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.

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 α -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 α-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.biomarkers | cyanobacteria | hopanoids | phylogeny | radical SAM

The rise of atmospheric O(2) was a milestone in the history of life. Although O(2) itself is not a climate-active gas, its appearance would have removed a methane greenhouse present on the early Earth and potentially led to dramatic cooling. Moreover, by fundamentally altering the biogeochemical cycles of C, N, S and Fe, its rise first in the atmosphere and later in the oceans would also have had important indirect effects on Earth's climate. Here, we summarize major lines of evidence from the geological literature that pertain to when and how O(2) first appeared in significant amounts in the atmosphere. On the early Earth, atmospheric O(2) would initially have been very low, probably <10(-5) of the present atmospheric level. Around 2.45 billion years ago, atmospheric O(2) rose suddenly in what is now termed the Great Oxidation Event. While the rise of oxygen has been the subject of considerable attention by Earth scientists, several important aspects of this problem remain unresolved. Our goal in this review is to provide a short summary of the current state of the field, and make the case that future progress towards solving the riddle of oxygen will benefit greatly from the involvement of molecular biologists.

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.

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are distinctive archaeal membrane-spanning lipids with up to eight cyclopentane rings and/or one cyclohexane ring. The number of rings added to the GDGT core structure can vary as a function of environmental conditions, such as changes in growth temperature. This physiological response enables cyclic GDGTs preserved in sediments to be employed as proxies for reconstructing past global and regional temperatures and to provide fundamental insights into ancient climate variability. Yet, confidence in GDGT-based paleotemperature proxies is hindered by uncertainty concerning the archaeal communities contributing to GDGT pools in modern environments and ambiguity in the environmental and physiological factors that affect GDGT cyclization in extant archaea. To properly constrain these uncertainties, a comprehensive understanding of GDGT biosynthesis is required. Here, we identify 2 GDGT ring synthases, GrsA and GrsB, essential for GDGT ring formation in Sulfolobus acidocaldarius. Both proteins are radical S-adenosylmethionine proteins, indicating that GDGT cyclization occurs through a free radical mechanism. In addition, we demonstrate that GrsA introduces rings specifically at the C-7 position of the core GDGT lipid, while GrsB cyclizes at the C-3 position, suggesting that cyclization patterns are differentially controlled by 2 separate enzymes and potentially influenced by distinct environmental factors. Finally, phylogenetic analyses of the Grs proteins reveal that marine Thaumarchaeota, and not Euryarchaeota, are the dominant source of cyclized GDGTs in open ocean settings, addressing a major source of uncertainty in GDGT-based paleotemperature proxy applications.

Sterols are essential components of eukaryotic cells whose biosynthesis and function has been studied extensively. Sterols are also recognized as the diagenetic precursors of steranes preserved in sedimentary rocks where they can function as geological proxies for eukaryotic organisms and/or aerobic metabolisms and environments. However, production of these lipids is not restricted to the eukaryotic domain as a few bacterial species also synthesize sterols. Phylogenomic studies have identified genes encoding homologs of sterol biosynthesis proteins in the genomes of several additional species, indicating that sterol production may be more widespread in the bacterial domain than previously thought. Although the occurrence of sterol synthesis genes in a genome indicates the potential for sterol production, it provides neither conclusive evidence of sterol synthesis nor information about the composition and abundance of basic and modified sterols that are actually being produced. Here, we coupled bioinformatics with lipid analyses to investigate the scope of bacterial sterol production. We identified oxidosqualene cyclase (Osc), which catalyzes the initial cyclization of oxidosqualene to the basic sterol structure, in 34 bacterial genomes from five phyla (Bacteroidetes, Cyanobacteria, Planctomycetes, Proteobacteria, and Verrucomicrobia) and in 176 metagenomes. Our data indicate that bacterial sterol synthesis likely occurs in diverse organisms and environments and also provides evidence that there are as yet uncultured groups of bacterial sterol producers. Phylogenetic analysis of bacterial and eukaryotic Osc sequences confirmed a complex evolutionary history of sterol synthesis in this domain. Finally, we characterized the lipids produced by Osc-containing bacteria and found that we could generally predict the ability to synthesize sterols. However, predicting the final modified sterol based on our current knowledge of sterol synthesis was difficult. Some bacteria produced demethylated and saturated sterol products even though they lacked homologs of the eukaryotic proteins required for these modifications emphasizing that several aspects of bacterial sterol synthesis are still completely unknown.

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.

Differentiating the contributions of photosynthesis and respiration to the global carbon cycle is critical for improving predictive climate models. Carbonic anhydrase (CA) activity in leaves is responsible for the largest biosphere-atmosphere trace gas fluxes of carbonyl sulfide (COS) and the oxygen-18 isotopologue of carbon dioxide (COO) that both reflect gross photosynthetic rates. However, CA activity also occurs in soils and will be a source of uncertainty in the use of COS and COO as carbon cycle tracers until process-based constraints are improved. In this study, we measured COS and COO exchange rates and estimated the corresponding CA activity in soils from a range of biomes and land use types. Soil CA activity was not uniform for COS and CO, and patterns of divergence were related to microbial community composition and CA gene expression patterns. In some cases, the same microbial taxa and CA classes catalyzed both COS and CO reactions in soil, but in other cases the specificity towards the two substrates differed markedly. CA activity for COS was related to fungal taxa and β-D-CA expression, whereas CA activity for CO was related to algal and bacterial taxa and α-CA expression. This study integrates gas exchange measurements, enzyme activity models, and characterization of soil taxonomic and genetic diversity to build connections between CA activity and the soil microbiome. Importantly, our results identify kinetic parameters to represent soil CA activity during application of COS and COO as carbon cycle tracers.