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    Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b6f Complex in Thermosynechococcus elongatus.
    Rexroth S, Rexroth D, Veit S, Plohnke N, Cormann KU, Nowaczyk MM, Rögner M. Plant Cell 2014.
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      Abstract The cyanobacterial cytochrome b6f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b6f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ?petP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700+ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b6f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ?petP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b6f complexes with different functions that might be correlated with supercomplex formation.

     

  • Proteomic analysis of mitochondria from senescent Podospora anserina casts new light on ROS dependent aging mechanisms.
    Plohnke N, Hamann A, Poetsch A, Osiewacz HD, Rögner M, Rexroth S. Exp Gerontol. 2014;56:13-25.
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      Abstract The mitochondrial free radical theory of aging (MFRTA) states that reactive oxygen species (ROS) generated at the respiratory electron transport chain are active in causing age-related damage of biomolecules like lipids, nucleic acids and proteins. Accumulation of this kind of damage results in functional impairments, aging and death of biological systems. Here we report data of an analysis to monitor the age-related quantitative protein composition of the mitochondria of the fungal aging model Podospora anserina. The impact of senescence on mitochondrial protein composition was analyzed by LC-MS. In an untargeted proteomic approach, we identified 795 proteins in samples from juvenile and senescent wild-type cultures and obtained quantitative information for 226 of these proteins by spectral counting. Despite the broad coverage of the proteome, no substantial changes in known age-related pathways could be observed. For a more detailed analysis, a targeted proteome analysis was applied focusing on 15 proteins from respiratory, ROS-scavenging and quality control pathways. Analyzing six distinct age-stages from juvenile to senescent P. anserina cultures revealed low, but statistically significant changes for the mitochondrial respiratory complexes. A P. anserina PaSod3 over-expression mutant with a phenotype of mitochondrial ROS over-production was used for biological evaluation of changes observed during aging. LC-MS analysis of the mutant revealed severe changes to the mitochondrial proteome - substantially larger than observed during senescence. Interestingly the amount of ATP synthase subunit g, involved in cristae formation is significantly decreased in the mutant implicating ROS-induced impairments in ATP synthase dimer and cristae formation. The difference between protein-profiles of aging wild type and ROS stressed mutant suggests that oxidative stress within the mitochondria is not the dominating mechanism for the aging process in P. anserina. Collectively, while our data do not exclude an effect of ROS on specific proteins and in signaling and control of pathways which are governing aging of P. anserina, it contradicts increasing ROS as a cause of a gross general and non-selective accumulation of damaged proteins during senescence. Instead, ROS may be effective by controlling specific regulators of mitochondrial function.

     

  • Rational design of cyanobacteria for hydrogen production.
    Rexroth S. EASAC-Workshop: Renewables – systems and storage, 19-20 September 2013
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      Abstract The solar-driven hydrogen production has tremendous potential as renewable and carbon-neutral energy source, since the substrate, water, and the energy source, sunlight, are virtually unlimited. Cyanobacteria, which perform oxygenic photosynthesis, can under certain conditions produce hydrogen using electrons extracted from water. Our goal is to improve the efficiency of hydrogen generation at the expense of biomass production. An important part is the efficient coupling of the linear photosynthetic electron transport from water to an imported, engineered hydrogenase. For this coupling the photosynthetic electron metabolism has to be engineered in many individual steps towards this goal. The result of each single engineering step (such as antenna size reduction, partial uncoupling of the thylakoid membrane, re-routing of electrons at the photosystem 1 acceptor site) has to be monitored by both functional as well as metabolic characterization on the whole cell level (for instance by an in depth quantitative proteome, lipidome- and metabolome analysis). Engineering of ferredoxin-dependent pathways – especially FNR-dependent steps – is a decisive step for re-routing electrons from water for hydrogen production instead of CO2-fixation. Studies of protein-protein-interactions in isolated model systems, as the affinity of ferredoxin for FNR and hydrogenase, are an important guide for the rational design of these regulatory elements. Optimization of photobioreactor systems and improved fermentation conditions are integral parts of the process design. Optimal culture conditions can be found and kept constant for several months by using continuous cultivation techniques which allow the systematic optimization of each individual parameter. Provided such systems are optimized both on the individual cell level and on the systems level, a more than 100-fold increase of hydrogen production in comparison with the most productive natural systems existing to date can be estimated, which would be a promising basis for an economically competitive H2 production.

     

  • Reduced light-harvesting antenna: Consequences on cyanobacterial metabolism and photosynthetic productivity.
    Kwon JH, Bernát G, Wagner H, Rögner M, Rexroth S. Algae Res. 2013;2:188-95.
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      Abstract Cyanobacteria are potential candidates for future photobiological hydrogen production. For this purpose, optimization of cyanobacterial metabolism and up-regulation of the linear electron flow are mandatory. One strategy to achieve this goal is the reduction of the photosynthetic antenna size. Here, we characterize the photosynthetic performance of two Synechocystis PCC 6803 antenna mutants in respect to culture density and light intensity under well-defined - continuous - cultivation conditions. The metabolic state of the mutants is defined by spectroscopic investigations and an in-depth proteomic analysis.Our results show that both biotic (i.e. balanced photosystem 2 to photosystem 1 ratios, light tolerance) and abiotic parameters (i.e. light intensity, cell density) are important for the optimization of photosynthetic efficiency which, in turn, is a prerequisite for high-yield photobiological hydrogen production. While a complete loss of light-harvesting antenna – as observed in the PAL mutant – has a significant negative impact on robustness and fitness, the Olive mutant lacking only the phycocyanin subunits reaches higher cell densities in our photobioreactor setup. This results in higher time-space-yields. For this reason the Olive mutant is a promising candidate for the design of future hydrogen production.

     

  • Light-induced oxidative stress, N-formylkynurenine, and oxygenic photosynthesis.
    Dreaden Kasson TM, Rexroth S, Barry BA. PLoS One. 2012;7:e42220.
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      Abstract Light stress in plants results in damage to the water oxidizing reaction center, photosystem II (PSII). Redox signaling, through oxidative modification of amino acid side chains, has been proposed to participate in this process, but the oxidative signals have not yet been identified. Previously, we described an oxidative modification, N-formylkynurenine (NFK), of W365 in the CP43 subunit. The yield of this modification increases under light stress conditions, in parallel with the decrease in oxygen evolving activity. In this work, we show that this modification, NFK365-CP43, is present in thylakoid membranes and may be formed by reactive oxygen species produced at the Mn(4)CaO(5) cluster in the oxygen-evolving complex. NFK accumulation correlates with the extent of photoinhibition in PSII and thylakoid membranes. A modest increase in ionic strength inhibits NFK365-CP43 formation, and leads to accumulation of a new, light-induced NFK modification (NFK317) in the D1 polypeptide. Western analysis shows that D1 degradation and oligomerization occur under both sets of conditions. The NFK modifications in CP43 and D1 are found 17 and 14 Angstrom from the Mn(4)CaO(5) cluster, respectively. Based on these results, we propose that NFK is an oxidative modification that signals for damage and repair in PSII. The data suggest a two pathway model for light stress responses. These pathways involve differential, specific, oxidative modification of the CP43 or D1 polypeptides.

     

  • Direct approach for bioprocess optimization in a continuous flat-bed photobioreactor system.
    Kwon JH, Rögner M, Rexroth S. J Biotechnol. 2012 Nov 30;162:156-62.
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      Abstract Application of photosynthetic micro-organisms, such as cyanobacteria and green algae, for the carbon neutral energy production raises the need for cost-efficient photobiological processes. Optimization of these processes requires permanent control of many independent and mutably dependent parameters, for which a continuous cultivation approach has significant advantages. As central factors like the cell density can be kept constant by turbidostatic control, light intensity and iron content with its strong impact on productivity can be optimized. Both are key parameters due to their strong dependence on photosynthetic activity. Here we introduce an engineered low-cost 5 L flat-plate photobioreactor in combination with a simple and efficient optimization procedure for continuous photo-cultivation of microalgae. Based on direct determination of the growth rate at constant cell densities and the continuous measurement of O? evolution, stress conditions and their effect on the photosynthetic productivity can be directly observed.

     

  • Unique properties vs. common themes: the atypical cyanobacterium Gloeobacter violaceus PCC 7421 is capable of state transitions and blue-light-induced fluorescence quenching.
    Bernát G, Schreiber U, Sendtko E, Stadnichuk IN, Rexroth S, Rögner M, Koenig F. Plant Cell Physiol. 2012;53:528-42.
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      Abstract The atypical unicellular cyanobacterium Gloeobacter violaceus PCC 7421, which diverged very early during the evolution of cyanobacteria, can be regarded as a key organism for understanding many structural, functional, regulatory and evolutionary aspects of oxygenic photosynthesis. In the present work, the performance of two basic photosynthetic adaptation/protection mechanisms, common to all other oxygenic photoautrophs, had been challenged in this ancient cyanobacterium which lacks thylakoid membranes: state transitions and non-photochemical fluorescence quenching. Both low temperature fluorescence spectra and room temperature fluorescence transients show that G. violaceus is capable of performing state transitions similar to evolutionarily more recent cyanobacteria, being in state 2 in darkness and in state 1 upon illumination by weak blue or far-red light. Compared with state 2, variable fluorescence yield in state 1 is strongly enhanced (almost 80%), while the functional absorption cross-section of PSII is only increased by 8%. In contrast to weak blue light, which enhances fluorescence yield via state 1 formation, strong blue light reversibly quenches Chl fluorescence in G. violaceus. This strongly suggests regulated heat dissipation which is triggered by the orange carotenoid protein whose presence was directly proven by immunoblotting and mass spectrometry in this primordial cyanobacterium. The results are discussed in the framework of cyanobacterial evolution.

     

  • Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress.
    Tamary E, Kiss V, Nevo R, Adam Z, Bernát G, Rexroth S, Rögner M, Reich Z. Biochim Biophys Acta. 2012;1817:319-27.
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      Abstract Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.

     

  • Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase.
    Rexroth S, Poetsch A, Rögner M, Hamann A, Werner A, Osiewacz HD, Schäfer ER, Seelert H, Dencher NA. Biochim. Biophys. Acta. 2012;1817:381-7.
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      Abstract The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimer's and Parkinson's Dementia. In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.

     

  • The plasma membrane of the cyanobacterium Gloeobacter violaceus contains segregated bioenergetic domains.
    Rexroth S, Mullineaux CW, Ellinger D, Sendtko E, Rögner M, Koenig F. Plant Cell. 2011;23:2379-90.
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      Abstract The light reactions of oxygenic photosynthesis almost invariably take place in the thylakoid membranes, a highly specialized internal membrane system located in the stroma of chloroplasts and the cytoplasm of cyanobacteria. The only known exception is the primordial cyanobacterium Gloeobacter violaceus, which evolved before the appearance of thylakoids and harbors the photosynthetic complexes in the plasma membrane. Thus, studies on G. violaceus not only shed light on the evolutionary origin and the functional advantages of thylakoid membranes but also might include insights regarding thylakoid formation during chloroplast differentiation. Based on biochemical isolation and direct in vivo characterization, we report here structural and functional domains in the cytoplasmic membrane of a cyanobacterium. Although G. violaceus has no internal membranes, it does have localized domains with apparently specialized functions in its plasma membrane, in which both the photosynthetic and the respiratory complexes are concentrated. These bioenergetic domains can be visualized by confocal microscopy, and they can be isolated by a simple procedure. Proteomic analysis of these domains indicates their physiological function and suggests a protein sorting mechanism via interaction with membrane-intrinsic terpenoids. Based on these results, we propose specialized domains in the plasma membrane as evolutionary precursors of thylakoids.

     

  • Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803.
    Schultze M, Forberich B, Rexroth S, Dyczmons NG, Rögner M, Appel J. Biochim Biophys Acta. 2009;1787:1479-85.
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      Abstract The light reactions of oxygenic photosynthesis almost invariably take place in the thylakoid membranes, a highly specialized internal membrane system located in the stroma of chloroplasts and the cytoplasm of cyanobacteria. The only known exception is the primordial cyanobacterium Gloeobacter violaceus, which evolved before the appearance of thylakoids and harbors the photosynthetic complexes in the plasma membrane. Thus, studies on G. violaceus not only shed light on the evolutionary origin and the functional advantages of thylakoid membranes but also might include insights regarding thylakoid formation during chloroplast differentiation. Based on biochemical isolation and direct in vivo characterization, we report here structural and functional domains in the cytoplasmic membrane of a cyanobacterium. Although G. violaceus has no internal membranes, it does have localized domains with apparently specialized functions in its plasma membrane, in which both the photosynthetic and the respiratory complexes are concentrated. These bioenergetic domains can be visualized by confocal microscopy, and they can be isolated by a simple procedure. Proteomic analysis of these domains indicates their physiological function and suggests a protein sorting mechanism via interaction with membrane-intrinsic terpenoids. Based on these results, we propose specialized domains in the plasma membrane as evolutionary precursors of thylakoids.

     

  • Segregated bioenergetic domains in the plasma membrane of Gloeobacter violaceus.
    Rexroth S. Gordon Research Conference, Photosynthesis, July 2009
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      Abstract The unusual cyanobacterium Gloeobacter violaceus lacking the system of internal thylakoid membranes found in all other known cyanobacteria was subjected to biochemical membrane fractionation, as well as in vivo imaging using confocal microscopy. With both approaches, the plasma membrane could be shown to be segregated in two distinct domains. Applying a crude membrane fractioning procedure consisting of French press treatment and sucrose gradient centrifugation, two membrane fractions could be separated out with green and orange colour, respectively. These two membrane fractions were subjected to analysis on proteome, pigment and lipid level and were shown to display properties typical of the thylakoid and plasma membrane fractions of normal cyanobacteria. Applying confocal microscopy, the segregation into two membrane phases was observed in living cells as patches displaying vast differences in chlorophyll autofluorescence intensities. Applying proteome analyses to the two membrane fractions in a LC/MS-approach, lead to the identification of 136 proteins with a proportion of 44 % membrane integral proteins. For both fractions display a distinct protein composition was detected with 80 and 29 proteins exclusively found in the green and orange membrane fractions, respectively. The population of protein found in the two fractions can be distinguished based on their distribution of isoelectrical points, which might hint towards a mechanism for the membrane domain formation. The green fraction contains a high chlorophyll to carotenoid ratio and the vast majority of bio-energetically active proteins, as it is usually observed for thylakoid membranes of typical cyanobacteria; the orange fraction might play a role in biogenesis of membrane protein complexes or in the assembly of cell wall components.

     



 

Kontakt

Dr. Sascha Rexroth



Ruhr-Universität Bochum
LS Biochemie der Pflanzen
Universitätsstraße 150
44780 Bochum

Raum: ND 3/133
Tel.: +49 234 32-29896
Fax : +49 234 32-14322
Sascha.Rexroth@rub.de