactions between Alphaproteobacteria and plants, they do not appear to facilitate symbiosis between N. punctiforme and the hornwort Anthoceros punctatus. Collectively, these findings support interpreting hopanes as general environmental stress biomarkers. If hopanoid‐mediated enhancement of nitrogen‐rich storage products turns out to be a conserved phenomenon in other organisms, a better understanding of this relationship may help us parse the enrichment of 2‐methylhopanes in the rock record during episodes of disrupted nutrient cycling.     

The Bacteriohopanepolyol Inventory of Novel Aerobic Methane Oxidising Bacteria Reveals New Biomarker Signatures of Aerobic Methanotrophy in Marine Systems

Aerobic methane oxidation (AMO) is one of the primary biologic pathways regulating the amount of methane (CH₄) released into the environment. AMO acts as a sink of CH₄, converting it into carbon dioxide before it reaches the atmosphere. It is of interest for (paleo)climate and carbon cycling studies to identify lipid biomarkers that can be used to trace AMO events, especially at times when the role of methane in the carbon cycle was more pronounced than today. AMO bacteria are known to synthesise bacteriohopanepolyol (BHP) lipids. Preliminary evidence pointed towards 35-aminobacteriohopane-30,31,32,33,34-pentol (aminopentol) being a characteristic biomarker for Type I methanotrophs. Here, the BHP compositions were examined for species of the recently described novel Type I methanotroph bacterial genera Methylomarinum and Methylomarinovum, as well as for a novel species of a Type I Methylomicrobium. Aminopentol was the most abundant BHP only in Methylomarinovum caldicuralii, while Methylomicrobium did not produce aminopentol at all. In addition to the expected regular aminotriol and aminotetrol BHPs, novel structures tentatively identified as methylcarbamate lipids related to C-35 amino-BHPs (MC-BHPs) were found to be synthesised in significant amounts by some AMO cultures. Subsequently, sediments and authigenic carbonates from methane-influenced marine environments were analysed. Most samples also did not contain significant amounts of aminopentol, indicating that aminopentol is not a useful biomarker for marine aerobic methanotophic bacteria. However, the BHP composition of the marine samples do point toward the novel MC-BHPs components being potential new biomarkers for AMO.     

Hopanoids,like sterols,modulate dynamics,compaction, phase segregation and permeability of membranes

In recent years, hopanoids, a group of pentacyclic compounds found in bacterial membranes, are in the spotlight since it was proposed that they induce order in lipid membranes in a similar way cholesterol do in eukaryotes, despite their structural differences. We studied here whether diplopterol (an abundant hopanoid) promoted similar effects on model membranes as sterols do. We analyzed the compaction, dynamics, phase segregation, permeability and compressibility of model membranes containing diplopterol, and compared with those containing sterols from animals, plants and fungi. We also tested the effect that the incubation with diplopterol had on hopanoid-lacking bacteria. Our results show that diplopterol induces phase segregation, increases lipid compaction, and decreases permeability on phospholipid membranes, while retaining membrane fluidity and compressibility. Furthermore, the exposition to this hopanoid decreases the permeability of the opportunistic pathogen Pseudomonas aeruginosa and increases the resistance to antibiotics. All effects promoted by diplopterol were similar to those generated by the sterols. Our observations add information on the functional significance of hopanoids as molecules that play an important role in membrane organization and dynamics in model membranes and in a bacterial system.     

Elucidation of the Burkholderia cenocepacia hopanoid biosynthesis pathway uncovers functions for conserved proteins in hopanoid‐producing bacteria

Hopanoids are bacterial surrogates of eukaryotic membrane sterols and among earth's most abundant natural products. Their molecular fossils remain in sediments spanning more than a billion years. However, hopanoid metabolism and function are not fully understood. Burkholderia species are environmental opportunistic pathogens that produce hopanoids and also occupy diverse ecological niches. We investigated hopanoids biosynthesis in Burkholderia cenocepacia by deletion mutagenesis and structural characterization of the hopanoids produced by the mutants. The enzymes encoded by hpnH and hpnG were essential for production of all C₃₅ extended hopanoids, including bacteriohopanetetrol (BHT), BHT glucosamine and BHT cyclitol ether. Deletion of hpnI resulted in BHT production, while ΔhpnJ produced only BHT glucosamine. Thus, HpnI is required for BHT glucosamine production while HpnJ is responsible for its conversion to the cyclitol ether. The ΔhpnH and ΔhpnG mutants could not grow under any stress condition tested, whereas ΔhpnI, ΔhpnJ and ΔhpnK displayed wild‐type growth rates when exposed to detergent, but varying levels of sensitivity to low pH and polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ and HpnK in other hopanoid‐producing bacteria.     

Interaction of hopanoids with phosphatidylcholines containing oleic and ω-cyclohexyldodecanoic acid in lipid bilayer membranes

Lipid bilayer membranes were made from hopanoid phosphatidylcholine mixtures dissolved in decane. The specific capacity of the mixed membranes was found to increase with increasing hopanoid content. This indicates an interaction between hopanoids and lipids which leads to a reduction of the chemical potential of the solvent in the membranes.The structural properties of mixtures of hopanoids and phosphatidylcholines were investigated using charged probe molecules, the negatively charged lipophilic ions dipicrylamine (DPA) and tetraphenylborate (TØB) and the positively charged potassium complex PV-K⁺ (PV, cyclo (D-Val-L-Pro-L-Val-D-Pro)₃). The transport properties of the lipophilic ions in the mixed membranes indicate that the electrical properties like dipolar potential and surface potentials of phosphatidylcholine membranes are not changed by the insertion of the hopanoids. The translocation rate constant K of the PV-K⁺ complex is drastically reduced in the hopanoid phosphatidylcholine membranes with increasing hopanoid content. This effect is discussed on the basis of an alteration of the microviscosity in the mixed membranes. There exists a close analogy between the action of cholesterol and hopanoids in bilayer membranes from phosphatidylcholines.A bilayer membrane composed of di-ω-cyclohexyldodecanoyl-phosphatidylcholine (DCPC) was found to possess a higher specific capacity as compared to other phosphatidylcholines. Also a lower translocation rate constant for PV-K⁺ was observed which may be caused by the relative high microviscosity of this lipid even above the phase transition temperature.     

Draft Genome Sequence of Rhodomicrobium udaipurense JA643T with Special Reference to Hopanoid Biosynthesis

Hopanoids are present in vast amounts as integral components of bacteria and plants with their primary function to strengthen rigidity of the plasma membrane. To establish their roles more precisely, we conducted sequencing of the whole genome of Rhodomicrobium udaipurense JA643^T isolated from a fresh water stream of Udaipur in Himachal Pradesh, India, by using the Illumina HiSeq pair end chemistry of 2 × 100 bp platform. Determined genome showed a high degree of similarity to the genome of R. vannielii ATCC17100^T and the 13.7 million reads generated a sequence of 3,649,277 bp possessing 3,611 putative genes. The genomic data were subsequently investigated with respect to genes involved in various features. The machinery required for the degradation of aromatic compounds and resistance to solvents as well as all that required for photosynthesis are present in this organism. Also, through extensive functional annotation, 18 genes involved in the biosynthesis of hopanoids are predicted, namely those responsible for the synthesis of diploptene, diplopterol, adenosylhopane, ribosylhopane, aminobacteriohopanetriol, glycosyl group containing hopanoids and unsaturated hopanoids. The hopanoid biosynthetic pathway was then inferred based on the genes identified and through experimental validation of individual hopanoid molecules. The genome data of R. udaipurense JA643^T will be useful in understanding the functional features of hopanoids in this bacterium.     

Hopanoid lipids in Bradyrhizobium and other plant-associated bacteria and cloning of the Bradyrhizobium japonicum squalene-hopene cyclase gene

Hopanoid lipids have been discovered recently in a number of nitrogen-fixing soil bacteria and in Bradyrhizobium bacteria which fix nitrogen in association with legume plants. We report here an investigation of the hopanoid content in an additional number of soil bacteria capable of living in close association with plants. Of the strains investigated, hopanoids were discovered in phototrophic, nitrogen-fixing bacteria and in an extended number of Bradyrhizobium strains. Strains in which hopanoids so far have not been found belong to the following genera: Rhizobium, Sinorhizobium, Phyllobacterium, Agrobacterium, and Azoarcus. To address the function of hopanoids in Bradyrhizobium, we cloned the gene coding for a key enzyme of hopanoid biosynthesis, the squalene-hopene cyclase, and expressed the gene in E. coli. The recombinant enzyme catalyzed in vitro the cyclization of squalene to hopanoid derivatives.Abbreviations SHC squalene-hopene cyclase - shc squalene-hopene cyclase gene     

Occurrence of hopanoid lipids in anaerobic Geobacter species

Geobacter metallireducens and G. sulfurreducens have been classified as strictly anaerobic bacteria which grow and thrive in subsurface and sediment environments. Hopanoids are pentacyclic triterpenoid lipids and are important for bacterial membrane stability and functioning. Hopanoids predominantly occur in aerobically growing bacteria of oxic environments. They rarely have been found in facultatively anaerobic bacteria and, to date, not at all in strict anaerobes. Our research shows that anaerobically grown G. metallireducens and G. sulfurreducens bacteria contain a range of hopanoid lipids, such as diploptene (i.e. hop-22(29)-ene) and hop-21-ene, and more complex, elongated hopanoids. In geological formations, diagenetic derivatives of hopanoids are widely used as biomarkers and are recognized as molecular fossils of bacterial origin. To date, these biomarkers have largely been interpreted as those derived from ancient oxic environments. Our findings presented here suggest that this interpretation needs to be re-evaluated. In addition to the origin in oxic environments, 'geohopanoids' may originate from ancient anaerobic environments as well.