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.

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.

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.