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Clear differences in metabolic and morphological adaptations of akinetes of two Nostocales living in different habitats
No full textAkinetes are resting spore-like cells formed by some heterocyst-forming filamentous cyanobacteria for surviving long periods of unfavourable conditions. We studied the development of akinetes in two model strains of cyanobacterial cell differentiation, the planktonic freshwater Anabaena variabilis ATCC 29413 and the terrestrial or symbiotic Nostoc punctiforme ATCC 29133, in response to low light and phosphate starvation. The best trigger of akinete differentiation of Anabaena variabilis was low light; that of N. punctiforme was phosphate starvation. Light and electron microscopy revealed that akinetes of both species differed from vegetative cells by their larger size, different cell morphology and large number of intracellular granules. Anabaena variabilis akinetes had a multilayer envelope; those of N. punctiforme had a simpler envelope. During akinete development of Anabaena variabilis, the amount of the storage compounds cyanophycin and glycogen increased transiently, whereas in N. punctiforme, cyanophycin and lipid droplets increased transiently. Photosynthesis and respiration decreased during akinete differentiation in both species, and remained at a low level in mature akinetes. The clear differences in the metabolic and morphological adaptations of akinetes of the two species could be related to their different lifestyles. The results pave the way for genetic and functional studies of akinete differentiation in these species.Fil: Perez, Rebeca. University of Tübingen; AlemaniaFil: Forchhammer, Karl. University of Tübingen; AlemaniaFil: Salerno, Graciela Lidia. Fundación para Investigaciones Biológicas Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; ArgentinaFil: Maldener, Iris. University of Tübingen; Alemani
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Interactions between the PII protein and its receptors revealed by NanoBiT technology
No full textThe PII proteins are notable members of a vast and ancient protein family involved in signal transduction. These
molecules are found in all living organisms and are primarily recognized for their ability to sense metabolites such
as ATP, ADP, and 2-oxoglutarate (2-OG). When the effector molecules are non-covalently bound by PII, they
cause several structural changes in PII proteins, particularly in their flexible T-loops, which serve as dynamic
modules for protein-protein interactions. The interpretation of metabolic data sent by PII is dependent on the
binding state of metabolites and the resulting conformation of PII receptors. To thoroughly investigate the complex
interactions between PII and target proteins, analytical methods that maintain the natural cellular milieu are
needed.
In light of the limitations inherent in alternative methodologies such as immobilization on sensor surfaces in
Surface-Plasmon-Resonance (SPR) and Biolayer Interferometry (BLI), as well as the reliance on sizable
fluorescence proteins in Förster Resonance Energy Transfer (FRET), our research endeavors focused on the
development of an innovative NanoBiT sensor. The focus of this sensor is on the interaction of the PII protein
derived from Synechocystis sp. PCC6803 with the PII-interacting protein X (PipX), N-acetyl-L glutamate kinase
(NAGK) and the PII-interacting regulator of arginine synthesis (PirA). Using the NanoBiT technology, we have
attained an advanced comprehension, enabling the calculation of KD values for the PII-NAGK and PII-PipX
complexes, which have not been previously reported. The test also demonstrated an increased level of sensitivity,
allowing for the detection of low-affinity interactions, such as the one seen between the PII-S49E variant and
NAGK. The study also highlights astounding proof indicating that the development of the PII-NAGK complex is
impacted by the presence of ADP, which reduces the complex affinity. Additional analysis by the NanoBiT
method and enzymatic assays provided further evidence that the PII-NAGK complex exhibits specific feed forward activation in response to increasing concentrations of NAG. These two sensors were also applied to
investigate the real time metabolic fluctuations in response to nitrogen upshift or nitrogen depletion treatments.
Furthermore, our exploration extended to a small protein encoded by the ssr0692 gene in Synechocystis sp. PCC
6803. The protein regulates the flux into the ornithine-ammonia cycle (OAC), a pivotal mechanism for the
accumulation and redistribution of nitrogen in cyanobacteria. The regulation described in this context arises from
the connection between the PII protein and the OAC cycle. PII traditionally regulates the key enzyme NAGK,
which catalyzes arginine production. The Ssr0692 protein competes with NAGK for PII binding, resulting in the
inhibition of NAGK activation and a consequent reduction in arginine synthesis. In light of its function, we have
identified it as the PII Interacting Regulator of Arginine Synthesis (PirA). The interaction between PirA and PII
depends on the presence of ADP and is hindered by mutations in PII that affect the structure of the T-loop.
Therefore, we propose that PirA serves as a crucial mediator, directing flux into nitrogen storage compounds by
considering both the availability of nitrogen and the energy level of the cell
Interaction of the Membrane-bound GlnK-AmtB Complex with the Master Regulator of Nitrogen Metabolism TnrA inBacillus subtilis
No full textP-II proteins are widespread and highly conserved signal transduction proteins occurring in bacteria, Archaea, and plants and play pivotal roles in controlling nitrogen assimilatory metabolism. This study reports on biochemical properties of the P-II-homologue GlnK (originally termed NrgB) in Bacillus subtilis (BsGlnK). Like other P-II proteins, the native BsGlnK protein has a trimeric structure and readily binds ATP in the absence of divalent cations, whereas 2-oxoglutarate is only weakly bound. In contrast to other P-II-like proteins, Mg2+ severely affects its ATP-binding properties. BsGlnK forms a tight complex with the membrane-bound ammonium transporter AmtB (NrgA), from which it can be relieved by millimolar concentrations of ATP. Immunoprecipitation and co-localization experiments identified a novel interaction between the BsGlnK-AmtB complex and the major transcription factor of nitrogen metabolism, TnrA. In vitro in the absence of ATP, TnrA is completely tethered to membrane (AmtB)-bound GlnK, whereas in extracts from BsGlnK- or AmtB-deficient cells, TnrA is entirely soluble. The presence of 4 mM ATP leads to concomitant solubilization of BsGlnK and TnrA. This ATP-dependent membrane re-localization of TnrA by BsGlnK/AmtB may present a novel mechanism to control the global nitrogen-responsive transcription regulator TnrA in B. subtilis under certain physiological conditions
Analyse der Funktion des Sll0783 Proteins in der PHB Bildung von Synechocystis PCC 6803: die entscheidene Rolle von NADPH im Stickstoffmangel
No full textNitrogen frequently is a limiting nutrient in natural habitats. Therefore, cyanobacteria as well as other autotrophic organisms have developed multiple strategies to adapt to nitrogen deficiency. Transcriptomic analyses of the strain Synechocystis PCC 6803 under nitrogen-deficient conditions revealed a highly induced gene (sll0783 ), which is annotated as conserved protein with unknown function. This gene is part of a cluster with seven genes and in the upstream region lies a predicted NtcA-binding site. Homologues of this cluster occur in some unicellular, non-diazotrophic cyanobacteria, in several alpha-, beta- and gamma-proteobacteria as well as in some gram-positives. The common link between the heterotrophic bacteria seems to be the ability of nitrogen fixation and production of polyhydroxybutyrate (PHB), whereas among the cyanobacteria only Synechocystis PCC 6803 can accumulate PHB.
In this work, a knockout mutant of this gene in Synechocystis PCC 6803 was
characterised. This mutant was unable to accumulate PHB, a carbon and energy storage compound. The levels of precursor metabolites such as glycogen and acetyl-CoA were not reduced. The impairment in PHB accumulation correlated with a loss of PHB synthase activity during prolonged nitrogen starvation.
We could show that the PHB synthase activity appeared to be a target
of activity regulation, which was influenced by the NADPH/NADP+ ratio. The
loss of PHB synthase activity in the Sll0783 mutant was caused by decreased
NADPH/NADP+ ratio, which plays a crucial role in PHB synthesis.Stickstoff ist häufig ein limitierender Nährstoff in natürlichen Lebensräumen. Aus diesem Grund haben Cyanobakterien und andere autotrophe Organismen verschiedene Strategien entwickelt, um sich an diese Mangelbedingung anzupassen. Transkriptomanalysen des Cyanobakteriums Synechocystis PCC 6803 zeigten, dass das Gen sll0783 unter Stickstoffmangelbedingungen besonders stark induziert wird. sll0783 codiert für ein konserviertes Protein mit unbekannter Funktion und ist Teil eines Clusters mit sieben Genen. Im Promotorbereich befindet sich ein NtcA-Bindemotiv. Homologe dieses Clusters sind in einigen einzelligen, nicht-diazotrophen Cyanobakterien, in mehreren alpha-, beta- und gamma-Proteobakterien, sowie in einigen grampositiven Bakterien nachgewiesen worden. Das gemeinsame Bindeglied zwischen den heterotrophen Bakterien ist die Fähigkeit Stickstoff zu fixieren und Polyhydroxybutyrat (PHB), ein Kohlenstoff- und Energiespeicher, einzulagern. Unter den Cyanobakterien ist nur Synechocystis PCC 6803 in der Lage PHB zu bilden.
In dieser Arbeit wurde eine Knockout-Mutante des Gens sll0783 in Synechocystis PCC 6803 charakterisiert. Diese Mutante konnte nach Stickstoffentzug kein PHB mehr bilden. Während die Glykogen- und Acetyl-CoA-Konzentrationen in der Mutante nicht verringert waren, korrelierte die verminderte PHB-Bildung mit dem Verlust der PHB-Synthase-Aktivität. Es konnte gezeigt werden, dass die PHB-Synthase einer Aktivitätsregulierung unterliegt, welche durch das NADPH/NADP+-Verhältnis beeinflusst wird. Der Verlust der PHB-Synthase-Aktivität in der Sll0783-Mutante wurde durch ein reduziertes NADPH/NADP+-Verhältnis verursacht. Dies spielt eine Entscheidende Rolle in der PHB-Synthese
Unraveling the Function of Sll0944 in the Regulation of Carbon and Nitrogen Metabolism in Synechocystis sp. PCC6803
Get PDFCyanobakterien sind als die Pioniere der oxygenen Photosynthese anerkannt und haben vor zwei Milliarden Jahren maßgeblich zur Umgestaltung der Erdat-mosphäre beigetragen. Im Laufe ihrer Evolution haben sie eine Vielzahl von Anpassungsmechanismen entwickelt, um sich an ständig wechselnde Umwelt-bedingungen zu gewöhnen. Ein zentraler Bestandteil dieser Anpassungen ist das regulatorische Netzwerk des Stickstoff-Regulations-Proteins PII. In dem nicht-diazotrophen Cyanobakterium Synechocystis sp. PCC 6803 steuert PII eine Vielzahl von Stoffwechselprozessen, die für die Aufrechterhaltung der Kohlenstoff- und Stickstoff-Homöostase essentiell sind. Die Regulation erfolgt über die Bindung von ATP, ADP und 2-Oxoglutarat, die je nach Energie- und Nährstoffverfügbarkeit als Signalmoleküle dienen. Unter Bedingungen eines ausgeglichenen Energiehaushalts und ausreichender Nährstoffversorgung ge-währleistet PII eine effiziente Bereitstellung von Aminosäurevorläufern und re-guliert die Fettsäuresynthese. Zudem hemmt es die Aktivierung des globalen Stickstoff-Transkriptionsregulators NtcA durch Bindung an das PII-interagierende Protein X.
Unsere Ergebnisse belegen eine entscheidende Rolle von PII bei der Regulation der Kohlenstoffspeicherung unter Stickstoffmangelbedingungen. Wir konnten zeigen, dass der PII-interagierende Regulator des Kohlenstoffstoffwechsels (PirC) die Umwandlung fixierten Kohlenstoffs von der Glykolyse zur Glykogen-synthese durch Hemmung der 2,3-Bisphosphoglycerat-unabhängigen Phos-phoglycerat-Mutase beeinflusst. Diese Hemmung wird durch PII reguliert, wobei die Bindung von PirC an PII von den intrazellulären Konzentrationen von ATP, ADP und 2-Oxoglutarat abhängt. Strukturanalysen deuten darauf hin, dass PirC spezifisch mit cyanobakterien-spezifischen Elementen der Phosphoglyce-rat-Mutase interagiert und somit deren Aktivität beeinflusst. Unsere Studie legt den Grundstein für die Entwicklung von Synechocystis sp. PCC 6803 als Chas-sisorganismus für die nachhaltige Produktion von Polyhydroxybutyrat (PHB) und anderen wertvollen Verbindungen. Durch gezielte genetische Modifikatio-nen konnten wir Stämme generieren, die bis zu 80% ihres Trockengewichts in Form von PHB speichern.Cyanobacteria are considered to be the inventors of oxygenic photosynthesis. Two billion years ago, they shaped the atmosphere by releasing oxygen. Through this time, they evolved various species with plenty of mechanisms to adapt to the constantly changing environment. Among all those mechanisms, some structures became established and further functions evolved around this basis. One of the most prominent examples of this is the regulatory network of the nitrogen-regulatory protein PII. In the non-diazotroph cyanobacterium Syn-echocystis sp. PCC 6803, PII regulates a plethora of reactions that maintain the Carbon/Nitrogen homeostasis. The regulations depend on the binding of either ATP or ADP during high energy and nitrogen availability and 2-oxoglutarate (2-OG) during low nitrogen availability. In a balanced proportion of energy and nutrition, PII ensures sufficient amounts of amino acid precursors by activating the phosphoenol pyruvate carboxylase and mitigating the fatty acid synthesis. It also binds the PII interacting protein X to prevent the activation of the global nitrogen transcriptional regulator NtcA. With the increase of 2-oxoglutarate, PII releases its binding partners, which cancels its regulations. It was suggested that PII also regulate carbon storage during low nitrogen availability.
This work clarified PII's involvement in carbon storage regulation during chloro-sis. The novel discovered that the PII interacting regulator of carbon metabo-lism (PirC) changes the direction of fixed CO2 from lower glycolysis to glycogen synthesis by inhibiting the 2,3-bisphosphoglycerate-independent phospho-glycerate mutase. PII regulates this inhibition by binding the PirC during high ADP and ATP and releasing it during high 2-OG levels. PirC mediates the inhi-bition by interacting with two cyanobacteria-exclusive structural elements with-in their phosphoglycerate mutase. Furthermore, the elements also have a strong influence on the activity of the enzyme. This work also created the basis on which Synechocystis sp. PCC 6803 can be edited to create a chassis for the sustainable production of polyhydroxybutyrate (PHB) or other metabo-lism-derived valuable compounds. A strain derived from this work produced 80 % PHB of their cell dry mass
Novel structures of PII signal transduction proteins from oxygenic phototropic organisms
Get PDFPII proteins constitute one of the most widely distributed families of signal transduction proteins, whose representatives are present in archaea, bacteria and plants. They play a pivotal role to control the nitrogen, carbon and energy status of the cell in response to the central metabolites ATP, ADP and 2-oxoglutarate (2-OG). These signals from central metabolites are integrated by PII proteins and transmitted to the regulatory targets (protein modifying enzymes, metabolic enzymes, transporters and transcription factors). In oxygenic phototrophic organisms, from cyanobacteria to higher plants, the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) is a major PII target, whose activity responds to the cellular metabolites via PII signalling. In this work, novel crystal structures of PII signal transduction proteins from oxygenic phototrophs (Synechococcus elongatus and Chlamydomonas reinhardtii) in the presence of signalling metabolites and in complex with NAGK are reported. These structures give deeper insights into PII-mediated mechanism and regulation which are in accordance with the obtained biochemical data. The novel role of glutamine as a signalling molecule in C. reinhardtii is elucidated for the first time, which highlights the nitrogen regulation at a different level. Further, the interpretation of these structures together with the comparison of aminoacid sequences sheds light on the evolutionary adaptation of PII signal transduction from cyanobacteria to plastids
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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