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.
- 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
Life on Earth depends on photosynthesis, the conversion of light energy into chemical energy. Plants collect photons by light harvesting complexes (LHC)-abundant membrane proteins containing chlorophyll and xanthophyll molecules. LHC-like proteins are similar in their amino acid sequence to true LHC antennae, however, they rather serve a photoprotective function. Whether the LHC-like proteins bind pigments has remained unclear. Here, we characterize plant LHC-like proteins (LIL3 and ELIP2) produced in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Both proteins were associated with chlorophyll a (Chl) and zeaxanthin and LIL3 was shown to be capable of quenching Chl fluorescence via direct energy transfer from the Chl Qy state to zeaxanthin S1 state. Interestingly, the ability of the ELIP2 protein to quench can be acquired by modifying its N-terminal sequence. By employing Synechocystis carotenoid mutants and site-directed mutagenesis we demonstrate that, although LIL3 does not need pigments for folding, pigments stabilize the LIL3 dimer.
- MeSH
- chlorofyl metabolismus MeSH
- karotenoidy metabolismus MeSH
- multimerizace proteinu MeSH
- mutace MeSH
- přenos energie MeSH
- proteiny chloroplastové chemie genetika metabolismus MeSH
- proteiny huseníčku chemie genetika metabolismus MeSH
- sbalování proteinů MeSH
- Synechocystis genetika metabolismus MeSH
- vazba proteinů MeSH
- xanthofyly metabolismus MeSH
- zeaxanthiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Low production rates are still one limiting factor for the industrial climate-neutral production of biovaluable compounds in cyanobacteria. Next to optimized cultivation conditions, new production strategies are required. Hence, the use of established molecular tools could lead to increased product yields in the cyanobacterial model organism Synechocystis sp. PCC6803. Its main storage compound glycogen was chosen to be increased by the use of these tools. In this study, the three genes glgC, glgA1 and glgA2, which are part of the glycogen synthesis pathway, were combined with the Pcpc560 promoter and the neutral cloning site NSC1. The complete genome integration, protein formation, biomass production and glycogen accumulation were determined to select the most productive transformants. The overexpression of glgA2 did not increase the biomass or glycogen production in short-term trials compared to the other two genes but caused transformants death in long-term trials. The transformants glgA1_11 and glgC_2 showed significantly increased biomass (1.6-fold - 1.7-fold) and glycogen production (3.5-fold - 4-fold) compared to the wild type after 96 h making them a promising energy source for further applications. Those could include for example a two-stage production process, with first energy production (glycogen) and second increased product formation (e.g. ethanol).
- MeSH
- glykogen MeSH
- Synechocystis * genetika MeSH
- Publikační typ
- časopisecké články MeSH
The biogenesis of the cyanobacterial photosystem II (PSII) complex requires a number of auxiliary assembly factors that improve efficiency of the process but their precise function is not well understood. To assess a possible synergic action of the Ycf48 and Ycf39 factors acting in early steps of the biogenesis via interaction with the nascent D1 subunit of PSII, we constructed and characterised a double mutant of the cyanobacterium Synechocystis PCC 6803 lacking both these proteins. In addition, we also deleted the ycf39 gene in the double mutant lacking Ycf48 and Pam68, the latter being a ribosomal factor promoting insertion of chlorophyll (Chl) into the CP47 subunit of PSII. The resulting double ΔYcf48/ΔYcf39 and triple ΔYcf48/ΔPam68/ΔYcf39 mutants were deficient in PSII and total Chl, and in contrast to the source mutants, they lost the capacity for autotrophy. Interestingly, autotrophic growth was restored in both of the new multiple mutants by enhancing Chl biosynthesis using a specific ferrochelatase inhibitor. Taking together with the weak radioactive labelling of the D1 protein, these findings can be explained by inhibition of the D1 synthesis caused by the lack and/or incorrect binding of Chl molecules. The results emphasise the key importance of the sufficient Chl supply for the PSII biogenesis and also support the existence of a so far enigmatic regulatory mechanism leading to the reduced overall Chl biosynthesis/accumulation when the PSII assembly is impaired.
- MeSH
- autotrofní procesy MeSH
- bakteriální proteiny genetika metabolismus MeSH
- chlorofyl metabolismus MeSH
- delece genu MeSH
- fotosystém II (proteinový komplex) genetika metabolismus MeSH
- mutace MeSH
- Synechocystis genetika růst a vývoj metabolismus MeSH
- vazba proteinů MeSH
- 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
Targeting mutations to specific genomic loci is invaluable for assessing in vivo the effect of these changes on the biological role of the gene in study. Here, we attempted to introduce a mutation that was previously implicated in an increased heat stability of the mesophilic cyanobacterium Synechocystis sp. PCC6803 via homologous recombination to the psbA gene of Chlamydomonas reinhardtii. For that, we established a strategy for targeted mutagenesis that was derived from the efficient genome-wide homologous-recombination-based methodology that was used to target individual genes of Saccharomyces cerevisiae. While the isolated mutants did not show any benefit under elevated temperature conditions, the new strategy proved to be efficient for C. reinhardtii even in the absence of direct positive selection.
- MeSH
- Chlamydomonas reinhardtii genetika MeSH
- fotosystém II (proteinový komplex) genetika MeSH
- geneticky modifikované rostliny genetika MeSH
- genom plastidový genetika MeSH
- homologní rekombinace MeSH
- mutageneze cílená metody MeSH
- rostlinné proteiny genetika MeSH
- selekce (genetika) MeSH
- serin genetika MeSH
- substituce aminokyselin MeSH
- Synechocystis genetika MeSH
- termotolerance genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phototrophic microorganisms are promising resources for green biotechnology. Compared to heterotrophic microorganisms, however, the cellular economy of phototrophic growth is still insufficiently understood. We provide a quantitative analysis of light-limited, light-saturated, and light-inhibited growth of the cyanobacterium Synechocystis sp. PCC 6803 using a reproducible cultivation setup. We report key physiological parameters, including growth rate, cell size, and photosynthetic activity over a wide range of light intensities. Intracellular proteins were quantified to monitor proteome allocation as a function of growth rate. Among other physiological acclimations, we identify an upregulation of the translational machinery and downregulation of light harvesting components with increasing light intensity and growth rate. The resulting growth laws are discussed in the context of a coarse-grained model of phototrophic growth and available data obtained by a comprehensive literature search. Our insights into quantitative aspects of cyanobacterial acclimations to different growth rates have implications to understand and optimize photosynthetic productivity.
Cyanobacteria possess a family of one-helix high-light-inducible proteins (HLIPs) that are widely viewed as ancestors of the light-harvesting antenna of plants and algae. HLIPs are essential for viability under various stress conditions, although their exact role is not fully understood. The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains four HLIPs named HliA-D, and HliD has recently been isolated in a small protein complex and shown to bind chlorophyll and β-carotene. However, no HLIP has been isolated and characterized in a pure form up to now. We have developed a protocol to purify large quantities of His-tagged HliC from an engineered Synechocystis strain. Purified His-HliC is a pigmented homo-oligomer and is associated with chlorophyll and β-carotene with a 2:1 ratio. This differs from the 3:1 ratio reported for HliD. Comparison of these two HLIPs by resonance Raman spectroscopy revealed a similar conformation for their bound β-carotenes, but clear differences in their chlorophylls. We present and discuss a structural model of HliC, in which a dimeric protein binds four chlorophyll molecules and two β-carotenes.
- MeSH
- bakteriální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- beta-karoten metabolismus MeSH
- chlorofyl metabolismus MeSH
- multimerizace proteinu MeSH
- Ramanova spektroskopie MeSH
- rekombinantní proteiny genetika izolace a purifikace metabolismus MeSH
- světlosběrné proteinové komplexy genetika metabolismus MeSH
- Synechocystis genetika metabolismus fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
The slow kinetic phases of the chlorophyll a fluorescence transient (induction) are valuable tools in studying dynamic regulation of light harvesting, light energy distribution between photosystems, and heat dissipation in photosynthetic organisms. However, the origin of these phases are not yet fully understood. This is especially true in the case of prokaryotic oxygenic photoautotrophs, the cyanobacteria. To understand the origin of the slowest (tens of minutes) kinetic phase, the M-T fluorescence decline, in the context of light acclimation of these globally important microorganisms, we have compared spectrally resolved fluorescence induction data from the wild type Synechocystis sp. PCC 6803 cells, using orange (λ = 593 nm) actinic light, with those of mutants, ΔapcD and ΔOCP, that are unable to perform either state transition or fluorescence quenching by orange carotenoid protein (OCP), respectively. Our results suggest a multiple origin of the M-T decline and reveal a complex interplay of various known regulatory processes in maintaining the redox homeostasis of a cyanobacterial cell. In addition, they lead us to suggest that a new type of regulatory process, operating on the timescale of minutes to hours, is involved in dissipating excess light energy in cyanobacteria.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- chlorofyl chemie genetika metabolismus MeSH
- diuron chemie MeSH
- fluorescence MeSH
- fluorescenční spektrometrie MeSH
- fykobilizomy genetika metabolismus MeSH
- kyanid draselný chemie MeSH
- luminiscenční měření MeSH
- světlo MeSH
- Synechocystis chemie genetika metabolismus MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
In the model cyanobacterium Synechocystis sp. PCC 6803, the terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), forms a complex with high light-inducible proteins, the photosystem II assembly factor Ycf39 and the YidC/Alb3/OxaI membrane insertase, co-ordinating chlorophyll delivery with cotranslational insertion of nascent photosystem polypeptides into the membrane. To gain insight into the ubiquity of this assembly complex in higher photosynthetic organisms, we produced functional foreign chlorophyll synthases in a cyanobacterial host. Synthesis of algal and plant chlorophyll synthases allowed deletion of the otherwise essential native cyanobacterial gene. Analysis of purified protein complexes shows that the interaction with YidC is maintained for both eukaryotic enzymes, indicating that a ChlG-YidC/Alb3 complex may be evolutionarily conserved in algae and plants.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- fotosyntéza účinky záření MeSH
- fotosystém II (proteinový komplex) genetika metabolismus MeSH
- fylogeneze MeSH
- ligasy tvořící vazby C-O klasifikace genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- světlo MeSH
- Synechocystis genetika metabolismus MeSH
- tylakoidy metabolismus účinky záření MeSH
- vazba proteinů účinky záření MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
