The biogenesis of Photosystem II is a complicated process requiring numerous auxiliary factors to assist in all steps of its assembly. The cyanobacterial protein Ycf39 forms a stress-induced complex with 2 small chlorophyll-binding, High-light-inducible proteins C and D (HliC and HliD), and has been reported to participate in the insertion of chlorophyll molecules into the central D1 subunit of Photosystem II. However, how this process is organized remains unknown. Here, we show that Ycf39 and both HliC and HliD can form distinct complexes with chlorophyll synthase (ChlG) in the model cyanobacterium Synechocystis sp. PCC 6803. We isolated and characterized ChlG complexes from various strains grown under different conditions and provide a mechanistic view of the docking of Ycf39 to ChlG via HliD and the structural role of HliC. In the absence of stress, chlorophyll is produced by the ChlG-HliD2-ChlG complex, which is stabilized by chlorophyll and zeaxanthin molecules bound to the HliD homodimer. The switch to high light leads to stress pressure and greatly elevated synthesis of HliC, resulting in the replacement of HliD homodimers with HliC-HliD heterodimers. Unlike HliD, HliC cannot interact directly with ChlG or Ycf39. Therefore, the original ChlG-HliD2-ChlG complex is converted into a ChlG-HliD-HliC hetero-trimer that presumably binds transiently to Ycf39 and the nascent D1 polypeptide. We speculate that this molecular machinery promotes the delivery of chlorophyll to D1 upon high-light-induced chlorophyll deficiency. The HliD homodimers formed under standard, nonstress growth conditions and attached to ChlG could serve as an emergency chlorophyll reserve.

• MeSH
• Bacterial Proteins * metabolism   genetics   MeSH
• Chlorophyll metabolism   MeSH
• Photosystem II Protein Complex * metabolism   MeSH
• Carbon-Oxygen Ligases * metabolism   genetics   MeSH
• Light * MeSH
• Light-Harvesting Protein Complexes MeSH
• Synechocystis * metabolism   radiation effects   genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins * MeSH
• Chlorophyll MeSH
• chlorophyll synthetase MeSH Browser
• Photosystem II Protein Complex * MeSH
• high light-inducible protein, cyanobacteria MeSH Browser
• Carbon-Oxygen Ligases * MeSH
• Light-Harvesting Protein Complexes MeSH

One potential approach to improve the productivity of cyanobacteria and microalgae is to enhance photosynthetic efficiency by introducing far-red absorbing pigment molecules (such as chlorophylls f and d) into the photosynthetic apparatus to expand the range of photosynthetically active radiation. We have shown previously that expressing the ChlF subunit of Chroococcidiopsis thermalis PCC 7203 in the model cyanobacterium Synechocystis sp. PCC 6803 (Syn6803) is sufficient to drive the production of chlorophyll f (Chl f), but only to low levels (0.24% Chl f/Chl a). By using the strong Pcpc560 promoter and an N-terminal truncated derivative of ChlF, we have been able to increase the yield of Chl f in white light by over 30-fold to about 8.2% Chl f/Chl a, close to the level displayed by far-red photoacclimated C. thermalis 7203. Additionally, we demonstrate that ChlF from Fisherella thermalis PCC 7521, like ChlF from C. thermalis 7203, assembles into a variant of the monomeric photosystem II (PSII) core complex termed the super-rogue PSII complex when expressed in Syn6803. This contrasts with the originally reported formation of a ChlF homodimeric complex in Synechococcus sp. PCC 7002. Overall, our work is an important starting point for mechanistic and structural studies of super-rogue PSII and for incorporating Chl f into the photosynthetic apparatus of Syn6803.

• MeSH
• Bacterial Proteins metabolism   genetics   MeSH
• Chlorophyll * analogs & derivatives   metabolism   biosynthesis   MeSH
• Photosynthesis MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Light MeSH
• Synechocystis * metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Chlorophyll * MeSH
• chlorophyll f MeSH Browser
• Photosystem II Protein Complex MeSH

The investigation of spatial heterogeneity within the thylakoid membrane (TM) proteins has gained increasing attention in photosynthetic research. The recent advances in live-cell imaging have allowed the identification of heterogeneous organisation of photosystems in small cyanobacterial cells. These sub-micrometre TM regions, termed microdomains in cyanobacteria, exhibit functional similarities with granal (Photosystem II dominant) and stromal (Photosystem I dominant) regions observed in TM of higher plants. This study delves into microdomain heterogeneity using super-resolution Airyscan-based microscopy enhancing resolution to approximately ~125 nm in x-y dimension. The new data reveal membrane areas rich in Photosystem I within the inner TM rings. Moreover, we identified analogous dynamics in the mobility of Photosystem II and phycobilisomes; countering earlier models that postulated differing mobility of these complexes. These novel findings thus hold significance for our understanding of photosynthesis regulation, particularly during state transitions.

• Keywords
• Airyscan, FRAP, cyanobacteria, microdomain, photosystem, protein mobility, super-resolution microscopy, thylakoid membrane heterogeneity,
• Publication type
• Journal Article MeSH

In natural environments, photosynthetic organisms adjust their metabolism to cope with the fluctuating availability of combined nitrogen sources, a growth-limiting factor. For acclimation, the dynamic degradation/synthesis of tetrapyrrolic pigments, as well as of the amino acid arginine, is pivotal; however, there has been no evidence that these processes could be functionally coupled. Using co-immunopurification and spectral shift assays, we found that in the cyanobacterium Synechocystis sp. PCC 6803, the arginine metabolism-related ArgD and CphB enzymes form protein complexes with Gun4, an essential protein for chlorophyll biosynthesis. Gun4 binds ArgD with high affinity, and the Gun4-ArgD complex accumulates in cells supplemented with ornithine, a key intermediate of the arginine pathway. Elevated ornithine levels restricted de novo synthesis of tetrapyrroles, which arrested the recovery from nitrogen deficiency. Our data reveal a direct crosstalk between tetrapyrrole biosynthesis and arginine metabolism that highlights the importance of balancing photosynthetic pigment synthesis with nitrogen homeostasis.

• Keywords
• CP: Plants, Synechocystis, arginine metabolism, bilins, chlorophyll, genome-uncoupled-4, nitrogen homeostasis, tetrapyrrole biosynthesis,
• MeSH
• Arginine metabolism   MeSH
• Chlorophyll metabolism   MeSH
• Nitrogen MeSH
• Ornithine MeSH
• Synechocystis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arginine MeSH
• Chlorophyll MeSH
• Nitrogen MeSH
• Ornithine MeSH

Synechocystis sp. PCC 6803 is a model cyanobacterium, glucose-tolerant substrains of which are commonly used as laboratory strains. In recent years, it has become evident that 'wild-type' strains used in different laboratories show some differences in their phenotypes. We report here the chromosome sequence of our Synechocystis sp. PCC 6803 substrain, named substrain GT-T. The chromosome sequence of GT-T was compared to those of two other commonly used laboratory substrains, GT-S and PCC-M. We identified 11 specific mutations in the GT-T substrain, whose physiological consequences are discussed. We also provide an update on evolutionary relationships between different Synechocystis sp. PCC 6803 substrains.

• Keywords
• Synechocystis sp. PCC 6803, chromosome sequence, cyanobacteria,
• MeSH
• Mutation MeSH
• Synechocystis * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

FtsH proteases are membrane-embedded proteolytic complexes important for protein quality control and regulation of various physiological processes in bacteria, mitochondria, and chloroplasts. Like most cyanobacteria, the model species Synechocystis sp. PCC 6803 contains four FtsH homologs, FtsH1-FtsH4. FtsH1-FtsH3 form two hetero-oligomeric complexes, FtsH1/3 and FtsH2/3, which play a pivotal role in acclimation to nutrient deficiency and photosystem II quality control, respectively. FtsH4 differs from the other three homologs by the formation of a homo-oligomeric complex, and together with Arabidopsis thaliana AtFtsH7/9 orthologs, it has been assigned to another phylogenetic group of unknown function. Our results exclude the possibility that Synechocystis FtsH4 structurally or functionally substitutes for the missing or non-functional FtsH2 subunit in the FtsH2/3 complex. Instead, we demonstrate that FtsH4 is involved in the biogenesis of photosystem II by dual regulation of high light-inducible proteins (Hlips). FtsH4 positively regulates expression of Hlips shortly after high light exposure but is also responsible for Hlip removal under conditions when their elevated levels are no longer needed. We provide experimental support for Hlips as proteolytic substrates of FtsH4. Fluorescent labeling of FtsH4 enabled us to assess its localization using advanced microscopic techniques. Results show that FtsH4 complexes are concentrated in well-defined membrane regions at the inner and outer periphery of the thylakoid system. Based on the identification of proteins that co-purified with the tagged FtsH4, we speculate that FtsH4 concentrates in special compartments in which the biogenesis of photosynthetic complexes takes place.

• Keywords
• FtsH4, high light-inducible protein, photosystem II biogenesis, proteolysis, thylakoid,
• MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Chloroplasts metabolism   MeSH
• Photosystem II Protein Complex genetics   metabolism   MeSH
• Phylogeny MeSH
• Metalloproteases genetics   metabolism   MeSH
• Peptide Hydrolases MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Synechocystis * genetics   metabolism   MeSH
• Thylakoids metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Photosystem II Protein Complex MeSH
• FtsH4 protein, Arabidopsis MeSH Browser
• Metalloproteases MeSH
• Peptide Hydrolases MeSH
• Arabidopsis Proteins * MeSH

Assembly of photosystem II (PSII), a water-splitting catalyst in chloroplasts and cyanobacteria, requires numerous auxiliary proteins which promote individual steps of this sequential process and transiently associate with one or more assembly intermediate complexes. In this study, we focussed on the role of a PSII-associated protein encoded by the ssl1498 gene in the cyanobacterium Synechocystis sp. PCC 6803. The N-terminal domain of this protein, which is here called Psb34, is very similar to the N-terminus of HliA/B proteins belonging to a family of high-light-inducible proteins (Hlips). Psb34 was identified in both dimeric and monomeric PSII, as well as in a PSII monomer lacking CP43 and containing Psb28. When FLAG-tagged, the protein is co-purified with these three complexes and with the PSII auxiliary proteins Psb27 and Psb28. However, the preparation also contained the oxygen-evolving enhancers PsbO and PsbV and lacked HliA/B proteins even when isolated from high-light-treated cells. The data suggest that Psb34 competes with HliA/B for the same binding site and that it is one of the components involved in the final conversion of late PSII assembly intermediates into functional PSII complexes, possibly keeping them free of Hlips. Unlike HliA/B, Psb34 does bind to the CP47 assembly module before its incorporation into PSII. Analysis of strains lacking Psb34 indicates that Psb34 mediates the optimal equilibrium of HliA/B binding among individual PSII assembly intermediates containing CP47, allowing Hlip-mediated photoprotection at all stages of PSII assembly.

• Keywords
• CP47, High-light-inducible protein, Photosynthesis, Photosystem II,
• MeSH
• Bacterial Proteins metabolism   MeSH
• Photosynthesis MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Tumor Necrosis Factor Ligand Superfamily Member 14 metabolism   MeSH
• Synechocystis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Photosystem II Protein Complex MeSH
• Tumor Necrosis Factor Ligand Superfamily Member 14 MeSH

High-light-inducible proteins (Hlips) are single-helix transmembrane proteins that are essential for the survival of cyanobacteria under stress conditions. The model cyanobacterium Synechocystis sp. PCC 6803 contains four Hlip isoforms (HliA-D) that associate with Photosystem II (PSII) during its assembly. HliC and HliD are known to form pigmented (hetero)dimers that associate with the newly synthesized PSII reaction center protein D1 in a configuration that allows thermal dissipation of excitation energy. Thus, it is expected that they photoprotect the early steps of PSII biogenesis. HliA and HliB, on the other hand, bind the PSII inner antenna protein CP47, but the mode of interaction and pigment binding have not been resolved. Here, we isolated His-tagged HliA and HliB from Synechocystis and show that these two very similar Hlips do not interact with each other as anticipated, rather they form HliAC and HliBC heterodimers. Both dimers bind Chl and β-carotene in a quenching conformation and associate with the CP47 assembly module as well as later PSII assembly intermediates containing CP47. In the absence of HliC, the cellular levels of HliA and HliB were reduced, and both bound atypically to HliD. We postulate a model in which HliAC-, HliBC-, and HliDC-dimers are the functional Hlip units in Synechocystis. The smallest Hlip, HliC, acts as a 'generalist' that prevents unspecific dimerization of PSII assembly intermediates, while the N-termini of 'specialists' (HliA, B or D) dictate interactions with proteins other than Hlips.

• Keywords
• CP47, Chlorophyll, High-light-inducible proteins, Photosystem II, Synechocystis,
• MeSH
• Bacterial Proteins metabolism   MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Tumor Necrosis Factor Ligand Superfamily Member 14 metabolism   MeSH
• Light-Harvesting Protein Complexes * metabolism   MeSH
• Synechocystis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Photosystem II Protein Complex MeSH
• Tumor Necrosis Factor Ligand Superfamily Member 14 MeSH
• Light-Harvesting Protein Complexes * MeSH

The repair of photosystem II is a key mechanism that keeps the light reactions of oxygenic photosynthesis functional. During this process, the PSII central subunit D1 is replaced with a newly synthesized copy while the neighbouring CP43 antenna with adjacent small subunits (CP43 module) is transiently detached. When the D2 protein is also damaged, it is degraded together with D1 leaving both the CP43 module and the second PSII antenna module CP47 unassembled. In the cyanobacterium Synechocystis sp. PCC 6803, the released CP43 and CP47 modules have been recently suggested to form a so-called no reaction centre complex (NRC). However, the data supporting the presence of NRC can also be interpreted as a co-migration of CP43 and CP47 modules during electrophoresis and ultracentrifugation without forming a mutual complex. To address the existence of NRC, we analysed Synechocystis PSII mutants accumulating one or both unassembled antenna modules as well as Synechocystis wild-type cells stressed with high light. The obtained results were not compatible with the existence of a stable NRC since each unassembled module was present as a separate protein complex with a mutually similar electrophoretic mobility regardless of the presence of the second module. The non-existence of NRC was further supported by isolation of the His-tagged CP43 and CP47 modules from strains lacking either D1 or D2 and their migration patterns on native gels.

• Keywords
• CP43, CP47, No reaction centre complex, Photosynthesis, Photosystem II,
• MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• Photosystem II Protein Complex metabolism   MeSH
• Oxygen metabolism   MeSH
• Synechocystis * genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Photosystem II Protein Complex MeSH
• Oxygen MeSH

Cytochrome (Cyt) b559 is a key component of the photosystem II complex (PSII) that is essential for its proper functioning and assembly. Site-directed mutants of the model cyanobacterium Synechocystis sp. PCC6803 with mutated heme axial ligands of Cyt b559 have little PSII and are therefore unable to grow photoautotrophically. Here we describe two types of Synechocystis autotrophic transformants that retained the same mutations in Cyt b559 but are able to accumulate PSII and grow photoautotrophically. Whole-genome sequencing revealed that all of these autotrophic transformants carried a variable number of tandem repeats (from 5 to 15) of chromosomal segments containing the psbEFLJ operon. RNA-seq analysis showed greatly increased transcript levels of the psbEFLJ operon in these autotrophic transformants. Multiple copies of the psbEFLJ operon in these transformants were only maintained during autotrophic growth, while its copy numbers gradually decreased under photoheterotrophic conditions. Two-dimensional PAGE analysis of membrane proteins revealed a strong deficiency in PSII complexes in the Cyt b559 mutants that was reversed in the autotrophic transformants. These results illustrate how tandem gene amplification restores PSII accumulation and photoautotrophic growth in Cyt b559 mutants of cyanobacteria, and may serve as an important adaptive mechanism for cyanobacterial survival.

• Keywords
• cyanobacterium, cytochrome b559, photosynthesis, photosystem II (PSII), tandem gene amplification,
• MeSH
• Gene Amplification MeSH
• Cytochromes b genetics   metabolism   MeSH
• Cytochrome b Group genetics   metabolism   MeSH
• Photosystem II Protein Complex * genetics   metabolism   MeSH
• Synechocystis * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytochromes b MeSH
• Cytochrome b Group MeSH
• Photosystem II Protein Complex * MeSH