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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.

Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes and to regulate their fluidity and permeability. To date, very few studies have evaluated the role of hopanoids in bacterial physiology. The synthesis of hopanoids depends on the enzyme squalene-hopene cyclase (Shc), which converts the linear squalene into the basic hopene structure. Deletion of the 2 genes encoding Shc enzymes in Burkholderia cenocepacia K56-2, BCAM2831 and BCAS0167, resulted in a strain that was unable to produce hopanoids, as demonstrated by gas chromatography and mass spectrometry. Complementation of the Δshc mutant with only BCAM2831 was sufficient to restore hopanoid production to wild-type levels, while introducing a copy of BCAS0167 alone into the Δshc mutant produced only very small amounts of the hopanoid peak. The Δshc mutant grew as well as the wild type in medium buffered to pH 7 and demonstrated no defect in its ability to survive and replicate within macrophages, despite transmission electron microscopy (TEM) revealing defects in the organization of the cell envelope. The Δshc mutant displayed increased sensitivity to low pH, detergent, and various antibiotics, including polymyxin B and erythromycin. Loss of hopanoid production also resulted in severe defects in both swimming and swarming motility. This suggests that hopanoid production plays an important role in the physiology of B. cenocepacia.

Hopanoids and sterols are members of a large group of cyclic triterpenoic compounds that have important functions in many prokaryotic and eukaryotic organisms. They are biochemically synthesized from linear precursors (squalene, 2,3-oxidosqualene) in only one enzymatic step that is catalyzed by squalene-hopene cyclase (SHC) or oxidosqualene cyclase (OSC). SHCs and OSCs are related in amino acid sequences and probably are derived from a common ancestor. The SHC reaction requires the formation of five ring structures, 13 covalent bonds, and nine stereo centers and therefore is one of the most complex one-step enzymatic reactions. We summarize the knowledge of the properties of triterpene cyclases and details of the reaction mechanism of Alicyclobacillus acidocaldarius SHC. Properties of other SHCs are included.

Oomycetes are a diverse group of filamentous eukaryotic microbes comprising devastating animal and plant pathogens. They share many characteristics with fungi, including polarized hyphal extension and production of spores, but phylogenetics studies have clearly placed oomycetes outside the fungal kingdom, in the kingdom Stramenopila which also includes marine organisms such as diatoms and brown algae. Oomycetes display various specific biochemical features, including sterol metabolism. Sterols are essential isoprenoid compounds involved in membrane function and hormone signaling. Oomycetes belonging to Peronosporales, such as Phytophthora sp., are unable to synthesize their own sterols and must acquire them from their plant or animal hosts. In contrast, a combination of biochemical and molecular approaches allowed us to decipher a nearly complete sterol biosynthetic pathway leading to fucosterol in the legume pathogen Aphanomyces euteiches, an oomycete belonging to Saprolegniales. Importantly, sterol demethylase, a key enzyme from this pathway, is susceptible to chemicals widely used in agriculture and medicine as antifungal drugs, suggesting that similar products could be used against plant and animal diseases caused by Saprolegniales.

The availability of complete genomes from a wide sampling of eukaryotic diversity has allowed the application of phylogenomics approaches to study the origin and evolution of unique eukaryotic cellular structures, but these are still poorly applied to study unique eukaryotic metabolic pathways. Sterols are a good example because they are an essential feature of eukaryotic membranes. The sterol pathway has been well dissected in vertebrates, fungi, and land plants. However, although different types of sterols have been identified in other eukaryotic lineages, their pathways have not been fully characterized. We have carried out an extensive analysis of the taxonomic distribution and phylogeny of the enzymes of the sterol pathway in a large sampling of eukaryotic lineages. This allowed us to tentatively indicate features of the sterol pathway in organisms where this has not been characterized and to point out a number of steps for which yet-to-discover enzymes may be at work. We also inferred that the last eukaryotic common ancestor already harbored a large panel of enzymes for sterol synthesis and that subsequent evolution over the eukaryotic tree occurred by tinkering, mainly by gene losses. We highlight a high capacity of sterol synthesis in the myxobacterium Plesiocystis pacifica, and we support the hypothesis that the few bacteria that harbor homologs of the sterol pathway have likely acquired these via horizontal gene transfer from eukaryotes. Finally, we propose a potential candidate for the elusive enzyme performing C-3 ketoreduction (ERG27 equivalent) in land plants and probably in other eukaryotic phyla.

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.

Pneumocystis carinii synthesizes sterols with a double bond at C-7 of the sterol nucleus and an alkyl group with one or two carbons at C-24 of the side chain. Also, some human-derived Pneumocystis carinii f. sp. hominis strains contain lanosterol derivatives with an alkyl group at C-24. These unique sterols have not been found in other pathogens of mammalian lungs. Thus, P. carinii may have important differences in its susceptibility to drugs known to block reactions in ergosterol biosynthesis in other fungi. In the present study, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, squalene epoxide-lanosterol cyclase, lanosterol demethylase, Δ8 to Δ7 isomerase, and S-adenosylmethionine:sterol methyltransferase were tested for their effects on P. carinii viability as determined by quantitation of cellular ATP levels in a population of organisms. Compounds within each category varied in inhibitory effect; the most effective included drugs targeted at squalene synthase, squalene epoxide-lanosterol cyclase, and Δ8 to Δ7 isomerase. Some drugs that are potent against ergosterol-synthesizing fungi had little effect against P. carinii, suggesting that substrates and/or enzymes in P. carinii sterol biosynthetic reactions are distinct. Amphotericin B is ineffective in clearing P. carinii infections at clinical doses; however, this drug apparently binds to sterols and causes permeability changes in P. carinii membranes, since it reduced cellular ATP levels in a dose-dependent fashion.

Life is a chemical reaction. Three major transitions in early evolution are considered without recourse to a tree of life. The origin of prokaryotes required a steady supply of energy and electrons, probably in the form of molecular hydrogen stemming from serpentinization. Microbial genome evolution is not a treelike process because of lateral gene transfer and the endosymbiotic origins of organelles. The lack of true intermediates in the prokaryote-to-eukaryote transition has a bioenergetic cause.

This article was reviewed by Dan Graur, W. Ford Doolittle, Eugene V. Koonin and Christophe Malaterre.

Sterols play multi-faceted roles in all eukaryotes. In plants, there are mounting evidences pointing to sterols, other than BRs, can act as signaling molecules. The Arabidopsis dry2/sqe1-5 mutant has multiple developmental defects caused by a point mutation in the SQE1 gene that generates a hypomorphic allele. SQE1 encodes a squalene epoxidase, which converts squalene into 2,3-oxidosqualene the precursor of plant sterols. Genetic, molecular and biochemical analyses suggest that dry2/sqe1-5 defective phenotypes cannot be simply explained by a depletion of bulk sterols but rather by altered ROS. It remains to be elucidated whether the altered ROS production of the mutant is caused by membrane composition, which in turn affect the lipid rafts composition and/or an altered signaling.

There is little doubt that genes can spread across unrelated prokaryotes, eukaryotes and even between these domains. It is expected that organisms inhabiting a common niche may exchange their genes even more often due to their physical proximity and similar demands. One such niche is anaerobic or microaerophilic environments in some sediments and intestines of animals. Indeed, enzymes advantageous for metabolism in these environments often exhibit an evolutionary history incoherent with the history of their hosts indicating potential transfers. The evolutionary paths of some very basic enzymes for energy metabolism of anaerobic eukaryotes (pyruvate formate lyase, pyruvate:ferredoxin oxidoreductase, [FeFe]hydrogenase and arginine deiminase) seems to be particularly intriguing and although their histories are not identical they share several unexpected features in common. Every enzyme mentioned above is present in groups of eukaryotes that are unrelated to each other. Although the enzyme phylogenies are not always robustly supported, they always suggest that the eukaryotic homologues form one or two clades, in which the relationships are not congruent with the eukaryotic phylogeny. Finally, these eukaryotic enzymes are never specifically related to homologues from α-proteobacteria, ancestors of mitochondria. The most plausible explanation for evolution of this pattern expects one or two interdomain transfers to one or two eukaryotes from prokaryotes, who were not the mitochondrial endosymbiont. Once the genes were introduced into the eukaryotic domain they have spread to other eukaryotic groups exclusively via eukaryote-to-eukaryote transfers. Currently, eukaryote-to-eukaryote gene transfers have been regarded as less common than prokaryote-to-eukaryote transfers. The fact that eukaryotes accepted genes for these enzymes solely from other eukaryotes and not prokaryotes present in the same environment is surprising.

Sterol biosynthesis in the yeast Saccharomyces cerevisiae is an energy-expensive, aerobic process, requiring heme and molecular oxygen. Heme, also synthesized exclusively during aerobic growth, not only acts as an enzymatic cofactor but also is directly and indirectly responsible for the transcriptional control of several yeast genes. Because of their biosynthetic similarities, we hypothesized that ergosterol, like heme, may have a regulatory function. Sterols are known to play a structural role in membrane integrity, but regulatory roles have not been characterized. To test possible regulatory roles of sterol, the promoter for the ERG3 gene, encoding the sterol C-5 desaturase, was fused to the bacterial lacZ reporter gene. This construct was placed in strains making aberrant sterols, and the effect of altered sterol composition on gene expression was monitored by beta-galactosidase activity. The absence of ergosterol resulted in a 35-fold increase in the expression of ERG3 as measured by beta-galactosidase activity. The level of ERG3 mRNA was increased as much as ninefold in erg mutant strains or wild-type strains inhibited in ergosterol biosynthesis by antifungal agents. The observed regulatory effects of ergosterol on ERG3 are specific for ergosterol, as several ergosterol derivatives failed to elicit the same controlling effect. These results demonstrate for the first time that ergosterol exerts a regulatory effect on gene transcription in S. cerevisiae.

Background

Ergosterol has been considered the “fungal sterol” for almost 125 years; however, additional sterol data superimposed on a recent molecular phylogeny of kingdom Fungi reveals a different and more complex situation.

Methodology/Principal Findings

The interpretation of sterol distribution data in a modern phylogenetic context indicates that there is a clear trend from cholesterol and other Δ5 sterols in the earliest diverging fungal species to ergosterol in later diverging fungi. There are, however, deviations from this pattern in certain clades. Sterols of the diverse zoosporic and zygosporic forms exhibit structural diversity with cholesterol and 24-ethyl -Δ5 sterols in zoosporic taxa, and 24-methyl sterols in zygosporic fungi. For example, each of the three monophyletic lineages of zygosporic fungi has distinctive major sterols, ergosterol in Mucorales, 22-dihydroergosterol in Dimargaritales, Harpellales, and Kickxellales (DHK clade), and 24-methyl cholesterol in Entomophthorales. Other departures from ergosterol as the dominant sterol include: 24-ethyl cholesterol in Glomeromycota, 24-ethyl cholest-7-enol and 24-ethyl-cholesta-7,24(28)-dienol in rust fungi, brassicasterol in Taphrinales and hypogeous pezizalean species, and cholesterol in Pneumocystis.

Conclusions/Significance

Five dominant end products of sterol biosynthesis (cholesterol, ergosterol, 24-methyl cholesterol, 24-ethyl cholesterol, brassicasterol), and intermediates in the formation of 24-ethyl cholesterol, are major sterols in 175 species of Fungi. Although most fungi in the most speciose clades have ergosterol as a major sterol, sterols are more varied than currently understood, and their distribution supports certain clades of Fungi in current fungal phylogenies. In addition to the intellectual importance of understanding evolution of sterol synthesis in fungi, there is practical importance because certain antifungal drugs (e.g., azoles) target reactions in the synthesis of ergosterol. These findings also invalidate use of ergosterol as an indicator of biomass of certain fungal taxa (e.g., Glomeromycota). Data from this study are available from the Assembling the Fungal Tree of Life (AFTOL) Structural and Biochemical Database: http://aftol.umn.edu.

The endosymbiotic origin of eukaryotes brought together two disparate genomes in the cell. Additionally, eukaryotic natural history has included other endosymbiotic events, phagotrophic consumption of organisms, and intimate interactions with viruses and endoparasites. These phenomena facilitated large-scale lateral gene transfer and biological conflicts. We synthesize information from nearly two decades of genomics to illustrate how the interplay between lateral gene transfer and biological conflicts has impacted the emergence of new adaptations in eukaryotes. Using apicomplexans as example, we illustrate how lateral transfer from animals has contributed to unique parasite-host interfaces comprised of adhesion- and O-linked glycosylation-related domains. Adaptations, emerging due to intense selection for diversity in the molecular participants in organismal and genomic conflicts, being dispersed by lateral transfer, were subsequently exapted for eukaryote-specific innovations. We illustrate this using examples relating to eukaryotic chromatin, RNAi and RNA-processing systems, signaling pathways, apoptosis and immunity. We highlight the major contributions from catalytic domains of bacterial toxin systems to the origin of signaling enzymes (e.g., ADP-ribosylation and small molecule messenger synthesis), mutagenic enzymes for immune receptor diversification and RNA-processing. Similarly, we discuss contributions of bacterial antibiotic/siderophore synthesis systems and intra-genomic and intra-cellular selfish elements (e.g., restriction-modification, mobile elements and lysogenic phages) in the emergence of chromatin remodeling/modifying enzymes and RNA-based regulation. We develop the concept that biological conflict systems served as evolutionary “nurseries” for innovations in the protein world, which were delivered to eukaryotes via lateral gene flow to spur key evolutionary innovations all the way from nucleogenesis to lineage-specific adaptations.

Background

Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis. However, details of the timing and number of symbiotic events are unclear. A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen. We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes.

Results

Eukaryotes were found to evolve faster than prokaryotes, with those eukaryotes derived from eubacteria evolving faster than those derived from archaebacteria. We found an early time of divergence (~4 billion years ago, Ga) for archaebacteria and the archaebacterial genes in eukaryotes. Our analyses support at least two horizontal gene transfer events in the origin of eukaryotes, at 2.7 Ga and 1.8 Ga. Time estimates for the origin of cyanobacteria (2.6 Ga) and the divergence of an early-branching eukaryote that lacks mitochondria (Giardia) (2.2 Ga) fall between those two events.

Conclusions

We find support for two symbiotic events in the origin of eukaryotes: one premitochondrial and a later mitochondrial event. The appearance of cyanobacteria immediately prior to the earliest undisputed evidence for the presence of oxygen (2.4–2.2 Ga) suggests that the innovation of oxygenic photosynthesis had a relatively rapid impact on the environment as it set the stage for further evolution of the eukaryotic cell.

A method has been devised to fractionate cells of Tetrahymena pyriformis, yielding pure or highly enriched preparations of cilia, cilia-associated soluble material, pellicles, mitochondria, microsomes, and postmicrosomal supernatant. The method prevents the destructive action of lipolytic enzymes commonly associated with this organism. Analysis of the membrane lipids of these fractions reveals significant differences in lipid composition. Most noteworthy are the high concentrations of phosphonolipid and tetrahymanol in the surface membranes.

Wild-type Saccharomyces cerevisiae do not accumulate exogenous sterols under aerobic conditions, and a mutant allele conferring sterol auxotrophy (erg7) could be isolated only in strains with a heme deficiency. delta-Aminolevulinic acid (ALA) fed to a hem1 (ALA synthetase-) erg7 (2,3-oxidosqualene cyclase-) sterol-auxotrophic strain of S. cerevisiae inhibited sterol uptake, and growth was negatively affected when intracellular sterol was depleted. The inhibition of sterol uptake (and growth of sterol auxotrophs) by ALA was dependent on the ability to synthesize heme from ALA. A procedure was developed which allowed selection of strains which would take up exogenous sterols but had no apparent defect in heme or ergosterol biosynthesis. One of these sterol uptake control mutants possessed an allele which allowed phenotypic expression of sterol auxotrophy in a heme-competent background.

Background

Theories about eukaryote origins (eukaryogenesis) need to provide unified explanations for the emergence of diverse complex features that define this lineage. Models that propose a prokaryote-to-eukaryote transition are gridlocked between the opposing "phagocytosis first" and "mitochondria as seed" paradigms, neither of which fully explain the origins of eukaryote cell complexity. Sex (outcrossing with meiosis) is an example of an elaborate trait not yet satisfactorily addressed in theories about eukaryogenesis. The ancestral nature of meiosis and its dependence on eukaryote cell biology suggest that the emergence of sex and eukaryogenesis were simultaneous and synergic and may be explained by a common selective pressure.

Presentation of the hypothesis

We propose that a local rise in oxygen levels, due to cyanobacterial photosynthesis in ancient Archean microenvironments, was highly toxic to the surrounding biota. This selective pressure drove the transformation of an archaeal (archaebacterial) lineage into the first eukaryotes. Key is that oxygen might have acted in synergy with environmental stresses such as ultraviolet (UV) radiation and/or desiccation that resulted in the accumulation of reactive oxygen species (ROS). The emergence of eukaryote features such as the endomembrane system and acquisition of the mitochondrion are posited as strategies to cope with a metabolic crisis in the cell plasma membrane and the accumulation of ROS, respectively. Selective pressure for efficient repair of ROS/UV-damaged DNA drove the evolution of sex, which required cell-cell fusions, cytoskeleton-mediated chromosome movement, and emergence of the nuclear envelope. Our model implies that evolution of sex and eukaryogenesis were inseparable processes.

Testing the hypothesis

Several types of data can be used to test our hypothesis. These include paleontological predictions, simulation of ancient oxygenic microenvironments, and cell biological experiments with Archaea exposed to ROS and UV stresses. Studies of archaeal conjugation, prokaryotic DNA recombination, and the universality of nuclear-mediated meiotic activities might corroborate the hypothesis that sex and the nucleus evolved to support DNA repair.

Implications of the hypothesis

Oxygen tolerance emerges as an important principle to investigate eukaryogenesis. The evolution of eukaryotic complexity might be best understood as a synergic process between key evolutionary innovations, of which meiosis (sex) played a central role.

Reviewers

This manuscript was reviewed by Eugene V. Koonin, Anthony M. Poole, and Gáspár Jékely.

Sterol regulatory element binding proteins (SREBPs) are membrane-bound transcription factors whose proteolytic activation is controlled by the cellular sterol concentration. Mammalian SREBPs are activated in cholesterol-depleted cells and serve to regulate cellular lipid homeostasis. Recent work demonstrates that SREBP is functionally conserved in fungi. While the ability to respond to sterols is conserved, fungal SREBPs are hypoxic transcription factors required for adaptation to a low-oxygen environment. In the fission yeast Schizosaccharomyces pombe, oxygen regulates the SREBP homolog Sre1 by independently controlling both its proteolytic activation and its degradation. SREBP is also required for adaptation to hypoxia in the human pathogens Cryptococcus neoformans and Aspergillus fumigatus. In these organisms, SREBP is required for virulence and resistance to antifungal drugs, making the SREBP pathway a potential target for antifungal therapy.

Abstract

All eukaryotic cells possess an endoplasmic reticulum (ER), which is the site for synthesizing proteins that populate the cell surface or extracellular space. The environment of the ER is oxidizing, which supports the formation of intra- and interchain disulfide bonds that serve to stabilize the folding and assembly of nascent proteins. Recent experimental data reveal that the formation of disulfide bonds does not occur spontaneously but results from the enzymatic transfer of disulfide bonds through a number of intermediate proteins, with molecular oxygen serving as the terminal electron acceptor. Thus, each disulfide bond that forms during oxidative folding should produce a single reactive oxygen species (ROS). Dedicated secretory tissues like the pancreas and plasma cells have been estimated to form up to 3–6 million disulfide bonds per minute, which would be expected to result in the production of the same number of molecules of ROS. Although the methods used to deal with this amount of oxidative stress are not well understood, recent research suggests that different types of cells use distinct strategies and that the unfolded protein response (UPR) is a critical component of the defense. Antioxid. Redox Signal. 11, 2317–2331.

Sterols are crucial lipid components that regulate membrane permeability and fluidity and are the precursors of bioactive steroids. The plant sterols exist as three major forms, free sterols, steryl glycosides and steryl esters. The storage of steryl esters in lipid droplets has been shown to contribute to cellular sterol homeostasis. To further document cellular aspects of sterol biosynthesis in plants, we addressed the question of the subcellular localization of the enzymes implicated in the final steps of the post-squalene biosynthetic pathway. In order to create a clear localization map of steroidogenic enzymes in cells, the coding regions of Δ7-sterol-C5-desaturase (STE1/DWARF7), Δ24-sterol-Δ24-reductase (DIMINUTO/DWARF1) and Δ5,7-sterol-Δ7-reductase (DWARF5) were fused to the yellow fluorescent protein (YFP) and transformed into Arabidopsis thaliana mutant lines deficient in the corresponding enzymes. All fusion proteins were found to localize in the endoplasmic reticulum in functionally complemented plants. The results show that both Δ5,7-sterol-Δ7-reductase and Δ24-sterol-Δ24-reductase are in addition localized to the plasma membrane, whereas Δ7-sterol-C5-desaturase was clearly detected in lipid particles. These findings raise new challenging questions about the spatial and dynamic cellular organization of sterol biosynthesis in plants.

Background

The Euglenozoa is a large group of eukaryotic flagellates with diverse modes of nutrition. The group consists of three main subclades – euglenids, kinetoplastids and diplonemids – that have been confirmed with both molecular phylogenetic analyses and a combination of shared ultrastructural characteristics. Several poorly understood lineages of putative euglenozoans live in anoxic environments, such as Calkinsia aureus, and have yet to be characterized at the molecular and ultrastructural levels. Improved understanding of these lineages is expected to shed considerable light onto the ultrastructure of prokaryote-eukaryote symbioses and the associated cellular innovations found within the Euglenozoa and beyond.

Results

We collected Calkinsia aureus from core samples taken from the low-oxygen seafloor of the Santa Barbara Basin (580 – 592 m depth), California. These biflagellates were distinctively orange in color and covered with a dense array of elongated epibiotic bacteria. Serial TEM sections through individually prepared cells demonstrated that C. aureus shares derived ultrastructural features with other members of the Euglenozoa (e.g. the same paraxonemal rods, microtubular root system and extrusomes). However, C. aureus also possessed several novel ultrastructural systems, such as modified mitochondria (i.e. hydrogenosome-like), an "extrusomal pocket", a highly organized extracellular matrix beneath epibiotic bacteria and a complex flagellar transition zone. Molecular phylogenies inferred from SSU rDNA sequences demonstrated that C. aureus grouped strongly within the Euglenozoa and with several environmental sequences taken from low-oxygen sediments in various locations around the world.

Conclusion

Calkinsia aureus possesses all of the synapomorphies for the Euglenozoa, but lacks traits that are specific to any of the three previously recognized euglenozoan subgroups. Molecular phylogenetic analyses of C. aureus demonstrate that this lineage is a member of a novel euglenozoan subclade consisting of uncharacterized cells living in low-oxygen environments. Our ultrastructural description of C. aureus establishes the cellular identity of a fourth group of euglenozoans, referred to as the "Symbiontida".

Coxiella burnetii, the etiological agent of human Q fever, occupies a unique niche inside the host cell, where it replicates in a modified acidic phagolysosome or parasitophorous vacuole (PV). The PV membrane is cholesterol-rich, and inhibition of host cholesterol metabolism negatively impacts PV biogenesis and pathogen replication. The precise source(s) of PV membrane cholesterol is unknown, as is whether the bacterium actively diverts and/or modifies host cell cholesterol or sterol precursors. C. burnetii lacks enzymes for de novo cholesterol biosynthesis; however, the organism encodes a eukaryote-like Δ24 sterol reductase homolog, CBU1206. Absent in other prokaryotes, this enzyme is predicted to reduce sterol double bonds at carbon 24 in the final step of cholesterol or ergosterol biosynthesis. In the present study, we examined the functional activity of CBU1206. Amino acid alignments revealed the greatest sequence identity (51.7%) with a Δ24 sterol reductase from the soil amoeba Naegleria gruberi. CBU1206 activity was examined by expressing the protein in a Saccharomyces cerevisiae erg4 mutant under the control of a galactose-inducible promoter. Erg4 is a yeast Δ24 sterol reductase responsible for the final reduction step in ergosterol synthesis. Like Erg4-green fluorescent protein (GFP), a CBU1206-GFP fusion protein localized to the yeast endoplasmic reticulum. Heterologous expression of CBU1206 rescued S. cerevisiae erg4 sensitivity to growth in the presence of brefeldin A and cycloheximide and resulted in new synthesis of ergosterol. These data indicate CBU1206 is an active sterol reductase and suggest the enzyme may act on host sterols during C. burnetii intracellular growth.

Background

The origin of eukaryotic cells was one of the most dramatic evolutionary transitions in the history of life. It is generally assumed that eukaryotes evolved later then prokaryotes by the transformation or fusion of prokaryotic lineages. However, as yet there is no consensus regarding the nature of the prokaryotic group(s) ancestral to eukaryotes. Regardless of this, a hardly debatable fundamental novel characteristic of the last eukaryotic common ancestor was the ability to exploit prokaryotic biomass by the ingestion of entire cells, i.e. phagocytosis. The recent advances in our understanding of the social life of prokaryotes may help to explain the origin of this form of total exploitation.

Presentation of the hypothesis

Here I propose that eukaryotic cells originated in a social environment, a differentiated microbial mat or biofilm that was maintained by the cooperative action of its members. Cooperation was costly (e.g. the production of developmental signals or an extracellular matrix) but yielded benefits that increased the overall fitness of the social group. I propose that eukaryotes originated as selfish cheaters that enjoyed the benefits of social aggregation but did not contribute to it themselves. The cheaters later evolved into predators that lysed other cells and eventually became professional phagotrophs. During several cycles of social aggregation and dispersal the number of cheaters was contained by a chicken game situation, i.e. reproductive success of cheaters was high when they were in low abundance but was reduced when they were over-represented. Radical changes in cell structure, including the loss of the rigid prokaryotic cell wall and the development of endomembranes, allowed the protoeukaryotes to avoid cheater control and to exploit nutrients more efficiently. Cellular changes were buffered by both the social benefits and the protective physico-chemical milieu of the interior of biofilms. Symbiosis with the mitochondial ancestor evolved after phagotrophy as alphaproteobacterial prey developed post-ingestion defence mechanisms to circumvent digestion in the food vacuole. Mitochondrial symbiosis triggered the origin of the nucleus. Cilia evolved last and allowed eukaryotes to predate also on planktonic prey. I will discuss how this scenario may possibly fit into the contrasting phylogenetic frameworks that have been proposed.

Testing the hypothesis

Some aspects of the hypothesis can be tested experimentally by studying the level of exploitation cheaters can reach in social microbes. It would be interesting to test whether absorption of nutrients from lysed fellow colony members can happen and if cheaters can evolve into predators that actively digest neighbouring cells.

Implications of the hypothesis

The hypothesis highlights the importance of social exploitation in cell evolution and how a social environment can buffer drastic cellular transformations that would be lethal for planktonic forms.

Reviewers

This article was reviewed by Eugene V Koonin, Purificación López-García, and Igor Zhulin.

The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.

Fungal pathogens of humans require molecular oxygen for several essential biochemical reactions, yet virtually nothing is known about how they adapt to the relatively hypoxic environment of infected tissues. We isolated mutants defective in growth under hypoxic conditions, but normal for growth in normoxic conditions, in Cryptococcus neoformans, the most common cause of fungal meningitis. Two regulatory pathways were identified: one homologous to the mammalian sterol-response element binding protein (SREBP) cholesterol biosynthesis regulatory pathway, and the other a two-component-like pathway involving a fungal-specific hybrid histidine kinase family member, Tco1. We show that cleavage of the SREBP precursor homolog Sre1—which is predicted to release its DNA-binding domain from the membrane—occurs in response to hypoxia, and that Sre1 is required for hypoxic induction of genes encoding for oxygen-dependent enzymes involved in ergosterol synthesis. Importantly, mutants in either the SREBP pathway or the Tco1 pathway display defects in their ability to proliferate in host tissues and to cause disease in infected mice, linking for the first time to our knowledge hypoxic adaptation and pathogenesis by a eukaryotic aerobe. SREBP pathway mutants were found to be a hundred times more sensitive than wild-type to fluconazole, a widely used antifungal agent that inhibits ergosterol synthesis, suggesting that inhibitors of SREBP processing could substantially enhance the potency of current therapies.

Author Summary

Opportunistic environmental pathogens adapt to hostile conditions within the host to cause disease. We describe two pathways in the pathogenic fungus Cryptococcus neoformans that are both necessary for adaptation to hypoxia and required for its virulence. One pathway uses a pathway homologous to the mammalian sterol-response element binding protein (SREBP) pathway to activate genes involved in sterol biosynthesis in response to low oxygen levels, while the other pathway involves the two-component hybrid histidine kinase protein Tco1. Mutant strains containing deletions of genes encoding components in either of these pathways were found to be less virulent in experimental mouse models. This study suggests that this pathogenic fungus experiences low levels of oxygen in the mammalian host, and that adaptation to these conditions is important for infection. Targeting components of the hypoxia response could yield more effective treatments for C. neoformans infections, which cause a large fraction of HIV/AIDS-related deaths worldwide. Notably, we find that mutants in the SREBP-like pathway are a hundred times more sensitive than wild-type cells to the widely used antifungal drug fluconazole.