Gábor Bernát

Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress

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.

Distinct Roles of Multiple NDH-1 Complexes in the Cyanobacterial Electron Transport Network as Revealed by Kinetic Analysis of P700+ Reduction in Various ndh-Deficient Mutants of Synechocystis sp. Strain PCC6803

While methyl viologen had only a small effect on P700(+) rereduction kinetics after far-red pulses in KCN-treated wild-type Synechocystis sp. strain PCC6803 and an NdhF3/NdhF4 (NdhF3/F4)-defective mutant, it involved a rather slow P700(+) rereduction in an NdhF1-defective mutant. This strongly indicates that (i) active electron flow from metabolites to plastoquinone is suppressed upon deletion of ndhF1 and (ii) photosystem 1-mediated cyclic electron transport is dependent on NdhF3/F4-type NDH-1 complexes.

Ssr2998 of Synechocystis sp. PCC 6803 is involved in regulation of cyanobacterial electron transport and associated with the cytochrome b6f Complex

To analyze the function of a protein encoded by the open reading frame ssr2998 in Synechocystis sp. PCC 6803, the corresponding gene was disrupted, and the generated mutant strain was analyzed. Loss of the 7.2-kDa protein severely reduced the growth of Synechocystis, especially under high light conditions, and appeared to impair the function of the cytochrome b6 f complex. This resulted in slower electron donation to cytochrome f and photosystem 1 and, concomitantly, over-reduction of the plastoquinone pool, which in turn had an impact on the photosystem 1 to photosystem 2 stoichiometry and state transition. Furthermore, a 7.2-kDa protein, encoded by the open reading frame ssr2998, was co-isolated with the cytochrome b6 f complex from the cyanobacterium Synechocystis sp. PCC 6803. ssr2998 seems to be structurally and functionally associated with the cytochrome b6 f complex from Synechocystis, and the protein could be involved in regulation of electron transfer processes in Synechocystis sp. PCC 6803.

Fluorescence quenching of the phycobilisome terminal emitter LCM from the cyanobacterium Synechocystis sp. PCC 6803 detected in vivo and in vitro

The fluorescence emission of the phycobilisome (PBS) core-membrane linker protein (L(CM)) can be directly quenched by photoactivated orange carotenoid protein (OCP) at room temperature both in vitro and in vivo, which suggests the crucial role of the OCP-L(CM) interaction in non-photochemical quenching (NPQ) of cyanobacteria. This implication was further supported (i) by low-temperature (77K) fluorescence emission and excitation measurements which showed a specific quenching of the corresponding long-wavelength fluorescence bands which belong to the PBS terminal emitters in the presence of photoactivated OCP, (ii) by systematic investigation of the fluorescence quenching and recovery in wild type and L(CM)-less cells of the model cyanobacterium Synechocystis sp. PCC 6803, and (iii) by the impact of dephosphorylation of isolated PBS on the quenching. The OCP binding site within the PBS and the most probable geometrical arrangement of the OCP-allophycocyanin (APC) complex was determined in silico using the crystal structures of OCP and APC. Geometrically modeled attachment of OCP to the PBS core is not at variance with the OCP-L(CM) interaction. It was concluded that besides being a very central element in the PBS to reaction center excitation energy transfer and PBS assembly, L(CM) also has an essential role in the photoprotective light adaptation processes of cyanobacteria.

Unique Properties vs. Common Themes: The Atypical Cyanobacterium Gloeobacter violaceus PCC 7421 is Capable of State Transitions and Blue-Light-Induced Fluorescence Quenching

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.

Properties of Mutants of Synechocystis sp. Strain PCC 6803 Lacking Inorganic Carbon Sequestration Systems

A mutant (Delta5) of Synechocystis sp. strain PCC 6803 constructed by inactivating five inorganic carbon sequestration systems did not take up CO(2) or HCO(3)(-) and was unable to grow in air with or without glucose. The Delta4 mutant in which BicA is the only active inorganic carbon sequestration system showed low activity of HCO(3)(-) uptake and grew under these conditions but more slowly than the wild-type strain. The Delta5 mutant required 1.7% CO(2) to attain half the maximal growth rate. Electron transport activity of the mutants was strongly inhibited under high light intensities, with the Delta5 mutant more susceptible to high light than the Delta4 mutant. The results implicated the significance of carbon sequestration in dissipating excess light energy.

Multiple Rieske proteins enable short- and long-term light adaptation of Synechocystis SP. PCC 6803

In contrast to eukaryotes, most cyanobacteria contain several isoforms of the Rieske iron-sulfur protein, PetC, resulting in heterogeneity in the composition of the cytochrome b6f complexes. Of three isoforms in the mesophilic cyanobacterium Synechocystis PCC 6803, PetC1 is the major Rieske protein in the cytochrome b6f complex, whereas the physiological function of PetC2 and PetC3 is still uncertain. Comparison of wild type and various petC-deficient strains under selected light conditions revealed distinct functional differences: high-light exposure of wild type cells resulted in a significantly enhanced petC2 transcript level, whereas a ΔpetC1 mutant showed a low cytochrome b6f content, low electron flux, and a considerably increased accumulation of cytochrome-bd oxidase. In contrast to wild type and ΔpetC1, ΔpetC2 and ΔpetC3 strains still grew fast under high-light conditions although all three Rieske proteins are required for maximal electron transport rates. Although the presence of PetC3 appears to be required for activation of the cyclic electron transport, state transitions were more effective in the absence of PetC2 and/or PetC3. In summary, our data suggest defined roles of the various PetC proteins in short- and long-term light adaptation.

Regulation of F0F1-ATPase from Synechocystis sp. PCC 6803 by γ and ϵ Subunits Is Significant for Light/Dark Adaptation

The γ and ϵ subunits of F0F1-ATP synthase from photosynthetic organisms display unique properties not found in other organisms. Although the γ subunit of both chloroplast and cyanobacterial F0F1 contains an extra amino acid segment whose deletion results in a high ATP hydrolysis activity (Sunamura, E., Konno, H., Imashimizu-Kobayashi, M., Sugano, Y., and Hisabori, T. (2010) Plant Cell Physiol. 51, 855–865), its ϵ subunit strongly inhibits ATP hydrolysis activity. To understand the physiological significance of these phenomena, we studied mutant strains with (i) a C-terminally truncated ϵ (ϵΔC), (ii) γ lacking the inserted sequence (γΔ198–222), and (iii) a double mutation of (i) and (ii) in Synechocystis sp. PCC 6803. Although thylakoid membranes from the ϵΔC strain showed higher ATP hydrolysis and lower ATP synthesis activities than those of the wild type, no significant difference was observed in growth rate and in intracellular ATP level both under light conditions and during light-dark cycles. However, both the ϵΔC and γΔ198–222 and the double mutant strains showed a lower intracellular ATP level and lower cell viability under prolonged dark incubation compared with the wild type. These data suggest that internal inhibition of ATP hydrolysis activity is very important for cyanobacteria that are exposed to prolonged dark adaptation and, in general, for the survival of photosynthetic organisms in an ever-changing environment.

Thylakoid Membrane Maturation and PSII Activation Are Linked in Greening Synechocystis sp. PCC 6803 Cells

Thylakoid membrane formation and photosynthetic electron transfer reactions are linked in greening cyanobacteria. Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. While they are crucial for phototrophic growth of cyanobacterial cells, biogenesis of thylakoid membranes is not well understood yet. Dark-grown Synechocystis sp. PCC 6803 cells contain only rudimentary thylakoid membranes but still a relatively high amount of phycobilisomes, inactive photosystem II and active photosystem I centers. After shifting dark-grown Synechocystis sp. PCC 6803 cells into the light, “greening” of Synechocystis sp. PCC 6803 cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions, was monitored. Complete restoration of a typical thylakoid membrane system was observed within 24 hours after an initial lag phase of 6 to 8 hours. Furthermore, activation of photosystem II complexes and restoration of a functional photosynthetic electron transport chain appears to be linked to the biogenesis of organized thylakoid membrane pairs.

Center of the Cyanobacterial Electron Transport Network: The Cytochrome b 6 f Complex

The cytochrome b 6 f complex is a shared, central component of both photosynthetic and respiratory electron- and proton transport in cyanobacteria. The following chapter provides an overview on the components of this complex and their function within the cyanobacterial electron transport network. This involves also regulatory aspects with special focus on (1) the role of non-essential subunits (PetL, PetM, PetP), (2) the multiplicity of the Rieske 2Fe–2S protein, and (3) the role of certain amino acid residues or small domains in the vicinity of catalytic sites and/or prosthetic groups. As complementary component of the plastoquinol oxidation which is highly dependent on the cytochrome b 6 f activity, the potential role of cytochrome bd oxidase in the electron transport processes is also reviewed.