Bacterial Hopanoids Research Articles

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.

From Molecular Fossils of Bacterial Hopanoids to the Formation of Isoprene Units: Discovery and Elucidation of the Methylerythritol Phosphate Pathway

Investigations on the biosynthesis of bacterial triterpenoids of the hopane series led to the unexpected discovery of an alternative mevalonate independent pathway for the formation of isoprene units. Methylerythritol phosphate, already presenting the C5 branched isoprene skeleton, is the key intermediate. This pathway was independently characterized in ginkgo embryos for the formation of diterpenoids. It is present in most bacteria and in the plastids of all organisms belonging to phototrophic phyla. The key steps of the discovery and elucidation of this metabolic route are presented in this review.

Lipid Biomarkers, Carbon Isotopes, and Phylogenetic Characterization of Bacteria in California and Nevada Hot Springs

Microbial mats were collected from hot springs in California (Eagleville) and Nevada (Paradise Valley and Crescent Valley) to determine bacterial community structure and pathways of carbon cycling in different geothermal environments of the western United States. Phospholipid fatty acids (PLFA) at Eagleville contained even-numbered fatty acids, with 16:0 being the most abundant (48.8%), followed by 18:1ω 9c (17.2%), 16:1ω 7c/t (6.3%), and 18:0 (6.2%), which are consistent with lipid profiles of cyanobacteria or other phototrophic bacteria. The PLFA profiles at Paradise Valley and Crescent Valley were dominated by similar even-numbered fatty acids; however, branched fatty acids such as iso- and anteiso- 15:0 and 17:0 were also abundant (up to 7.1% compared to 2.0% at Eagleville), suggesting greater relative abundance of heterotrophic bacteria in these springs. Analysis of neutral lipids was only performed on Eagleville and Paradise Valley springs, which revealed abundant bacterial hopanoids including the 2–methylbacteriohopane-32,33,34,35-tetrol (2-methylBHT) that is specific to cyanobacteria; however, the diversity of hopanoid compounds was significantly lower at Eagleville than at Paradise Valley. The carbon-isotope composition of individual PLFA averaged −30.7 ± 1.3‰ (n = 7) at Eagleville, −28.0 ± 1.8‰ (n = 3) at Crescent Valley, and −29.7 ± 3.1‰ (n = 12) at Paradise Valley. Carbon isotope fractionation between PLFA and CO 2 was only available for Eagleville (−11.7‰) and Paradise Valley (−21.7‰), which indicated the predominance of the Calvin cycle for CO 2 fixation in these hot springs. Bacterial 16S rRNA genes were extracted from environmental samples at Eagleville and Paradise Valley but not Crescent Valley. Clone libraries indicated the predominance of cyanobacteria (50–75%) at these locations, which is consistent with the lipid profiles. Phylogenetic tree of the 16S rRNA genes indicated that most of the cyanobacterial sequences are unknown and may be specific to the Nevada and California hot springs. Phototrophic green non-sulfur bacteria were also present at Eagleville (13%) and Paradise Valley (7%). The remaining sequences were related to α-, β -, and γ -Proteobacteria, Acidobacteria, Deinococcus/Thermus, Bacteroidetes, and Spirochaetes. However, not all of these sequences were present at each of the springs. Results of this study demonstrate the consistency among lipid profiles (phenotypes), carbon isotopes (biogeochemistry), and 16S rRNA genes (genotypes) of the bacterial community in these hot springs, which cumulatively suggest the importance of cyanobacteria in primary production of biomass under the environmental conditions examined.

Structural characterisation of unsaturated bacterial hopanoids by atmospheric pressure chemical ionisation liquid chromatography/ion trap mass spectrometry

The production of bacteriohopanepolyols (BHPs) is widespread in many different groups of prokaryotes; however, unsaturated components are less common except amongst the acetic acid bacteria. Here we describe the characterisation of mono- (Delta(6) or Delta(11)) and diunsaturated (Delta(6,11)) bacteriohopanetetrols isolated from the acetic acid bacterium Gluconacetobacter xylinus (formerly Acetobacter aceti ssp. xylinum) by atmospheric pressure chemical ionisation ion trap mass spectrometry (APCI-MS(n)). APCI-MS(2) spectra are compared with equivalent electron ionisation (EI) spectra and differences in fragmentation pathways are discussed. Having established characteristic spectral features for a range of unsaturated BHPs we now have the ability to rapidly detect the presence of unsaturated BHPs in both natural environmental samples (soils, sediments, water columns) as well as in microbial cultures.

First evidence for the Cenomanian–Turonian oceanic anoxic event (OAE2, ‘Bonarelli’ event) from the Ionian Zone, western continental Greece

Integrated biostratigraphic (planktonic foraminifera, calcareous nannofossils), chemostratigraphic (bulk C and O isotopes) and compound-specific organic geochemical studies of a mid-Cretaceous pelagic carbonate—black shale succession of the Ionian Zone (western Greece), provide the first evidence for the Cenomanian–Turonian oceanic anoxic event (OAE2, ‘Bonarelli’ event) in mainland Greece. The event is manifested by the occurrence of a relatively thin (35 cm), yet exceptionally organic carbon-rich (44.5 wt% TOC), carbonate-free black shale, near the Cenomanian–Turonian boundary within the Vigla limestone formation (Berriasian–Turonian). Compared to the ‘Bonarelli’ black-shale interval from the type locality of OAE2 in Marche–Umbria, Italy, this black shale exhibits greatly reduced stratigraphic thickness, coupled with a considerable relative enrichment in TOC. Isotopically, enriched δ13C values for both bulk organic matter (−22.2‰) and specific organic compounds are up to 5‰ higher than those of underlying organic-rich strata of the Aptian-lower Albian Vigla Shale member, and thus compare very well with similar values of Cenomanian–Turonian black shale occurrences elsewhere. The relative predominance of bacterial hopanoids in the saturated, apolar lipid fraction of the OAE2 black shale of the Ionian Zone supports recent findings suggesting the abundance of N2-fixing cyanobacteria in Cretaceous oceans during the Cenomanian–Turonian and early Aptian oceanic anoxic events.

Investigation of bacterial hopanoid inputs to soils from Western Canada

Hopanoids have been widely used as characteristic biomarkers to study inputs of bacterial biomass to sediments because they are preserved in the geologic record. A limited number of studies have been performed on hopanoid biomarkers in soils. The present study examined the distribution and potential preservation of hopanoids in soils that are developed under different climatic conditions and varying vegetative inputs. Solvent extraction and sequential chemical degradation methods were employed to extract both “free” and “bound” hopanoids, from three grassland soils, a grassland–forest transition soil, and a forest soil from Western Canada. Identification and quantification of hopanoids in the soil samples were carried out by gas chromatography–mass spectrometry. Methylbishomohopanol, bishomohopanol and bishomohopanoic acid were detected in all solvent extracts. The base hydrolysis and ruthenium tetroxide extracts contained only bishomohopanoic acid at a concentration range of 0.8–8.8 μg/gC and 2.2–28.3 μg/gC, respectively. The acid hydrolysis procedure did not release detectable amounts of hopanoids. The solvent extraction yielded the greatest amounts of “free” hopanoids in two of the grassland soils (Dark Brown and Black Chernozems) and in the forest soil (Gray Luvisol). In contrast, the chemical degradation methods resulted in higher amounts of “bound” hopanoids in the third grassland soil (Brown Chernozem) and the transition soil (Dark Gray Chernozem), indicating that more hopanoids exist in the “bound” form in these soils. Overall, the forest and the transition soils contained more hopanoids than the grassland soils. This is hypothesized to be due to the greater degradation of hopanoids in the grassland soils and or sorption to clay minerals, as compared to the forest and transition soils.

Well preserved Palaeogene and Cretaceous biomarkers from the Kilwa area, Tanzania

We have investigated the biomarker composition of sediments ranging in age from 30 to 70 Ma from the Kilwa area of Tanzania. They are dominated by material of terrestrial ( n-alkanols, n-alkanoic acids, n-alkanes and triterpenoids) and [bacterial hopanoids and a dipentadecyl glycerol diether (DGD)] origin with only a minor contribution of marine origin (crenarchaeol and some algal steroids). This indicates a marine setting dominated by higher plant input, which most likely stimulated sedimentary microbial activity, including sulfate reduction. The lipid composition, specifically the presence of bacteriohopanepolyols (BHPs) and the stereochemistry of hopanoids, suggests a low degree of thermal maturity. Many biomarkers have still retained functionality even after 30–70 million years. This is in good agreement with the exceptional preservation of foraminifera and nannofossils and can probably be attributed to the very low sediment permeability and burial depth. There is a clear distinction in the biomarker distributions between the Oligocene-age sediments collected at the end of the peninsula near Kilwa Masoko and the older sediments collected from elsewhere, indicating differences in depositional environment. Sediments from the end of the peninsula have higher branched and isoprenoid tetraether index values, indicating a relatively higher terrestrial input. They also contain two tentatively assigned C 28 hopenes (28,30-dinorneohop-13(18)-ene and 28,30-dinorhop-17(21)-ene), (aromatic) des- A-triterpenes, des- E-hopenes, aromatic pentacyclic triterpenoids, C 33 DGD, archaeol and BHPs, which are absent from or only present in small amounts in the other sediments. These differences likely reflect an evolution of the depositional area from a relative open shelf environment with substantial water depth to a shallower setting during the early Oligocene.

Synthesis of the Aminocyclopentitol Moieties of the Hopanoids of Zymomonas mobilis and “Anacystis montana”

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis of the aminocyclopentitol moieties of the hopanoids of Zymomonas mobilis and 'Anacystis montana'.

The first synthesis of the cyclopentitol units in bacterial hopanoids has been accomplished from D-glucosamine and the possible biological activity of the free cyclitols as glycosidase inhibitors has been studied.

Morphologic and spectral investigation of exceptionally well-preserved bacterial biofilms from the Oligocene Enspel formation, Germany

The fossilised soft tissues of a tadpole and an associated coprolitic structure from the organic-rich volcanoclastic lacustrine Upper Oligocene Enspel sediments (Germany) were investigated using high-resolution imaging techniques and nondestructive in situ surface analysis. Total organic carbon analysis of the coprolite and the sediment revealed values of 28.9 and 8.9% respectively. The soft tissues from the tadpole and the coprolite were found to be composed of 0.5 to 1 μm-sized spheres and rod shapes. These features are interpreted as the fossil remains of bacterial biofilms consisting probably of heterotrophic bacteria and fossilised extracellular polymeric substances. They became fossilised while in the process of degrading the organic matter of the organism and the coprolite. Comparison with a modern marine biofilm revealed morphologic details identical to those observed in the fossil bacterial biofilms. Although the fossil biofilms on both macrofossils exhibited identical microtextures, their mode of preservation was inhomogeneous and varied between calcium phosphate and an as yet unidentified mineral phase consisting mainly of Si, Ca, Ti, P, and S, but also showing the presence of Mg, Al, and Fe. The coprolite consists purely of fossilised bacterial cells in a densely packed arrangement and associated fossilised extracellular polymeric substances.In addition to preliminary imaging and energy-dispersive X-ray analysis, both the fossil biofilms and the sediment were investigated by nondestructive in situ analysis using time of flight-secondary ion mass spectroscopy (ToF-SIMS). The mass spectra obtained on the coprolite in mass-resolved chemical mapping mode allowed the tentative identification of a number of organic secondary ion species. Some spectra appear to indicate the presence of bacterial hopanoids, but further work using standard techniques such as gas chromatography mass spectroscopy is needed to conclusively verify the presence of these substances. Nevertheless, ToF-SIMS chemical maps were successfully correlated with electron microscopy images, allowing the correlation of molecular spectra, the spatial distribution of individual organic species, and specific morphologic features to demonstrate the potential of this approach in the analysis of microfossils.

ABSTRACT: Bacterial Hopanoids as Markers for Depositional Environments

ABSTRACT: Bacterial Hopanoids as Markers for Depositional Environments

Novel hopanoids from Frankia spp. and related soil bacteria. Squalene cyclization and significance of geological biomarkers revisited.

Three series of hopanoids, differing by their configurations at C-17 and C-21, have been identified in several Frankia spp. and other related soil bacteria. The widespread bacterial hopanoids of the 17beta(H),21beta(H) series were accompanied by their isomers of the 17beta(H),21alpha(H) (moretane) and 17alpha(H), 21beta(H) series. The latter series has not previously been found in living organisms and is considered to be a result of the abiotic isomerization of the thermodynamically less stable 17beta(H),21beta(H) hopanoids. This simultaneous presence of three isomeric hopanoid series highlights intriguing problems in the biogenesis of the bacteriohopane skeleton and partly questions the significance of hopanic biomarkers in sediments.

Time of flight secondary ion mass spectrometry (ToFSIMS) of a number of hopanoids

Time of flight secondary ion mass spectrometry (ToFSIMS) has been applied to a number of bacterial hopanoids in an attempt to characterise these geologically important molecules in situ by a surface sensitive technique. Our results show that these molecules can be detected using this instrumentation to a high degree of mass accuracy. We believe that ToFSIMS can, therefore, be used to identify these molecules in environmental samples where sample size may be an issue and contraindicate the use of more traditional techniques such as GC–MS.

Analysis of bacteriohopanepolyols in sediment and bacterial extracts by high performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry

We report the new application of negative ion atmospheric pressure chemical ionization (APCI) to the detection of composite bacteriohopanepolyols in bacterial and sedimentary (or other natural environmental) samples. The method employs the addition of dichloromethane to the high performance liquid chromatography eluent prior to APCI to stimulate formation of chlorine addition/association ions from the hopanoid alcohols. This approach provided the first direct analysis of composite bacterial hopanoids in a sediment, identifying bacteriohopanetrol and bacteriohopanetetrol cyclitol ether. © 1998 John Wiley & Sons, Ltd.

Modulation of lipoxygenase activity by bacterial hopanoids.

Tetrahydroxybacteriohopane (1), a bacterial hopanoid, inhibited soybean 15-lipoxygenase with an IC50 of about 10 microM. After per-O-acetylation of 1 no inhibition of the 15-lipoxygenase was observed. Two other bacterial hopanoids, tetrahydroxybacteriohopane glucosamine (2) and tetrahydroxybacteriohopane ether (3), stimulated the activity of soybean 15-lipoxygenase. The activities of two other arachidonic acid-metabolizing enzymes, human 5-lipoxygenase and prostaglandin H synthase, were unaffected by 1.

Molecular taphonomy of membrane constituents the search for the most primitive membranes

Taphonomy is the study of fossilization processes, and includes "reconstruction" of past animals from their fossils. At the molecular level, the study of the organic constituents of sediments (crude oils and coals, but also clays, shales, etc) has yielded hundreds of structures, essentially in the hands of P.Albrecht and his co-workers in Strasbourg. These are sometimes hardly modified molecular fossils of known natural products. For instance, cholestane "must" come from cholesterol : reductions are frequent molecular taphonomic processes. Diacholestane, also frequently present in sediments, might be derived from "diasteror', an unknown but biosynthetically reasonable rearranged cholesterol ; however, the ease of rearrangement of cholesterol to diasterane derivatives by moderate heating with clays suggests rather that diacholestane derives also from cholesterol ; Wagner-Meerwein rearrangements are reasonable taphonomic processes. To interpret data from organic geochemistry, one must therefore collect trustworthy precise structural data, rely on the extensive catalogs of natural products structures and of biosynthetic process, and know which molecular taphonomic processes are plausible. Known molecular fossils belong to several natural products families, including porphyrins obviously derived from bacterial pigments ; by far most of them are derived from terpenoid precursors, as expected as biodegradation of highly branched structures is notoriously difficult. It was also not unexpected to find, frequently, molecular fossils of some arch~eal lipids : phytane and pristane, which could also have come from chlorophyll or from tocopherols, but also bisphytane, the structure of which became meaningful only once the C40 chains of phospholipid ethers of thermophilic and of methanogenic arch~ea became known. In other cases, while the structures of the precursors could be derived without doubt, they did not tally with any known one. The most spectacular case of these "orphan fossils" has been that of the ubiquitous geohopanoids, triterpene derivatives carrying an additional n-C 5 chain, for which more than 250 structures have been fully demonstrated ; their ubiquity and abundance in sediments make them probably the most abundant natural products on Earth ! Their precursors, the bacterial hopanoids, were discovered initially in two particular bacteria, but later, by M.Rohmer, in many very varied bacteria, with very varied structures (more than 40 established so far, including the fantastic adenosylhopane). Based on the molecular dimensions and amphiphilic character of bacterial hopanoids, we postulated that they reinforce bacterial membranes like

Isotope and molecular evidence for the diverse origins of carboxylic acids in leaf fossils and sediments from the Miocene Lake Clarkia deposit, Idaho, U.S.A.

The normal fatty acids (C 14C 32), ω-hydroxy acids (C 12C 32), (ω-1)-hydroxy acids (C 28 to C 32), terpenoid acids and hopanoid acids in higher plant leaf fossils and enclosing sediments from the Miocene lacustrine Clarkia deposit (17–20 Ma) show a wide range (−18.5 to −55.2‰ vs PDB) of carbon isotopic compositions. Both molecular distributions and isotopic compositions display highly informative similarities (e.g., C 22C 32 n-acids) and differences (e.g., C 16 and C 18 n-acids, C 22 ω- and C 28 and C 30 (ω-1) hydroxy acids) between the individual fossils and sediment matrix. These δ 13C values are distinctive among compound classes. The molecular distributions and the ranges of δ 13C values (−27.2 to −32.2‰) for long chain n-fatty acids (C 22C 32), terpenoid acids and ω-hydroxy acids (except for C 22 ω-hydroxy acids) are in accord with derivation from mainly C 3 terrestrial higher plants. The (ω-1) hydroxy acids (C 28 and C 30 homologues), however, show a significant 13C enrichment (−18.5 to −22.8‰) in the sediments, consistent with an origin in aquatic photosynthetic organisms whose habitats are severely CO 2 limited. The C 22 ω-hydroxy acid shows high abundance in sediments but is not detected in association with leaf fossils and has distinctive δ 13C values (ca. −33‰), depleted in 13C by about 4–5‰ compared with the other ω-hydroxy acid homologues (C 24C 28), and is probably derived from micro-organisms living below the oxycline. Bacterial hopanoids are dominated by isomers with 22 R-17β(H),21β(H) configuration, consistent with the low thermal maturity of the sediment, and also display rather negative δ 13C values (−38 to −55‰), suggesting their derivation from methanotrophic bacteria. From the isotopic data we infer the operation of a methane cycle, which is also supported by the palaeontological evidence for anoxia in the water column. A novel compound, des-A-olean-28-oic acid was also tentatively identified.

Structure and origin of two triterpene-derived aromatic hydrocarbons in Messel shale

Two triterpene-derived aromatic hydrocarbons of the gammacerane-type have been isolated from Messel shale; structural (MS, NMR) and carbon isotopic data indicate that these biological markers are derived from tetrahymanol from protozoa or purple phototrophic bacteria, and from a bacterial hopanoid.

Prokaryotic triterpenoids: new hopanoids from the nitrogen-fixing bacteria Azotobacter vinelandii, Beijerinckia indica and Beijerinckia mobilis

Three nitrogen-fixing bacteria, Azotobacter vinelandii, Beijerinckia indica and Beijerinckia mobilis, were shown to contain large amounts of triterpenoids of the hopane series. In A. vinelandii, the major compound was a novel bacteriohopanepentol ether accompanied by a similar bacteriohopanetetrol derivative: in both compounds, the hopanoids are linked via an ether bond to a carbapseudopentose moiety often found in bacterial hopanoids. In the two Beijerinckia species, diplopterol and 2β-methyldiplopterol were accumulated in much larger amounts than those usually recorded in hopanoid-producing eubacteria, while 2β-methyldiploptene was isolated for the first time from B. mobilis. Whereas in B. mobilis aminobacteriohopanetriol was the only C35 hopanoid, the simultaneous presence of bacteriohopanetetrol and aminobacteriohopanetriol in B. indica is a rather unusual feature.

Bacterial hopanoids from pink-pigmented facultative methylotrophs (PPFMs) and from green plant surfaces

Six strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) isolated from phylloplane surfaces of different plants were analysed for the presence of triterpenoids of the hopane series. All of the cultures produced hopanoids in abundant quantities and contained the same compounds as the type strain of Methylobacterium organophilum: diplopterol, 2β-methyldiplopterol, bacteriohopanetetrol, a tetrol glycoside and two tetrol ethers. The presence of a guanidinium group on the carbapseudopentose moiety of one of these ethers and/or of 2β-methyldiplopterol seems to be restricted to the genus Methylobacterium.Small amounts of bacteriohopanepolyols were detected in three of seven plants studied. Since no bacterial C35hopanoids have been reported in eukaryotes, we believe they are probably derived from eubacterial epibionts present on the phylloplane surfaces, the most numerous of which are Methylobacteriumspp.