ATP synthase: evolution, energetics, and membrane interactions

The synthesis of ATP, life's 'universal energy currency', is the most prevalent chemical reaction in biological systems, and is responsible for fueling nearly all cellular processes, from nerve impulse propagation to DNA synthesis. ATP synthases, the family of enzymes that carry out this endless task, are nearly as ubiquitous as the energy-laden molecule they are responsible for making. The F-type ATP synthase (F-ATPase) is found in every domain of life, and is believed to predate the divergence of these lineages over 1.5 billion years ago. These enzymes have therefore facilitated the survival of organisms in a wide range of habitats, ranging from the deep-sea thermal vents to the human intestine. In this review, we present an overview of the current knowledge of the structure and function of F-type ATPases, highlighting several adaptations that have been characterized across taxa. We emphasize the importance of studying these features within the context of the enzyme's particular lipid environment: Just as the interactions between an organism and its physical environment shape its evolutionary trajectory, ATPases are impacted by the membranes within which they reside. We argue that a comprehensive understanding of the structure, function, and evolution of membrane proteins -- including ATP synthase -- requires such an integrative approach.Comment: Review article; 29 pages, 6 figures/1 tabl

Proton gradients at the origin of life

Chemiosmotic coupling − the harnessing of electrochemical ion gradients across membranes to drive metabolism − is as universally conserved as the genetic code. As argued previously in these pages, such deep conservation suggests that ion gradients arose early in evolution, and might have played a role in the origin of life. Alkaline hydrothermal vents harbour pH gradients of similar polarity and magnitude to those employed by modern cells, one of many properties that make them attractive models for life's origin. Their congruence with the physiology of anaerobic autotrophs that use the acetyl CoA pathway to fix CO2 gives the alkaline vent model broad appeal to biologists. Recently, however, a paper by Baz Jackson criticized the hypothesis, concluding that natural pH gradients were unlikely to have played any role in the origin of life. Unfortunately, Jackson mainly criticized his own interpretations of the theory, not what the literature says. This counterpoint is intended to set the record straight

Microbial energy and matter transformation in agricultural soils

Low bioavailability of organic carbon (C) and energy are key constraints to microbial biomass and activity. Microbial biomass, biodiversity and activity are all involved in regulating soil ecosystem services such as plant productivity, nutrient cycling and greenhouse gas emissions. A number of agricultural practices, of which tillage and fertiliser application are two examples, can increase the availability of soil organic C (SOC). Such practices often lead to reductions in soil aggregation and increases in SOC loss and greenhouse gas emissions. This review focuses on how the bioavailability of SOC and energy influence the ecology and functioning of microorganisms in agricultural soils. Firstly we consider how management practices affect the bioavailability of SOC and energy at the ecosystem level. Secondly we consider the interaction between SOC bioavailability and ecological principles that shape microbial community composition and function in agricultural systems. Lastly, we discuss and compare several examples of physiological differences that underlie how microbial species respond to C availability and management practices. We present evidence whereby management practices that increase the bioavailability of SOC alter community structure and function to favour microbial species likely to be associated with increased rates of SOC loss compared to natural ecosystems. We argue that efforts to restore stabilised, sequestered SOC stocks and improve ecosystem services in agricultural systems should be directed toward the manipulation of the microbial community composition and function to favour species associated with reduced rates of SOC loss. We conclude with several suggestions regarding where improvements in multi-disciplinary approaches concerning soil microbiology can be made to improve the sustainability of agricultural systems

Supercomplexes of Prokaryotic Aerobic Respiratory Chains-Escherichia coli and Bacillus subtilis supramolecular assemblies

Dissertation presented to obtain the Ph.D degree in Biochemistry.Aerobic respiratory chains are composed of a series of membrane complexes that catalyze the electron transfer from reducing substrates to oxygen. The energy released through this process is used to translocate protons across the membranes, thus generating a proton motive force that activates F1FO-ATP synthase to synthesize ATP. The arrangement of these enzymes in the inner mitochondrial membrane is well characterized in mammalian mitochondria, where different sets of supramolecular assemblies, or supercomplexes, involving the majority of the respiratory complexes were described, reinforcing the idea of an operational solid state model wherein the oxidative phosphorylation processes are optimized.(...

In silico analysis of membrane transport/permeability mechanisms

Lipid membranes are a fundamental component of living cells, mediating the physical separation of intracellular components from the external environment, as well as the different cellular organelles from cytoplasm. Transmembrane transport proteins confer permeability to lipid membranes, which is essential for nutrient translocation and energy metabolism. Crystallography of transmembrane proteins is a particularly challenging problem. Due to their natural localization and chemical properties only a limited number of structures are to date available at atomic resolution. In silico analysis can be successfully applied to address the structure and to propose testable models of transporters and pores and of their function. My PhD work focused on two main models: Pendrin (SLC26A4) and the Permeability Transition Pore (PTP). These two systems allowed me to investigate different membrane types and permeation mechanisms, i.e. the plasma membrane-specific anion exchange (SLC26A4) and the inner mitochondrial membrane (IMM) unselective PTP. Pendrin mutations are estimated to be the second most common genetic cause of human deafness, but a precise 3D structure of the protein is still missing. Aim of my work was to obviate the absence of structural information for pendrin transmembrane domain and to give a functional explanation for mutations collected in the MORL Deafness Variation Database. The human pendrin 3D model was inferred by homology with SLC26Dg and then validated analyzing the surface distribution of hydrophobic residues. The resulting high quality model was used to map 147 pathogenic human mutations. Three mutation clusters were found, while their localization suggested an innovative 14 transmembrane domain structure for pendrin. The nature of PTP has long remained a mystery. In 2013 Giorgio et. al. suggested dimers of F1FO (F)-ATP synthase to form the pore, however the exact PTP composition and how can a pore form from the energy-conserving enzyme is still matter of debate. PTP opening is triggered by an increased Ca2+ concentration in the mitochondrial matrix, and is favored by oxidative stress. To shed light on PTP function, I investigated the effect of Ca2+ binding to the Me2+ binding site of the F1 domain of F-ATP synthase through molecular dynamics (MD) simulations. A similar approach was also applied to the F-ATP synthase β subunit mutation T163S, which alters the relative affinity for Mg2+ and Ca2+. Experimental data show that Ca2+ binding stiffens the complex structure and that the T163S mutation induces resistance to PTP opening. Further, catalytic site rearrangement induced from different ion occupancy, as well as the mutation T163S, yields relevant variation of the interaction between F1 domain and OSCP subunit. I suggest that an unstructured loop between residues 82-131 of the β subunit transmits the structural rearrangement originated into catalytic site to the OSCP subunit and then to the inner membrane through the rigid lateral stalk. The critical role emerging for OSCP in the PTP regulation opens two parallel questions, i.e. (i) how the OSCP-mediated opening signal is transmitted to the trans-membrane region and (ii) what are the transmembrane PTP components. Variation in pore conductivity among species suggested that the putative pore-forming subunits may be different in different species. Sequence alignment was performed for all the subunits of F-ATP synthase, but we mainly focused on subunits e, g and b due to their localization in the complex and sequence conservation. Specific mutations affecting F-ATP synthase were collected and their functional effect is currently under analysis. In parallel, the presence and features of e, g and f subunits across eukaryotes was investigated by mean of phylogenetic analysis. Protein homologues of these specific subunits were found to be widespread in eukaryotes from yeast to plants while we found that Oomycetes lack subunits e and g and green algae subunit e. This observation suggest an ancient evolution for the F-ATP synthase dimerization subunits and possibly for the PTP. Further analysis and experimental validation are planned to clarify this aspect

STRUCTURAL BASIS FOR A UNIQUE ATP SYNTNASE CORE COMPLEX FROM NANOARCHAEUM EQUITANS

Ph.DDOCTOR OF PHILOSOPH

Diversity of understudied archaeal and bacterial populations of Yellowstone National Park: from genes to genomes

Yellowstone National Park (YNP) thermal springs have been a crucial resource for the discovery and characterization of microbial biodiversity. The use of cultivation-independent methods has documented many new phyla of uncultured Archaea and Bacteria within thermal springs. Here, I describe the phylogenetic diversity and distribution of Archaea within the YNP thermal spring ecosystem and the phylogenetic and physiologic characterization of novel, uncultured hyperthermophilic bacterial lineages from metagenomic data. In chapter two, I evaluated the efficacy of commonly used, \u27universal\u27 archaeal-specific 16S rRNA gene PCR primers in detecting archaeal phylogenetic diversity. In chapter three, using the PCR primers that would provide the best representation of archaeal communities, I used high-throughput 454 pyrosequencing to analyze the phylogenetic diversity and distribution of Archaea and Bacteria among 33 YNP springs. The results indicated that Archaea were ubiquitously distributed across YNP springs and exhibited significant taxonomic diversity across springs but were overall less phylogenetically diverse in the YNP system than Bacteria. pH, followed by temperature primarily explained the distribution of both archaeal and bacterial taxonomic distribution. Co-occurence analysis suggested a substantial number of putative interactions across the YNP system between and within domains. The results from these two chapters provide the largest survey of Archaea in any thermal system to date and contribute to our understanding of their phylogenetic diversity and ecology in such systems. In Chapter 4, I report the phylogenetic and physiologic characterization of novel, deep-branching bacterial phylotypes from metagenomic data from two YNP springs. Genome assemblies representing four populations were recovered from Aquificaceae-dominated community metagenomes from two high-temperature, circumneutral YNP springs. Phylogenetic analyses indicated they belonged to two distinct, deep-branching bacterial lineages, one of which has no currently characterized genome references. The lineages appeared to be heteroorganotrophs based on metabolic reconstructions and also were both putatively capable of using energy conserved from organic carbon degradation to fuel aerobic respiration. Analysis of the ecological distribution of these populations confirmed that they are currently restricted to high-temperature circumneutral terrestrial springs, largely within YNP. The characterization of these populations provides important physiologic context for the deepest-branching bacterial lineages and valuable genomic references for uncultured, ubiquitously distributed hyperthermophilic bacteria

Genetics of Halophilic Microorganisms

Halophilic microorganisms are found in all domains of life and thrive in hypersaline (high salt content) environments. These unusual microbes have been a subject of study for many years due to their interesting properties and physiology. Studies of the genetics of halophilic microorganisms (from gene expression and regulation to genomics) have provided understanding into the mechanisms of how life can exist at high salinity levels. Here, we highlight recent studies that advance the knowledge of biological function through examination of the genetics of halophilic microorganisms and their viruses

Organization, dynamics and adaptation of the photosynthetic machinery in cyanobacteria

Ye