Characterization of pigment-protein complexes in the cyanobacterium Anacystis Nidulans R2

Includes vita.Intrinsic membrane proteins associated with photosynthetic electron transfer have highly-conserved analogs in both cyanobacteria and chloroplasts. However, the protein complexes required for light-harvesting are quite different. Cyanobacteria contain extrinsic phycobilisomes, whereas chloroplasts have integral membrane light-harvesting Chl-protein complexes. We show that cyanobacteria are capable of synthesizing an intrinsic light-harvesting structure (termed CPVI-4) similar in many respects to that found in chloroplasts. In normally grown A. nidulans R2, the main light-harvesting structure is the extrinsic phyco- bilisome, whereas under iron stress, phycobilisome quantities decrease and the cells accumulate CPVI-4. CPVI-4 was biochemically purified and characterized with respect to its spectral and biochemical properties. Antisera were raised against an apoprotein of CPVI-4 (34 kD) as well as several other membrane components regulated by iron. The antibodies were used to follow the kinetics of the disappearance of each of these components following addition of iron to iron-depleted cultures. Phycobilisome linker and anchor polypeptides were found to be specifically glycosylated, and purified phyco- bilisomes were found to contain significant quantities of carbohydrates (particularly glucose). The glycosylated sub-units contained differential quantities of glucose and N acetylgalactosamine, and these sugars were present on regions of the linker proteins previously shown to be important for PBsome assembly. These findings have broad implications regarding both the function and assembly of phycobilisomes as well as the response of A. nidulans R2 to a variety of different stress conditions.Includes bibliographical references

Interactions Between Light and Production of Microcystins in the Toxic Cyanobacterium Microcystis

Cyanobacterial harmful algal blooms (cHABs) are characterized by the formation of toxins that can impact animal health, cause water quality issues, and recreational hazards. Microcystis, a common genus of cyanobacteria, produces the potent protein phosphatase inhibitor microcystins. Microcystins are nitrogen-rich and have an associated metabolic cost for production. Some outstanding questions in the study of cHABs is why are microcystins produced, what are the benefits of toxin formation, and why only some Microcystis strains produce microcystins? We examined a potential biochemical role for microcystins in the cyanobacterial photosystem regulation in response to various light conditions. Single-celled culture strains of toxic and non- toxic Microcystis aeruginosa were grown under different light irradiances. High-light conditions caused light stress based on decreased photosynthetic efficiency. Cells responded over 2-3 days by decreasing their chlorophyll and phycobilisome content per cell in unison. Looking at a natural system over a diurnal cycle of changing light intensities, Microcystis responded to high-light environments via vertical migration deeper in the water column to avoid light stress. In one culture and in situ, there were no changes in microcystins concentration per cell, but one strain showed decreased microcystins under low-light. Therefore, microcystins protein phosphatase activity may not be involved with phosphorylation/dephosphorylation under high-light. High-light environments are also associated reactive oxygen species (ROS) production. We investigated if microcystins were linked to ROS sensitivity by examining how three chlorophytes and seven cyanobacteria responded to the ROS compound hydrogen peroxide (H2O2). There was no evidence that toxic cyanobacteria were more sensitive to ROS than non-toxic cyanobacteria or chlorophytes. Addition of H2O2 did not change the microcystins concentration per cell. While these experiments did not elucidate the biochemical function of microcystins in Microcystis, they provided valuable information for water quality managers. cHAB monitoring programs must carefully consider vertical migration away from the surface during high-light conditions. The use of H2O2 as a control mechanism may not selectively remove toxic cyanobacteria. Despite visual similarities, Microcystis is composed of diverse species with a wide-range of responses to light and ROS. Care must be taken when applying conclusions using a limited number of strains to broader populations

CyanoNews (Vol. 8, No. 1, February 1992)

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

Characterisation and functional analysis of a lumenal proline isomerase from photosynthetic membranes of higher plants and cyanobacteria

TLP40 is the first complex immunophilin which was described from plant chloroplasts (Fulgosi et al., 1998). For this protein a role in regulation of PSII protein phosphorylation has been suggested (Fulgosi et al., 1998; Vener et al., 1999; Rokka et al., 2000). Homologous proteins were found in Arabidopsis thaliana and in Synechocystis. In Synechocystis, different from higher plants, the relevant PSII proteins are not phosphorylated. The main topic of this work was therefore to characterise the homologous protein of TLP40 in Synechocystis (named cTLP40, cyanobacterial TLP40). The investigation shows: • cTLP40 contains the major structural domains of TLP40 both at the N- and at the C-termini. A higher homology is found in the immunophilin domain located at the C-terminal end. • As in chloroplasts, cTLP40 is also located in the thylakoid lumen where it is present in free form or associated to the membrane. • A Synechocystis strain lacking cTLP40 ( ∆sll0408) could grow photoautotrophycally under normal grown conditions in a way comparable to wild-type. However, after adaptation to strong light the mutant strain showed higher photosensibility. Under these conditions, there was a decrease in oxygen evolution. • The total amount of PSII dimer was reduced in the mutant under high light. The lower amount of PSII can be attributed to a slower assembly rate of the complex and/or to higher degradation rate. Protein synthesis was not impaired under any of the tested conditions. • The PPIAse activity of cTLP40 was tested in vitro on synthetic prolinecontaining peptides of PSII proteins which are exposed to the lumenal face in thylakoids. The in vitro assays showed that cTLP40 possesses PPIAse activity on control peptides but can not efficiently isomerise the specific synthetic peptides

KEGG orthology-based annotation of the predicted proteome of Acropora digitifera: ZoophyteBase - an open access and searchable database of a coral genome

BACKGROUND: Contemporary coral reef research has firmly established that a genomic approach is urgently needed to better understand the effects of anthropogenic environmental stress and global climate change on coral holobiont interactions. Here we present KEGG orthology-based annotation of the complete genome sequence of the scleractinian coral Acropora digitifera and provide the first comprehensive view of the genome of a reef-building coral by applying advanced bioinformatics. DESCRIPTION: Sequences from the KEGG database of protein function were used to construct hidden Markov models. These models were used to search the predicted proteome of A. digitifera to establish complete genomic annotation. The annotated dataset is published in ZoophyteBase, an open access format with different options for searching the data. A particularly useful feature is the ability to use a Google-like search engine that links query words to protein attributes. We present features of the annotation that underpin the molecular structure of key processes of coral physiology that include (1) regulatory proteins of symbiosis, (2) planula and early developmental proteins, (3) neural messengers, receptors and sensory proteins, (4) calcification and Ca2+-signalling proteins, (5) plant-derived proteins, (6) proteins of nitrogen metabolism, (7) DNA repair proteins, (8) stress response proteins, (9) antioxidant and redox-protective proteins, (10) proteins of cellular apoptosis, (11) microbial symbioses and pathogenicity proteins, (12) proteins of viral pathogenicity, (13) toxins and venom, (14) proteins of the chemical defensome and (15) coral epigenetics. CONCLUSIONS: We advocate that providing annotation in an open-access searchable database available to the public domain will give an unprecedented foundation to interrogate the fundamental molecular structure and interactions of coral symbiosis and allow critical questions to be addressed at the genomic level based on combined aspects of evolutionary, developmental, metabolic, and environmental perspectives

Characterization of a gene encoding an RNA-binding protein (rbpA) in the cyanobacterium Synechococcus sp. PCC 7942

Many species of cyanobacteria possess genes whose products are highly similar to the RNP family of RNA-binding proteins found in eukaryotes. This work describes the characterization of rbpA, one of two RNA-binding protein {rbp) genes now known to exist in the unicellular cyanobacterium Synechococcus sp. PCC 7942. This gene codes for a protein of 107 amino acids. It contains a single RNA Recognition Motif (RRM) as well as an auxiliary domain rich in glycine residues. -- Mutation of the rbpA gene by insertional inactivation using the spectinomycin resistance omega cassette resulted in a temperature-sensitive phenotype with an altered pigment composition when compared with the wild type organism. This phenotype was not observed in a control mutant, in which the omega cassette was inserted outside of the rbpA gene. Complementation experiments demonstrated that it was possible to rescue the phenotype of the "knock-out" mutant by insertion of a wild type copy of the rbp A gene into a neutral site in the cyanobacterial genome. -- The function of cyanobacterial RNA-binding proteins is not known. A histidine-tagged form of RbpA (HeRbpA) was purified using metal chelate affinity chromatography. RNA binding experiments demonstrated that this protein showed a preference for poly(A), poly(G) and poly(U) RNA but not poly(C). This specificity did not appear to be significantly affected by removal of the auxiliary domain. Overall, work presented here suggests that the RbpA protein may affect content of the phycobilisome components in the photosynthetic apparatus. It also appears to be a protein which is required for growth at lower temperatures

Structural revelations of photosynthesis' membrane protein complexes

Photosynthetic organisms appeared early in evolution and their photosynthetic apparatus has evolved along. The first bacteria carried out only anoxygenic photosynthesis catalyzed by one type of reaction center, type I or II, which somehow came together in cyanobacteria, and evolved into photosystems I and II. This was an evolutionary step that enabled cyanobacteria to carry out oxygenic photosynthesis. The photosystems have the unique capacity to perform and fix energy in a process where water splitting and oxygen evolution takes place, providing planet Earth with an essential molecule for development of life, i.e. Oxygen. Throughout evolution, primordial organisms became more complex upon colonizing diverse environments resulting into the current day sophisticated systems. Nevertheless, the photosystems have preserved their vital mechanisms of sunlight conversion with PSI at almost 100% efficiency, and PSII’s unique water splitting property. Important about photosynthesis systems are the high-energy conversion efficiency and oxygen evolution besides hydrogen generation by some organisms like cyanobacteria. These features are precious global demands for efficient sun utilizing devices, environmental concerns and current economics of alternative energy source to fossil fuel depletion. The diversity of the photosynthesis proteins due to evolution upon adaptation and exploitability is intriguing for researchers from all fields of science to understand aspects of structural diversity, function and dynamics. This work is highly complementary and has been carried out in multidisciplinary collaborations to get more impact for understanding the photosynthesis systems that evolved early or later. The results of which can be integrated into applied technology.

Biochemische Charakterisierung von Photosystem-I-Komplexen aus Diatomeen

Photosystem (PS) I is a huge membrane protein complex which coordinates around 200 co-factors. Upon light excitation a charge separation at the PS I reaction centre is induced which leads to an electron transport across the thylakoid membrane and the generation of redox equivalents needed for several biochemical reactions, e.g. the synthesis of sugars. For higher plants and cyanobacteria the crystal structure of PS I complexes were resolved to resolutions of 4.4 Å and 2.5 Å. Furthermore, supramolecular structures of PS I of eukaryotic algae, mainly of the green line, were obtained recently. However, up to now, no structure of diatoms is available yet. Diatoms are key players in global primary production and derived from a secondary endosymbiosis event. Their chloroplasts are surrounded by four envelope membranes and their thylakoids are evenly arranged in bands of three, i.e. no separation in grana and stroma regions is apparent. In this thesis a protocol was developed to isolate a functional PS I complex of diatoms which can be used for structural analysis by transmissional electron microscopy (TEM). A photosystem I-fucoxanthin chlorophyll protein (PS I-FCP) complex was isolated from the pennate diatom Phaeodactylum tricornutum by ion exchange chromatography. Spectroscopic analysis proved that bound Fcp polypeptides function as a light-harvesting complex. An active light energy transfer from Fcp associated pigments, Chl c and fucoxanthin, towards the PS I core was proven by fluorescence spectroscopy. Oxidised minus reduced difference spectroscopy evidenced the activity of the PS I reaction centre P700 and yielded a chlorophyll a/P700 ratio of approximately 200:1. These data indicate that the isolated PS I-FCP complex exceeds the PS I cores from cyanobacteria and higher plants in the numbers of chlorophyll a molecules. Because of the strict conservation of PS I cores among organisms the additional 100 chlorophyll a molecules must either be coordinated by Fcps or function as linker molecules between the Fcp antenna and the PS I core as shown for the PS I-LHC I complex of higher plants. To tell something about the structural organisation, the PS I-FCP complex was compared with its cyanobacterial and higher plant counterparts. Whereas cyanobacterial PS I cores aggregate to trimers, usually without associated antennae, higher plant PS I is a monomer and binds additionally two LHC I heterodimers. BN-PAGE and gel filtration experiments showed that also diatoms contain PS I monomers associated with Fcps as light-harvesting antenna. First TEM studies evidenced these observations. Negatively stained PS I-FCP particles had an increased size compared to PS I cores of other organisms. No PS I trimers or higher oligomers have been found. The calculated diameter and shape of the particles correspond to PS I-LHC I particles obtained from green algae, which also comprise of a higher number of LHC I polypeptides compared to the higher plant x-ray structure. Additionally, the analysis of polypeptides indicates that the PS I associated Fcps differ from the free Fcp pool and also from Fcps of a PS II enriched fraction. The assumption that diatoms harbour just one Fcp antenna that serve both Photosystems equally seems to be wrong. To further study the association of Fcps with the two Photosystems, both complexes plus the free FCP complexes were isolated from the centric diatom Cyclotella meneghiniana. Because of the availability of antibodies directed against specific Fcp polypeptides of Cyclotella the PS I-FCP complex of Phaeodactylum could not be used. A trimeric FCP complex, FCPa, and a higher FCP oligomer, FCPb, have already been described for C. meneghiniana. The latter is assumed to be composed of only Fcp5, whereas the FCPa contains Fcp2 and Fcp6. Biochemical and spectroscopical evidences revealed a different subset of associated Fcp polypeptides within the isolated photosystem complexes. Whereas the PS II associated Fcp antenna resembles FCPa, at least three different Fcp polypeptides are associated with PS I. By re-solubilisation of the PS I complex and a further purification step Fcp polypeptides were partially removed from PS I and both fractions were analysed again by biochemical and spectroscopical means, as well as by HPLC. Thereby Fcp4 and a so far undescribed 17 kDa Fcp were found to be strongly coupled to PS I, whereas another Fcp, presumably Fcp5, is only loosely bound to the PS I core. Thus an association of FCPb and PS I is assumed.Bei Photosystem (PS) I handelt es sich um einen großen Membranproteinkomplex, welcher ca. 200 Cofaktoren koordiniert. Die Anregung mit Licht verursacht eine Ladungstrennung am Reaktionszentrum von PS I die zu einem Elektronentransport über die Thylakoidmembran führt und letztendlich Redoxäquivalente generiert. Die Struktur von PS I Komplexen höherer Pflanzen sowie Cyanobakterien wurden mit Auflösungen von 4.4 Å bzw. 2.5 Å durch Röntgenstrukturanalyse bestimmt. Bis heute liegen keinerlei Strukturen von photosynthetischen Membranproteinkomplexen aus Diatomeen vor. Diatomeen nehmen eine Schlüsselrolle in der globalen Primärproduktion ein und entstanden durch sekundäre Endosymbiose. Dabei wurden Verwandte der heutigen Rotalgen von einem unbekannten eukaryotischen Einzeller aufgenommen. Die Chloroplasten der Diatomeen sind infolgedessen von vier Membranen umschlossen. Ihre Thylakoide sind gleichmäßig in Form von Dreierbändern organisiert, d.h. es werden keine Grana- und Stromaregionen ausgebildet. Ziel dieser Arbeit war es, ein Protokoll zu entwickeln, welches die Isolierung eines funktionsfähigen PS I Komplexes aus Diatomeen ermöglicht und der für die Strukturanalyse mittels Transmissionselektronenmikroskopie (TEM) geeignet ist. Mittels Ionenaustauschchromatographie wurde ein Komplex aus PS I und Fucoxanthin-Chlorophyll bindenden Proteinen (PS I-FCP Komplex) aus der pennaten Diatomee Phaeodactylum tricornutum gereinigt. Spektroskopische Analysen wiesen auf die Funktion der Fcps als funktioneller Lichtsammelkomplex für PS I hin. Aktiver Energietransfer von Fcp assoziierten Pigmenten, wie Fucoxanthin und Chlorophyll c, zum PS I Reaktionszentrum konnte durch Fluoreszenzspektroskopie nachgewiesen werden. Weiterhin bestätigten differenzspektroskopische Messungen von oxidierten minus reduzierten Proben die Aktivität des P700 Reaktionszentrums. Zudem konnte ein Verhältnis von 200 Chlorophyll a Molekülen pro Reaktionszentrum ermittelt werden. Im Vergleich zu monomeren PS I-Core Komplexen aus Cyanobakterien oder höheren Pflanzen (etwa 100:1) deutet dieses Verhältnis auf eine höhere Anzahl von Chlorophyll a im PS I-FCP Komplex von Diatomeen hin. Da die Grundstruktur von PS I stark konserviert ist, werden die ca. 100 zusätzlichen Chlorophylle entweder von den gebundenen Fcps koordiniert oder sie fungieren als Verbindungschlorophylle zwischen der Fcp Antenne und dem PS I Core, ähnlich den Verbindungschlorophyllen zwischen dem LHC I und dem PS I-Core höherer Pflanzen. Um etwas über den strukturellen Aufbau des PS I-FCP Komplexes zu erfahren, fand ein Vergleich mit den entsprechenden Komplexen aus Cyanobakterien und höheren Pflanzen statt. Die Methoden der Wahl waren hierbei BN-PAGE sowie Gelfiltrationsexperimente. Während PS I in Cyanobakterien Trimere ausbildet, welche üblicherweise keine weiteren Lichtsammelkomplexe binden, liegt in höheren Pflanzen ein PS I Monomer assoziiert mit zwei Heterodimeren LHC I Komplexen vor. Die Vergleiche zeigten, dass PS I in Diatomeen ebenfalls als Monomer vorliegt und mit Fcp Lichtsammelkomplexen assoziiert ist. Erste TEM Studien bestätigten diese Beobachtungen. Negativ kontrastierte PS I-FCP Partikel deuteten darauf hin, daß PS I-Cores anderer Organismen eine geringere Größe aufweisen. Keine PS I Trimere oder gar höhere Oligomere wurden beobachtet. Der berechnete Durchmesser und die Form der PS I-FCP Partikel ähnelten PS I-LHC I Partikel aus Grünalgen. Letztere weisen im Vergleich zu der PS I-LHC I Struktur höherer Pflanzen ebenfalls eine höhere Anzahl an gebundenen LHC I Proteinen auf. Zusätzlich deutete die Untersuchung der Polypeptide darauf hin, daß sich die PS I assoziierten Fcps von ungebundenen Fcps sowie Fcps einer PS II angereicherten Fraktion unterscheiden. Aus diesem Grund erscheint die Annahme falsch, dass Diatomeen über eine einzige Fcp Antenne verfügen, die sowohl für PS I und PS II gleichermaßen die Funktion als Lichtsammelkomplex übernimmt. Um die Assoziation von Fcps mit den zwei Photosystemen zu untersuchen, wurden PS I- und PS II-Komplexe sowie ungebundene FCP Komplexe aus der zentrischen Diatomee Cyclotella meneghiniana isoliert. Aufgrund der Tatsache, daß spezifische Cyclotella Fcp Antikörper zur Verfügung standen, konnte der PS I-FCP Komplex aus P. tricornutum nicht verwendet werden. Trimere, sogenannte FCPa Komplexe sowie höhere FCPb Oligomere wurden für C. meneghiniana bereits beschrieben. Während der letztere vermutlich ausschließlich aus Fcp5 zusammengesetzt ist, besteht der FCPa Komplex zumindest aus Fcp2 und Fcp6 Untereinheiten. Spektroskopische und biochemische Nachweismethoden zeigten, daß unterschiedliche Fcp Polypeptide mit den zwei Photosystemen assoziiert sind. Die PS II assoziierten Fcps entsprechen den Eigenschaften und dem Aufbau des FCPa Komplexes, während PS I mindestens drei unterschiedliche Fcp Polypeptide bindet. Demnach scheinen Fcp4 sowie ein bislang unbekanntes 17 kDa Fcp Polypeptid relativ stark mit PS I gekoppelt zu sein

Investigation of RNA degradation in the cyanobacterium Synechocystis sp. PCC6803

Cyanobacteria occupy very diverse habitats with rapidly changing environmental conditions, which forces them to develop effective response mechanisms in order to survive. Post-transcriptional control of gene expression, which is mostly determined by the function of regulatory RNA molecules and the RNA degradation apparatus, provides an important mechanism for adaptation to environmental demands. Investigation of major players in RNA degradation and maturation in the model cyanobacterium Synechocystis sp. PCC6803, namely homologs of RNase E/G (Rne) and RNase III (Rnc2), was the main focus of the present work. As RNA chaperone Hfq, which facilitates otherwise imperfect sRNA-­mRNA base pairing, functions as a post-­transcriptional regulator of gene expression in many bacteria, we also studied two Hfq-­‐dependent sRNAs Hpr8 and Hpr10 with a closer look on their degradation patterns. In order to clarify protein-­RNA interactions between studied RNases and their possible RNA targets in vivo a genome wide analysis of binding sites for Rne and Rnc2 was performed using individual-­nucleotide resolution crosslinking and immunoprecipitation (iCLIP) combined with Solexa high-throughput sequencing. This novel approach confirmed that Rne binds to the stem loop structure in the 5’ UTR of rne gene and therefore most likely regulates its own synthesis in a similar manner as it has been shown for E. coli. Discovery of Rne binding sites within the rRNA precursor between 23S and 5S rRNAs led to the assumption that the maturation of 5S rRNA in Synechocystis is analogous to the one in E. coli. Conducted in vitro cleavage assays and a 3’ RACE experiment substantiated this hypothesis and proved the accuracy of results provided by iCLIP method. We also revealed interaction of Rne with a number of sRNAs. In vitro cleavage assays were performed to verify Rne-dependent processing of some of the putative targets. Interestingly, we could see a clear pattern in Rne interaction with tRNAs: analysis of the location of the binding site determined that Rne always binds to the anticodon loop of tRNAs; an additional binding site at the variable loop of some tRNAs was also discovered. Evaluation of Rnc2 binding properties was completed by implementing iCLIP approach as well. Detection of Rnc2 binding sites within rRNAs and tRNAs suggested involvement of this RNase in maturation of their precursors in Synechocystis as it has been shown for other bacteria. We could also observe that the two studied RNases Rne and Rnc2 in some cases have binding sites mapped to the same transcripts and therefore might act together. In addition we could demonstrate using in vitro cleavage assays that the sRNA Hpr10 is a true substrate for Rnc2. iCLIP experiment revealed a binding site next to a long double-stranded region within this sRNA, where processing most likely occurs. In summary, we could show that the iCLIP method can be used for the study of RNase-­RNA interactions in bacteria. Verification of iCLIP data using in vitro assays confirmed that several RNAs are true targets of the respective RNases. Clearly, more comprehensive studies are needed in the future to analyse the specific functions of these ribonucleases in post-­‐transcriptional gene regulation.Cyanobakterien besiedeln sehr vielfältige Habitate, in denen sich Umweltbedingungen sehr schnell ändern können. Dadurch sind Cyanobakterien gezwungen effektive Mechanismen zu entwickeln um sich an die jeweiligen Bedingungen anzupassen. Die posttranskriptionale Regulation der Genexpression, welche überwiegend durch kleine regulatorische RNAs und RNA-­Abbau bestimmt wird, stellt einen Mechanismus für die Anpassung an umweltbedingte Veränderungen dar. Die Untersuchung der wesentlichen Enzyme beim RNA-­Abbau und der RNA-­Reifung im Modelcyanobakterium Synechocystis sp. PCC6803, Homologe von RNase E/G (Rne) und RNase III (Rnc2), stellt den Kern dieser Arbeit dar. Da in vielen Bakterien das RNA-­Chaperon Hfq eine wichtige Rolle für die posttranskriptionale Regulation der Genexpression durch kleine nicht-­kodierende RNAs hat, wurden in dieser Arbeit auch die zwei Hfq-­abhängigen sRNAs, Hpr8 and Hpr10, vor allem bezüglich ihres Degradationsmuster näher untersucht. Für die Darstellung der RNA-­Proteininteraktionen zwischen untersuchten RNasen und deren möglichen RNA-­Zielen wurde eine genomweite Analyse der Bindungsstellen von Rne und Rnc2 in vivo -­ unter Verwendung der Methode der Individual Nucleotide Resolution Crosslinking und Immunoprecipitation (iCLIP), kombiniert mit Solexa-­High-­Throughput-Sequenzierung-­ durchgeführt. Dieser neuartige Untersuchungsansatz bestätigte, dass Rne an eine Stem-­Loop-­Struktur der 5’ UTR der rne mRNA bindet und daher sehr wahrscheinlich die eigene Synthese in einer ähnlichen Weise, wie auch bei E. coli bekannt, reguliert. Die Entdeckung von Rne-Bindungsstellen in rRNA-­Vorstufen zwischen den 23S und 5S rRNAs führte zur Annahme, dass die Reifung der 5S rRNA in Synechocystis analog zu E. coli ist. Die durchgeführten in vitro Untersuchungen zur Prozessierung der rRNA und ein 3’-RACE-Experiment bestätigten die vorgenannte Hypothese und die Genauigkeit der Ergebnisse, welche durch die iCLIP-­Methode erlangt wurden. Zudem wurde eine potenzielle Interaktion zwischen Rne und einigen sRNAs identifiziert und durch in vitro Untersuchungen belegt. Interessanterweise wurde ein deutliches Muster in potenziellen Rne-­Interaktionen mit tRNAs deutlich: Die Analyse offenbarte, dass Rne an die Antikodon-Schleife verschiedener tRNAs bindet; eine zusätzliche Bindungsstelle an der variablen Schleife einiger tRNAs wurde ebenfalls postuliert. Die iCLIP-­Methode wurde auch für die Identifizierung von Rnc2-­RNA-­Bindestellen verwendet. Die detektierten Rnc2-Bindungsstellen in rRNAs und tRNAs legen die Beteiligung der RNase III an der Reifung dieser Produkte in Synechocystis, wie dies bereits für andere Bakterien bekannt ist, nahe. In dieser Arbeit wird auch ersichtlich, dass die RNasen Rne und Rnc2 teilweise an die gleichen Transkripte binden und daher sehr wahrscheinlich gemeinsam an der Prozessierung verschiedener RNAs beteiligt sind. Zusätzlich wurde durch in vitro-­RNA-­Spaltung verifiziert, dass die sRNA Hpr10 ein Substrat für Rnc2 darstellt. Die iCLIP-­Untersuchungen haben gezeigt, dass eine RNase-­Bindungsstelle neben einer langen doppelsträngigen Region in der sRNA besteht, dort, wo die Prozessierung sehr wahrscheinlich stattfindet. Zusammenfassend lässt sich festhalten, dass die iCLIP-­Methode erfolgreich für die Untersuchung von RNase-­RNA Interaktionen in Bakterien verwendet werden kann. Die Verifizierung von iCLIP-­Daten unter Verwendung der in vitro-­Spaltungsuntersuchungen hat bestätigt, dass einige RNAs echte Ziele der untersuchten RNasen sind. Sicherlich sind zukünftig noch weitere umfassende Analysen erforderlich, um die spezifischen Funktionen der hier untersuchten Ribonukleasen in der post-­transkriptionalen Genregulation besser zu verstehen