Energy metabolism of Heliobacterium modesticaldum during phototrophic and chemotrophic growth

<p>Abstract</p> <p>Background</p> <p><it>Heliobacterium modesticaldum </it>is a gram-positive nitrogen-fixing phototrophic bacterium that can grow either photoheterotrophically or chemotrophically but not photoautotrophically. Surprisingly, this organism is lacking only one gene for the complete reverse tricarboxylic acid (rTCA) cycle required for autotrophic carbon fixation. Along with the genomic information reported recently, we use multiple experimental approaches in this report to address questions regarding energy metabolic pathways in darkness, CO₂fixation, sugar assimilation and acetate metabolism.</p> <p>Results</p> <p>We present the first experimental evidence that D-ribose, D-fructose and D-glucose can be photoassimilated by <it>H. modesticaldum </it>as sole carbon sources in newly developed defined growth medium. Also, we confirm two non-autotrophic CO₂-fixation pathways utilized by <it>H. modesticaldum</it>: reactions catalyzed by pyruvate:ferredoxin oxidoreductase and phosphoenolpyruvate carboxykinase, and report acetate excretion during phototrophic and chemotrophic growth. Further, genes responsible for pyruvate fermentation, which provides reducing power for nitrogen assimilation, carbon metabolism and hydrogen production, are either active or up-regulated during chemotrophic growth. The discovery of ferredoxin-NADP⁺oxidoreductase (FNR) activity in cell extracts provides the reducing power required for carbon and nitrogen metabolisms. Moreover, we show that photosynthetic pigments are produced by <it>H. modesticaldum </it>during the chemotrophic growth, and demonstrate that <it>H. modesticaldum </it>performs nitrogen fixation during both phototrophic and chemotrophic growth.</p> <p>Conclusion</p> <p>Collectively, this report represents the first comprehensive studies for energy metabolism in heliobacteria, which have the simplest known photosynthetic machinery among the entire photosynthetic organisms. Additionally, our studies provide new and essential insights, as well as broaden current knowledge, on the energy metabolism of the thermophilic phototrophic bacterium <it>H. modesticaldum </it>during phototrophic and chemotrophic growth.</p

"The Evolution of Photosynthesis and the Transition from an Anaerobic to an Aerobic World"

This project was focused on elucidating the evolution of photosynthesis, in particular the evolutionary developments that preceded and accompanied the transition from anoxygenic to oxygenic photosynthesis. Development of this process has clearly been of central importance to evolution of life on Earth. Photosynthesis is the mechanism that ultimately provides for the energy needs of most surface-dwelling organisms. Eukaryotic organisms are absolutely dependent on the molecular oxygen that has been produced by oxygenic photosynthesis. In this project we have employed a multidisciplinary approach to understand some of the processes that took place during the evolution of photosynthesis. In this project, we made excellent progress in the overall area of understanding the origin and evolution of photosynthesis. Particular progress has been made on several more specific research questions, including the molecular evolutionary analysis of photosynthetic components and biosynthetic pathways (2,3, 5, 7, 10), as well as biochemical characterization of electron transfer proteins related to photosynthesis and active oxygen protection (4,6,9). Finally, several review and commentary papers have been published (1, 8, 1 1). A total of twelve publications arose out of this grant, references to which are given below. Some specific areas of progress are highlighted and discussed in more detail

Correlated interaction fluctuations in photosynthetic complexes

The functioning and efficiency of natural photosynthetic complexes is strongly influenced by their embedding in a noisy protein environment, which can even serve to enhance the transport efficiency. Interactions with the environment induce fluctuations of the transition energies of and interactions between the chlorophyll molecules, and due to the fact that different fluctuations will partially be caused by the same environmental factors, correlations between the various fluctuations will occur. We argue that fluctuations of the interactions should in general not be neglected, as these have a considerable impact on population transfer rates, decoherence rates and the efficiency of photosynthetic complexes. Furthermore, while correlations between transition energy fluctuations have been studied, we provide the first quantitative study of the effect of correlations between interaction fluctuations and transition energy fluctuations, and of correlations between the various interaction fluctuations. It is shown that these additional correlations typically lead to changes in interchromophore transfer rates, population oscillations and can lead to a limited enhancement of the light harvesting efficiency

A unique photosynthetic reaction center from Heliobacterium chlorum

AbstractA previously unknown type of photosynthetic reaction center in the brownish-green bacterium Heliobacterium chlorum is described. The reaction center is tightly bound in a highly proteinaceous undifferentiated plasma membrane and contains bacteriochlorophyll g, which has major in vivo absorbancies at 788, 576 and 370 nm. The purified membrane shows a reversible photobleaching at 798 nm; the reaction center bacteriochlorophyll g is designated P798. A reversible photobleaching at 553 nm is assigned to photooxidation of a membrane-bound c-type cytochrome. The membrane structure, pigment composition and photochemical properties suggest that H. chlorum may represent a fifth family of anoxygenic photosynthetic procaryotes

Degree-Day Requirements for Alfalfa Weevil (Coleoptera: Curculionidae) Development in Eastern Nebraska

The alfalfa weevil, Hypera postica (Gyllenhal), is a serious, yet sporadic defoliator of alfalfa, Medicago sativa L., in Nebraska. A 2-yr study was conducted in 2005 and 2006 to test for variation in degree-day requirements by location in eastern Nebraska. Sampling took place along a latitudinal gradient in three regions of eastern Nebraska. Three fields were sampled in each region during the 2 yr of the study. Alfalfa weevil larval degree-day requirements were found to vary by latitude in eastern Nebraska. Alfalfa weevil larvae were discovered in southern regions after fewer developmental degree-days had accumulated than in fields in the northern regions. Alfalfa weevils may be more damaging to alfalfa in southern regions than in northern regions of eastern Nebraska because they emerge earlier relative to alfalfa growth. Management implications of this shift in alfalfa weevil phenology are discussed

ASPECTOS EVOLUTIVOS DO METABOLISMO ÁCIDO DAS CRASSULACEAS

Crassulacean acid metabolism (CAM) is a carbon concentrating metabolism present in more than 30 botanical families. This metabolism is often associated with water stressed environments although it can be found among aquatic plants. CAM's main feature is nighttime stomatal opening and acid accumulation due to CO2 fixation into a four carbon organic acid, often malate. CAM is present among pteridophytes, gymnosperms and angiosperms, in monocotyledonous and dicotyledonous species. Within some families, such as Crassulaceae, Orchidaceae and Bromeliaceae, CAM is widely represented. The genus Clusia has both facultative and constitutive CAM species. Regulation of the diel cycle of CAM hinges on the activation status of phosphoenolpyruvate carboxylase, responsible for fixation of CO2 at  night. This enzyme is in turn regulated by phosphoenolpyruvate carboxykinase, a dedicated phosphorylating enzyme which is under circadian control. As CAM is widely distributed among botanical families, its origin is believed to be polyphyletic; however, the evolutionary mechanisms which allowed reappearance of this complex metabolism are not yet understood. Perhaps the answer relies on viewing CAM as a network that evolved by gene duplication from the pre-existing non photosynthetic C4 cycle.El metabolismo ácido de las Crassuláceas (CAM) es un metabolismo fotosintético de concentración de CO2 presente en más de 30 familias botánicas. Está frecuentemente asociado a la economía de agua, a pesar de estar presente también en especies acuáticas. El  CAM se caracteriza por la abertura de los estomas en la noche, y con la acumulación de ácidos en función de la fijación de carbono en compuestos de cuatro carbonos, en general, o malato. El CAM está presente en Pteridófitas, Gimnospermas e angiospermas, tanto mono como dicotiledóneas. En algunas familias, como Crassulaceae, Orquidaceae y Bromeliaceae, el metabolismo ácido de las Crassuláceas está fuertemente representado. En el género Clusia, el CAM puede estar presente en las especies de forma facultativa o constitutiva. La regulación del ciclo diurno del metabolismo ácido de las Crassuláceas es fuertemente determinada pela regulación de la enzima fosfoenolpiruvato carboxilasa, responsable por la fijación de CO2 durante la noche. A su vez, la regulación es realizada a través de la fosforilación que ejecuta la enzima fosfoenolpiruvato carboxiquinasa que presenta ritmo circadiano. Como la distribución de CAM es discontinua se cree que su origen sea polifilético, sin embargo, no están claros los mecanismos evolutivos que tornaron el reapareciendo de este complejo metabolismo posible. Tal vez CAM posa ser visto como un complejo que evoluciono por duplicación de genes del pré-existente no fotosintético ciclo C4.O metabolismo ácido das crassuláceas (CAM) é um metabolismo fotossintético de concentração de CO2 presente em mais de 30 famílias botânicas. Está frequentemente associado à economia de água, embora esteja presente também em espécies aquáticas. CAM é caracterizado pela abertura dos estômatos à noite com acúmulo de ácidos em função da fixação de carbono em compostos de quatro carbonos, em geral, o malato. CAM está presente em pteridófitas, gimnospermas e angiospermas, tanto mono quanto dicotiledôneas. Em algumas famílias, como Crassulaceae, Orquidaceae e Bromeliaceae, o metabolismo ácido das crassuláceas está fortemente representado. No gênero Clusia, CAM pode estar presente nas espécies de forma facultativa ou constitutiva. A regulação do ciclo diurno do metabolismo ácido das crassuláceas é fortemente determinada pela regulação da enzima fosfoenolpiruvato carboxilase, responsável pela fixação do CO2 durante a noite. Por sua vez, sua regulação é feita através da fosforilação pela enzima fosfoenolpiruvato carboxiquinase que apresenta ritmo circadiano. Como a distribuição de CAM é descontínua acredita-se que sua origem seja polifilética, contudo, não estão claros os mecanismos evolutivos que tornaram o reaparecimento desse complexo metabolismo possível. Talvez CAM possa ser visto como um complexo que evoluiu por duplicação de genes do pré-existente não fotossintético ciclo C4

Carbohydrate Metabolism and Carbon Fixation in Roseobacter denitrificans OCh114

The Roseobacter clade of aerobic marine proteobacteria, which compose 10–25% of the total marine bacterial community, has been reported to fix CO2, although it has not been determined what pathway is involved. In this study, we report the first metabolic studies on carbohydrate utilization, CO2 assimilation, and amino acid biosynthesis in the phototrophic Roseobacter clade bacterium Roseobacter denitrificans OCh114. We develop a new minimal medium containing defined carbon source(s), in which the requirements of yeast extract reported previously for the growth of R. denitrificans can be replaced by vitamin B12 (cyanocobalamin). Tracer experiments were carried out in R. denitrificans grown in a newly developed minimal medium containing isotopically labeled pyruvate, glucose or bicarbonate as a single carbon source or in combination. Through measurements of 13C-isotopomer labeling patterns in protein-derived amino acids, gene expression profiles, and enzymatic activity assays, we report that: (1) R. denitrificans uses the anaplerotic pathways mainly via the malic enzyme to fix 10–15% of protein carbon from CO2; (2) R. denitrificans employs the Entner-Doudoroff (ED) pathway for carbohydrate metabolism and the non-oxidative pentose phosphate pathway for the biosynthesis of histidine, ATP, and coenzymes; (3) the Embden-Meyerhof-Parnas (EMP, glycolysis) pathway is not active and the enzymatic activity of 6-phosphofructokinase (PFK) cannot be detected in R. denitrificans; and (4) isoleucine can be synthesized from both threonine-dependent (20% total flux) and citramalate-dependent (80% total flux) pathways using pyruvate as the sole carbon source

Native Electrospray and Electron-Capture Dissociation in FTICR Mass Spectrometry Provide Top-Down Sequencing of a Protein Component in an Intact Protein Assembly

The intact yeast alcohol dehydrogenase (ADH) tetramer of 147 kDa was introduced into a FTICR mass spectrometer by native electrospray. Electron capture dissociation of the entire 23+ to 27+ charge state distribution produced the expected charge-reduced ions and, more unexpectedly, 39 c-type peptide fragments that identified N-terminus acetylation and the first 55 amino acids. The results are in accord with the crystal structure of yeast ADH, which shows that the C-terminus is buried at the assembly interface, whereas the N-terminus is exposed, allowing ECD to occur. This remarkable observation shows promise that a top-down approach for intact protein assemblies will be effective for characterizing their components, inferring their interfaces, and obtaining both proteomics and structural biology information in one experiment

Dynamics of Gene Duplication in the Genomes of Chlorophyll d-Producing Cyanobacteria: Implications for the Ecological Niche

Gene duplication may be an important mechanism for the evolution of new functions and for the adaptive modulation of gene expression via dosage effects. Here, we analyzed the fate of gene duplicates for two strains of a novel group of cyanobacteria (genus Acaryochloris) that produces the far-red light absorbing chlorophyll d as its main photosynthetic pigment. The genomes of both strains contain an unusually high number of gene duplicates for bacteria. As has been observed for eukaryotic genomes, we find that the demography of gene duplicates can be well modeled by a birth–death process. Most duplicated Acaryochloris genes are of comparatively recent origin, are strain-specific, and tend to be located on different genetic elements. Analyses of selection on duplicates of different divergence classes suggest that a minority of paralogs exhibit near neutral evolutionary dynamics immediately following duplication but that most duplicate pairs (including those which have been retained for long periods) are under strong purifying selection against amino acid change. The likelihood of duplicate retention varied among gene functional classes, and the pronounced differences between strains in the pool of retained recent duplicates likely reflects differences in the nutrient status and other characteristics of their respective environments. We conclude that most duplicates are quickly purged from Acaryochloris genomes and that those which are retained likely make important contributions to organism ecology by conferring fitness benefits via gene dosage effects. The mechanism of enhanced duplication may involve homologous recombination between genetic elements mediated by paralogous copies of recA