Biosynthesis of Chlorophyll Regulates Reconstruction of Thylakoid Membrane in Cyanobacterium Synechocystis sp. PCC 6803

By DNA recombination technology in vitro, ORF469- mutant of cynobacterium Synechocystis sp. PCC 6803 was constructed, in which the ORF469 fragment relative to the light-inde-pendent protochlorophyllide (Pchlide) reduction was deleted. In BG-11 medium with 5 mmol/L glucose, the mutant was grown in darkness with a brief period (10 min) of illumination everyday (light-activated heterotrophic growth, LAHG) for 2 weeks to delete chlorophyll (Chl). The 665 mn Chl peak was replaced by the 629 nm Pchlide peak in the absorption spectra of the methanol extracts. The absorption spectra of the intact cells showed only shoulder peak at 620 nm (representing phyco- biliprotein). The thylakoid membrane disappeared, but the amount of phycobilisome did not decrease. When the mutant was transferred from LAHG condition to continuous light illumination for 3 h, the absorbance at 665 nm became higher than that at 629 nm and two peaks at 620 nm and 440 nm,representing phycobiliprotein and Chi-protein complex respectively, appeared in the absorption spectra of the intact cells. Mter exposure to the light for 8 h, the thylakoid membrane was visible in the cells. And for 24 h, a shoulder peak was present at 680 nm in the absorption spectra of the intact cells. Meanwhile the absorption spectra of the methanol extracts had no difference from that of cells grown in the light. Mter 48 h, the shape of the absorption spectra of the intact cells became the same as that of cells grown in the light. The layers of thylakoid membranes were as clear as those of the cells grown in the light. The results indicated that the biosynthesis of chlorophyll regulates the reconstmction of thylakoid membrane rendering the Chl protein complex to play its functional role in photosystems.     

Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803

The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that “light” plasma membranes have a high carotenoid/protein ratio, when compared to “heavier” plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitope tagging and western blots. Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone.     

Competitive Inhibitions of the Chlorophyll Synthase of Synechocystis sp. Strain PCC 6803 by Bacteriochlorophyllide a and the Bacteriochlorophyll Synthase of Rhodobacter sphaeroides by Chlorophyllide a

The photosynthetic growth of Synechocystis sp. strain PCC 6803 is hampered by exogenously added bacteriochlorophyllide a (Bchlide a) in a dose-dependent manner. The growth inhibition caused by Bchlide a, however, is relieved by an increased level of exogenously added chlorophyllide a (Chlide a). The results are explained by the competitive inhibition of chlorophyll synthase by Bchlide a, with inhibition constants (K_Is) of 0.3 mM and 1.14 mM in the presence of sufficient geranylgeranyl pyrophosphate (GGPP) and phytyl pyrophosphate (PPP), respectively. Surprisingly, the bacteriochlorophyll synthase of Rhodobacter sphaeroides is inhibited competitively by Chlide a, with K_Is of 0.54 mM and 0.77 mM in the presence of sufficient GGPP and PPP, respectively. Consistently, exogenously added Chlide a inhibits the metabolic conversion of exogenously added Bchlide a to bacteriochlorophyll a by an R. sphaeroides bchFNB-bchZ mutant that neither synthesizes nor metabolizes Chlide a. The metabolic inhibition by Chlide a, however, is relieved by the elevated level of Bchlide a. Thus, the chlorophyll synthase of Synechocystis sp. PCC 6803 and the bacteriochlorophyll synthase of R. sphaeroides, both of which perform ping-pong-type reactions, are inhibited by Bchlide a and Chlide a, respectively. Although neither inhibitor is catalyzed by the target enzyme, inhibitions in the competitive mode suggest a structural similarity between their active sites.The biosynthetic pathways for bacteriochlorophyll a (Bchl a) and chlorophyll a (Chl a) share the metabolic steps from protoporphyrin IX to chlorophyllide a (Chlide a) (Fig. ​(Fig.1).1). The C₂₀ moiety from geranylgeranyl pyrophosphate (GGPP) can be directly esterified to ring D of Chlide a by chlorophyll synthase (ChlG) to yield geranylgeranylated Chl a (Chl a_gg), which is subsequently reduced (at positions 6, 10, and 14 of GG) by chlorophyll reductase (ChlP) to yield phytylated Chl a (Chl a_p, but it is usually abbreviated as Chl a) (2, 7). The chlorophyll synthase of Avena sativa has a broad substrate specificity for C₂₀, and it may accept either GGPP or phytyl pyrophosphate (PPP) as the first substrate in its ping-pong-type reaction (27). Either a geranylgeranylated or phytylated enzyme esterifies the second substrate Chlide a, yielding Chl a_gg or Chl a, respectively (5, 24). Chlorophyll reductase reduces either the GG moiety of Chl a_gg or free GGPP, yielding Chl a or free PPP, respectively (25).Open in a separate windowFIG. 1.Chl a and Bchl a biosynthetic pathways (6, 7). The chemical structures of Chlide a and Bchlide a are shown. bchF codes for 3-vinyl bacteriochlorophyllide hydratase; bchXYZ for three subunits comprising COR; bchC for 3-hydroxyethyl bacteriochlorophyllide dehydrogenase; chlG and bchG for chlorophyll synthase and bacteriochlorophyll synthase, respectively; and chlP and bchP for chlorophyll reductase and bacteriochlorophyll reductase, respectively.Chlide a may be further metabolized to bacteriochlorophyllide a (Bchlide a) (Fig. ​(Fig.1).1). Chlide a reductase (COR) reduces ring B of Chlide a to form 3-vinyl bacteriochlorophyllide a, whose C-3-vinyl group on ring A is then converted into an acetyl group through the activities of hydratase (BchF) and dehydrogenase (BchC) to form Bchlide a (6). The hydratase reaction may alternatively precede that of COR (Fig. ​(Fig.1).1). Once Bchlide a is formed, its ring D is esterified with the C₂₀ geranylgeranyl moiety by bacteriochlorophyll synthase (BchG), yielding geranylgeranylated Bchl a (Bchl a_gg) (3). The C₂₀ moiety is subsequently reduced by bacteriochlorophyll reductase (BchP), yielding the phytylated Bchl a (Bchl a_p, but it is usually abbreviated as Bchl a) (1).The biosynthesis of Chl a has been regarded as a metabolism that evolved after Bchl a (33). ChlG and ChlP have been thought to emerge through the gene duplication of BchG and BchP, respectively. Recently, we found that the COR reaction, which is specific to Bchl a biosynthesis, generates superoxide at low levels of oxygen (16), and we further proposed that the degeneration of the superoxide-generating COR step may be associated with the emergence of cyanobacterium-based Chl a biosynthesis (15).The predicted sequence of ChlG of Synechocystis sp. strain PCC 6803 bears 35% identity with that of Rhodobacter sphaeroides BchG. Nonetheless, chlorophyll synthase and bacteriochlorophyll synthase exhibit a high degree of substrate specificity to distinguish their own Mg-tetrapyrrole substrates from that of the other enzyme (23, 28). We further examined whether chlorophyll synthase and bacteriochlorophyll synthase are affected by Bchlide a and Chlide a, respectively, which are structurally similar to each other. In this work, we found that the chlorophyll synthase of Synechocystis sp. PCC 6803 is competitively inhibited by Bchlide a. We further found that the bacteriochlorophyll synthase of R. sphaeroides is competitively inhibited by Chlide a. Thus, the active site of chlorophyll synthase is recognized by Bchlide a, while that of bacteriochlorophyll synthase is recognized by Chlide a. The results suggest a structural similarity between the active sites of the two enzymes.     

Genomic Structure of the Cyanobacterium Synechocystis sp. PCC 6803 Strain GT-S

Synechocystis sp. PCC 6803 is the most popular cyanobacterial strain, serving as a standard in the research fields of photosynthesis, stress response, metabolism and so on. A glucose-tolerant (GT) derivative of this strain was used for genome sequencing at Kazusa DNA Research Institute in 1996, which established a hallmark in the study of cyanobacteria. However, apparent differences in sequences deviating from the database have been noticed among different strain stocks. For this reason, we analysed the genomic sequence of another GT strain (GT-S) by 454 and partial Sanger sequencing. We found 22 putative single nucleotide polymorphisms (SNPs) in comparison to the published sequence of the Kazusa strain. However, Sanger sequencing of 36 direct PCR products of the Kazusa strains stored in small aliquots resulted in their identity with the GT-S sequence at 21 of the 22 sites, excluding the possibility of their being SNPs. In addition, we were able to combine five split open reading frames present in the database sequence, and to remove the C-terminus of an ORF. Aside from these, two of the Insertion Sequence elements were not present in the GT-S strain. We have thus become able to provide an accurate genomic sequence of Synechocystis sp. PCC 6803 for future studies on this important cyanobacterial strain.     

Multiple light inputs control phototaxis in Synechocystis sp. strain PCC6803

The phototactic behavior of individual cells of the cyanobacterium Synechocystis sp. strain PCC6803 was studied with a glass slide-based phototaxis assay. Data from fluence rate-response curves and action spectra suggested that there were at least two light input pathways regulating phototaxis. We observed that positive phototaxis in wild-type cells was a low fluence response, with peak spectral sensitivity at 645 and 704 nm. This red-light-induced phototaxis was inhibited or photoreversible by infrared light (760 nm). Previous work demonstrated that a taxD1 mutant (Cyanobase accession no. sll0041; also called pisJ1) lacked positive but maintained negative phototaxis. Therefore, the TaxD1 protein, which has domains that are similar to sequences found in both bacteriophytochrome and the methyl-accepting chemoreceptor protein, is likely to be the photoreceptor that mediates positive phototaxis. Wild-type cells exhibited negative phototaxis under high-intensity broad-spectrum light. This phenomenon is predominantly blue light responsive, with a maximum sensitivity at approximately 470 nm. A weakly negative phototactic response was also observed in the spectral region between 600 and 700 nm. A deltataxD1 mutant, which exhibits negative phototaxis even under low-fluence light, has a similar action maximum in the blue region of the spectrum, with minor peaks from green to infrared (500 to 740 nm). These results suggest that while positive phototaxis is controlled by the red light photoreceptor TaxD1, negative phototaxis in Synechocystis sp. strain PCC6803 is mediated by one or more (as yet) unidentified blue light photoreceptors.     

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10⁵ molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.     

Three-Piece-Ligation PCR and Application in Disruption of Chlorophyll Synthesis Genes in Synechocystis sp. PCC 6803

Linear DNA, consisting of a drug-resistance marker and long flanking sequences, was synthesized by one-step polymerase chain reaction after a three-piece ligating reaction. Chlorophyll synthesis genes, chlH and chlL in Synechocystis sp. PCC 6803, were replaced by a kanamycin-resistance marker through double recombinations with flanking homology regions. Under LAHG conditions, the chlL but not chlH mutant stopped chlorophyll synthesis, while both synthesized chlorophyll in the light. Received: 20 June 2001 / Accepted: 30 July 2001     

Chlorophyll b inhibits the formation of photosystem I trimer in Synechocystis sp. PCC6803

Chlorophyllide a oxygenase (CAO) catalyzes two-step oxygenation reactions and converts chlorophyllide a to chlorophyllide b. When CAO was introduced into the Synechocystis sp. PCC6803 genome, chlorophyll b was synthesized and incorporated into P700-chlorophyll a-protein complexes. Curve analysis of photosystem I particles showed that Ca687 was decreased with a concomitant increase in Cb652 suggesting that chlorophyll b was incorporated into Ca687-binding sites. When the level of chlorophyll b was high, Ca704, which is known as red chlorophyll and photosystem I trimers were decreased. Formation of photosystem I trimers is discussed in relation to red chlorophyll and chlorophyll b accumulation.     

Prediction of Translation Initiation Sites on the Genome of Synechocystis sp. Strain PCC6803 by Hidden Markov Model

We developed a computer program, GeneHackerTL, which predictsthe most probable translation initiation site for a given nucleotidesequence. The program requires that information be extractedfrom the nucleotide sequence data surrounding the translationinitiation sites according to the framework of the Hidden MarkovModel. Since the translation initiation sites of 72 highly abundantproteins have already been assigned on the genome of Synechocystissp. strain PCC6803 by amino-terminal analysis, we extractednecessary information for GeneHackerTL from the nucleotide sequencedata. The prediction rate of the GeneHackerTL for these proteinswas estimated to be 86.1%. We then used GeneHackerTL for predictionof the translation initiation sites of 24 other proteins, ofwhich the initiation sites were not assigned experimentally,because of the lack of a potential initiation codon at the amino-terminalposition. For 20 out of the 24 proteins, the initiation siteswere predicted in the upstream of their amino-terminal positions.According to this assignment, the processed regions representa typical feature of signal peptides. We could also predictmultiple translation initiation sites for a particular genefor which at least two initiation sites were experimentallydetected. This program would be e.ective for the predictionof translation initiationsites of other proteins, not only inthis species but also in other prokaryotes as well.     

Pitt,a Novel Tetratricopeptide Repeat Protein Involved in Light-Dependent Chlorophyll Biosynthesis and Thylakoid Membrane Biogenesis in Synechocystis sp. PCC 6803

Biogenesis of photosynthetic pigment/protein complexes is a highly regulated process that requires various assisting factors. Here, we report on the molecular analysis of the Pitt gene （sir1644） from the cyanobacterium Synechocystis sp. PCC 6803 （Synechocystis 6803） that encodes a membrane-bound tetratricopeptide repeat （TPR） protein of formerly unknown function. Targeted inactivation of Pitt affected photosynthetic performance and light-dependent chlorophyll synthesis. Yeast two-hybrid analyses and native PAGE strongly suggest a complex formation between Pitt and the light-dependent protochlorophyllide oxidoreductase （POR）. Consistently, POR levels are approximately threefold reduced in the pitt insertion mutant. The membrane sublocalization of Pitt was found to be dependent on the presence of the periplasmic photosystem Ⅱ （PSⅡ） biogenesis factor PratA, supporting the idea that Pitt is involved in the early steps of photosynthetic pigment/protein complex formation.     

Photoheterotrophic Fluxome in Synechocystis sp. Strain PCC 6803 and Its Implications for Cyanobacterial Bioenergetics

This study investigated metabolic responses in Synechocystis sp. strain PCC 6803 to photosynthetic impairment. We used 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU; a photosystem II inhibitor) to block O₂ evolution and ATP/NADPH generation by linear electron flow. Based on ¹³C-metabolic flux analysis (¹³C-MFA) and RNA sequencing, we have found that Synechocystis sp. PCC 6803 employs a unique photoheterotrophic metabolism. First, glucose catabolism forms a cyclic route that includes the oxidative pentose phosphate (OPP) pathway and the glucose-6-phosphate isomerase (PGI) reaction. Glucose-6-phosphate is extensively degraded by the OPP pathway for NADPH production and is replenished by the reversed PGI reaction. Second, the Calvin cycle is not fully functional, but RubisCO continues to fix CO₂ and synthesize 3-phosphoglycerate. Third, the relative flux through the complete tricarboxylic acid (TCA) cycle and succinate dehydrogenase is small under heterotrophic conditions, indicating that the newly discovered cyanobacterial TCA cycle (via the γ-aminobutyric acid pathway or α-ketoglutarate decarboxylase/succinic semialdehyde dehydrogenase) plays a minimal role in energy metabolism. Fourth, NAD(P)H oxidation and the cyclic electron flow (CEF) around photosystem I are the two main ATP sources, and the CEF accounts for at least 40% of total ATP generation from photoheterotrophic metabolism (without considering maintenance loss). This study not only demonstrates a new topology for carbohydrate oxidation but also provides quantitative insights into metabolic bioenergetics in cyanobacteria.     

A gene of Synechocystis sp. Strain PCC 6803 encoding a novel iron transporter

A mutant of Synechocystis sp. strain PCC 6803 disrupted for sll1878 exhibited greatly reduced Fe(3+) transport activity. The K(m) value of sll1878-dependent Fe(3+) transport in cells grown in iron-replete medium was 0.5 microM. Both the maximal rate and K(m) value were increased in iron-starved cells.     

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.     

Accumulation of poly-beta-hydroxybutyrate in cyanobacterium Synechocystis sp. PCC6803

Accumulation of poly-beta-hydroxybutyrate (PHB) by photoautotrophic microorganisms makes it possible to reduce the production cost of PHB. The Synechocystis sp. PCC6803 cells grown in BG11 medium under balanced, nitrogen-starved or phosphorus-starved conditions were observed by transmission electron microscope. Many electron-transparent granules in the nitrogen-starved cells had a diameter up to 0.8 micron. In contrast, the number of granules in the normally cultured cells decreased obviously and only zero to three much smaller granules were in each cell. These granules were similar to those in bacteria capable of synthesizing PHB. They were proved to be PHB by gas chromatography after subjecting the cells to methanolysis. Effects of glucose as carbon source and light intensity on PHB accumulation in Synechocystis sp. PCC6803 under nitrogen-starved cultivation were further studied. Glucose and illumination promoted cell growth but did not favor PHB synthesis. After 7 days of growth under nitrogen-starved photoautotrophic conditions, the intracellular level of PHB was up to 4.1% of cellular dry weight and the PHB concentration in the culture broth was 27 mg/l.     

Proteomic analysis of heterotrophy in Synechocystis sp. PCC 6803

To provide an insight into the heterotrophic metabolism of cyanobacteria, a proteomic approach has been employed with the model organism Synechocystis sp. PCC 6803. The soluble proteins from Synechocystis grown under photoautotrophic and light-activated heterotrophic conditions were separated by 2-DE and identified by MALDI-MS or LC-MS/MS analysis. 2-DE gels made using narrow- and micro-range IPG strips allowed quantitative comparison of more than 900 spots. Out of 67 abundant protein spots identified, 13 spots were increased and 9 decreased under heterotrophy, representing all the major fold changes. Proteomic alterations and activity levels of selected enzymes indicate a shift in the central carbon metabolism in response to trophic change. The significant reduction in light-saturated rate of photosynthesis as well as in the expression levels of rubisco and CO(2)-concentrating mechanism proteins under heterotrophy indicates the down-regulation of the photosynthetic machinery. Alterations in the expression level of proteins involved in carbon utilization pathways refer to enhanced glycolysis, oxidative pentose phosphate pathway as well as tricarboxylic acid cycle under heterotrophy. Proteomic evidences also suggest an enhanced biosynthesis of amino acids such as histidine and serine during heterotrophic growth.     

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.     

Putrescine transport in a cyanobacterium Synechocystis sp. PCC 6803

The transport of putrescine into a moderately salt tolerant cyanobacterium Synechocystis sp. PCC 6803 was characterized by measuring the uptake of radioactively-labeled putrescine. Putrescine transport showed saturation kinetics with an apparent K(m) of 92 +/- 10 microM and V(max) of 0.33 +/- 0.05 nmol/min/mg protein. The transport of putrescine was pH-dependent with highest activity at pH 7.0. Strong inhibition of putrescine transport was caused by spermine and spermidine whereas only slight inhibition was observed by the addition of various amino acids. These results suggest that the transport system in Synechocystis sp. PCC 6803 is highly specific for polyamines. Putrescine transport is energy-dependent as evidenced by the inhibition by various metabolic inhibitors and ionophores. Slow growth was observed in cells grown under salt stress. Addition of low concentration of putrescine could restore growth almost to the level observed in the absence of salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased putrescine transport with an optimum 2-fold increase at 20 mosmol/kg. The stimulation of putrescine transport mediated by osmotic upshift was abolished in chloramphenicol-treated cells, suggesting possible involvement of an inducible transport system.