CyanoNews (Vol. 4, No. 3, December 1988)

CyanoNews was a newsletter that served the cyanobacteriological community from 1985 to 2003, with content provided by readers (sort of a blog before there were blogs). The newsletter reported new findings from the lab, summaries of recent meetings (often provided by graduate students and post-docs entering the field), positions sought or available, life transitions, a compendium of recent cyanobacteria-related articles, and other items of interest to those who study cyanobacteria

An insight into the role of 9360FtsH protease in photoprotection in the cyanobacterium Synechococcus PCC7942

PhDThe multisubunit complex of photosystem-II, present in thylakoid membranes of all oxygen evolving photosynthetic organisms has some unique features. It drives one of the most thermodynamically demanding reactions, the oxidation of water. Yet it turns over more rapidly than any other protein complex of the photosynthetic apparatus and its D1 core protein, binding the majority of electron transport cofactors, is the most frequently damaged subunit of all. The repair mechanism operating to restore photosystem II to its functional state requires proteolytic activity to degrade the photoinactivated D1 subunit before replacing it with a newly synthesized copy. In our model organism, the cyanobacterium Synechococcus 7942, we explored the possibility that an FtsH protease (JGI ID 637799360) plays an important role in the early stages of the repair cycle by subjecting the gene encoding it to insertional mutagenesis. The phenotype of the resulted mutant was subsequently compared to that of the wild type, as both types of cells were simultaneously assessed using various biochemical and biophysical techniques. The assay produced results correlating well with the present knowledge about the role of the particular protease in other model organisms such as Synechocystis 6803 and Arabidopsis thaliana and thus substantiated the significance of the protease in the repair cycle of photosystem II and yet proposed an evolutionary conserved role among oxygenic phototrophs. We identified a possible role in the degradation of functional photosystem II under stress conditions and its dynamics within thylakoid membranes since absence of this protease profoundly affects the diffusion of the complex in the membranes. Besides, computational analysis of FtsH proteins, present as multigene families in all oxygenic phototrophs, brought forth the discovery of a unique domain present in cyanobacterial peptidases.

Alternative electron transfer routes involved in photoprotection of cyanobacteria

In oxygenic photosynthesis, the highly oxidizing reactions of water splitting produce reactive oxygen species (ROS) and other radicals that could damage the photosynthetic apparatus and affect cell viability. Under particular environmental conditions, more electrons are produced in water oxidation than can be harmlessly used by photochemical processes for the reduction of metabolic electron sinks. In these circumstances, the excess of electrons can be delivered, for instance, to O2, resulting in the production of ROS. To prevent detrimental reactions, a diversified assortment of photoprotection mechanisms has evolved in oxygenic photosynthetic organisms. In this thesis, I focus on the role of alternative electron transfer routes in photoprotection of the cyanobacterium Synechocystis sp. PCC 6803. Firstly, I discovered a novel subunit of the NDH-1 complex, NdhS, which is necessary for cyclic electron transfer around Photosystem I, and provides tolerance to high light intensities. Cyclic electron transfer is important in modulating the ATP/NADPH ratio under stressful environmental conditions. The NdhS subunit is conserved in many oxygenic phototrophs, such as cyanobacteria and higher plants. NdhS has been shown to link linear electron transfer to cyclic electron transfer by forming a bridge for electrons accumulating in the Ferredoxin pool to reach the NDH-1 complexes. Secondly, I thoroughly investigated the role of the entire flv4-2 operon in the photoprotection of Photosystem II under air level CO2 conditions and varying light intensities. The operon encodes three proteins: two flavodiiron proteins Flv2 and Flv4 and a small Sll0218 protein. Flv2 and Flv4 are involved in a novel electron transport pathway diverting electrons from the QB pocket of Photosystem II to electron acceptors, which still remain unknown. In my work, it is shown that the flv4-2 operon-encoded proteins safeguard Photosystem II activity by sequestering electrons and maintaining the oxidized state of the PQ pool. Further, Flv2/Flv4 was shown to boost Photosystem II activity by accelerating forward electron flow, triggered by an increased redox potential of QB. The Sll0218 protein was shown to be differentially regulated as compared to Flv2 and Flv4. Sll0218 appeared to be essential for Photosystem II accumulation and was assigned a stabilizing role for Photosystem II assembly/repair. It was also shown to be responsible for optimized light-harvesting. Thus, Sll0218 and Flv2/Flv4 cooperate to protect and enhance Photosystem II activity. Sll0218 ensures an increased number of active Photosystem II centers that efficiently capture light energy from antennae, whilst the Flv2/Flv4 heterodimer provides a higher electron sink availability, in turn, promoting a safer and enhanced activity of Photosystem II. This intertwined function was shown to result in lowered singlet oxygen production. The flv4-2 operon-encoded photoprotective mechanism disperses excess excitation pressure in a complimentary manner with the Orange Carotenoid Protein-mediated non-photochemical quenching. Bioinformatics analyses provided evidence for the loss of the flv4-2 operon in the genomes of cyanobacteria that have developed a stress inducible D1 form. However, the occurrence of various mechanisms, which dissipate excitation pressure at the acceptor side of Photosystem II was revealed in evolutionarily distant clades of organisms, i.e. cyanobacteria, algae and plants.Siirretty Doriast

Biochemical characterization of an intermediate membrane subfraction in cyanobacteria involved in an assembly network for photosystem II

Oxygenic photosynthesis converts light energy into chemical energy and is responsible for generating most of our atmosphere’s oxygen and biomass on earth. Several multimeric protein complexes are involved in the underlying photosynthetic electron transfer chain with photosystem II (PSII) representing the initial complex mediating the extraction of electrons from water molecules, thus generating molecular oxygen as a by-product. During recent years, the structural details and components of the PSII complex, including its inorganic and organic cofactors, have been elucidated in great detail. However, little is known about the assembly pathway of this at least 20 protein subunits containing machinery. Previous work indicated that PSII assembly occurs in a step-wise fashion and requires a number of facilitating factors, which interact transiently with nascent PSII complexes. Earlier studies of one of those assembly factors, the cyanobacterial PratA protein, suggested that PSII biogenesis does not only underlie a temporal order but is also organized at the spatial level, as PratA was shown to mark a special intermediate membrane subfraction (PDMs), hypothesized to represent regions for initial steps of PSII biogenesis. The presented work focused on a more detailed characterization of PDMs, clearly supporting their significance not only with regard to early protein assembly, but also concerning pigment synthesis and integration into the PSII precomplexes. The PDMs could further be allocated to special membrane regions, named biogenesis centers, at sites where thylakoid membranes converge to the plasma membrane, thus demonstrating the spatial organization of PSII assembly at the cellular level. Moreover, a novel function of PratA in preloading of PSII with Mn2+ ions, necessary for construction of the water-splitting complex, was discovered. Concomitantly with progression of the assembly, the nascent PSII complexes are transported from the PDMs to the thylakoid membrane system, where Sll0933 – a novel PSII assembly factor identified in this thesis – mediates the integration of the PSII inner antenna proteins followed by completion of the assembly process. Additionally, it could be shown that many of the so far identified facilitating factors interact with each other and, thus, form a complex network for PSII assembly. Especially the interaction between the two assembly factors YCF48 and Sll0933 was characterized in more detail, revealing a successive mode of action with YCF48 operating upstream of Sll0933. Taken together, the presented results enable the development of an extended and elaborated model of PSII assembly, which is a concerted process connecting protein and cofactor synthesis/integration in a spatiotemporal manner, and thus contribute to a more profound understanding of photosynthesis itself.Oxygene Photosynthese ermöglicht die Umwandlung von Lichtenergie in chemische Energie. Aufgrund des bei den zugrunde liegenden Reaktionen gebildeten molekularen Sauerstoffs (O2) stellt sie die Basis für den in unserer Atmosphäre angesammelten Sauerstoff und somit die Grundlage für höheres Leben auf der Erde dar. Die Entstehung von O2 wird von Photosystem II (PSII) katalysiert, dem ersten Komplex in einer Reihe von aus verschiedenen Bestandteilen aufgebauten Proteinkomplexen, welche den Transport von Elektronen zur Energiegewinnung vermitteln. Während die Zusammensetzung von PSII aus mindestens 20 verschiedenen Protein-Untereinheiten und diversen Co-Faktoren in den letzten Jahren weitgehend aufgeklärt wurde, ist das Wissen über den zugrunde liegenden Assemblierungsweg verhältnismäßig beschränkt. Bislang konnte gezeigt werden, dass dieser Ablauf schrittweise erfolgt und eine Reihe von Assemblierungsfaktoren erfordert. Dass die Biogenese von PSII neben ihrer zeitlichen Abfolge auch auf räumlicher Ebene organisiert ist, lässt sich aus früheren Studien eines dieser Assemblierungsfaktoren, des cyanobakteriellen PratA-Proteins schließen, welches eine spezielle Membranfraktion (PDMs) kennzeichnet, die offenbar an den frühen Schritten der PSII-Biogenese beteiligt ist. Die hier gezeigten Ergebnisse einer detaillierteren Charakterisierung der PDMs unterstreichen ihre Bedeutung nicht nur im Hinblick auf die frühen Schritte der Assemblierung der Proteinuntereinheiten, sondern auch auf die Pigmentsynthese und deren Integration in PSII-Präkomplexe. Strukturell konnten PDMs sogenannten Biogenesezentren zugeordnet werden, welche sich an Stellen, an denen sich die Thylakoidmembranen der Plasmamembran annähern, befinden. Somit wurde die Frage der subzellulären Lokalisierung der PSII-Assemblierung beantwortet. Außerdem konnte eine Beteiligung von PratA an der Beladung von PSII mit Mn2+-Ionen zum Aufbau des Wasserspaltungsapparates gezeigt werden. Weitere Daten weisen darauf hin, dass mit zunehmender Assemblierung ein Transport der entstehenden PSII-Komplexe von den PDMs in Richtung der Thylakoidmembranen erfolgt. Unmittelbar nach Erreichen der Thylakoide scheint dann Sll0933 – ein neuer, in dieser Arbeit identifizierter PSII-Assemblierungsfaktor – die Integration der inneren PSII-Antennenproteine zu vermitteln. Der Abschluss der Assemblierung von PSII erfolgt ebenfalls im Thylakoidmembransystem. Es wurden außerdem Hinweise dafür erhalten, dass sich viele der bisher identifizierten Assemblierungsfaktoren gegenseitig beeinflussen und somit ein komplexes Netzwerk bilden. Insbesondere die Interaktion zwischen den beiden Assemblierungsfaktoren YCF48 und Sll0933 wurde detaillierter untersucht und führte zur Schlussfolgerung, dass YCF48 an früheren Schritten als Sll0933 an der PSII-Assemblierung beteiligt ist. Alles in allem handelt es sich bei der Assemblierung von PSII also um einen Prozess mit einer auf räumlicher und zeitlicher Ebene organisierten Verknüpfung von Integration der Protein-Untereinheiten und Synthese/Insertion der Co-Faktoren. Die dargestellten Ergebnisse ermöglichen eine deutliche Ausweitung des bisher geltenden Modells der PSII-Assemblierung und tragen somit zu einem besseren Gesamtverständnis des Prozesses der oxygenen Photosynthese an sich bei

Investigating the Life-cycle of Photosystem II Using Mass Spectrometry

Photosystem II (PSII) is a protein complex found embedded in the thylakoid membranes of all organisms that perform oxygenic photosynthesis. PSII converts sunlight into chemical energy, filling our atmosphere with molecular oxygen in the process and supporting nearly all life on Earth. PSII undergoes frequent light-induced damage as an unavoidable result of the electron transfer reactions it catalyzes. When damaged, PSII is disassembled, repaired, and reassembled in an intricate, tightly regulated process. The structure and mechanism of function of active PSII are relatively well-understood, due to the available crystal structures of the active complex and many years of biochemical and biophysical investigation. However, many aspects of the broader PSII life-cycle are less clear. In this work, several structural aspects of the PSII life-cycle are investigated, with an approach that emphasizes mass spectrometry (MS)-based tools. The field of protein MS is developing rapidly, and, especially in the last several years, MS has become a key tool for addressing a variety of questions in the area of photosynthesis. Chapter 1 provides an in-depth review of the ways in which MS has been, and can be, applied to PSII life-cycle research. This chapter presents the relevant MS-based techniques, as well as the knowledge that has been gained about the PSII life-cycle through their application. The work in Chapter 2 used cross-linking and MS to identify the binding site of the Psb28 protein to PSII. Psb28 binds transiently to a PSII assembly intermediate complex, exerting a protective effect on this complex. However, since Psb28 dissociates before assembly is complete, it is not found in the crystal structure and its structural location within the complex has remained unknown. We used isotope-encoded chemical cross-linking followed by MS to identify the binding partners of Psb28 in the model cyanobacterium Synechocystis sp. PCC 6803, the organism used throughout this work. We identified three cross-links between Psb28 and the α- and β- subunits of cytochrome b559 (PsbE and PsbF), pinpointing the structural location of Psb28 on the cytosolic surface of PSII in close association with these subunits. Our results allow us to propose several mechanisms by which Psb28 could exert its protective effect. In Chapter 3, we used high-resolution tandem MS to identify oxidative modifications in PSII. We found that the total number of modified residues increased by over 50% following light incubation, with the D1 protein showing the most marked increase (3.3-fold) of the proteins we monitored. These results strongly support the idea that ROS are generated as a byproduct of PSII photochemistry and that they damage PSII subunits, especially D1, which has the fastest turnover rate of all the subunits. By mapping the modified residues onto the PSII crystal structure, we found that the lumen-side residues form two nearly continuous, roughly linear arms starting at the Mn4Ca cluster and radiating outward all the way to the surface of PSII. We propose that these two arms are oxygen/ROS exit channels that protect PSII by removing ROS from the complex after they are generated at the Mn4Ca cluster. It has long been believed that PSII must contain such channels, and this study provides the most complete and descriptive molecular-level evidence yet for their existence and location. Chapter 4 describes a study that used cross-linking and MS to identify the binding location of PsbQ on the lumenal surface of PSII. Though PsbQ is a necessary component of PSII complexes with highest oxygen-evolving activity, it is not found in the available cyanobacterial crystal structures. Our results show that PsbQ helps stabilize the PSII dimer, providing a structural basis to explain our biochemical data and previous findings. A novel PSII subcomplex with multiple copies of the PsbQ protein was also discovered, and its characterization is described in this chapter. Based on our results, we propose it is a late PSII assembly intermediate that stabilizes the active PSII dimer just before association of the other lumenal extrinsic proteins PsbU and PsbV

Localisation of key proteases involved in the assembly and repair of Photosystem II in cyanobacterium Synechocystis sp. PCC 6803.

PhDAll photosynthetic organisms use light as a source of energy, however prolonged excessively high light causes irreversible damage to the main photosynthetic complexes. In particular the D1 polypeptide of Photosystem II is susceptible to damage and must be degraded and replaced. While the concept of PSII repair has attracted intensive research, important details remain to be determined. The sub-cellular localisation of proteases involved in PSII repair and assembly is investigated here in the model cyanobacterium Synechocystis sp. PCC 6803, by employing fluorescent protein tagging and fluorescence imaging in vivo. Results show that all FtsH protease homologues in Synechocystis are localised to distinct regions of the plasma membrane (FtsH1) and thylakoids (FtsH2, FtsH3, FtsH4). Importantly, FtsH2, involved in PSII repair, remains within distinct thylakoid membrane zones when activated by high light, leading to the hypothesis of localised PSII repair centres in the thylakoid membranes. In order to assess composition of the FtsH2-defined membrane zones, a novel technique for isolating membrane sub-fractions by anti-GFP pulldowns was employed. Mass spectrometry identified potentially interacting and neighbouring proteins within the repair centres, whose content changes under different light exposure. Furthermore, observed changes in FtsH2 and FtsH4 distributions under iron and copper deprivation suggest functions in responses to other stress conditions. To find the locations of D1 synthesis during PSII repair and de novo assembly, the D1 C-terminal processing peptidase CtpA was similarly GFP-tagged and observed in vivo. Results suggest that D1 synthesis for PSII repair takes place in the thylakoid membranes, while D1 synthesis for de novo PSII biogenesis takes place in specialised regions at both edges of the thylakoid system, adjacent to the plasma membrane and protruding into the central cytoplasm. By localising crucial cellular enzymes in vivo, this study demonstrates functional compartmentalisation and membrane heterogeneity in a prokaryote.HARVEST Marie Curie ACTION

Organization, dynamics and adaptation of the photosynthetic machinery in cyanobacteria

Ye

Identifizierung und Charakterisierung nukleärer Mutanten mit defekter Photoakklimation in Arabidopsis thaliana

Borgstädt R. Identifizierung und Charakterisierung nukleärer Mutanten mit defekter Photoakklimation in Arabidopsis thaliana. Bielefeld (Germany): Bielefeld University; 2004.Die Organismen der oxygenen Photosynthese passen sich mit Photoakklimationsprozessen wechselnden Lichtqualitäten und -quantitäten an. Durch Analyse der Redox-Zustände der photosynthetischen Elektronentransportkette werden Sensor-/Regulator-Elemente aktiviert, welche die Photosynthese an die neuen Umweltbedingungen anpassen. In einem dieser Prozesse, der "State Transition", migrieren die mobilen LHCII-Proteine zwischen den Photosystemen und passen so die Lichtenergieversorgung den Umweltbedingungen (Bonaventura und Meyers 1969; Murata 1969) und den Bedürfnissen des Organismus an Reduktions-/Energie-Äquivalenten (Finazzi et al., 1999, 2000, 2001) an. Durch Reduktion des PQ-Pools wird eine Regulationskaskade ausgehend von dem Cytb6f-Komplex induziert. Diese phosphoryliert die mit dem PSII assoziierten mobilen LHCII-Proteine, die zu PSI migrieren (Kruse, 2001, Wollman, 2001, Allen, 2003). Der genaue Mechanismus dieser Signalkaskade ist noch unbekannt. Zur Analyse der Regulation des Prozesses der "State Transition" wurde ein "forward genetics"-Ansatz in Arabidopsis thaliana verwendet. Mit der Entwicklung eines "State Transition-Screening"-Systems wurden drei Mutanten aus 10.000 Arabidopsis thaliana-EMS und "Neutron bombardtment"-Linien mit einer defekten "State Transition" identifiziert. Eine dieser Mutanten, Stmu10, wurde eingehend genotypisch und phänotypisch charakterisiert. Durch die Sequenzierung des Arabidopsis thaliana-Genoms (The Arabidopsis Genome Initiative, 2001) konnte ein "mapping"-Ansatz zur Eingrenzung des Locus der Mutation verwendet werden. Die Deletion der Transkription des Gens At4G08870 in Stmu10 konnte mit den Methoden der RT-PCR und des Northern-Blot identifiziert werden. Bei dem Genprodukt handelt es sich um eine Arginase2, die im Mitochondrium lokalisiert ist und deren Deletion in Stmu10 die Reduktion der Arginase-Aktivität hervorruft. Durch Hemmung der Respiration kommt es in Stmu10 zur dauerhaften Reduktion des PQ-Pools und damit zu dem Verbleib in State2. Die Hemmung von Stoffwechselwegen durch die Deletion der Arginase2, sowie eine potentielle regulatorische Funktion in der NO-Signaltransduktion als Anpassung an wechselnde Umweltbedingungen wird diskutiert. Die Reduktion von Thiol-Resten der "State Transition"-induzierenden LHCII-Kinase durch das Thiordoxin-System hemmt bei Starklicht-Bedingungen die Phosphorylierung der mobilen LHCII-Proteine. Unter diesen Bedingungen wird der Prozess der Photoinhibition induziert. Bei einer vollständigen Reduktion des PQ-Pools wird eine Signalkaskade induziert, an deren Ende PSII-Proteine als Substrat phosphoryliert werden. Die Phosphorylierung induziert eine Stabilisierung des Dimeren-Zustandes des PSII und hemmt so den "PSII-Repair-Cycle". Dieser Mechanismus soll dadurch eine Schutzfunktion gegen ständige Neusynthese und Degradierung des D1-Proteins sein (Kruse, 2001, Aro und Ohad, 2003). Mit einem "reverse genetics"-Ansatz wurde das Arabidopsis thaliana-Genom auf potentielle Mitglieder photosynthetischer Signalkaskaden analysiert. Bei diesen Mitgliedern sollte es sich um chloroplastidäre Serin-Threonin-Membrankinasen handeln, die potentiell durch Phosphorylierung die Prozesse der "State Transition" oder der Photoinhibition induzieren. Durch "Screening" der Arabidopsis thaliana-Datenbank (TAIR, Garcia-Hernandez, 2002) konnten 12 potentielle chloroplastidäre Serin-Threonin-Membrankinasen identifiziert werden, von denen eine, mit der Tair-Nr. At3g24550, charakterisiert wurde. Von der als STK1 benannten Kinase wurden "in silico" T-DNA-Mutanten identifiziert, die phänotypisch charakterisiert wurden. Bei den Mutanten konnte eine Hemmung der D1/D2-Phosphorylierung und bei andauernden Starklicht-Bedingungen (3 Wochen 700µE) eine einsetzende Seneszenz detektiert werden. Die Kinase STK1 wurde in E. coli exprimiert und gegen Kaninchen immunisiert. Mit Immunodetektion durch das STK1-Antiserum wurden erste Lokalisierungsstudien durchgeführt. Dabei konnte durch vergleichende Maldi-Analysen eine potentiell chloroplastidäre Lokalisierung unterstützt werden. Über die Funktion der STK1 in dem Prozess der Photoinhibition wird diskutiert