Phycobilisome rod mutants in Synechocystis sp. strain PCC6803

The phycobilisome is a large pigment-protein assembly that harvests light energy for photosynthesis. This supramolecular complex is composed of two main structures: a core substructure and peripheral rods. Linker polypeptides assemble phycobiliproteins within these structures and optimize light absorption and energy transfer. Mutations have been constructed in three rod-linker-coding genes located in the cpc operon of Synechocystis sp. strain PCC6803. The cpcC1 gene encoding the 33 kDa linker is found to be epistatic to cpcC2 encoding the 30 kDa linker, indicating a specific role for each of these two linkers in rod growth. This corroborates studies on the sequential degradation of phycobilisomes upon nitrogen starvation. Three allelic mutants affecting cpcC2 revealed a polar effect of commonly used cassettes (aphI, aadA) on the operon steady-state transcripts and an effect of rod linker availability on the amount of phycocyanin incorporated in the phycobilisome. This led to the proposal that regulation of rod length could occur through processing of transcripts upstream of the cpcC2 gene

Biomedical potential of cyanobacteria and algae

Cyanobacteria have appeared on the primordial Earth over three billion years ago and still thrive in most habitats. These photosynthetic microbes have remarkable genetic plasticity and variability and have evolved an amazing arsenal of biochemical pathways that exert defence mechanisms and produce metabolites unique to them. By forming plastids, endosymbiont cyanobacteria contributed to the development of plants. Algae, the simplest plants, thrive in similar habitats and face the same challenges of the ever changing environment as cyanobacteria; and they have maintained similarity to them, with respect to production of unique metabolites and utilizing unique pathways. The exploration of these natural compounds and the biochemical pathways leading to their production provide excellent tools in fighting some major challenge that mankind needs to face in our days. In this contribution we briefly list the benefits that the genetics of these microbes and the produced compounds can offer, with emphasis on possible medical relevance. We mention applications in basic science, industry and agriculture, and list the potentials in medical drug development, therapy and nutrition of some enzymes, polysaccharides, polyphenols, pigments, peptides and lipids, among others, in the current state of the world-wide research on the topic

Controlling Cellular P-TEFb Activity by the HIV-1 Transcriptional Transactivator Tat

The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5′-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5′-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR

Modulation of non-bilayer lipid phases and the structure and functions of thylakoid membranes: effects on the water-soluble enzyme violaxanthin de-epoxidase

The role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. Non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content-yet their functional state is a bilayer. In vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. P-31-NMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH- and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. These reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase

Chloroplast Acetyltransferase GNAT2 is Involved in the Organization and Dynamics of Thylakoid Structure

Higher plants acclimate to changes in light conditions by adjusting the thylakoid membrane ultrastructure. Additionally, excitation energy transfer between photosystem II (PSII) and photosystem I (PSI) is balanced in a process known as state transition. These modifications are mediated by reversible phosphorylation of Lhcb1 and Lhcb2 proteins in different pools of light-harvesting complex (LHCII) trimers. Our recent study demonstrated that chloroplast acetyltransferase NUCLEAR SHUTTLE INTERACTING (NSI)/GNAT2 (general control non-repressible 5 (GCN5)-related N-acetyltransferase 2) is also needed for the regulation of light harvesting, evidenced by the inability of the gnat2 mutant to perform state transitions although there are no defects in LHCII phosphorylation. Here, we show that despite contrasting phosphorylation states of LHCII, grana packing in the gnat2 and state transition 7 (stn7) mutants possesses similar features, as the thylakoid structure of the mutants does not respond to the shift from darkness to light, which is in striking contrast to wild type (Wt). Circular dichroism and native polyacrylamide gel electrophoresis analyses further revealed that the thylakoid protein complex organization of gnat2 and stn7 resembles each other, but differ from that of Wt. Also, the location of the phosphorylated Lhcb2 as well as the LHCII antenna within the thylakoid network in gnat2 mutant is different from that of Wt. In gnat2, the LHCII antenna remains largely in grana stacks, where the phosphorylated Lhcb2 is found in all LHCII trimer pools, including those associated with PSII. These results indicate that in addition to phosphorylation-mediated regulation through STN7, the GNAT2 enzyme is involved in the organization and dynamics of thylakoid structure, probably through the regulation of chloroplast protein acetylation.</p

Rate-limiting steps in the dark-to-light transition of Photosystem II - revealed by chlorophyll-a fluorescence induction

Photosystem II (PSII) catalyses the photoinduced oxygen evolution and, by producing reducing equivalents drives, in concert with PSI, the conversion of carbon dioxide to sugars. Our knowledge about the architecture of the reaction centre (RC) complex and the mechanisms of charge separation and stabilisation is well advanced. However, our understanding of the processes associated with the functioning of RC is incomplete: the photochemical activity of PSII is routinely monitored by chlorophyll-a fluorescence induction but the presently available data are not free of controversy. In this work, we examined the nature of gradual fluorescence rise of PSII elicited by trains of single-turnover saturating flashes (STSFs) in the presence of a PSII inhibitor, permitting only one stable charge separation. We show that a substantial part of the fluorescence rise originates from light-induced processes that occur after the stabilisation of charge separation, induced by the first STSF; the temperature-dependent relaxation characteristics suggest the involvement of conformational changes in the additional rise. In experiments using double flashes with variable waiting times (tau) between them, we found that no rise could be induced with zero or short tau, the value of which depended on the temperature - revealing a previously unknown rate-limiting step in PSII

A foszfatidilglicerol szerepe fotoszintetikus membránok szerkezetének kialakításában = The essential role of phosphatidylglycerol in the formation of photosynthetic membrane structure

A negatív töltéssel rendelkező lipidek közül a foszfatidilglicerol (PG) jelentős szerepet tölt be. 1. Létrehoztuk egy FG szintézisben gátolt Synechocytis PCC6803 mutánst melyben megnéztük a CP43 kapcsolódását a kettes fotokémiai rendszer reakció centrumához. Ezt a mutánst egy, a fénybegyűjtő rendszer nélküli mutánssal hoztuk létre, a cdsA gén inaktiválásával. Megállapítottuk, hogy az FG kiürülés hatására a CP43 fehérje bekötődése gátolt. Megállapítottuk, hogy az FG kiürülés csökkenti a cianobaktérium sejtek oxigénfejlesztő képességét. 2. Megállapítottuk, hogy az FG kiürülés megváltoztatja a fotoszintetikus membrán felszíni töltését. 3. Az FG molekulák hatására bekövetkező fotoszintetikus aktivitást a reakció centrumok megváltozása okozza. A reakció centrumok fényérzékennyé vállnak. Ez ellen a sejtek megnövekedett karotin tartalommal védekeznek. A myxoxantin és az echinenon mennyisége nő meg. 4. A Syenechococcus PCC PCC7942 cdsA mutáns létrehozásával igazoltuk, hogy az FG molekulák szerepe a fotoszintetikus szervezetekben általánosítható. Megállapítottuk, hogy az FG kiürülés a különböző törzseken különböző mértékben hat. 5 Az FG molekulák kiürülésének hatására bekövetkező stressz elleni protekció a karotinoid molekulák megjelenése fotoszintetikus reakció centrumokbanmellett megfigyeltük azt is, hogy az FG molekulák zsírsav összetétele megváltozik. A molekulák átalakulását tömegspektometriásan igazoltuk. Az eredményeinket egy áttekintő cikkünkben foglaltuk össze. | Among the negatively charged lipids phosphatidylglycerol (PG) plays an important role. We used transformable cyanobacterial strains.1n We used a Phycobilisome-less mutant of Synechocytis PCC6803 was transformed with inactivated cdsA and the synthesis of PG was blocked. Binding of CP43 to the reaction center complex was investigated. The oxygen evolving activity of mutant was reduced. 2. The PG-depletion affected the surface charge of membranes. 3. PG-depletion induced sensitivity of cells which was compensated by elevated carotenoid content, mainly myxoxanthophyll and echinenone. 4. We generated Syenechococcus PCC PCC7942 cdsA mutant with which we could generalize the importance of PG in photosynthetic organizms. However, we could observe slight differencein different mutant. 5. Westudied PG remodeling by mas spectrometry

Lipid polymorphism in chloroplast thylakoid membranes - as revealed by 31P-NMR and time-resolved merocyanine fluorescence spectroscopy

Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three nonbilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes