A polycyclic terpenoid that alleviates oxidative stress.
ABSTRACT: Polycyclic terpenoid lipids such as hopanes and steranes have been widely used to understand ancient biology, Earth history, and the oxygenation of the ocean-atmosphere system. Some of these lipids are believed to be produced only by aerobic organisms, whereas others actually require molecular oxygen for their biosynthesis. A persistent question remains: Did some polycyclic lipids initially evolve in response to certain environmental or metabolic stresses, including the presence of oxygen? Here, we identify tetracyclic isoprenoids in spores of the bacterium Bacillus subtilis. We call them sporulenes. They are produced by cyclization of regular polyprenes, a reaction that is more favorable chemically than the formation of terpenoids such as hopanoids and steroids from squalene. The simplicity of the reaction suggests that the B. subtilis cyclase may be analogous to evolutionarily ancient cyclases. We show that these molecules increase the resistance of spores to a reactive oxygen species, demonstrating a specific physiological role for a nonpigment bacterial lipid biomarker. Geostable derivatives of these compounds in sediments could thus be used as direct indicators of oxidative stress and aerobic environments.
Project description:Several studies suggest that petroleum biodegradation can be achieved by either aerobic or anaerobic microorganisms, depending on oxygen input or other electron acceptors and appropriate nutrients. Evidence from in vitro experiments with samples of petroleum formation water and oils from Pampo Field indicate that petroleum biodegradation is more likely to be a joint achievement of both aerobic and anaerobic bacterial consortium, refining our previous observations of aerobic degradation. The aerobic consortium depleted, in decreasing order, hydrocarbons > hopanes > steranes > tricyclic terpanes while the anaerobic consortium depleted hydrocarbons > steranes > hopanes > tricyclic terpanes. The oxygen content of the mixed consortia was measured from time to time revealing alternating periods of microaerobicity (O2 ~0.8 mg.L-1) and of aerobicity (O2~6.0 mg.L-1). In this experiment, the petroleum biodegradation changed from time to time, alternating periods of biodegradation similar to the aerobic process and periods of biodegradation similar to the anaerobic process. The consortia showed preferences for metabolizing hydrocarbons > hopanes > steranes > tricyclic terpanes during a 90-day period, after which this trend changed and steranes were more biodegraded than hopanes. The analysis of aerobic oil degrading microbiota by the 16S rRNA gene clone library detected the presence of Bacillus, Brevibacterium, Mesorhizobium and Achromobacter, and the analysis of the anaerobic oil degrading microbiota using the same technique detected the presence of Bacillus and Acinetobacter (facultative strains). In the mixed consortia Stenotrophomonas, Brevibacterium, Bacillus, Rhizobium, Achromobacter and 5% uncultured bacteria were detected. This is certainly a new contribution to the study of reservoir biodegradation processes, combining two of the more important accepted hypotheses.
Project description:Sedimentary hopanes are pentacyclic triterpenoids that serve as biomarker proxies for bacteria and certain bacterial metabolisms, such as oxygenic photosynthesis and aerobic methanotrophy. Their parent molecules, the bacteriohopanepolyols (BHPs), have been hypothesized to be the bacterial equivalent of sterols. However, the actual function of BHPs in bacterial cells is poorly understood. Here, we report the physiological study of a mutant in Rhodopseudomonas palustris TIE-1 that is unable to produce any hopanoids. The deletion of the gene encoding the squalene-hopene cyclase protein (Shc), which cyclizes squalene to the basic hopene structure, resulted in a strain that no longer produced any polycyclic triterpenoids. This strain was able to grow chemoheterotrophically, photoheterotrophically, and photoautotrophically, demonstrating that hopanoids are not required for growth under normal conditions. A severe growth defect, as well as significant morphological damage, was observed when cells were grown under acidic and alkaline conditions. Although minimal changes in shc transcript expression were observed under certain conditions of pH shock, the total amount of hopanoid production was unaffected; however, the abundance of methylated hopanoids significantly increased. This suggests that hopanoids may play an indirect role in pH homeostasis, with certain hopanoid derivatives being of particular importance.
Project description:Diesel exhaust particulate matter contains many semivolatile organic compounds (SVOCs) of environmental and health significance. This study investigates the composition, emission rates, and integrity of 25 SVOCs, including polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs (NPAHs), and diesel biomarkers hopanes and steranes. Diesel engine particulate matter (PM), generated using an engine test bench, three engine conditions, and ultra-low sulfur diesel (ULSD), was collected on borosilicate glass fiber filters. Under high engine load, the PM emission rate was 0.102 g/kWh, and emission rate of ?PAHs (10 compounds), ?NPAHs (6 compounds), ?hopanes (2 compounds), and ?steranes (2 compounds) were 2.52, 0.351, 0.02 ~ 2 and 1?g/kWh, respectively. Storage losses were evaluated for three cases: conditioning filters in clean air at 25 °C and 33% relative humidity (RH) for 24 h; storing filter samples (without extraction) wrapped in aluminum foil at 4 °C for up to one month; and storing filter extracts in glass vials capped with Teflon crimp seals at 4 °C for up to six months. After conditioning filters for 24 h, 30% of the more volatile PAHs were lost, but lower volatility NPAHs, hopanes and steranes showed negligible changes. Storing wrapped filters and extracts at 4 °C for up to one month did not lead to significant losses, but storing extracts for five months led to significant losses of PAHs and NPAHs; hopanes and steranes demonstrated greater integrity. These results suggest that even relatively brief filter conditioning periods, needed for gravimetric measurements of PM mass, and extended storage of filter extracts can lead to underestimates of SVOC concentrations. Thus, SVOC sampling and analysis protocols should utilize stringent criteria and performance checks to identify and limit possible biases occurring during filter and extract processing.
Project description:Hopanes and steranes found in Archean rocks have been presented as key evidence supporting the early rise of oxygenic photosynthesis and eukaryotes, but the syngeneity of these hydrocarbon biomarkers is controversial. To resolve this debate, we performed a multilaboratory study of new cores from the Pilbara Craton, Australia, that were drilled and sampled using unprecedented hydrocarbon-clean protocols. Hopanes and steranes in rock extracts and hydropyrolysates from these new cores were typically at or below our femtogram detection limit, but when they were detectable, they had total hopane (<37.9 pg per gram of rock) and total sterane (<32.9 pg per gram of rock) concentrations comparable to those measured in blanks and negative control samples. In contrast, hopanes and steranes measured in the exteriors of conventionally drilled and curated rocks of stratigraphic equivalence reach concentrations of 389.5 pg per gram of rock and 1,039 pg per gram of rock, respectively. Polycyclic aromatic hydrocarbons and diamondoids, which exceed blank concentrations, exhibit individual concentrations up to 80 ng per gram of rock in rock extracts and up to 1,000 ng per gram of rock in hydropyrolysates from the ultraclean cores. These results demonstrate that previously studied Archean samples host mixtures of biomarker contaminants and indigenous overmature hydrocarbons. Therefore, existing lipid biomarker evidence cannot be invoked to support the emergence of oxygenic photosynthesis and eukaryotes by ? 2.7 billion years ago. Although suitable Proterozoic rocks exist, no currently known Archean strata lie within the appropriate thermal maturity window for syngenetic hydrocarbon biomarker preservation, so future exploration for Archean biomarkers should screen for rocks with milder thermal histories.
Project description: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.
Project description:Quasi-ultrafine (quasi-UF) particulate matter (PM(0.25)) and its components were measured in indoor and outdoor environments at four retirement communities in Los Angeles Basin, California, as part of the Cardiovascular Health and Air Pollution Study (CHAPS). The present paper focuses on the characterization of the sources, organic constituents and indoor and outdoor relationships of quasi-UF PM. The average indoor/outdoor ratios of most of the measured polycyclic aromatic hydrocarbons (PAHs), hopanes, and steranes were close to or slightly lower than 1, and the corresponding indoor-outdoor correlation coefficients (R) were always positive and, for the most part, moderately strong (median R was 0.60 for PAHs and 0.74 for hopanes and steranes). This may reflect the possible impact of outdoor sources on indoor PAHs, hopanes, and steranes. Conversely, indoor n-alkanes and n-alkanoic acids were likely to be influenced by indoor sources. A chemical mass balance model was applied to both indoor and outdoor speciated chemical measurements of quasi-UF PM. Among all apportioned sources of both indoor and outdoor particles, vehicular emissions was the one contributing the most to the PM(0.25) mass concentration measured at all sites (24-47% on average).Although people (particularly the elderly retirees of our study) generally spend most of their time indoors, a major portion of the PM(0.25) particles they are exposed to comes from outdoor mobile sources. This is important because, an earlier investigation, also conducted within the Cardiovascular Health and Air Pollution Study (CHAPS), showed that indoor-infiltrated particles from mobile sources are more strongly correlated with adverse health effects observed in the elderly subjects living in the studied retirement communities compared with other particles found indoors (Delfino et al., 2008).
Project description:The rise of atmospheric oxygen has driven environmental change and biological evolution throughout much of Earth's history and was enabled by the evolution of oxygenic photosynthesis in the cyanobacteria. Dating this metabolic innovation using inorganic proxies from sedimentary rocks has been difficult and one important approach has been to study the distributions of fossil lipids, such as steranes and 2-methylhopanes, as biomarkers for this process. 2-methylhopanes arise from degradation of 2-methylbacteriohopanepolyols (2-MeBHPs), lipids thought to be synthesized primarily by cyanobacteria. The discovery that 2-MeBHPs are produced by an anoxygenic phototroph, however, challenged both their taxonomic link with cyanobacteria and their functional link with oxygenic photosynthesis. Here, we identify a radical SAM methylase encoded by the hpnP gene that is required for methylation at the C-2 position in hopanoids. This gene is found in several, but not all, cyanobacteria and also in alpha -proteobacteria and acidobacteria. Thus, one cannot extrapolate from the presence of 2-methylhopanes alone, in modern environments or ancient sedimentary rocks, to a particular taxonomic group or metabolism. To understand the origin of this gene, we reconstructed the evolutionary history of HpnP. HpnP proteins from cyanobacteria, Methylobacterium species, and other alpha-proteobacteria form distinct phylogenetic clusters, but the branching order of these clades could not be confidently resolved. Hence,it is unclear whether HpnP, and 2-methylhopanoids, originated first in the cyanobacteria. In summary, existing evidence does not support the use of 2-methylhopanes as biomarkers for oxygenic photosynthesis.
Project description:Hopanoids methylated at the C-3 position are a subset of bacterial triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids by extant aerobic methanotrophs and their common occurrence in modern and fossil methane seep communities, in conjunction with carbon isotope analysis, has led to their use as biomarker proxies for aerobic methanotrophy. In addition, these lipids are also produced by aerobic acetic acid bacteria and, lacking carbon isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have brought into question our current understanding of the taxonomic diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock record requires a deeper understanding of their cellular function. In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they appear to be required for the maintenance of intracytoplasmic membranes and cell survival in late stationary phase. Therefore, 3-methylhopanoids may prove more useful as proxies for specific environmental conditions encountered during stationary phase rather than a particular bacterial group.
Project description:Hopanoids are steroid-like lipids from the isoprenoid family that are produced primarily by bacteria. Hopanes, molecular fossils of hopanoids, offer the potential to provide insight into environmental transitions on the early Earth, if their sources and biological functions can be constrained. Semiquantitative methods for mass spectrometric analysis of hopanoids from cultures and environmental samples have been developed in the last two decades. However, the structural diversity of hopanoids, and possible variability in their ionization efficiencies on different instruments, have thus far precluded robust quantification and hindered comparison of results between laboratories. These ionization inconsistencies give rise to the need to calibrate individual instruments with purified hopanoids to reliably quantify hopanoids. Here, we present new approaches to obtain both purified and synthetic quantification standards. We optimized 2-methylhopanoid production in Rhodopseudomonas palustris TIE-1 and purified 2Me-diplopterol, 2Me-bacteriohopanetetrol (2Me-BHT), and their unmethylated species (diplopterol and BHT). We found that 2-methylation decreases the signal intensity of diplopterol between 2 and 34% depending on the instrument used to detect it, but decreases the BHT signal less than 5%. In addition, 2Me-diplopterol produces 10× higher ion counts than equivalent quantities of 2Me-BHT. Similar deviations were also observed using a flame ionization detector for signal quantification in GC. In LC-MS, however, 2Me-BHT produces 11× higher ion counts than 2Me-diplopterol but only 1.2× higher ion counts than the sterol standard pregnane acetate. To further improve quantification, we synthesized tetradeuterated (D4) diplopterol, a precursor for a variety of hopanoids. LC-MS analysis on a mixture of (D4)-diplopterol and phospholipids showed that under the influence of co-eluted phospholipids, the D4-diplopterol internal standard quantifies diplopterol more accurately than external diplopterol standards. These new quantitative approaches permit meaningful comparisons between studies, allowing more accurate hopanoid pattern detection in both laboratory and environmental samples.
Project description: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.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.