New Insights on Rotenone Resistance of Complex I Induced by the m.11778G>A/MT-ND4 Mutation Associated with Leber's Hereditary Optic Neuropathy.

The finding that the most common mitochondrial DNA mutation m.11778G>A/MT-ND4 (p.R340H) associated with Leber's hereditary optic neuropathy (LHON) induces rotenone resistance has produced a long-standing debate, because it contrasts structural evidence showing that the ND4 subunit is far away from the quinone-reaction site in complex I, where rotenone acts. However, recent cryo-electron microscopy data revealed that rotenone also binds to the ND4 subunit. We investigated the possible structural modifications induced by the LHON mutation and found that its amino acid replacement would disrupt a possible hydrogen bond between native R340 and Q139 in ND4, thereby destabilizing rotenone binding. Our analysis thus explains rotenone resistance in LHON patients as a biochemical signature of its pathogenic effect on complex I.

Current phylogeny of Rhodospirillaceae: A multi-approach study.

Rhodospirillaceae represents a major family of the class alphaproteobacteria that includes an increasing number of functionally diverse taxa. The aim of this work is to evaluate the present phylogenetic diversity of the Rhodospirillaceae, which includes several metagenome-assembled genomes of uncultivated bacteria, as well as cultivated bacteria that were previously classified in different families. Various methodological approaches have been followed to discern the phylogenetic diversity of the taxa associated with the Rhodospirillaceae, which are grouped in three major sub-divisions and several other taxonomic entities that are currently confined to the genus rank. These genera include Tistrella, Elstera, Dongia and Ferrovibrio among cultivated organisms and alphaproteobacteria bacterium 41-28 among uncultivated bacteria. Overall, this study adds at least 11 genera and over 40 species to the current set of taxa belonging to the Rhodospirillaceae, a taxonomic term that clearly requires amendment. We propose to re-classify all taxa associated with the Rhodospirillaceae family under the new order, Diaforabacterales ord. nov. (from the Greek word for diversity, διάφορα). This study also uncovers the likely root of Rhodospirillaceae among recently reported metagenome-assembled genomes of uncultivated marine and groundwater bacteria.

Changes in membrane lipids drive increased endocytosis following Fas ligation.

Once activated, some surface receptors promote membrane movements that open new portals of endocytosis, in part to facilitate the internalization of their activated complexes. The prototypic death receptor Fas (CD95/Apo1) promotes a wave of enhanced endocytosis that induces a transient intermixing of endosomes with mitochondria in cells that require mitochondria to amplify death signaling. This initiates a global alteration in membrane traffic that originates from changes in key membrane lipids occurring in the endoplasmic reticulum (ER). We have focused the current study on specific lipid changes occurring early after Fas ligation. We analyzed the interaction between endosomes and mitochondria in Jurkat T cells by nanospray-Time-of-flight (ToF) Mass Spectrometry. Immediately after Fas ligation, we found a transient wave of lipid changes that drives a subpopulation of early endosomes to merge with mitochondria. The earliest event appears to be a decrease of phosphatidylcholine (PC), linked to a metabolic switch enhancing phosphatidylinositol (PI) and phosphoinositides, which are crucial for the formation of vacuolar membranes and endocytosis. Lipid changes occur independently of caspase activation and appear to be exacerbated by caspase inhibition. Conversely, inhibition or compensation of PC deficiency attenuates endocytosis, endosome-mitochondria mixing and the induction of cell death. Deficiency of receptor interacting protein, RIP, also limits the specific changes in membrane lipids that are induced by Fas activation, with parallel reduction of endocytosis. Thus, Fas activation rapidly changes the interconversion of PC and PI, which then drives enhanced endocytosis, thus likely propagating death signaling from the cell surface to mitochondria and other organelles.

Did mitophagy follow the origin of mitochondria?

Mitophagy is the process of selective autophagy that removes superfluous and dysfunctional mitochondria. Mitophagy was first characterized in mammalian cells and is now recognized to follow several pathways including basal forms in specific organs. Mitophagy pathways are regulated by multiple, often interconnected factors. The present review aims to streamline this complexity and evaluate common elements that may define the evolutionary origin of mitophagy. Key issues surrounding mitophagy signaling at the mitochondrial surface may fundamentally derive from mitochondrial membrane dynamics. Elements of such membrane dynamics likely originated during the endosymbiosis of the alphaproteobacterial ancestor of our mitochondria but underwent an evolutionary leap forward in basal metazoa that determined the currently known variations in mitophagy signaling.Abbreviations: AGPAT, 1-acylglycerol-3-phosphate O-acyltransferase; ATG, autophagy related; BCL2L13, BCL2 like 13; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; CALCOCO, calcium binding and coiled-coil domain; CL, cardiolipin; ER, endoplasmic reticulum; ERMES, ER-mitochondria encounter structure; FBXL4, F-box and leucine rich repeat protein 4; FUNDC1, FUN14 domain containing 1; GABARAPL1, GABA type A receptor associated protein like 1; HIF, hypoxia inducible factor; IMM, inner mitochondrial membrane; LBPA/BMP, lysobisphosphatidic acid; LIR, LC3-interacting region; LPA, lysophosphatidic acid; MAM, mitochondria-associated membranes; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MCL, monolysocardiolipin; ML, maximum likelihood; NBR1, NBR1 autophagy cargo receptor; OMM, outer mitochondrial membrane; PA, phosphatidic acid; PACS2, phosphofurin acidic cluster sorting protein 2; PC/PLC, phosphatidylcholine; PE, phosphatidylethanolamine; PHB2, prohibitin 2; PINK1, PTEN induced kinase 1; PtdIns, phosphatidylinositol; SAR, Stramenopiles, Apicomplexa and Rhizaria; TAX1BP1, Tax1 binding protein 1; ULK1, unc-51 like autophagy activating kinase 1; VDAC/porin, voltage dependent anion channel.

Three enzymes governed the rise of O2 on Earth.

Current views of O2 accumulation in Earth history depict three phases: The onset of O2 production by ∼2.4 billion years ago; 2 billion years of stasis at ∼1 % of modern atmospheric levels; and a rising phase, starting about 500 million years ago, in which oxygen eventually reached modern values. Purely geochemical mechanisms have been proposed to account for this tripartite time course of Earth oxygenation. In particular the second phase, the long period of stasis between the advent of O2 and the late rise to modern levels, has posed a puzzle. Proposed solutions involve Earth processes (geochemical, ecosystem, day length). Here we suggest that Earth oxygenation was not determined by geochemical processes. Rather it resulted from emergent biological innovations associated with photosynthesis and the activity of only three enzymes: 1) The oxygen evolving complex of cyanobacteria that makes O2; 2) Nitrogenase, with its inhibition by O2 causing two billion years of oxygen level stasis; 3) Cellulose synthase of land plants, which caused mass deposition and burial of carbon, thus removing an oxygen sink and therefore increasing atmospheric O2. These three enzymes are endogenously produced by, and contained within, cells that have the capacity for exponential growth. The catalytic properties of these three enzymes paved the path of Earth's atmospheric oxygenation, requiring no help from Earth other than the provision of water, CO2, salts, colonizable habitats, and sunlight.

The bacterial origin of mitochondria: Incorrect phylogenies and the importance of metabolic traits.

This article provides an updated review on the evolution of mitochondria from bacteria, which were likely related to extant alphaproteobacteria. Particular attention is given to the timeline of oxygen history on Earth and the entwined phases of eukaryotic evolution that produced the animals that still populate our planet. Mitochondria of early-branching unicellular eukaryotes and plants appear to retain partial or vestigial traits that were directly inherited from the alphaproteobacterial ancestors of the organelles. Most of such traits define the current aerobic physiology of mitochondria. Conversely, the anaerobic traits that would be essential in the syntrophic associations postulated for the evolution of eukaryotic cells are scantly present in extant alphaproteobacteria, and therefore cannot help defining from which bacterial lineage the ancestors of mitochondria originated. This question has recently been addressed quantitatively, reaching the novel conclusion that marine bacteria related to Iodidimonas may be the living relatives of protomitochondria. Additional evidence is presented that either support or does not contrast this novel view of the bacterial origin of mitochondria.

Multiple approaches of cellular metabolism define the bacterial ancestry of mitochondria.

We breathe at the molecular level when mitochondria in our cells consume oxygen to extract energy from nutrients. Mitochondria are characteristic cellular organelles that derive from aerobic bacteria and carry out oxidative phosphorylation and other key metabolic pathways in eukaryotic cells. The precise bacterial origin of mitochondria and, consequently, the ancestry of the aerobic metabolism of our cells remain controversial despite the vast genomic information that is now available. Here, we use multiple approaches to define the most likely living relatives of the ancestral bacteria from which mitochondria originated. These bacteria live in marine environments and exhibit the highest frequency of aerobic traits and genes for the metabolism of fundamental lipids that are present in the membranes of eukaryotes, sphingolipids, and cardiolipin.

The phylogeny of Acetobacteraceae: photosynthetic traits and deranged respiratory enzymes.

IMPORTANCE: Acetobacteraceae are one of the best known and most extensively studied groups of bacteria, which nowadays encompasses a variety of taxa that are very different from the vinegar-producing species defining the family. Our paper presents the most detailed phylogeny of all current taxa classified as Acetobacteraceae, for which we propose a taxonomic revision. Several of such taxa inhabit some of the most extreme environments on the planet, from the deserts of Antarctica to the Sinai desert, as well as acidic niches in volcanic sites like the one we have been studying in Patagonia. Our work documents the progressive variation of the respiratory chain in early branching Acetobacteraceae into the different respiratory chains of acidophilic taxa such as Acidocella and acetous taxa such as Acetobacter. Remarkably, several genomes retain remnants of ancestral photosynthetic traits and functional bc 1 complexes. Thus, we propose that the common ancestor of Acetobacteraceae was photosynthetic.

New Alphaproteobacteria Thrive in the Depths of the Ocean with Oxygen Gradient.

We survey here the Alphaproteobacteria, a large class encompassing physiologically diverse bacteria which are divided in several orders established since 2007. Currently, there is considerable uncertainty regarding the classification of an increasing number of marine metagenome-assembled genomes (MAGs) that remain poorly defined in their taxonomic position within Alphaproteobacteria. The traditional classification of NCBI taxonomy is increasingly complemented by the Genome Taxonomy Database (GTDB), but the two taxonomies differ considerably in the classification of several Alphaproteobacteria, especially from ocean metagenomes. We analyzed the classification of Alphaproteobacteria lineages that are most common in marine environments, using integrated approaches of phylogenomics and functional profiling of metabolic features that define their aerobic metabolism. Using protein markers such as NuoL, the largest membrane subunit of complex I, we have identified new clades of Alphaproteobacteria that are specific to marine niches with steep oxygen gradients (oxycline). These bacteria have relatives among MAGs found in anoxic strata of Lake Tanganyika and together define a lineage that is distinct from either Rhodospirillales or Sneathiellales. We characterized in particular the new 'oxycline' clade. Our analysis of Alphaproteobacteria also reveals new clues regarding the ancestry of mitochondria, which likely evolved in oxycline marine environments.

Respiratory Heme A-Containing Oxidases Originated in the Ancestors of Iron-Oxidizing Bacteria.

Respiration is a major trait shaping the biology of many environments. Cytochrome oxidase containing heme A (COX) is a common terminal oxidase in aerobic bacteria and is the only one in mammalian mitochondria. The synthesis of heme A is catalyzed by heme A synthase (CtaA/Cox15), an enzyme that most likely coevolved with COX. The evolutionary origin of COX in bacteria has remained unknown. Using extensive sequence and phylogenetic analysis, we show that the ancestral type of heme A synthases is present in iron-oxidizing Proteobacteria such as Acidithiobacillus spp. These bacteria also contain a deep branching form of the major COX subunit (COX1) and an ancestral variant of CtaG, a protein that is specifically required for COX biogenesis. Our work thus suggests that the ancestors of extant iron-oxidizers were the first to evolve COX. Consistent with this conclusion, acidophilic iron-oxidizing prokaryotes lived on emerged land around the time for which there is the earliest geochemical evidence of aerobic respiration on earth. Hence, ecological niches of iron oxidation have apparently promoted the evolution of aerobic respiration.

Genomic evolution of the class Acidithiobacillia: deep-branching Proteobacteria living in extreme acidic conditions.

Members of the genus Acidithiobacillus, now ranked within the class Acidithiobacillia, are model bacteria for the study of chemolithotrophic energy conversion under extreme conditions. Knowledge of the genomic and taxonomic diversity of Acidithiobacillia is still limited. Here, we present a systematic analysis of nearly 100 genomes from the class sampled from a wide range of habitats. Some of these genomes are new and others have been reclassified on the basis of advanced genomic analysis, thus defining 19 Acidithiobacillia lineages ranking at different taxonomic levels. This work provides the most comprehensive classification and pangenomic analysis of this deep-branching class of Proteobacteria to date. The phylogenomic framework obtained illuminates not only the evolutionary past of this lineage, but also the molecular evolution of relevant aerobic respiratory proteins, namely the cytochrome bo3 ubiquinol oxidases.

Oxygen Reductases in Alphaproteobacterial Genomes: Physiological Evolution From Low to High Oxygen Environments.

Oxygen reducing terminal oxidases differ with respect to their subunit composition, heme groups, operon structure, and affinity for O2. Six families of terminal oxidases are currently recognized, all of which occur in alphaproteobacterial genomes, two of which are also present in mitochondria. Many alphaproteobacteria encode several different terminal oxidases, likely reflecting ecological versatility with respect to oxygen levels. Terminal oxidase evolution likely started with the advent of O2 roughly 2.4 billion years ago and terminal oxidases diversified in the Proterozoic, during which oxygen levels remained low, around the Pasteur point (ca. 2 µM O2). Among the alphaproteobacterial genomes surveyed, those from members of the Rhodospirillaceae reveal the greatest diversity in oxygen reductases. Some harbor all six terminal oxidase types, in addition to many soluble enzymes typical of anaerobic fermentations in mitochondria and hydrogenosomes of eukaryotes. Recent data have it that O2 levels increased to current values (21% v/v or ca. 250 µM) only about 430 million years ago. Ecological adaptation brought forth different lineages of alphaproteobacteria and different lineages of eukaryotes that have undergone evolutionary specialization to high oxygen, low oxygen, and anaerobic habitats. Some have remained facultative anaerobes that are able to generate ATP with or without the help of oxygen and represent physiological links to the ancient proteobacterial lineage at the origin of mitochondria and eukaryotes. Our analysis reveals that the genomes of alphaproteobacteria appear to retain signatures of ancient transitions in aerobic metabolism, findings that are relevant to mitochondrial evolution in eukaryotes as well.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced mitochondrial pathway to apoptosis and caspase activation is potentiated by phospholipid scramblase-3.

Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) initiate pathways of cell death in which caspase activation is mediated either directly (without mitochondrial amplification), or indirectly via the release of apoptogenic factors from mitochondria. Phospholipid scramblases (PLS) are enzymes that play a key role in cellular function by inducing bidirectional movement of membrane lipids. Changes in mitochondrial membrane lipids, cardiolipin, are critical for mediating apoptotic response in many cell-types. PLS3 is a phospholipid scramblase that is localized to mitochondria and is thought to be involved in the regulation of apoptotic signals. Here we report that exogenous-expression of PLS3 enhances apoptotic death induced by TRAIL. This is acheived by potentiating the mitochondrial arm of the death pathway. Thereby, PLS3 expression facilitates changes in mitochondrial membrane lipids that promote the release of apoptogenic factors and consequent full activation and processing of the caspase-9 and effector caspase-3. Moreover, we show that knock-down of endogenous PLS3 suppresses TRAIL-induced changes in cardiolipin. Finally, we demonstrate that TRAIL-induced activation of PKC-delta mediates regulation of the PLS3-induced changes in cardiolipin.

Tumor necrosis factor-related apoptosis-inducing ligand alters mitochondrial membrane lipids.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to have selective antitumor activity. TRAIL induces ubiquitous pathways of cell death in which caspase activation is mediated either directly or via the release of apoptogenic factors from mitochondria; however, the precise components of the mitochondrial signaling pathway have not been well defined. Notably, mitochondria constitute an important target in overcoming resistance to TRAIL in many types of tumors. Bid is considered to be fundamental in engaging mitochondria during death receptor-mediated apoptosis, but this action is dependent on mitochondrial lipids. Here, we report that TRAIL signaling induces an alteration in mitochondrial membrane lipids, particularly cardiolipin. This occurs independently of caspase activation and primes mitochondrial membranes to the proapoptotic action of Bid. We unveil a link between TRAIL signaling and alteration of membrane lipid homeostasis that occurs in parallel to apical caspase activation but does not take over the mode of cell death because of the concurrent activation of caspase-8. In particular, TRAIL-induced alteration of mitochondrial lipids follows an imbalance in the cellular homeostasis of phosphatidylcholine, which results in an elevation in diacylglycerol (DAG). Elevated DAG in turn activates the delta isoform of phospholipid-dependent serine/threonine protein kinase C, which then accelerates the cleavage of caspase-8. We also show that preservation of phosphatidylcholine homeostasis by inhibition of lipid-degrading enzymes almost completely impedes the activation of pro-caspase-9 while scarcely changing the activation of caspase-8.

A Journey across Genomes Uncovers the Origin of Ubiquinone in Cyanobacteria.

Ubiquinone (Q) is an isoprenoid quinone that functions as membrane electron carrier in mitochondria and bacterial organisms belonging to the alpha, beta, and gamma class of proteobacteria. The biosynthesis of Q follows various biochemical steps catalyzed by diverse proteins that are, in general, homologous in mitochondria and bacteria. Nonorthologous proteins can also contribute to some biochemical steps as originally uncovered in Escherichia coli, which is the best studied organism for Q biosynthesis in prokaryotes. However, the origin of the biosynthetic pathway of Q has remained obscure. Here, I show by genome analysis that Q biosynthesis originated in cyanobacteria and then diversified in anaerobic alpha proteobacteria which have extant relatives in members of the Rhodospirillaceae family. Two distinct biochemical pathways diverged when ambient oxygen reached current levels on earth, one leading to the well-known series of Ubi genes found in E. coli, and the other containing CoQ proteins originally found in eukaryotes. Extant alpha proteobacteria show Q biosynthesis pathways that are more similar to that present in mitochondria than to that of E. coli. Hence, this work clarifies not only the origin but also the evolution of Q biosynthesis from bacteria to mitochondria.

The long story of mitochondrial DNA and respiratory complex I.

This article examines the long story of the relationship between mitochondrial DNA (mtDNA) and respiratory complex I, NADH:Ubiquinone Oxidoreductase, from its beginning  in the genome of the bacterial endosymbiont which then evolved into the mitochondria of our cells. The story begins with the evolution of ancient forms of bacterial complex I into the Nuo14 complex I that was present in the alpha proteobacterial ancestor of mitochondria. The story then becomes complicated in the diversity of eukaryotic organisms that are currently recognized. Therefore, it does not have a clear end, because currently available information shows different situations of metabolic adaptation and gene loss, indicating cases of de-evolution of the original protonmotive complex into a system that may fundamentally assist [FeFe]-hydrogenases in re-oxidising metabolically produced NADH under anaerobic conditions. The history of complex I is thus a never ending story of molecular and physiological evolution producing new perspectives for studying the enzyme complex that occupies the largest proportion of mitochondrial DNA.

The functional microbiome of arthropods.

Many studies on the microbiome of animals have been reported but a comprehensive analysis is lacking. Here we present a meta-analysis on the microbiomes of arthropods and their terrestrial habitat, focusing on the functional profile of bacterial communities derived from metabolic traits that are essential for microbial life. We report a detailed analysis of probably the largest set of biochemically defined functional traits ever examined in microbiome studies. This work deals with the phylum proteobacteria, which is usually dominant in marine and terrestrial environments and covers all functions associated with microbiomes. The considerable variation in the distribution and abundance of proteobacteria in microbiomes has remained fundamentally unexplained. This analysis reveals discrete functional groups characteristic for adaptation to anaerobic conditions, which appear to be defined by environmental filtering of taxonomically related taxa. The biochemical diversification of the functional groups suggests an evolutionary trajectory in the structure of arthropods' microbiome, from metabolically versatile to specialized proteobacterial organisms that are adapted to complex environments such as the gut of social insects. Bacterial distribution in arthropods' microbiomes also shows taxonomic clusters that do not correspond to functional groups and may derive from other factors, including common contaminants of soil and reagents.

Candidatus Dactylopiibacterium carminicum, a Nitrogen-Fixing Symbiont of Dactylopius Cochineal Insects (Hemiptera: Coccoidea: Dactylopiidae).

The domesticated carmine cochineal Dactylopius coccus (scale insect) has commercial value and has been used for more than 500 years for natural red pigment production. Besides the domesticated cochineal, other wild Dactylopius species such as Dactylopius opuntiae are found in the Americas, all feeding on nutrient poor sap from native cacti. To compensate nutritional deficiencies, many insects harbor symbiotic bacteria which provide essential amino acids or vitamins to their hosts. Here, we characterized a symbiont from the carmine cochineal insects, Candidatus Dactylopiibacterium carminicum (betaproteobacterium, Rhodocyclaceae family) and found it in D. coccus and in D. opuntiae ovaries by fluorescent in situ hybridization, suggesting maternal inheritance. Bacterial genomes recovered from metagenomic data derived from whole insects or tissues both from D. coccus and from D. opuntiae were around 3.6 Mb in size. Phylogenomics showed that dactylopiibacteria constituted a closely related clade neighbor to nitrogen fixing bacteria from soil or from various plants including rice and other grass endophytes. Metabolic capabilities were inferred from genomic analyses, showing a complete operon for nitrogen fixation, biosynthesis of amino acids and vitamins and putative traits of anaerobic or microoxic metabolism as well as genes for plant interaction. Dactylopiibacterium nif gene expression and acetylene reduction activity detecting nitrogen fixation were evidenced in D. coccus hemolymph and ovaries, in congruence with the endosymbiont fluorescent in situ hybridization location. Dactylopiibacterium symbionts may compensate for the nitrogen deficiency in the cochineal diet. In addition, this symbiont may provide essential amino acids, recycle uric acid, and increase the cochineal life span.

Late Mitochondrial Acquisition, Really?

This article provides a timely critique of a recent Nature paper by Pittis and Gabaldón that has suggested a late origin of mitochondria in eukaryote evolution. It shows that the inferred ancestry of many mitochondrial proteins has been incorrectly assigned by Pittis and Gabaldón to bacteria other than the aerobic proteobacteria from which the ancestor of mitochondria originates, thereby questioning the validity of their suggestion that mitochondrial acquisition may be a late event in eukaryote evolution. The analysis and approach presented here may guide future studies to resolve the true ancestry of mitochondria.

A survey of the energy metabolism of nodulating symbionts reveals a new form of respiratory complex I.

A limiting factor in agriculture is the availability of nitrogen in the soil, which may be compensated by biological N2 fixation catalysed by bacteria. Most biological N2 fixation occurs in root nodules of plants that respond to bacterial infection by establishing symbiosis with selected prokaryotes. The plants provide energy metabolites and a microoxic environment to the bacterial symbionts to facilitate their capacity of N2 fixation. Despite enormous advances in the molecular genetics of this symbiosis between plants and nodulating bacteria, we still do not understand fundamental aspects which determine the efficiency of N2 fixation in the nodules, and therefore the capacity to biologically enrich agricultural soils. Here we provide an overview of the central features of the energy metabolism that sustains N2 fixation, with emphasis on the bacterial respiratory chain supplying the electrons and ATP required for the nitrogenase reaction. We discuss common trends and specific variations in the integrated process of respiratory electron transport and N2 fixation. Among such variations we introduce green complex I, an ancient version of NADH:ubiquinone oxidoreductase that is present in several Rhizobiaceae and may facilitate N2 fixation.