Activation energies for two steps in the S_2 → S_3 transition of photosynthetic water oxidation from time-resolved single-frequency infrared spectroscopy

The mechanism of water oxidation by the Photosystem II (PSII) protein–cofactor complex is of high interest, but specifically, the crucial coupling of protonation dynamics to electron transfer (ET) and dioxygen chemistry remains insufficiently understood. We drove spinach-PSII membranes by nanosecond-laser flashes synchronously through the water-oxidation cycle and traced the PSII processes by time-resolved single-frequency infrared (IR) spectroscopy in the spectral range of symmetric carboxylate vibrations of protein side chains. After the collection of IR-transients from 100 ns to 1 s, we analyzed the proton-removal step in the S2 ⇒ S3 transition, which precedes the ET that oxidizes the Mn4CaOx-cluster. Around 1400 cm−1, pronounced changes in the IR-transients reflect this pre-ET process (∼40 µs at 20 °C) and the ET step (∼300 µs at 20 °C). For transients collected at various temperatures, unconstrained multi-exponential simulations did not provide a coherent set of time constants, but constraining the ET time constants to previously determined values solved the parameter correlation problem and resulted in an exceptionally high activation energy of 540 ± 30 meV for the pre-ET step. We assign the pre-ET step to deprotonation of a group that is re-protonated by accepting a proton from the substrate–water, which binds concurrently with the ET step. The analyzed IR-transients disfavor carboxylic-acid deprotonation in the pre-ET step. Temperature-dependent amplitudes suggest thermal equilibria that determine how strongly the proton-removal step is reflected in the IR-transients. Unexpectedly, the proton-removal step is only weakly reflected in the 1400 cm−1 transients of PSII core complexes of a thermophilic cyanobacterium (T. elongatus)

Highly plastic genome of Microcystis aeruginosa PCC 7806, a ubiquitous toxic freshwater cyanobacterium

Background The colonial cyanobacterium Microcystis proliferates in a wide range of freshwater ecosystems and is exposed to changing environmental factors during its life cycle. Microcystis blooms are often toxic, potentially fatal to animals and humans, and may cause environmental problems. There has been little investigation of the genomics of these cyanobacteria. Results Deciphering the 5,172,804 bp sequence of Microcystis aeruginosa PCC 7806 has revealed the high plasticity of its genome: 11.7% DNA repeats containing more than 1,000 bases, 6.8% putative transposases and 21 putative restriction enzymes. Compared to the genomes of other cyanobacterial lineages, strain PCC 7806 contains a large number of atypical genes that may have been acquired by lateral transfers. Metabolic pathways, such as fermentation and a methionine salvage pathway, have been identified, Conclusion Microcystis aeruginosa PCC 7806 appears to have adopted an evolutionary strategy relying on unusual genome plasticity to adapt to eutrophic freshwater ecosystems, a property shared by another strain of M. aeruginosa (NIES-843). Comparisons of the genomes of PCC 7806 and other cyanobacterial strains indicate that a similar strategy may have also been used by the marine strain Crocosphaera watsonii WH8501 to adapt to other ecological niches, such as oligotrophic open oceans.

The Cyanobacterial Hepatotoxin Microcystin Binds to Proteins and Increases the Fitness of Microcystis under Oxidative Stress Conditions

Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that microcystin fulfils an important function within cells of its natural producer Microcystis. The microcystin deficient mutant ΔmcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of microcystin in the wild type. Finally, the mutant defective in microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites

Molekulare Funktionsanalyse von Microcystin in Microcystis aeruginosa PCC 7806

Microcystine sind die wohl bekanntesten cyanobakteriellen Toxine. Sie werden im Wesentlichen durch die im Süßwasser weltweit verbreitete, koloniebildende Gattung Microcystis synthetisiert. Die biologische Funktion dieser Peptide ist jedoch ungeklärt. In dieser Studie wurde die Fragestellung erstmals über einen globalen Ansatz auf molekularer Ebene analysiert. Die proteomischen Analysen zwischen M. aeruginosa PCC 7806/ Wildtyp und einigen Microcystin-freien Mutanten deuten auf eine physiologische Rolle der Microcystine. Microcystine beeinflussen die Abundanz zahlreicher Proteine. Prominentester Vertreter ist RubisCO – Schlüsselenzym des Calvin Zyklus. RubisCO und andere im 2D selektierte Proteine konnten außerdem als mögliche zelluläre Bindepartner des Microcystins identifiziert werden. Möglicherweise bindet MC an bestimmte Cysteinreste dieser Proteine. Mit dem Knockout der mcy-Gene geht außerdem eine Überexpression eines Morphotyp-spezifischen Proteins einher, das MrpC genannt wurde. Dieses Protein vermittelt möglicherweise Zell-Zell-Interaktionen in Microcystis.Microcystins are the most common cyanobacterial toxins found in freshwater lakes and reservoirs throughout the world. They are frequently produced by the unicellular, colonial cyanobacterium Microcystis; however, the role of the peptide for the producing organismen is poorly understood. In this study we describe the first global approach to investigate this topic on a molecular level. Proteomic studies with M. aeruginosa PCC 7806 wild-type and several microcystin-deficient mutants indicated a physiological function for microcystin. Microcystin was shown to influence the abundance of several proteins which have an intra- or extracellular function. A prominent candidate is RubisCO, the key enzyme of the calvin cycle. RubisCO and other proteins, initially selected by 2D analysis, are putative cellular binding partners of microcystin. A potentially interaction mechanismen is the kovalent binding of microcystin to cysteine residues of the protein. Moreover, several knockouts of microcystin biosynthesis genes result in an overexpression of a putative morpho-type specific factor, named MrpC. This protein possibly mediates cell-cell interactions in Microcystis

The Multiple Functions of Common Microbial Carbon Polymers, Glycogen and PHB, during Stress Responses in the Non-Diazotrophic Cyanobacterium Synechocystis sp. PCC 6803

Classical microbial carbon polymers such as glycogen and polyhydroxybutyrate (PHB) have a crucial impact as both a sink and a reserve under macronutrient stress conditions. Most microbial species exclusively synthesize and degrade either glycogen or PHB. A few bacteria such as the phototrophic model organism Synechocystis sp. PCC 6803 surprisingly produce both physico-chemically different polymers under conditions of high C to N ratios. For the first time, the function and interrelation of both carbon polymers in non-diazotrophic cyanobacteria are analyzed in a comparative physiological study of single- and double-knockout mutants (ΔglgC; ΔphaC; ΔglgC/ΔphaC), respectively. Most of the observed phenotypes are explicitly related to the knockout of glycogen synthesis, highlighting the metabolic, energetic, and structural impact of this process whenever cells switch from an active, photosynthetic ‘protein status’ to a dormant ‘glycogen status’. The carbon flux regulation into glycogen granules is apparently crucial for both phycobilisome degradation and thylakoid layer disassembly in the presence of light. In contrast, PHB synthesis is definitely not involved in this primary acclimation response. Moreover, the very weak interrelations between the two carbon-polymer syntheses indicate that the regulation and role of PHB synthesis in Synechocystis sp. PCC 6803 is different from glycogen synthesis

Flux balance analysis of cyanobacterial metabolism: the metabolic network of Synechocystis sp. PCC 6803.

Cyanobacteria are versatile unicellular phototrophic microorganisms that are highly abundant in many environments. Owing to their capability to utilize solar energy and atmospheric carbon dioxide for growth, cyanobacteria are increasingly recognized as a prolific resource for the synthesis of valuable chemicals and various biofuels. To fully harness the metabolic capabilities of cyanobacteria necessitates an in-depth understanding of the metabolic interconversions taking place during phototrophic growth, as provided by genome-scale reconstructions of microbial organisms. Here we present an extended reconstruction and analysis of the metabolic network of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Building upon several recent reconstructions of cyanobacterial metabolism, unclear reaction steps are experimentally validated and the functional consequences of unknown or dissenting pathway topologies are discussed. The updated model integrates novel results with respect to the cyanobacterial TCA cycle, an alleged glyoxylate shunt, and the role of photorespiration in cellular growth. Going beyond conventional flux-balance analysis, we extend the computational analysis to diurnal light/dark cycles of cyanobacterial metabolism

Experimental validation of the glyoxylate shunt.

<p>(A) Enzymatic test to determine isocitrate lyase activity in cell-free extract of <i>Synechocystis</i> PCC 6803. Phenylhydrazon formation were measured as increase of A324 nm with time. Trace A - crude cell extract of <i>Synechocystis</i> cells, Trace B - crude cell extract passed over a PD-10 gel filtration column both corresponding to a protein concentration of reaction volume. Arrows mark the stepwise addition of phenylhydrazin (Phe, 5 mM), isocitrate (IC, 1 mM) and NADP ( mM), the latter as a control for isocitrate dehydrogenase activity. Increase in A324 nm in Trace A after adding of phenylhydrazin results from phenylhydrazine reactive metabolites, further increase after addition of IC results from internal NADP as a co-substrate of isocitrate dehydrogenase within the crude extract. Both traces show the same trend after addition of NADP, corresponding to the same isocitrate dehydrogenase activity. (B) The capability for utilization of acetate as sole carbon source was tested in spot assays in the presence of the photosystem II inhibitor DCMU (photoheterotrophic growth). Respective controls were conducted without the addition of DCMU (putative photomixotrophic growth) and without both sodium acetate and DCMU, respectively (photoautotrophic growth) to BG11 agar. 1∶1, 1.10 and 1∶100 represent dilution factors of the stock cell suspension of <i>Synechocystis</i> sp. PCC 6803 that contains chlorophyll a per ml cell suspension.</p

Overview of network properties.

<p>(A) We distinguish between a core network and all remaining genes annotated with enzymatic function. (B) Distribution of pathway annotations across the 760 reactions. (C) The distribution of components that define the biomass objective function (BOF), excluding storage and maintenance (ATP). Abbreviations: Terpenoids (Terp.), Carotenoids (Carot.), Plastoquinone (PQ), Tocopherol (Toco.).</p

A flux map for phototrophic growth under constant light.

<p>The flux distribution was optimized for maximal biomass yield. Shown are flux values in units of . Flux values marked with an asterisk indicate non-unique values. The solution is characterized by a large flux through the CBB cycle and non-cyclic operation of the TCA cycle. Aerobic respiration and oxygenic photosynthesis share common components in the electron transport chain. During phototrophic growth, electrons originate from water splitting in PS II and are transferred to NADPH via PS I and FNR. Abbreviations are defined in the <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003081#s3" target="_blank">Materials and Methods</a>.</p