Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In Synechocystis 6803, the DNA methyltransferase M.Ssp6803II modifies the first cytosine in the GGCC motif, forming N4-methylcytosine (GGm4CC). Deletion of the sll0729 gene encoding M.Ssp6803II (∆sll0729) caused a bluish phenotype due to reduced chlorophyll levels, which was reversed by suppressor mutations. Re-sequencing of 7 suppressor clones revealed a common GGCC to GGTC mutation in the slr1790 promoter's discriminator sequence, encoding protoporphyrinogen IX oxidase, HemJ, crucial for tetrapyrrole biosynthesis. Transcriptomic and qPCR analyses indicated aberrant slr1790 expression in ∆sll0729 mutants. This aberration led to the accumulation of coproporphyrin III and protoporphyrin IX, indicative of impaired HemJ activity. To confirm the importance of DNA methylation in hemJ expression, hemJ promoter variants with varying discriminator sequences were introduced into the wild type, followed by sll0729 deletion. The sll0729 deletion segregated in strains with the GGTC discriminator motif, resulting in wild-type-like pigmentation, whereas freshly prepared ∆sll0729 mutants with the native hemJ promoter exhibited the bluish phenotype. These findings demonstrate that hemJ is tightly regulated in Synechocystis and that N4-methylcytosine is essential for proper hemJ expression. Thus, cytosine N4-methylation is a relevant epigenetic marker in Synechocystis and likely other cyanobacteria.

• Klíčová slova
• DNA methyltransferase, HemJ, cyanobacteria, epigenetic modifications, tetrapyrrole biosynthesis,
• MeSH
• bakteriální proteiny metabolismus   genetika   MeSH
• epigeneze genetická * MeSH
• metylace DNA * MeSH
• mutace MeSH
• promotorové oblasti (genetika) * MeSH
• regulace genové exprese u bakterií MeSH
• Synechocystis * genetika   metabolismus   MeSH
• tetrapyrroly * metabolismus   biosyntéza   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• tetrapyrroly * MeSH

In most eukaryotic phototrophs, the entire heme synthesis is localized to the plastid, and enzymes of cyanobacterial origin dominate the pathway. Despite that, porphobilinogen deaminase (PBGD), the enzyme responsible for the synthesis of hydroxymethybilane in the plastid, shows phylogenetic affiliation to α-proteobacteria, the supposed ancestor of mitochondria. Surprisingly, no PBGD of such origin is found in the heme pathway of the supposed partners of the primary plastid endosymbiosis, a primarily heterotrophic eukaryote, and a cyanobacterium. It appears that α-proteobacterial PBGD is absent from glaucophytes but is present in rhodophytes, chlorophytes, plants, and most algae with complex plastids. This may suggest that in eukaryotic phototrophs, except for glaucophytes, either the gene from the mitochondrial ancestor was retained while the cyanobacterial and eukaryotic pseudoparalogs were lost in evolution, or the gene was acquired by non-endosymbiotic gene transfer from an unspecified α-proteobacterium and functionally replaced its cyanobacterial and eukaryotic counterparts.

• Klíčová slova
• evolution, gene replacement, heme biosynthesis, horizontal gene transfer, hydroxymethylbilane synthase, mitochondrion, porphobilinogen deaminase,
• Publikační typ
• časopisecké články 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
• bakteriální proteiny genetika   metabolismus   MeSH
• biologické pigmenty izolace a purifikace   MeSH
• chlorofyl biosyntéza   MeSH
• fotosyntéza fyziologie   MeSH
• fotosystém I (proteinový komplex) metabolismus   MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• mutace MeSH
• rubredoxiny chemie   genetika   metabolismus   MeSH
• Synechocystis genetika   růst a vývoj   metabolismus   MeSH
• tylakoidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• biologické pigmenty MeSH
• chlorofyl MeSH
• fotosystém I (proteinový komplex) MeSH
• fotosystém II (proteinový komplex) MeSH
• rubredoxiny MeSH

Ferrochelatase (FeCh) is an essential enzyme catalyzing the synthesis of heme. Interestingly, in cyanobacteria, algae, and plants, FeCh possesses a conserved transmembrane chlorophyll a/b binding (CAB) domain that resembles the first and the third helix of light-harvesting complexes, including a chlorophyll-binding motif. Whether the FeCh CAB domain also binds chlorophyll is unknown. Here, using biochemical and radiolabeled precursor experiments, we found that partially inhibited activity of FeCh in the cyanobacterium Synechocystis PCC 6803 leads to overproduction of chlorophyll molecules that accumulate in the thylakoid membrane and, together with carotenoids, bind to FeCh. We observed that pigments bound to purified FeCh are organized in an energy-dissipative conformation and further show that FeCh can exist in vivo as a monomer or a dimer depending on its own activity. However, pigmented FeCh was purified exclusively as a dimer. Separately expressed and purified FeCH CAB domain contained a pigment composition similar to that of full-length FeCh and retained its quenching properties. Phylogenetic analysis suggested that the CAB domain was acquired by a fusion between FeCh and a single-helix, high light-inducible protein early in the evolution of cyanobacteria. Following this fusion, the FeCh CAB domain with a functional chlorophyll-binding motif was retained in all currently known cyanobacterial genomes except for a single lineage of endosymbiotic cyanobacteria. Our findings indicate that FeCh from Synechocystis exists mostly as a pigment-free monomer in cells but can dimerize, in which case its CAB domain creates a functional pigment-binding segment organized in an energy-dissipating configuration.

• Klíčová slova
• Synechocystis, carotenoid, chlorophyll, chloroplast, ferrochelatase, heme, light harvesting complex (LHC)-like proteins, membrane protein, photosynthesis, photosynthetic pigment, pigment binding, plant biochemistry,
• MeSH
• chlorofyl a metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• dimerizace MeSH
• ferrochelatasa chemie   metabolismus   MeSH
• fylogeneze MeSH
• karotenoidy metabolismus   MeSH
• konformace proteinů MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• Synechocystis enzymologie   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl a MeSH
• chlorofyl MeSH
• chlorophyll b MeSH Prohlížeč
• ferrochelatasa MeSH
• karotenoidy MeSH
• světlosběrné proteinové komplexy 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.

• Klíčová slova
• Gun4, Mgprotoporphyrin IX, Synechocystis 6803, chlorophyll, cyanobacteria, docking, molecular modeling, porphyrin,
• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• chlorofyl biosyntéza   MeSH
• cirkulární dichroismus MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• mutace MeSH
• porfyriny metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• Synechocystis genetika   růst a vývoj   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chlorofyl MeSH
• porfyriny MeSH

The negatively charged lipid phosphatidylglycerol (PG) constitutes up to 10% of total lipids in photosynthetic membranes, and its deprivation in cyanobacteria is accompanied by chlorophyll (Chl) depletion. Indeed, radioactive labeling of the PG-depleted ΔpgsA mutant of Synechocystis sp. strain PCC 6803, which is not able to synthesize PG, proved the inhibition of Chl biosynthesis caused by restriction on the formation of 5-aminolevulinic acid and protochlorophyllide. Although the mutant accumulated chlorophyllide, the last Chl precursor, we showed that it originated from dephytylation of existing Chl and not from the block in the Chl biosynthesis. The lack of de novo-produced Chl under PG depletion was accompanied by a significantly weakened biosynthesis of both monomeric and trimeric photosystem I (PSI) complexes, although the decrease in cellular content was manifested only for the trimeric form. However, our analysis of ΔpgsA mutant, which lacked trimeric PSI because of the absence of the PsaL subunit, suggested that the virtual stability of monomeric PSI is a result of disintegration of PSI trimers. Interestingly, the loss of trimeric PSI was accompanied by accumulation of monomeric PSI associated with the newly synthesized CP43 subunit of photosystem II. We conclude that the absence of PG results in the inhibition of Chl biosynthetic pathway, which impairs synthesis of PSI, despite the accumulation of chlorophyllide released from the degraded Chl proteins. Based on the knowledge about the role of PG in prokaryotes, we hypothesize that the synthesis of Chl and PSI complexes are colocated in a membrane microdomain requiring PG for integrity.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• chlorofyl biosyntéza   metabolismus   MeSH
• chlorofylidy metabolismus   MeSH
• fosfatidylglyceroly genetika   metabolismus   MeSH
• fotosystém I (proteinový komplex) metabolismus   MeSH
• ligasy tvořící vazby C-O metabolismus   MeSH
• protochlorofylid metabolismus   MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• Synechocystis genetika   metabolismus   MeSH
• transferasy pro jiné substituované fosfátové skupiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase MeSH Prohlížeč
• chlorofyl MeSH
• chlorofylidy MeSH
• chlorophyll A binding protein CP43, Cyanobacteria MeSH Prohlížeč
• chlorophyll synthetase MeSH Prohlížeč
• fosfatidylglyceroly MeSH
• fotosystém I (proteinový komplex) MeSH
• ligasy tvořící vazby C-O MeSH
• protochlorofylid MeSH
• světlosběrné proteinové komplexy MeSH
• transferasy pro jiné substituované fosfátové skupiny MeSH

Chlorophyll (Chl) is an essential component of the photosynthetic apparatus. Embedded into Chl-binding proteins, Chl molecules play a central role in light harvesting and charge separation within the photosystems. It is critical for the photosynthetic cell to not only ensure the synthesis of a sufficient amount of new Chl-binding proteins but also avoids any misbalance between apoprotein synthesis and the formation of potentially phototoxic Chl molecules. According to the available data, Chl-binding proteins are translated on membrane bound ribosomes and their integration into the membrane is provided by the SecYEG/Alb3 translocon machinery. It appears that the insertion of Chl molecules into growing polypeptide is a prerequisite for the correct folding and finishing of Chl-binding protein synthesis. Although the Chl biosynthetic pathway is fairly well-described on the level of enzymatic steps, a link between Chl biosynthesis and the synthesis of apoproteins remains elusive. In this review, I summarize the current knowledge about this issue putting emphasis on protein-protein interactions. I present a model of the Chl biosynthetic pathway organized into a multi-enzymatic complex and physically attached to the SecYEG/Alb3 translocon. Localization of this hypothetical large biosynthetic centre in the cyanobacterial cell is also discussed as well as regulatory mechanisms coordinating the rate of Chl and apoprotein synthesis.

• MeSH
• bakteriální proteiny metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• fotosyntéza MeSH
• proteiny vázající chlorofyl biosyntéza   MeSH
• sinice cytologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chlorofyl MeSH
• proteiny vázající chlorofyl MeSH

Ferrochelatase (FeCH) catalyzes the insertion of Fe(2+) into protoporphyrin, forming protoheme. In photosynthetic organisms, FeCH and magnesium chelatase lie at a biosynthetic branch point where partitioning down the heme and chlorophyll (Chl) pathways occurs. Unlike their mammalian, yeast, and other bacterial counterparts, cyanobacterial and algal FeCHs as well as FeCH2 isoform from plants possess a carboxyl-terminal Chl a/b-binding (CAB) domain with a conserved Chl-binding motif. The CAB domain is connected to the FeCH catalytic core by a proline-rich linker sequence (region II). In order to dissect the regulatory, catalytic, and structural roles of the region II and CAB domains, we analyzed a FeCH ΔH347 mutant that retains region II but lacks the CAB domain and compared it with the ΔH324-FeCH mutant that lacks both these domains. We found that the CAB domain is not required for catalytic activity but is essential for dimerization of FeCH; its absence causes aberrant accumulation of Chl-protein complexes under high light accompanied by high levels of the Chl precursor chlorophyllide. Thus, the CAB domain appears to serve mainly a regulatory function, possibly in balancing Chl biosynthesis with the synthesis of cognate apoproteins. Region II is essential for the catalytic function of the plastid-type FeCH enzyme, although the low residual activity of the ΔH324-FeCH is more than sufficient to furnish the cellular demand for heme. We propose that the apparent surplus of FeCH activity in the wild type is critical for cell viability under high light due to a regulatory role of FeCH in the distribution of Chl into apoproteins.

• MeSH
• aklimatizace MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• chlorofyl biosyntéza   MeSH
• ferrochelatasa genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• světlo MeSH
• Synechocystis enzymologie   genetika   růst a vývoj   MeSH
• tetrapyrroly biosyntéza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chlorofyl MeSH
• ferrochelatasa MeSH
• tetrapyrroly MeSH

Gun4 is a porphyrin-binding protein that activates magnesium chelatase, a multimeric enzyme catalyzing the first committed step in chlorophyll biosynthesis. In plants, GUN4 has been implicated in plastid-to-nucleus retrograde signaling processes that coordinate both photosystem II and photosystem I nuclear gene expression with chloroplast function. In this work we present the functional analysis of Gun4 from the cyanobacterium Synechocystis sp. PCC 6803. Affinity co-purification of the FLAG-tagged Gun4 with the ChlH subunit of the magnesium chelatase confirmed the association of Gun4 with the enzyme in cyanobacteria. Inactivation of the gun4 gene abolished photoautotrophic growth of the resulting gun4 mutant strain that exhibited a decreased activity of magnesium chelatase. Consequently, the cellular content of chlorophyll-binding proteins was highly inadequate, especially that of proteins of photosystem II. Immunoblot analyses, blue native polyacrylamide gel electrophoresis, and radiolabeling of the membrane protein complexes suggested that the availability of the photosystem II antenna protein CP47 is a limiting factor for the photosystem II assembly in the gun4 mutant.

• MeSH
• buněčná membrána metabolismus   MeSH
• chlorofyl chemie   metabolismus   MeSH
• chloroplasty metabolismus   MeSH
• elektronová mikroskopie MeSH
• fenotyp MeSH
• fluorescenční spektrometrie metody   MeSH
• fotosyntetická reakční centra (proteinové komplexy) chemie   fyziologie   MeSH
• fotosyntéza MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• intracelulární signální peptidy a proteiny genetika   metabolismus   fyziologie   MeSH
• lyasy chemie   MeSH
• mutace MeSH
• porfyriny chemie   MeSH
• sinice metabolismus   MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• transportní proteiny genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chlorofyl MeSH
• fotosyntetická reakční centra (proteinové komplexy) MeSH
• fotosystém II (proteinový komplex) MeSH
• intracelulární signální peptidy a proteiny MeSH
• lyasy MeSH
• magnesium chelatase MeSH Prohlížeč
• photosystem II, chlorophyll-binding protein, CP-47 MeSH Prohlížeč
• porfyriny MeSH
• světlosběrné proteinové komplexy MeSH
• transportní proteiny MeSH