Dark fermentation and microalgae cultivation coupled systems: Outlook and challenges.

The implementation of a sustainable bio-based economy is considered a top priority today. There is no doubt about the necessity to produce renewable bioenergy and bio-sourced chemicals to replace fossil-derived compounds. Under this scenario, strong efforts have been devoted to efficiently use organic waste as feedstock for biohydrogen production via dark fermentation. However, the technoeconomic viability of this process needs to be enhanced by the valorization of the residual streams generated. The use of dark fermentation effluents as low-cost carbon source for microalgae cultivation arises as an innovative approach for bioproducts generation (e.g., biodiesel, bioactive compounds, pigments) that maximizes the carbon recovery. In a biorefinery context, after value-added product extraction, the spent microalgae biomass can be further valorised as feedstock for biohydrogen production. This integrated process would play a key role in the transition towards a circular economy. This review covers recent advances in microalgal cultivation on dark fermentation effluents (DFE). BioH2 via dark fermentation processes and the involved metabolic pathways are detailed with a special focus on the main aspects affecting the effluent composition. Interesting traits of microalgae and current approaches to solve the challenges associated to the integration of dark fermentation and microalgae cultivation are also discussed.

Proteomics unveil a central role for peroxisomes in butyrate assimilation of the heterotrophic Chlorophyte alga Polytomella sp.

Volatile fatty acids found in effluents of the dark fermentation of biowastes can be used for mixotrophic growth of microalgae, improving productivity and reducing the cost of the feedstock. Microalgae can use the acetate in the effluents very well, but butyrate is poorly assimilated and can inhibit growth above 1 gC.L-1. The non-photosynthetic chlorophyte alga Polytomella sp. SAG 198.80 was found to be able to assimilate butyrate fast. To decipher the metabolic pathways implicated in butyrate assimilation, quantitative proteomics study was developed comparing Polytomella sp. cells grown on acetate and butyrate at 1 gC.L-1. After statistical analysis, a total of 1772 proteins were retained, of which 119 proteins were found to be overaccumulated on butyrate vs. only 46 on acetate, indicating that butyrate assimilation necessitates additional metabolic steps. The data show that butyrate assimilation occurs in the peroxisome via the ß-oxidation pathway to produce acetyl-CoA and further tri/dicarboxylic acids in the glyoxylate cycle. Concomitantly, reactive oxygen species defense enzymes as well as the branched amino acid degradation pathway were strongly induced. Although no clear dedicated butyrate transport mechanism could be inferred, several membrane transporters induced on butyrate are identified as potential condidates. Metabolic responses correspond globally to the increased needs for central cofactors NAD, ATP and CoA, especially in the peroxisome and the cytosol.

Traumatic Injuries in Sexual Assault Patients in the Emergency Department.

INTRODUCTION: The emergency department (ED) is at the forefront for treatment of sexual assault patients. Many require treatment for injuries sustained during the assault, ranging from mild to severe. Our objective in this study was to characterize types of injuries associated with sexual assault and identify associated factors. METHODS: We reviewed ED charts from an inner-city trauma center and nearby community hospital from 2019-2020 for patients age ≥13 years with a chief complaint of sexual assault. We used descriptive statistics, chi square, and logistic regression to characterize demographics and identify factors associated with trauma. RESULTS: A total of 157 patients met inclusion criteria. The mean age was 27.9 years old (range 13-79 years) and 92.4% were female. Adult patients (age >18 years) comprised 77.5% of assaults vs adolescents (age 13-18 years) at 22.3%. Most patients presented to the trauma center compared to the community hospital (69.4% vs 30.6%). The assailants were reported as 61.2% acquaintance, 22.9% stranger, and 15.9% intimate partner. A forensic rape kit was performed in 92 (58.6%) cases. The patient was intoxicated with alcohol in 39 (24.8%) cases, and 22 (14%) patients reported drug-facilitated assault where an unknown substance was given to them. Alcohol (P = 0.95) and drug-facilitated assault (P = 0.64) did not change the occurrence of injuries. Fifty-seven (36.3%) patients exhibited physical trauma on presentation. Forty-five (28.6%) patients had minor injuries of abrasions, lacerations, or contusions. Major trauma was defined as fracture, brain injury, hemorrhage, strangulation, or injury requiring surgical consultation. There were 12 patients with major trauma consisting of fracture injury or nonfatal strangulation. None of the patients required admission. Sexual assault by an intimate partner (odds ratio [OR] 2.6; 95% CI: 1.1-6.5) and being an adult patient compared to adolescent (OR 3.0; 95% CI, 1.1-7.7) was significantly associated with physical trauma. Sexual assault by an intimate partner was also associated with nonfatal strangulation (OR 4.0; 95% CI, 1.1-15.4). CONCLUSION: Physical injuries that resulted from sexual assault were mostly minor and occurred in 36% of rape victims. Intimate partner violence was found to be associated with physical trauma as well as nonfatal strangulation. Overall, this study helps us to understand key factors associated with sexual violence.

Mixotrophic Growth of Chlorella sorokiniana on Acetate and Butyrate: Interplay Between Substrate, C:N Ratio and pH.

Microalgae can be cultivated on waste dark fermentation effluents containing volatile fatty acids (VFA) such as acetate or butyrate. These VFA can however inhibit microalgae growth at concentrations above 0.5-1 gC.L-1. This study used the model strain Chlorella sorokiniana to investigate the effects of acetate or butyrate concentration on biomass growth rates and yields alongside C:N:P ratios and pH control. Decreasing undissociated acid levels by raising the initial pH to 8.0 allowed growth without inhibition up to 5 gC.L-1 VFAs. However, VFA concentration strongly affected biomass yields irrespective of pH control or C:N:P ratios. Biomass yields on 1.0 gC.L-1 acetate were around 1.3-1.5 gC.gC -1 but decreased by 26-48% when increasing initial acetate to 2.0 gC.L-1. This was also observed for butyrate with yields decreasing up to 25%. This decrease in yield in suggested to be due to the prevalence of heterotrophic metabolism at high organic acid concentration, which reduced the amount of carbon fixed by autotrophy. Finally, the effects of C:N:P on biomass, lipids and carbohydrates production dynamics were assessed using a mixture of both substrates. In nutrient replete conditions, C. sorokiniana accumulated up to 20.5% carbohydrates and 16.4% lipids while nutrient limitation triggered carbohydrates accumulation up to 45.3%.

Phylogenetic and functional diversity of aldehyde-alcohol dehydrogenases in microalgae.

KEY MESSAGE: The study shows the biochemical and enzymatic divergence between the two aldehyde-alcohol dehydrogenases of the alga Polytomella sp., shedding light on novel aspects of the enzyme evolution amid unicellular eukaryotes. Aldehyde-alcohol dehydrogenases (ADHEs) are large metalloenzymes that typically perform the two-step reduction of acetyl-CoA into ethanol. These enzymes consist of an N-terminal acetylating aldehyde dehydrogenase domain (ALDH) and a C-terminal alcohol dehydrogenase (ADH) domain. ADHEs are present in various bacterial phyla as well as in some unicellular eukaryotes. Here we focus on ADHEs in microalgae, a diverse and polyphyletic group of plastid-bearing unicellular eukaryotes. Genome survey shows the uneven distribution of the ADHE gene among free-living algae, and the presence of two distinct genes in various species. We show that the non-photosynthetic Chlorophyte alga Polytomella sp. SAG 198.80 harbors two genes for ADHE-like enzymes with divergent C-terminal ADH domains. Immunoblots indicate that both ADHEs accumulate in Polytomella cells growing aerobically on acetate or ethanol. ADHE1 of ~ 105-kDa is found in particulate fractions, whereas ADHE2 of ~ 95-kDa is mostly soluble. The study of the recombinant enzymes revealed that ADHE1 has both the ALDH and ADH activities, while ADHE2 has only the ALDH activity. Phylogeny shows that the divergence occurred close to the root of the Polytomella genus within a clade formed by the majority of the Chlorophyte ADHE sequences, next to the cyanobacterial clade. The potential diversification of function in Polytomella spp. unveiled here likely took place after the loss of photosynthesis. Overall, our study provides a glimpse at the complex evolutionary history of the ADHE in microalgae which includes (i) acquisition via different gene donors, (ii) gene duplication and (iii) independent evolution of one of the two enzymatic domains.

Hybrid cluster proteins in a photosynthetic microalga.

Hybrid cluster proteins (HCPs) are metalloproteins characterized by the presence of an iron-sulfur-oxygen cluster. These proteins occur in all three domains of life. In eukaryotes, HCPs have so far been found only in a few anaerobic parasites and photosynthetic microalgae. With respect to all species harboring an HCP, the green microalga Chlamydomonas reinhardtii stands out by the presence of four HCP genes. The study of the gene and protein structures as well as the phylogenetic analyses strongly support a model in which the HCP family in the alga has emerged from a single gene of alpha proteobacterial origin and then expanded by several rounds of duplications. The spectra and redox properties of HCP1 and HCP3, produced heterologously in Escherichia coli, were analyzed by electron paramagnetic resonance spectroscopy on redox-titrated samples. Both proteins contain a [4Fe-4S]-cluster as well as a [4Fe-2O-2S]-hybrid cluster with paramagnetic properties related to those of HCPs from Desulfovibrio species. Immunoblotting experiments combined with mass spectrometry-based proteomics showed that both nitrate and darkness contribute to the strong upregulation of the HCP levels in C. reinhardtii growing under oxic conditions. The link to the nitrate metabolism is discussed in the light of recent data on the potential role of HCP in S-nitrosylation in bacteria.

Concerted Up-regulation of Aldehyde/Alcohol Dehydrogenase (ADHE) and Starch in Chlamydomonas reinhardtii Increases Survival under Dark Anoxia.

Aldehyde/alcohol dehydrogenases (ADHEs) are bifunctional enzymes that commonly produce ethanol from acetyl-CoA with acetaldehyde as intermediate and play a key role in anaerobic redox balance in many fermenting bacteria. ADHEs are also present in photosynthetic unicellular eukaryotes, where their physiological role and regulation are, however, largely unknown. Herein we provide the first molecular and enzymatic characterization of the ADHE from the photosynthetic microalga Chlamydomonas reinhardtii Purified recombinant ADHE catalyzed the reversible NADH-mediated interconversions of acetyl-CoA, acetaldehyde, and ethanol but seemed to be poised toward the production of ethanol from acetaldehyde. Phylogenetic analysis of the algal fermentative enzyme supports a vertical inheritance from a cyanobacterial-related ancestor. ADHE was located in the chloroplast, where it associated in dimers and higher order oligomers. Electron microscopy analysis of ADHE-enriched stromal fractions revealed fine spiral structures, similar to bacterial ADHE spirosomes. Protein blots showed that ADHE is regulated under oxic conditions. Up-regulation is observed in cells exposed to diverse physiological stresses, including zinc deficiency, nitrogen starvation, and inhibition of carbon concentration/fixation capacity. Analyses of the overall proteome and fermentation profiles revealed that cells with increased ADHE abundance exhibit better survival under dark anoxia. This likely relates to the fact that greater ADHE abundance appeared to coincide with enhanced starch accumulation, which might reflect ADHE-mediated anticipation of anaerobic survival.

Coccomyxa actinabiotis sp. nov. (Trebouxiophyceae, Chlorophyta), a new green microalga living in the spent fuel cooling pool of a nuclear reactor.

Life can thrive in extreme environments where inhospitable conditions prevail. Organisms which resist, for example, acidity, pressure, low or high temperature, have been found in harsh environments. Most of them are bacteria and archaea. The bacterium Deinococcus radiodurans is considered to be a champion among all living organisms, surviving extreme ionizing radiation levels. We have discovered a new extremophile eukaryotic organism that possesses a resistance to ionizing radiations similar to that of D. radiodurans. This microorganism, an autotrophic freshwater green microalga, lives in a peculiar environment, namely the cooling pool of a nuclear reactor containing spent nuclear fuels, where it is continuously submitted to nutritive, metallic, and radiative stress. We investigated its morphology and its ultrastructure by light, fluorescence and electron microscopy as well as its biochemical properties. Its resistance to UV and gamma radiation was assessed. When submitted to different dose rates of the order of some tens of mGy · h-1 to several thousands of Gy · h-1 , the microalga revealed to be able to survive intense gamma-rays irradiation, up to 2,000 times the dose lethal to human. The nuclear genome region spanning the genes for small subunit ribosomal RNA-Internal Transcribed Spacer (ITS) 1-5.8S rRNA-ITS2-28S rRNA (beginning) was sequenced (4,065 bp). The phylogenetic position of the microalga was inferred from the 18S rRNA gene. All the revealed characteristics make the alga a new species of the genus Coccomyxa in the class Trebouxiophyceae, which we name Coccomyxa actinabiotis sp. nov.

The evolution of respiratory O2/NO reductases: an out-of-the-phylogenetic-box perspective.

Complex life on our planet crucially depends on strong redox disequilibria afforded by the almost ubiquitous presence of highly oxidizing molecular oxygen. However, the history of O2-levels in the atmosphere is complex and prior to the Great Oxidation Event some 2.3 billion years ago, the amount of O2 in the biosphere is considered to have been extremely low as compared with present-day values. Therefore the evolutionary histories of life and of O2-levels are likely intricately intertwined. The obvious biological proxy for inferring the impact of changing O2-levels on life is the evolutionary history of the enzyme allowing organisms to tap into the redox power of molecular oxygen, i.e. the bioenergetic O2 reductases, alias the cytochrome and quinol oxidases. Consequently, molecular phylogenies reconstructed for this enzyme superfamily have been exploited over the last two decades in attempts to elucidate the interlocking between O2 levels in the environment and the evolution of respiratory bioenergetic processes. Although based on strictly identical datasets, these phylogenetic approaches have led to diametrically opposite scenarios with respect to the history of both the enzyme superfamily and molecular oxygen on the Earth. In an effort to overcome the deadlock of molecular phylogeny, we here review presently available structural, functional, palaeogeochemical and thermodynamic information pertinent to the evolution of the superfamily (which notably also encompasses the subfamily of nitric oxide reductases). The scenario which, in our eyes, most closely fits the ensemble of these non-phylogenetic data, sees the low O2-affinity SoxM- (or A-) type enzymes as the most recent evolutionary innovation and the high-affinity O2 reductases (SoxB or B and cbb3 or C) as arising independently from NO-reducing precursor enzymes.

On the universal core of bioenergetics.

Living cells are able to harvest energy by coupling exergonic electron transfer between reducing and oxidising substrates to the generation of chemiosmotic potential. Whereas a wide variety of redox substrates is exploited by prokaryotes resulting in very diverse layouts of electron transfer chains, the ensemble of molecular architectures of enzymes and redox cofactors employed to construct these systems is stunningly small and uniform. An overview of prominent types of electron transfer chains and of their characteristic electrochemical parameters is presented. We propose that basic thermodynamic considerations are able to rationalise the global molecular make-up and functioning of these chemiosmotic systems. Arguments from palaeogeochemistry and molecular phylogeny are employed to discuss the evolutionary history leading from putative energy metabolisms in early life to the chemiosmotic diversity of extant organisms. Following the Occam's razor principle, we only considered for this purpose origin of life scenarios which are contiguous with extant life. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Arsenics as bioenergetic substrates.

Although at low concentrations, arsenic commonly occurs naturally as a local geological constituent. Whereas both arsenate and arsenite are strongly toxic to life, a number of prokaryotes use these compounds as electron acceptors or donors, respectively, for bioenergetic purposes via respiratory arsenate reductase, arsenite oxidase and alternative arsenite oxidase. The recent burst in discovered arsenite oxidizing and arsenate respiring microbes suggests the arsenic bioenergetic metabolisms to be anything but exotic. The first goal of the present review is to bring to light the widespread distribution and diversity of these metabolizing pathways. The second goal is to present an evolutionary analysis of these diverse energetic pathways. Taking into account not only the available data on the arsenic metabolizing enzymes and their phylogenetical relatives but also the palaeogeochemical records, we propose a crucial role of arsenite oxidation via arsenite oxidase in primordial life. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Anaerobic energy metabolism in unicellular photosynthetic eukaryotes.

Anaerobic metabolic pathways allow unicellular organisms to tolerate or colonize anoxic environments. Over the past ten years, genome sequencing projects have brought a new light on the extent of anaerobic metabolism in eukaryotes. A surprising development has been that free-living unicellular algae capable of photoautotrophic lifestyle are, in terms of their enzymatic repertoire, among the best equipped eukaryotes known when it comes to anaerobic energy metabolism. Some of these algae are marine organisms, common in the oceans, others are more typically soil inhabitants. All these species are important from the ecological (O(2)/CO(2) budget), biotechnological, and evolutionary perspectives. In the unicellular algae surveyed here, mixed-acid type fermentations are widespread while anaerobic respiration, which is more typical of eukaryotic heterotrophs, appears to be rare. The presence of a core anaerobic metabolism among the algae provides insights into its evolutionary origin, which traces to the eukaryote common ancestor. The predicted fermentative enzymes often exhibit an amino acid extension at the N-terminus, suggesting that these proteins might be compartmentalized in the cell, likely in the chloroplast or the mitochondrion. The green algae Chlamydomonas reinhardtii and Chlorella NC64 have the most extended set of fermentative enzymes reported so far. Among the eukaryotes with secondary plastids, the diatom Thalassiosira pseudonana has the most pronounced anaerobic capabilities as yet. From the standpoints of genomic, transcriptomic, and biochemical studies, anaerobic energy metabolism in C. reinhardtii remains the best characterized among photosynthetic protists. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Chlamydomonas reinhardtii chloroplasts contain a homodimeric pyruvate:ferredoxin oxidoreductase that functions with FDX1.

Eukaryotic algae have long been known to live in anoxic environments, but interest in their anaerobic energy metabolism has only recently gained momentum, largely due to their utility in biofuel production. Chlamydomonas reinhardtii figures remarkably in this respect, because it efficiently produces hydrogen and its genome harbors many genes for anaerobic metabolic routes. Central to anaerobic energy metabolism in many unicellular eukaryotes (protists) is pyruvate:ferredoxin oxidoreductase (PFO), which decarboxylates pyruvate and forms acetyl-coenzyme A with concomitant reduction of low-potential ferredoxins or flavodoxins. Here, we report the biochemical properties of the homodimeric PFO of C. reinhardtii expressed in Escherichia coli. Electron paramagnetic resonance spectroscopy of the recombinant enzyme (Cr-rPFO) showed three distinct [4Fe-4S] iron-sulfur clusters and a thiamine pyrophosphate radical upon reduction by pyruvate. Purified Cr-rPFO exhibits a specific decarboxylase activity of 12 µmol pyruvate min⁻¹ mg⁻¹ protein using benzyl viologen as electron acceptor. Despite the fact that the enzyme is very oxygen sensitive, it localizes to the chloroplast. Among the six known chloroplast ferredoxins (FDX1-FDX6) in C. reinhardtii, FDX1 and FDX2 were the most efficient electron acceptors from Cr-rPFO, with comparable apparent K(m) values of approximately 4 µm. As revealed by immunoblotting, anaerobic conditions that lead to the induction of CrPFO did not increase levels of either FDX1 or FDX2. FDX1, being by far the most abundant ferredoxin, is thus likely the partner of PFO in C. reinhardtii. This finding postulates a direct link between CrPFO and hydrogenase and provides new opportunities to better study and engineer hydrogen production in this protist.

Phylogeny of Rieske/cytb complexes with a special focus on the Haloarchaeal enzymes.

Rieske/cytochrome b (Rieske/cytb) complexes are proton pumping quinol oxidases that are present in most bacteria and Archaea. The phylogeny of their subunits follows closely the 16S-rRNA phylogeny, indicating that chemiosmotic coupling was already present in the last universal common ancestor of Archaea and bacteria. Haloarchaea are the only organisms found so far that acquired Rieske/cytb complexes via interdomain lateral gene transfer. They encode two Rieske/cytb complexes in their genomes; one of them is found in genetic context with nitrate reductase genes and has its closest relatives among Actinobacteria and the Thermus/Deinococcus group. It is likely to function in nitrate respiration. The second Rieske/cytb complex of Haloarchaea features a split cytochrome b sequence as do Cyanobacteria, chloroplasts, Heliobacteria, and Bacilli. It seems that Haloarchaea acquired this complex from an ancestor of the above-mentioned phyla. Its involvement in the bioenergetic reaction chains of Haloarchaea is unknown. We present arguments in favor of the hypothesis that the ancestor of Haloarchaea, which relied on a highly specialized bioenergetic metabolism, that is, methanogenesis, and was devoid of quinones and most enzymes of anaerobic or aerobic bioenergetic reaction chains, integrated laterally transferred genes into its genome to respond to a change in environmental conditions that made methanogenesis unfavorable.

Heterologously expressed arsenite oxidase: a system to study biogenesis and structure/function relationships of the enzyme family.

Studies of native arsenite oxidases from Ralstonia sp. S22 and Rhizobium sp. NT-26 raised two major questions. The first one concerns the mode of the enzyme's membrane-association. It has been suggested that a hypothetical not conserved protein could account for this variable association. Expression of the wild type arsenite oxidase in Escherichia coli allowed us to study the cellular localization of this enzyme in the absence of such a hypothetical partner. The results with the Ralstonia sp. S22 enzyme suggest that no additional protein is required for membrane association. The second question addresses the influence of the disulfide bridge in the small Rieske subunit, conspicuously absent in the Rhizobium sp. NT-26 enzyme, on the properties of the [2Fe-2S] center. The disulfide bridge is considered to be formed only after translocation of the enzyme to the periplasm. To address this question we thus first expressed the enzyme in the absence of its Twin-arginine translocation signal sequence. The spectral and redox properties of the cytoplasmic enzyme are unchanged compared to the periplasmic one. We finally studied a disulfide bridge mutant, Cys106Ala, devoid of the first Cys involved in the disulfide bridge formation. This mutation, proposed to have a strong effect on redox and catalytic properties of the Rieske protein in Rieske/cytb complexes, had no significant effect on properties of the Rieske protein from arsenite oxidase. Our present results demonstrate that the effects attributed to the disulfide bridge in the Rieske/cytb complexes are likely to be secondary effects due to conformational changes.

The ineluctable requirement for the trans-iron elements molybdenum and/or tungsten in the origin of life.

An evolutionary tree of key enzymes from the Complex-Iron-Sulfur-Molybdoenzyme (CISM) superfamily distinguishes "ancient" members, i.e. enzymes present already in the last universal common ancestor (LUCA) of prokaryotes, from more recently evolved subfamilies. The majority of the presented subfamilies and, as a consequence, the Molybdo-enzyme superfamily as a whole, appear to have existed in LUCA. The results are discussed with respect to the nature of bioenergetic substrates available to early life and to problems arising from the low solubility of molybdenum under conditions of the primordial Earth.

Arsenite oxidase from Ralstonia sp. 22: characterization of the enzyme and its interaction with soluble cytochromes.

We characterized the aro arsenite oxidation system in the novel strain Ralstonia sp. 22, a beta-proteobacterium isolated from soil samples of the Salsigne mine in southern France. The inducible aro system consists of a heterodimeric membrane-associated enzyme reacting with a dedicated soluble cytochrome c(554). Our biochemical results suggest that the weak association of the enzyme to the membrane probably arises from a still unknown interaction partner. Analysis of the phylogeny of the aro gene cluster revealed that it results from a lateral gene transfer from a species closely related to Achromobacter sp. SY8. This constitutes the first clear cut case of such a transfer in the Aro phylogeny. The biochemical study of the enzyme demonstrates that it can accommodate in vitro various cytochromes, two of which, c(552) and c(554,) are from the parent species. Cytochrome c(552) belongs to the sox and not the aro system. Kinetic studies furthermore established that sulfite and sulfide, substrates of the sox system, are both inhibitors of Aro activity. These results reinforce the idea that sulfur and arsenic metabolism are linked.

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-L-cysteine in tumor derived cell lines.

The kinesin Eg5 plays an essential role in bipolar spindle formation. A variety of structurally diverse inhibitors of the human kinesin Eg5, including monastrol and STLC, share the same binding pocket on Eg5, composed by helix alpha2/loop L5, and helix alpha3 of the Eg5 motor domain. Previous biochemical analysis in the inhibitor binding pocket of Eg5 identified key residues in the inhibitor binding pocket of Eg5 that in the presence of either monastrol or STLC exhibited ATPase activities similar to the untreated wild type Eg5. Here we evaluated the ability of full-length human Eg5 carrying point mutations in the drug binding pocket to confer resistance in HeLa and U2OS cells to either monastrol or STLC, as measured by the formation of bipolar spindles. Both transfected cells expressing wild type Eg5 and untransfected cells were equally sensitive to both inhibitors. Expression of Eg5 single point mutants R119A, D130A, L132A, I136A, L214A and E215A conferred significant resistance to monastrol. Certain mutations inducing monastrol resistance such as R119A, D130A and L214A also conferred significant resistance to STLC. For the first time at a cellular level, the propensity of selected Eg5 point mutants to confer drug resistance confirms the target specificity of monastrol and STLC for Eg5. These data also suggest a possible mechanism by which drug resistance may occur in tumors treated with agents targeting Eg5.

A proteomic survey of Chlamydomonas reinhardtii mitochondria sheds new light on the metabolic plasticity of the organelle and on the nature of the alpha-proteobacterial mitochondrial ancestor.

Mitochondria play a key role in the life and death of eukaryotic cells, yet the full spectrum of mitochondrial functions is far from being fully understood, especially in photosynthetic organisms. To advance our understanding of mitochondrial functions in a photosynthetic cell, an extensive proteomic survey of Percoll-purified mitochondria from the metabolically versatile, hydrogen-producing green alga Chlamydomonas reinhardtii was performed. Different fractions of purified mitochondria from Chlamydomonas cells grown under aerobic conditions were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry after protein separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis or on blue-native polyacrylamide gel electrophoresis. Of the 496 nonredundant proteins identified, 149 are known or predicted to reside in other cellular compartments and were thus excluded from the molecular and evolutionary analyses of the Chlamydomonas proteome. The mitochondrial proteome of the photosynthetic alga reveals important lineage-specific differences with other mitochondrial proteomes, reflecting the high metabolic diversity of the organelle. Some mitochondrial metabolic pathways in Chlamydomonas appear to combine typical mitochondrial enzymes and bacterial-type ones, whereas others are unknown among mitochondriate eukaryotes. The comparison of the Chlamydomonas proteins to their identifiable homologs predicted from 354 sequenced genomes indicated that Arabidopsis is the most closely related nonalgal eukaryote. Furthermore, this phylogenomic analysis shows that free-living alpha-proteobacteria from the metabolically versatile orders Rhizobiales and Rhodobacterales better reflect the gene content of the ancestor of the chlorophyte mitochondria than parasitic alpha-proteobacteria with reduced and specialized genomes.

Was nitric oxide the first deep electron sink?

Evolutionary histories of enzymes involved in chemiosmotic energy conversion indicate that a strongly oxidizing substrate was available to the last universal common ancestor before the divergence of Bacteria and Archaea. According to palaeogeochemical evidence, O(2) was not present beyond trace amounts on the early Earth. Based on recent phylogenetic, enzymatic and geochemical results, we propose that, in the earliest Archaean, nitric oxide (NO) and its derivatives nitrate and nitrite served as strongly oxidizing substrates driving the evolution of a bioenergetic pathway related to modern dissimilatory denitrification. Aerobic respiration emerged later from within this ancestral pathway via adaptation of the enzyme NO reductase to its new substrate, dioxygen.