Disproportionate Carbon Dioxide Efflux in Bacterial Metabolic Pathways for Different Organic Substrates Leads to Variable Contribution to Carbon-Use Efficiency.

Microbial organic matter turnover is an important contributor to the terrestrial carbon dioxide (CO2) budget. Partitioning of organic carbons into biomass relative to CO2 efflux, termed carbon-use efficiency (CUE), is widely used to characterize organic carbon cycling by soil microorganisms. Recent studies challenge proposals of CUE dependence on the oxidation state of the substrate carbon and implicate instead metabolic strategies. Still unknown are the metabolic mechanisms underlying variability in CUE. We performed a multiomics investigation of these mechanisms in Pseudomonas putida, a versatile soil bacterium of the Gammaproteobacteria, processing a mixture of plant matter derivatives. Our 13C-metabolomics data captured substrate carbons into different metabolic pathways: cellulose-derived sugar carbons in glycolytic and pentose-phosphate pathways; lignin-related aromatic carbons in the tricarboxylic acid cycle. Subsequent 13C-metabolic flux analysis revealed a 3-fold lower investment of sugar carbons in CO2 efflux compared to aromatic carbons, in agreement with reported substrate-dependent CUE. Proteomics analysis revealed enzyme-level regulation only for substrate uptake and initial catabolism, which dictated downstream fluxes through CO2-producing versus biomass-synthesizing reactions. Metabolic partitioning as shown here explained the substrate-dependent CUE calculated from reported metabolic flux analyses of other bacteria, further supporting a metabolism-guided perspective for predicting the microbial conversion of accessible organic matter to CO2 efflux.

Seasonal shifts in community composition and proteome expression in a sulphur-cycling cyanobacterial mat.

Seasonal changes in light and physicochemical conditions have strong impacts on cyanobacteria, but how they affect community structure, metabolism, and biogeochemistry of cyanobacterial mats remains unclear. Light may be particularly influential for cyanobacterial mats exposed to sulphide by altering the balance of oxygenic photosynthesis and sulphide-driven anoxygenic photosynthesis. We studied temporal shifts in irradiance, water chemistry, and community structure and function of microbial mats in the Middle Island Sinkhole (MIS), where anoxic and sulphate-rich groundwater provides habitat for cyanobacteria that conduct both oxygenic and anoxygenic photosynthesis. Seasonal changes in light and groundwater chemistry were accompanied by shifts in bacterial community composition, with a succession of dominant cyanobacteria from Phormidium to Planktothrix, and an increase in diatoms, sulphur-oxidizing bacteria, and sulphate-reducing bacteria from summer to autumn. Differential abundance of cyanobacterial light-harvesting proteins likely reflects a physiological response of cyanobacteria to light level. Beggiatoa sulphur oxidation proteins were more abundant in autumn. Correlated abundances of taxa through time suggest interactions between sulphur oxidizers and sulphate reducers, sulphate reducers and heterotrophs, and cyanobacteria and heterotrophs. These results support the conclusion that seasonal change, including light availability, has a strong influence on community composition and biogeochemical cycling of sulphur and O2 in cyanobacterial mats.

Metaproteomics reveals functional partitioning and vegetational variation among permafrost-affected Arctic soil bacterial communities.

Microbial activity in Arctic soils controls the cycling of significant stores of organic carbon and nutrients. We studied in situ processes in Alaskan soils using original metaproteomic methods in order to relate important heterotrophic functions to microbial taxa and to understand the microbial response to Arctic greening. Major bacterial groups show strong metabolic specialization in organic topsoils. α-/ß-/γ-Proteobacteria specialized in the acquisition of small, soluble compounds, whereas Acidobacteria, Actinobacteria, and other detritosphere groups specialized in the degradation of plant-derived polymers. α-/ß-/γ-Proteobacteria dominated the expression of transporters for common root exudates and limiting nitrogenous compounds, supporting an ecological model of dependence upon plants for carbon and competition with plants for nitrogen. Detritosphere groups specialized in distinct substrates, with Acidobacteria producing the most enzymes for hemicellulose depolymerization. Acidobacteria was the most active group across the three plant ecotypes sampled-the largely nonvascular, lower biomass intertussock and the largely vascular, higher biomass tussock and shrub. Functional partitioning among bacterial groups was stable between plant ecotypes, but certain functions associated with α-/ß-/γ-Proteobacteria were more strongly expressed in higher biomass ecotypes. We show that refined metaproteomic approaches can elucidate soil microbial ecology as well as biogeochemical trajectories of major carbon stocks. IMPORTANCE The Arctic is warming twice as fast as the rest of the planet, and Arctic soils currently store twice as much carbon as the entire atmosphere-two facts that make understanding how Arctic soil microbial communities are responding to climate change particularly urgent. Greening of vegetation cover across the Arctic landscape is one of the most prominent climate-driven shifts in Arctic terrestrial ecology, with potentially profound effects on biogeochemical cycling by the soil microbiome. Here we use metaproteomics to document microbial metabolic functions that drive soil carbon and nutrient cycling processes in an Arctic tundra landscape. We identify functional roles among bacterial taxonomic groups that are largely stable across vegetation types, with certain functions strongly expressed by rhizosphere groups reflecting a community metabolic response to greening.

Chitin utilization by marine picocyanobacteria and the evolution of a planktonic lifestyle.

Marine picocyanobacteria Prochlorococcus and Synechococcus, the most abundant photosynthetic cells in the oceans, are generally thought to have a primarily single-celled and free-living lifestyle. However, while studying the ability of picocyanobacteria to supplement photosynthetic carbon fixation with the use of exogenous organic carbon, we found the widespread occurrence of genes for breaking down chitin, an abundant source of organic carbon that exists primarily as particles. We show that cells that encode a chitin degradation pathway display chitin degradation activity, attach to chitin particles, and show enhanced growth under low light conditions when exposed to chitosan, a partially deacetylated soluble form of chitin. Marine chitin is largely derived from arthropods, which underwent major diversifications 520 to 535 Mya, close to when marine picocyanobacteria are inferred to have appeared in the ocean. Phylogenetic analyses confirm that the chitin utilization trait was acquired at the root of marine picocyanobacteria. Together this leads us to postulate that attachment to chitin particles allowed benthic cyanobacteria to emulate their mat-based lifestyle in the water column, initiating their expansion into the open ocean, seeding the rise of modern marine ecosystems. Subsequently, transitioning to a constitutive planktonic life without chitin associations led to cellular and genomic streamlining along a major early branch within Prochlorococcus. Our work highlights how the emergence of associations between organisms from different trophic levels, and their coevolution, creates opportunities for colonizing new environments. In this view, the rise of ecological complexity and the expansion of the biosphere are deeply intertwined processes.

Tracking nitrogen allocation to proteome biosynthesis in a marine microbial community.

Microbial growth in many environments is limited by nitrogen availability, yet there is limited understanding of how complex communities compete for and allocate this resource. Here we develop a broadly applicable approach to track biosynthetic incorporation of 15N-labelled nitrogen substrates into microbial community proteomes, enabling quantification of protein turnover and N allocation to specific cellular functions in individual taxa. Application to oligotrophic ocean surface water identifies taxa-specific substrate preferences and a distinct subset of protein functions undergoing active biosynthesis. The cyanobacterium Prochlorococcus is the most effective competitor for acquisition of ammonium and urea and shifts its proteomic allocation of N over the day/night cycle. Our approach reveals that infrastructure and protein-turnover functions comprise substantial biosynthetic demand for N in Prochlorococcus and a range of other microbial taxa. The direct interrogation of the proteomic underpinnings of N limitation with 15N-tracking proteomics illuminates how nutrient stress differentially influences metabolism in co-existing microbes.

DsrC is involved in fermentative growth and interacts directly with the FlxABCD-HdrABC complex in Desulfovibrio vulgaris Hildenborough.

DsrC is a key protein in dissimilatory sulfur metabolism, where it works as co-substrate of the dissimilatory sulfite reductase DsrAB. DsrC has two conserved cysteines in a C-terminal arm that are converted to a trisulfide upon reduction of sulfite. In sulfate-reducing bacteria, DsrC is essential and previous works suggested additional functions beyond sulfite reduction. Here, we studied whether DsrC also plays a role during fermentative growth of Desulfovibrio vulgaris Hildenborough, by studying two strains where the functionality of DsrC is impaired by a lower level of expression (IPFG07) and additionally by the absence of one conserved Cys (IPFG09). Growth studies coupled with metabolite and proteomic analyses reveal that fermentation leads to lower levels of DsrC, but impairment of its function results in reduced growth by fermentation and a shift towards more fermentative metabolism during sulfate respiration. In both respiratory and fermentative conditions, there is increased abundance of the FlxABCD-HdrABC complex and Adh alcohol dehydrogenase in IPFG09 versus the wild type, which is reflected in higher production of ethanol. Pull-down experiments confirmed a direct interaction between DsrC and the FlxABCD-HdrABC complex, through the HdrB subunit. Dissimilatory sulfur metabolism, where sulfur compounds are used for energy generation, is a key process in the ecology of anoxic environments, and is more widespread among bacteria than previously believed. Two central proteins for this type of metabolism are DsrAB dissimilatory sulfite reductase and its co-substrate DsrC. Using physiological, proteomic and biochemical studies of Desulfovibrio vulgaris Hildenborough and mutants affected in DsrC functionality, we show that DsrC is also relevant for fermentative growth of this model organism and that it interacts directly with the soluble FlxABCD-HdrABC complex that links the NAD(H) pool with dissimilatory sulfite reduction.

Proteome Expression and Survival Strategies of a Proteorhodopsin-Containing Vibrio Strain under Carbon and Nitrogen Limitation.

Photoheterotrophy is a widespread mode of microbial metabolism, notably in the oligotrophic surface ocean, where microbes experience chronic nutrient limitation. One especially widespread form of photoheterotrophy is based on proteorhodopsin (PR), which uses light to generate proton motive force that can drive ATP synthesis, flagellar movement, or nutrient uptake. To clarify the physiological benefits conferred by PR under nutrient stress conditions, we quantified protein-level gene expression of Vibrio campbellii CAIM 519 under both carbon and nitrogen limitation and under both light and dark conditions. Using a novel membrane proteomics strategy, we determined that PR expression is higher under C limitation than N limitation but is not light regulated. Despite expression of PR photosystems, V. campbellii does not exhibit any growth or survival advantages in the light and only a few proteins show significant expression differences between light and dark conditions. While protein-level proteorhodopsin expression in V. campbellii is clearly responsive to nutrient limitation, photoheterotrophy does not appear to play a central role in the survival physiology of this organism under these nutrient stress conditions. C limitation and N limitation, however, result in very different survival responses: under N-limited conditions, viability declines, cultivability is lost rapidly, central carbon flux through the Entner-Doudoroff pathway is increased, and ammonium is assimilated via the GS-GOGAT pathway. In contrast, C limitation drives cell dwarfing with maintenance of viability, as well as utilization of the glyoxylate shunt, glutamate dehydrogenase and anaplerotic C fixation, and a stringent response mediated by the Pho regulon. IMPORTANCE Understanding the nutrient stress responses of proteorhodopsin-bearing microbes like Vibrio campbellii yields insights into microbial contributions to nutrient cycling, lifestyles of emerging pathogens in aquatic environments, and protein-level adaptations implemented during times of nutrient limitation. In addition to its broad taxonomic and geographic prevalence, the physiological role of PR is diverse, so we developed a novel proteomics strategy to quantify its expression at the protein level. We found that proteorhodopsin expression levels in this wild-type photoheterotroph under these experimental conditions, while higher under C than under N limitation, do not afford measurable light-driven growth or survival advantages. Additionally, this work links differential protein expression patterns between C- and N-limited cultures to divergent stationary-phase survival phenotypes.

Carbon substrate re-orders relative growth of a bacterium using Mo-, V-, or Fe-nitrogenase for nitrogen fixation.

Biological nitrogen fixation is catalyzed by the molybdenum (Mo), vanadium (V) and iron (Fe)-only nitrogenase metalloenzymes. Studies with purified enzymes have found that the 'alternative' V- and Fe-nitrogenases generally reduce N2 more slowly and produce more byproduct H2 than the Mo-nitrogenase, leading to an assumption that their usage results in slower growth. Here we show that, in the metabolically versatile photoheterotroph Rhodopseudomonas palustris, the type of carbon substrate influences the relative rates of diazotrophic growth based on different nitrogenase isoforms. The V-nitrogenase supports growth as fast as the Mo-nitrogenase on acetate but not on the more oxidized substrate succinate. Our data suggest that this is due to insufficient electron flux to the V-nitrogenase isoform on succinate compared with acetate. Despite slightly faster growth based on the V-nitrogenase on acetate, the wild-type strain uses exclusively the Mo-nitrogenase on both carbon substrates. Notably, the differences in H2 :N2 stoichiometry by alternative nitrogenases (~1.5 for V-nitrogenase, ~4-7 for Fe-nitrogenase) and Mo-nitrogenase (~1) measured here are lower than prior in vitro estimates. These results indicate that the metabolic costs of V-based nitrogen fixation could be less significant for growth than previously assumed, helping explain why alternative nitrogenase genes persist in diverse diazotroph lineages and are broadly distributed in the environment.

Metabolic and biogeochemical consequences of viral infection in aquatic ecosystems.

Ecosystems are controlled by 'bottom-up' (resources) and 'top-down' (predation) forces. Viral infection is now recognized as a ubiquitous top-down control of microbial growth across ecosystems but, at the same time, cell death by viral predation influences, and is influenced by, resource availability. In this Review, we discuss recent advances in understanding the biogeochemical impact of viruses, focusing on how metabolic reprogramming of host cells during lytic viral infection alters the flow of energy and nutrients in aquatic ecosystems. Our synthesis revealed several emerging themes. First, viral infection transforms host metabolism, in part through virus-encoded metabolic genes; the functions performed by these genes appear to alleviate energetic and biosynthetic bottlenecks to viral production. Second, viral infection depends on the physiological state of the host cell and on environmental conditions, which are challenging to replicate in the laboratory. Last, metabolic reprogramming of infected cells and viral lysis alter nutrient cycling and carbon export in the oceans, although the net impacts remain uncertain. This Review highlights the need for understanding viral infection dynamics in realistic physiological and environmental contexts to better predict their biogeochemical consequences.

Nitrogen sourcing during viral infection of marine cyanobacteria.

The building blocks of a virus derived from de novo biosynthesis during infection and/or catabolism of preexisting host cell biomass, and the relative contribution of these 2 sources has important consequences for understanding viral biogeochemistry. We determined the uptake of extracellular nitrogen (N) and its biosynthetic incorporation into both virus and host proteins using an isotope-labeling proteomics approach in a model marine cyanobacterium Synechococcus WH8102 infected by a lytic cyanophage S-SM1. By supplying dissolved N as 15N postinfection, we found that proteins in progeny phage particles were composed of up to 41% extracellularly derived N, while proteins of the infected host cell showed almost no isotope incorporation, demonstrating that de novo amino acid synthesis continues during infection and contributes specifically and substantially to phage replication. The source of N for phage protein synthesis shifted over the course of infection from mostly host derived in the early stages to more medium derived later on. We show that the photosystem II reaction center proteins D1 and D2, which are auxiliary metabolic genes (AMGs) in the S-SM1 genome, are made de novo during infection in an apparently light-dependent manner. We also identified a small set of host proteins that continue to be produced during infection; the majority are homologs of AMGs in S-SM1 or other viruses, suggesting selective continuation of host protein production during infection. The continued acquisition of nutrients by the infected cell and their utilization for phage replication are significant for both evolution and biogeochemical impact of viruses.

Closely related viruses of the marine picoeukaryotic alga Ostreococcus lucimarinus exhibit different ecological strategies.

In marine ecosystems, viruses are major disrupters of the direct flow of carbon and nutrients to higher trophic levels. Although the genetic diversity of several eukaryotic phytoplankton virus groups has been characterized, their infection dynamics are less understood, such that the physiological and ecological implications of their diversity remain unclear. We compared genomes and infection phenotypes of the two most closely related cultured phycodnaviruses infecting the widespread picoprasinophyte Ostreococcus lucimarinus under standard- (1.3 divisions per day) and limited-light (0.41 divisions per day) nutrient replete conditions. OlV7 infection caused early arrest of the host cell cycle, coinciding with a significantly higher proportion of infected cells than OlV1-amended treatments, regardless of host growth rate. OlV7 treatments showed a near-50-fold increase of progeny virions at the higher host growth rate, contrasting with OlV1's 16-fold increase. However, production of OlV7 virions was more sensitive than OlV1 production to reduced host growth rate, suggesting fitness trade-offs between infection efficiency and resilience to host physiology. Moreover, although organic matter released from OlV1- and OlV7-infected hosts had broadly similar chemical composition, some distinct molecular signatures were observed. Collectively, these results suggest that current views on viral relatedness through marker and core gene analyses underplay operational divergence and consequences for host ecology.

Postnovo: Postprocessing Enables Accurate and FDR-Controlled de Novo Peptide Sequencing.

De novo sequencing offers an alternative to database search methods for peptide identification from mass spectra. Since it does not rely on a predetermined database of expected or potential sequences in the sample, de novo sequencing is particularly appropriate for samples lacking a well-defined or comprehensive reference database. However, the low accuracy of many de novo sequence predictions has prevented the widespread use of the variety of sequencing tools currently available. Here, we present a new open-source tool, Postnovo, that postprocesses de novo sequence predictions to find high-accuracy results. Postnovo uses a predictive model to rescore and rerank candidate sequences in a manner akin to database search postprocessing tools such as Percolator. Postnovo leverages the output from multiple de novo sequencing tools in its own analyses, producing many times the length of amino acid sequence information (including both full- and partial-length peptide sequences) at an equivalent false discovery rate (FDR) compared to any individual tool. We present a methodology to reliably screen the sequence predictions to a desired FDR given the Postnovo sequence score. We validate Postnovo with multiple data sets and demonstrate its ability to identify proteins that are missed by database search even in samples with paired reference databases.

Distinct molecular signatures in dissolved organic matter produced by viral lysis of marine cyanobacteria.

Dissolved organic matter (DOM) plays a central role in the microbial ecology and biogeochemistry of aquatic environments, yet little is known about how the mechanism of DOM release from its ultimate source, primary producer biomass, affects the molecular composition of the inputs to the dissolved pool. Here we used a model marine phytoplankton, the picocyanobacterium Synechococcus WH7803, to compare the composition of DOM released by three mechanisms: exudation, mechanical cell lysis and infection by the lytic phage S-SM1. A broad, untargeted analytical approach reveals the complexity of this freshly sourced DOM, and comparative analysis between DOM produced by the different mechanisms suggests that exudation and viral lysis are sources of unsaturated, oxygen-rich and possibly novel biomolecules. Furthermore, viral lysis of WH7803 by S-SM1 releases abundant peptides derived from specific proteolysis of the major light-harvesting protein phycoerythrin, raising the possibility that phage infection of these abundant cyanobacteria could be a significant source of high molecular weight dissolved organic nitrogen compounds.

Global tRNA misacylation induced by anaerobiosis and antibiotic exposure broadly increases stress resistance in Escherichia coli.

High translational fidelity is commonly considered a requirement for optimal cellular health and protein function. However, recent findings have shown that inducible mistranslation specifically with methionine engendered at the tRNA charging level occurs in mammalian cells, yeast and archaea, yet it was unknown whether bacteria were capable of mounting a similar response. Here, we demonstrate that Escherichia coli misacylates non-methionyl-tRNAs with methionine in response to anaerobiosis and antibiotic exposure via the methionyl-tRNA synthetase (MetRS). Two MetRS succinyl-lysine modifications independently confer high tRNA charging fidelity to the otherwise promiscuous, unmodified enzyme. Strains incapable of tRNA mismethionylation are less adept at growth in the presence of antibiotics and stressors. The presence of tRNA mismethionylation and its potential role in mistranslation within the bacterial domain establishes this response as a pervasive biological mechanism and connects it to diverse cellular functions and modes of fitness.

Deciphering ocean carbon in a changing world.

Dissolved organic matter (DOM) in the oceans is one of the largest pools of reduced carbon on Earth, comparable in size to the atmospheric CO2 reservoir. A vast number of compounds are present in DOM, and they play important roles in all major element cycles, contribute to the storage of atmospheric CO2 in the ocean, support marine ecosystems, and facilitate interactions between organisms. At the heart of the DOM cycle lie molecular-level relationships between the individual compounds in DOM and the members of the ocean microbiome that produce and consume them. In the past, these connections have eluded clear definition because of the sheer numerical complexity of both DOM molecules and microorganisms. Emerging tools in analytical chemistry, microbiology, and informatics are breaking down the barriers to a fuller appreciation of these connections. Here we highlight questions being addressed using recent methodological and technological developments in those fields and consider how these advances are transforming our understanding of some of the most important reactions of the marine carbon cycle.

Transcriptome and proteome dynamics of a light-dark synchronized bacterial cell cycle.

BACKGROUND: Growth of the ocean's most abundant primary producer, the cyanobacterium Prochlorococcus, is tightly synchronized to the natural 24-hour light-dark cycle. We sought to quantify the relationship between transcriptome and proteome dynamics that underlie this obligate photoautotroph's highly choreographed response to the daily oscillation in energy supply. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA-sequencing transcriptomics and mass spectrometry-based quantitative proteomics, we measured timecourses of paired mRNA-protein abundances for 312 genes every 2 hours over a light-dark cycle. These temporal expression patterns reveal strong oscillations in transcript abundance that are broadly damped at the protein level, with mRNA levels varying on average 2.3 times more than the corresponding protein. The single strongest observed protein-level oscillation is in a ribonucleotide reductase, which may reflect a defense strategy against phage infection. The peak in abundance of most proteins also lags that of their transcript by 2-8 hours, and the two are completely antiphase for some genes. While abundant antisense RNA was detected, it apparently does not account for the observed divergences between expression levels. The redirection of flux through central carbon metabolism from daytime carbon fixation to nighttime respiration is associated with quite small changes in relative enzyme abundances. CONCLUSIONS/SIGNIFICANCE: Our results indicate that expression responses to periodic stimuli that are common in natural ecosystems (such as the diel cycle) can diverge significantly between the mRNA and protein levels. Protein expression patterns that are distinct from those of cognate mRNA have implications for the interpretation of transcriptome and metatranscriptome data in terms of cellular metabolism and its biogeochemical impact.

Microaerobic steroid biosynthesis and the molecular fossil record of Archean life.

The power of molecular oxygen to drive many crucial biogeochemical processes, from cellular respiration to rock weathering, makes reconstructing the history of its production and accumulation a first-order question for understanding Earth's evolution. Among the various geochemical proxies for the presence of O(2) in the environment, molecular fossils offer a unique record of O(2) where it was first produced and consumed by biology: in sunlit aquatic habitats. As steroid biosynthesis requires molecular oxygen, fossil steranes have been used to draw inferences about aerobiosis in the early Precambrian. However, better quantitative constraints on the O(2) requirement of this biochemistry would clarify the implications of these molecular fossils for environmental conditions at the time of their production. Here we demonstrate that steroid biosynthesis is a microaerobic process, enabled by dissolved O(2) concentrations in the nanomolar range. We present evidence that microaerobic marine environments (where steroid biosynthesis was possible) could have been widespread and persistent for long periods of time prior to the earliest geologic and isotopic evidence for atmospheric O(2). In the late Archean, molecular oxygen likely cycled as a biogenic trace gas, much as compounds such as dimethylsulfide do today.

The carbon cycle and associated redox processes through time.

Earth's biogeochemical cycle of carbon delivers both limestones and organic materials to the crust. In numerous, biologically catalysed redox reactions, hydrogen, sulphur, iron, and oxygen serve prominently as electron donors and acceptors. The progress of these reactions can be reconstructed from records of variations in the abundance of 13C in sedimentary carbonate minerals and organic materials. Because the crust is always receiving new CO2 from the mantle and a portion of it is being reduced by photoautotrophs, the carbon cycle has continuously released oxidizing power. Most of it is represented by Fe3+ that has accumulated in the crust or been returned to the mantle via subduction. Less than 3% of the estimated, integrated production of oxidizing power since 3.8 Gyr ago is represented by O2 in the atmosphere and dissolved in seawater. The balance is represented by sulphate. The accumulation of oxidizing power can be estimated from budgets summarizing inputs of mantle carbon and rates of organic-carbon burial, but levels of O2 are only weakly and indirectly coupled to those phenomena and thus to carbon-isotopic records. Elevated abundances of 13C in carbonate minerals ca 2.3 Gyr old, in particular, are here interpreted as indicating the importance of methanogenic bacteria in sediments rather than increased burial of organic carbon.

Steroids, triterpenoids and molecular oxygen.

There is a close connection between modern-day biosynthesis of particular triterpenoid biomarkers and presence of molecular oxygen in the environment. Thus, the detection of steroid and triterpenoid hydrocarbons far back in Earth history has been used to infer the antiquity of oxygenic photosynthesis. This prompts the question: were these compounds produced similarly in the past? In this paper, we address this question with a review of the current state of knowledge surrounding the oxygen requirement for steroid biosynthesis and phylogenetic patterns in the distribution of steroid and triterpenoid biosynthetic pathways. The hopanoid and steroid biosynthetic pathways are very highly conserved within the bacterial and eukaryotic domains, respectively. Bacteriohopanepolyols are produced by a wide range of bacteria, and are methylated in significant abundance at the C2 position by oxygen-producing cyanobacteria. On the other hand, sterol biosynthesis is sparsely distributed in distantly related bacterial taxa and the pathways do not produce the wide range of products that characterize eukaryotes. In particular, evidence for sterol biosynthesis by cyanobacteria appears flawed. Our experiments show that cyanobacterial cultures are easily contaminated by sterol-producing rust fungi, which can be eliminated by treatment with cycloheximide affording sterol-free samples. Sterols are ubiquitous features of eukaryotic membranes, and it appears likely that the initial steps in sterol biosynthesis were present in their modern form in the last common ancestor of eukaryotes. Eleven molecules of O2 are required by four enzymes to produce one molecule of cholesterol. Thermodynamic arguments, optimization of function and parsimony all indicate that an ancestral anaerobic pathway is highly unlikely. The known geological record of molecular fossils, especially steranes and triterpanes, is notable for the limited number of structural motifs that have been observed. With a few exceptions, the carbon skeletons are the same as those found in the lipids of extant organisms and no demonstrably extinct structures have been reported. Furthermore, their patterns of occurrence over billion year time-scales correlate strongly with environments of deposition. Accordingly, biomarkers are excellent indicators of environmental conditions even though the taxonomic affinities of all biomarkers cannot be precisely specified. Biomarkers are ultimately tied to biochemicals with very specific functional properties, and interpretations of the biomarker record will benefit from increased understanding of the biological roles of geologically durable molecules.