The growth of plants, algae, and cyanobacteria relies on the catalytic activity of the oxygen-evolving PSII complex, which uses solar energy to extract electrons from water to feed into the photosynthetic electron transport chain. PSII is proving to be an excellent system to study how large multi-subunit membrane-protein complexes are assembled in the thylakoid membrane and subsequently repaired in response to photooxidative damage. Here we summarize recent developments in understanding the biogenesis of PSII, with an emphasis on recent insights obtained from biochemical and structural analysis of cyanobacterial PSII assembly/repair intermediates. We also discuss how chlorophyll synthesis is synchronized with protein synthesis and suggest a possible role for PSI in PSII assembly. Special attention is paid to unresolved and controversial issues that could be addressed in future research.

• MeSH
• Chlorophyll metabolism   MeSH
• Photosynthesis MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Cyanobacteria * metabolism   MeSH
• Thylakoids metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Chlorophyll MeSH
• Photosystem II Protein Complex * MeSH

Type IV pili are bacterial surface-exposed filaments that are built up by small monomers called pilin proteins. Pilins are synthesized as longer precursors (prepilins), the N-terminal signal peptide of which must be removed by the processing protease PilD. A mutant of the cyanobacterium Synechocystis sp. PCC 6803 lacking the PilD protease is not capable of photoautotrophic growth because of the impaired function of Sec translocons. Here, we isolated phototrophic suppressor strains of the original ΔpilD mutant and, by sequencing their genomes, identified secondary mutations in the SigF sigma factor, the γ subunit of RNA polymerase, the signal peptide of major pilin PilA1, and in the pilA1-pilA2 intergenic region. Characterization of suppressor strains suggests that, rather than the total prepilin level in the cell, the presence of non-glycosylated PilA1 prepilin is specifically harmful. We propose that the restricted lateral mobility of the non-glycosylated PilA1 prepilin causes its accumulation in the translocon-rich membrane domains, which attenuates the synthesis of membrane proteins.

• Keywords
• PilD peptidase, Synechocystis, Type IV pili, photosystem II, suppressor mutations,
• Publication type
• Journal Article MeSH

Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.

• Keywords
• chlorophyll, cyanobacteria, cyclase, microevolution, photosynthesis,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Bacteriochlorophylls biosynthesis   genetics   MeSH
• Gene Deletion * MeSH
• Oxygenases genetics   metabolism   MeSH
• Prochlorococcus genetics   metabolism   MeSH
• Synechocystis genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Bacteriochlorophylls MeSH
• Oxygenases MeSH

Oxygenic photosynthesis relies on accessory factors to promote the assembly and maintenance of the photosynthetic apparatus in the thylakoid membranes. The highly conserved membrane-bound rubredoxin-like protein RubA has previously been implicated in the accumulation of both PSI and PSII, but its mode of action remains unclear. Here, we show that RubA in the cyanobacterium Synechocystis sp PCC 6803 is required for photoautotrophic growth in fluctuating light and acts early in PSII biogenesis by promoting the formation of the heterodimeric D1/D2 reaction center complex, the site of primary photochemistry. We find that RubA, like the accessory factor Ycf48, is a component of the initial D1 assembly module as well as larger PSII assembly intermediates and that the redox-responsive rubredoxin-like domain is located on the cytoplasmic surface of PSII complexes. Fusion of RubA to Ycf48 still permits normal PSII assembly, suggesting a spatiotemporal proximity of both proteins during their action. RubA is also important for the accumulation of PSI, but this is an indirect effect stemming from the downregulation of light-dependent chlorophyll biosynthesis induced by PSII deficiency. Overall, our data support the involvement of RubA in the redox control of PSII biogenesis.

• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Pigments, Biological isolation & purification   MeSH
• Chlorophyll biosynthesis   MeSH
• Photosynthesis physiology   MeSH
• Photosystem I Protein Complex metabolism   MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Mutation MeSH
• Rubredoxins chemistry   genetics   metabolism   MeSH
• Synechocystis genetics   growth & development   metabolism   MeSH
• Thylakoids metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Pigments, Biological MeSH
• Chlorophyll MeSH
• Photosystem I Protein Complex MeSH
• Photosystem II Protein Complex MeSH
• Rubredoxins MeSH

Protoporphyrinogen IX oxidase (PPO), the last enzyme that is common to both chlorophyll and heme biosynthesis pathways, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. PPO has several isoforms, including the oxygen-dependent HemY and an oxygen-independent enzyme, HemG. However, most cyanobacteria encode HemJ, the least characterized PPO form. We have characterized HemJ from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) as a bona fide PPO; HemJ down-regulation resulted in accumulation of tetrapyrrole precursors and in the depletion of chlorophyll precursors. The expression of FLAG-tagged Synechocystis 6803 HemJ protein (HemJ.f) and affinity isolation of HemJ.f under native conditions revealed that it binds heme b The most stable HemJ.f form was a dimer, and higher oligomeric forms were also observed. Using both oxygen and artificial electron acceptors, we detected no enzymatic activity with the purified HemJ.f, consistent with the hypothesis that the enzymatic mechanism for HemJ is distinct from those of other PPO isoforms. The heme absorption spectra and distant HemJ homology to several membrane oxidases indicated that the heme in HemJ is redox-active and involved in electron transfer. HemJ was conditionally complemented by another PPO, HemG from Escherichia coli. If grown photoautotrophically, the complemented strain accumulated tripropionic tetrapyrrole harderoporphyrin, suggesting a defect in enzymatic conversion of coproporphyrinogen III to protoporphyrinogen IX, catalyzed by coproporphyrinogen III oxidase (CPO). This observation supports the hypothesis that HemJ is functionally coupled with CPO and that this coupling is disrupted after replacement of HemJ by HemG.

• Keywords
• HemJ, Synechocystis sp. PCC 6803, coproporphyrinogen III oxidase, cyanobacteria, enzyme purification, heme, membrane protein, photosynthesis, protoporphyrinogen IX oxidase,
• MeSH
• Heme chemistry   metabolism   MeSH
• Coproporphyrinogen Oxidase chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Oxidation-Reduction MeSH
• Protoporphyrinogen Oxidase chemistry   metabolism   MeSH
• Synechocystis enzymology   MeSH
• Tetrapyrroles chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Heme MeSH
• Coproporphyrinogen Oxidase MeSH
• Protoporphyrinogen Oxidase MeSH
• Tetrapyrroles MeSH

Photosystem II (PSII) is a large enzyme complex embedded in the thylakoid membrane of oxygenic phototrophs. The biogenesis of PSII requires the assembly of more than 30 subunits, with the assistance of a number of auxiliary proteins. In plants and cyanobacteria, the photosynthesis-affected mutant 68 (Pam68) is important for PSII assembly. However, its mechanisms of action remain unknown. Using a Synechocystis PCC 6803 strain expressing Flag-tagged Pam68, we purified a large protein complex containing ribosomes, SecY translocase, and the chlorophyll-binding PSII inner antenna CP47. Using 2D gel electrophoresis, we identified a pigmented Pam68-CP47 subcomplex and found Pam68 bound to ribosomes. Our results show that Pam68 binds to ribosomes even in the absence of CP47 translation. Furthermore, Pam68 associates with CP47 at an early phase of its biogenesis and promotes the synthesis of this chlorophyll-binding polypeptide until the attachment of the small PSII subunit PsbH. Deletion of both Pam68 and PsbH nearly abolishes the synthesis of CP47, which can be restored by enhancing chlorophyll biosynthesis. These results strongly suggest that ribosome-bound Pam68 stabilizes membrane segments of CP47 and facilitates the insertion of chlorophyll molecules into the translated CP47 polypeptide chain.

• MeSH
• Electrophoresis, Gel, Two-Dimensional MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Phosphoproteins genetics   metabolism   MeSH
• Photosystem II Protein Complex genetics   metabolism   MeSH
• Mutation MeSH
• Ribosomes metabolism   MeSH
• Light-Harvesting Protein Complexes genetics   metabolism   MeSH
• Synechocystis genetics   metabolism   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Chlorophyll MeSH
• Phosphoproteins MeSH
• Photosystem II Protein Complex MeSH
• photosystem II, chlorophyll-binding protein, CP-47 MeSH Browser
• photosystem II, psbH subunit MeSH Browser
• Light-Harvesting Protein Complexes MeSH

Arsenic (As) pollution is a serious concern worldwide. Recent studies under environmentally relevant conditions revealed that, in the aquatic plant Ceratophyllum demersum, pigments are the first observable target of toxicity, prior to any effect on photosynthetic parameters or to oxidative stress. Lethal toxicity was initiated by a change of As species and their distribution pattern in various tissues. Here, the localization of As was investigated at the subcellular level through X-ray fluorescence using a submicron beam and a Maia detector. Further, it was possible to obtain useful tissue structural information from the ratio of the tomogram of photon flux behind the sample to the tomogram of Compton scattering. The micro-X-ray fluorescence tomograms showed that As predominantly accumulated in the nucleus of the epidermal cells in young mature leaves exposed to sublethal 1 µM As. This suggests that As may exert toxic effects in the nucleus, for example, by interfering with nucleic acid synthesis by replacing phosphorous with As. At higher cellular concentrations, As was mainly stored in the vacuole, particularly in mature leaves. An analysis of precursors of chlorophyll and degradation metabolites revealed that the observed decrease in chlorophyll concentration was associated with hindered biosynthesis, and was not due to degradation. Coproporphyrinogen III could not be detected after exposure to only 0.5 µM As. Levels of subsequent precursors, for example, protoporphyrin IX, Mg-protoporphyrin, Mg-protoporphyrin methyl ester, and divinyl protochlorophyllide, were significantly decreased at this concentration as well, indicating that the pathway was blocked upstream of tetrapyrrole synthesis.

• Keywords
• Arsenic, arsenic toxicity, chlorophyll biosynthesis, chlorophyll precursors, subcellular distribution of arsenic, synchrotron micro-X-ray fluorescence,
• MeSH
• Arsenic toxicity   MeSH
• Chlorophyll biosynthesis   metabolism   MeSH
• Plant Leaves drug effects   metabolism   MeSH
• Magnoliopsida drug effects   metabolism   MeSH
• Spectrometry, X-Ray Emission MeSH
• Subcellular Fractions metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arsenic MeSH
• Chlorophyll MeSH

Cyanobacterium Synechocystis PCC 6803 represents a favored model organism for photosynthetic studies. Its easy transformability allowed construction of a vast number of Synechocystis mutants including many photosynthetically incompetent ones. However, it became clear that there is already a spectrum of Synechocystis "wild-type" substrains with apparently different phenotypes. Here, we analyzed organization of photosynthetic membrane complexes in a standard motile Pasteur collection strain termed PCC and two non-motile glucose-tolerant substrains (named here GT-P and GT-W) previously used as genetic backgrounds for construction of many photosynthetic site directed mutants. Although, both the GT-P and GT-W strains were derived from the same strain constructed and described by Williams in 1988, only GT-P was similar in pigmentation and in the compositions of Photosystem II (PSII) and Photosystem I (PSI) complexes to PCC. In contrast, GT-W contained much more carotenoids but significantly less chlorophyll (Chl), which was reflected by lower level of dimeric PSII and especially trimeric PSI. We found that GT-W was deficient in Chl biosynthesis and contained unusually high level of unassembled D1-D2 reaction center, CP47 and especially CP43. Another specific feature of GT-W was a several fold increase in the level of the Ycf39-Hlip complex previously postulated to participate in the recycling of Chl molecules. Genome re-sequencing revealed that the phenotype of GT-W is related to the tandem duplication of a large region of the chromosome that contains 100 genes including ones encoding D1, Psb28, and other PSII-related proteins as well as Mg-protoporphyrin methylester cyclase (Cycl). Interestingly, the duplication was completely eliminated after keeping GT-W cells on agar plates under photoautotrophic conditions for several months. The GT-W strain without a duplication showed no obvious defects in PSII assembly and resembled the GT-P substrain. Although, we do not exactly know how the duplication affected the GT-W phenotype, we hypothesize that changed stoichiometry of protein components of PSII and Chl biosynthetic machinery encoded by the duplicated region impaired proper assembly and functioning of these multi-subunit complexes. The study also emphasizes the crucial importance of a proper control strain for evaluating Synechocystis mutants.

• Keywords
• Synechocystis 6803, chlorophyll, large tandem duplication, photosystem I, photosystem II assembly,
• Publication type
• Journal Article MeSH

In the chlorophyll (Chl) biosynthesis pathway the formation of protochlorophyllide is catalyzed by Mg-protoporphyrin IX methyl ester (MgPME) cyclase. The Ycf54 protein was recently shown to form a complex with another component of the oxidative cyclase, Sll1214 (CycI), and partial inactivation of the ycf54 gene leads to Chl deficiency in cyanobacteria and plants. The exact function of the Ycf54 is not known, however, and further progress depends on construction and characterization of a mutant cyanobacterial strain with a fully inactivated ycf54 gene. Here, we report the complete deletion of the ycf54 gene in the cyanobacterium Synechocystis 6803; the resulting Δycf54 strain accumulates huge concentrations of the cyclase substrate MgPME together with another pigment, which we identified using nuclear magnetic resonance as 3-formyl MgPME. The detection of a small amount (~13%) of Chl in the Δycf54 mutant provides clear evidence that the Ycf54 protein is important, but not essential, for activity of the oxidative cyclase. The greatly reduced formation of protochlorophyllide in the Δycf54 strain provided an opportunity to use (35)S protein labeling combined with 2D electrophoresis to examine the synthesis of all known Chl-binding protein complexes under drastically restricted de novo Chl biosynthesis. We show that although the Δycf54 strain synthesizes very limited amounts of photosystem I and the CP47 and CP43 subunits of photosystem II (PSII), the synthesis of PSII D1 and D2 subunits and their assembly into the reaction centre (RCII) assembly intermediate were not affected. Furthermore, the levels of other Chl complexes such as cytochrome b 6 f and the HliD- Chl synthase remained comparable to wild-type. These data demonstrate that the requirement for de novo Chl molecules differs completely for each Chl-binding protein. Chl traffic and recycling in the cyanobacterial cell as well as the function of Ycf54 are discussed.

• Keywords
• Mg-protoporphyrin IX methylester cyclase, Synechocystis 6803, Ycf54, chlorophyll, photosystem II, protochlorophyllide,
• Publication type
• Journal Article MeSH

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

• Keywords
• Gun4, Mgprotoporphyrin IX, Synechocystis 6803, chlorophyll, cyanobacteria, docking, molecular modeling, porphyrin,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Chlorophyll biosynthesis   MeSH
• Circular Dichroism MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Mutation MeSH
• Porphyrins metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Synechocystis genetics   growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Chlorophyll MeSH
• Porphyrins MeSH