Thylakoid Membrane-Bound, NADPH-Specific Pyridine Nucleotide Dehydrogenase Complex Mediates Cyclic Electron Transport in the Cyanobacterium Synechocystis sp. PCC 6803

The donation of electrons from NADPH to the intersystem chain,as monitored by an increase in Chl fluorescence, occurred inthe isolated thylakoid membranes of Synechocystis PCC 6803.The stimulation by NADPH of the methyl viologen-dependent photoreductionof dioxygen and of the reduction of P700⁺ after photooxidationin the presence of DCMU also confirmed the donation of electronsfrom NADPH to the electron carriers in the intersystem. Thesereactions were sensitive to rotenone, capsaicin, l-(2-thenoyl)-3,3,3-trifluoroacetoneand HgCl₂ but not to antimycin A or flavone. In contrast tothe thylakoid membranes from the wild type, those from a mutant,designated M55, in which a gene of a subunit of the pyridinenucleotide dehydrogenase complex (NDH) had been inactivated,did not show evidence of such reactions. These results supportour previous hypothesis that the transport of electrons fromNADPH to the intersystem chain is mediated by NDH [Mi et al.(1994) Plant Cell Physiol. 35: 163] and indicate the bindingof an NADPH-specific NDH to the thylakoid membranes. The Chlfluorescence was quenched transiently by addition of ferredoxinand NADP₊ to the thylakoid membranes but showed a subsequentincrease. This result suggests the reduction of plastoquinoneby the photoreduced NADP₊ and initiation of the NADPH-mediatedcyclic flow of electrons around PSI. Furthermore, a similarresponse of Chl fluorescence was observed upon the additionof ferredoxin only, demonstrating the ferredoxin-dependent cyclicflow of electrons. Both pathways of cyclic electron transportwere inhibited by rotenone, and were not detected in the NDH-defectedthylakoid membranes from M55, indicating the participation ofthe NDH complex. These results confirm that, in Synechocystis,the thylakoid-bound NDH complex mediates the ferredoxin-dependentcyclic electron flow, as well as the NADPH-dependent cyclicelectron flow. (Received November 24, 1994; Accepted March 16, 1995)     

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.     

Thylakoid Membrane Reduction Affects the Photosystem Stoichiometry in the Cyanobacterium Synechocystis sp. PCC 6803

Biogenesis of thylakoid membranes in both chloroplasts and cyanobacteria is largely not understood today. The vesicle-inducing protein in plastids 1 (Vipp1) has been suggested to be essential for thylakoid membrane formation in Arabidopsis (Arabidopsis thaliana), as well as in the cyanobacterium Synechocystis sp. PCC 6803, although its exact physiological function remains elusive so far. Here, we report that, upon depletion of Vipp1 in Synechocystis cells, the number of thylakoid layers in individual Synechocystis cells decreased, and that, in particular, the content of photosystem I (PSI) complexes was highly diminished in thylakoids. Furthermore, separation of native photosynthetic complexes indicated that PSI trimers are destabilized and the monomeric species is enriched. Therefore, depletion of thylakoid membranes specifically affects biogenesis and/or stabilization of PSI in cyanobacteria.In chloroplasts and cyanobacteria the energy transfer between PSI and PSII is regulated in a light-dependent manner (for a recent review, see Kramer et al., 2004). The two photosystems are connected by the cytochrome b₆f complex, and electron transfer from PSII via the cytochrome b₆f complex to PSI is believed to be regulated by the redox state of the plastoquinol pool potentially also involving the cytochrome b₆f complex (Fujita et al., 1987; Murakami and Fujita, 1993; Schneider et al., 2001, 2004; Pfannschmidt, 2003; Volkmer et al., 2007). Transfer of light energy to the two photosystems is mediated by light-harvesting complexes, and in cyanobacteria light is harvested by the soluble extramembranous phycobilisomes. The efficient energy transfer to PSI and PSII has to be balanced to synchronize the function of the two photosystems. In response to changing light intensities and qualities, energy coupling between the phycobilisomes and the photosystems changes, which allows a rapid adjustment of light absorbance by the individual photosystems. Furthermore, besides this short-term adaptation mechanism, it has been shown in many studies that on a longer term in cyanobacteria the ratio of the two photosystems changes depending on the light conditions (Manodori and Melis, 1986; Murakami and Fujita, 1993; Murakami et al., 1997). Upon shifting cyanobacterial cells from low-light to high-light growth conditions, the PSI-to-PSII ratio decreases due to selective suppression of the amount of functional PSI. In recent years, some genes have already been identified that are involved in this regulation of the photosystem stoichiometry (Hihara et al., 1998; Sonoike et al., 2001; Fujimori et al., 2005; Ozaki et al., 2007).Whereas in chloroplasts of higher plants and green algae the amounts of the two photosystems change in response to changing light conditions (Melis, 1984; Chow et al., 1990; Smith et al., 1990; Kim et al., 1993), it has already been noted a long time ago that the chloroplast ultrastructure also adapts to high-light and low-light conditions (Melis, 1984). Chloroplasts of plants grown under low light or far-red light have more thylakoid membranes than chloroplasts of plants grown under high light or blue light (Anderson et al., 1973; Lichtenthaler et al., 1981; Melis and Harvey, 1981). There appears to be a direct correlation between the chlorophyll content and the amount of thylakoids per chloroplast because light harvesting is increased by enhanced chlorophyll and thylakoid membrane content per chloroplast. Thus, chloroplasts adapt to high light both by a reduction of thylakoid membranes and by a decrease in the PSI-to-PSII ratio.Thylakoid membranes are exclusive features of both cyanobacteria and chloroplasts, and it still remains mysterious how formation of thylakoid membranes is organized. Many cellular processes, like lipid biosynthesis, membrane formation, protein synthesis in the cytoplasm and/or at a membrane, protein transport, protein translocation, and protein folding have to be organized and aligned for formation of internal thylakoid membranes. The recent observation that deletion of the vipp1 gene in Arabidopsis (Arabidopsis thaliana) results in complete loss of thylakoid membranes has indicated that Vipp1 is involved in biogenesis of thylakoid membranes. Further analysis has suggested that Vipp1 could be involved in vesicle trafficking between the inner envelope and the thylakoid membrane of chloroplasts (Kroll et al., 2001). Because of this, the protein was named Vipp1, for vesicle-inducing protein in plastids 1. Depletion of Vipp1 strongly affected the ability of cyanobacterial cells to form proper thylakoid membranes (Westphal et al., 2001) and, consequently, also in cyanobacteria Vipp1 appears to be involved in formation of thylakoid membranes. A Vipp1 depletion strain of Arabidopsis is deficient in photosynthesis, although the defect could not be assigned to a deficiency of a single photosynthetic complex, but appeared to be caused by dysfunction of the entire photosynthetic electron transfer chain (Kroll et al., 2001). Therefore, depletion of Vipp1 in Arabidopsis seems to affect thylakoid membrane formation rather than the assembly of thylakoid membrane protein complexes (Aseeva et al., 2007). However, for cyanobacteria, it is not clear yet how diminishing the amount of thylakoid membrane layers would affect the amount and stoichiometry of the two photosystems.Here, we present the generation and characterization of a Vipp1 depletion strain of the cyanobacterium Synechocystis sp. PCC 6803. Upon depletion of Vipp1, a decrease in thylakoid membrane pairs in the generated mutant strain and, furthermore, a significant decrease in active PSI centers was observed. Moreover, trimerization of PSI also appeared to be impaired in the mutant strain. These results suggest that thylakoid membrane perturbations caused by the Vipp1 depletion directly affects PSI assembly and stability in cyanobacterial thylakoid membranes.     

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.     

Thylakoid Membrane Maturation and PSII Activation Are Linked in Greening Synechocystis sp. PCC 6803 Cells

Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. While they are crucial for phototrophic growth of cyanobacterial cells, biogenesis of thylakoid membranes is not well understood yet. Dark-grown Synechocystis sp. PCC 6803 cells contain only rudimentary thylakoid membranes but still a relatively high amount of phycobilisomes, inactive photosystem II and active photosystem I centers. After shifting dark-grown Synechocystis sp. PCC 6803 cells into the light, “greening” of Synechocystis sp. PCC 6803 cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions, was monitored. Complete restoration of a typical thylakoid membrane system was observed within 24 hours after an initial lag phase of 6 to 8 hours. Furthermore, activation of photosystem II complexes and restoration of a functional photosynthetic electron transport chain appears to be linked to the biogenesis of organized thylakoid membrane pairs.Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. The intracellular thylakoid membranes of cyanobacteria harbor the protein complexes of the photosynthetic electron transport chain (Nowaczyk et al., 2010; Bernat and Rögner, 2011). The photosynthetic electron transport chain is composed of three large membrane protein complexes, i.e. PSII, the cytochrome b₆f complex, and PSI. Excitation energy trapping by PSII results in water splitting at the PSII donor side within the thylakoid lumen and transport of electrons to the primary and secondary electron accepting quinone molecules Q_A and Q_B, respectively. Following double reduction and protonation, Q_B is released from PSII into the plastoquinone (PQ) pool and delivers electrons to the cytochrome b₆f complex. The cytochrome b₆f complex transfers the electrons to the soluble electron carrier plastocyanin or cytochrome c₆, which subsequently reduces PSI. For efficient light harvesting, cyanobacteria contain soluble light-harvesting antenna proteins, the phycobilisomes (PBSs), which transfer light energy to the photosynthetic reaction centers. In cyanobacteria, the PSI-to-PSII ratio is controlled by light and by the redox state of the PQ/PQH₂-pool (Fujita et al., 1987), and under high-light growth conditions, typically less PSI is present in cyanobacterial thylakoid membranes compared with low-light conditions (Fujita et al., 1994).Thylakoid membranes and photosynthetic electron transport are essential for phototrophic growth of cyanobacterial cells. Despite their importance for survival of cyanobacteria, biogenesis of thylakoid membranes is yet not well understood. It still is an ongoing debate whether the internal membrane systems (cytoplasmic and thylakoid membranes) are connected in cyanobacteria or not, and thus whether thylakoids represent a completely separated membrane entity (Liberton et al., 2006; van de Meene et al., 2006, 2012; Schneider et al., 2007). Up to now, only few proteins have been described to be involved in thylakoid membrane biogenesis. Among them the Vipp1 protein (vesicle inducing protein in plastids1) seems to play an important role in thylakoid membrane biogenesis, as in chloroplasts of Arabidopsis (Arabidopsis thaliana) and in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), depletion of Vipp1 results in a reduced thylakoid membrane system (Kroll et al., 2001; Westphal et al., 2001). While the exact physiological function of the protein is not yet known (Vothknecht et al., 2012), depletion of Vipp1 in Synechocystis not only results in reduced thylakoid membrane formation, but also affects the activity and structure of components of the photosynthetic electron transport chain (Fuhrmann et al., 2009; Gao and Xu, 2009).As complexes of the respiratory electron transport chain are also localized in cyanobacterial thylakoids, the photosynthetic and respiratory electron transport pathways are highly interconnected and both contribute to formation of an electrochemical gradient across the thylakoid membrane and energy production. Due to this, Synechocystis is able to grow completely heterotrophically under light-activated photoheterotrophic growth (LAHG) conditions in the presence of high Glc concentrations (Anderson and McIntosh, 1991; Smart et al., 1991).In this study, we have used dark-grown Synechocystis cells to investigate “greening” of Synechocystis cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions. Following transfer of Synechocystis cells into the light, complete restoration of a typical thylakoid membrane system was observed within 24 h. While dark-grown Synechocystis cells contained only rudimentary thylakoid membranes, they still contained a high concentration of PBSs, active PSI as well as inactive PSII complexes. Activation of PSII complexes appears to be linked to the biogenesis of organized thylakoid membrane pairs.     

NdhV Is a Subunit of NADPH Dehydrogenase Essential for Cyclic Electron Transport in Synechocystis sp. Strain PCC 6803

Two mutants sensitive to heat stress for growth and impaired in NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET) were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in the same sll0272 gene, encoding a protein highly homologous to NdhV identified in Arabidopsis (Arabidopsis thaliana). Deletion of the sll0272 gene (ndhV) did not influence the assembly of NDH-1 complexes and the activities of CO₂ uptake and respiration but reduced the activity of NDH-CET. NdhV interacted with NdhS, a ferredoxin-binding subunit of cyanobacterial NDH-1 complex. Deletion of NdhS completely abolished NdhV, but deletion of NdhV had no effect on the amount of NdhS. Reduction of NDH-CET activity was more significant in ΔndhS than in ΔndhV. We therefore propose that NdhV cooperates with NdhS to accept electrons from reduced ferredoxin.Cyanobacterial NADPH dehydrogenase (NDH-1) complexes are localized in the thylakoid membrane (Ohkawa et al., 2001, 2002; Zhang et al., 2004; Xu et al., 2008; Battchikova et al., 2011b) and participate in a variety of bioenergetic reactions, such as respiration, cyclic electron transport around photosystem I (NDH-CET), and CO₂ uptake (Ogawa, 1991; Mi et al., 1992; Ohkawa et al., 2000). Structurally, the cyanobacterial NDH-1 complexes closely resemble energy-converting complex I in eubacteria and the mitochondrial respiratory chain regardless of the absence of homologs of three subunits in cyanobacterial genomes that constitute the catalytically active core of complex I (Friedrich et al., 1995; Friedrich and Scheide, 2000; Arteni et al., 2006). Over the past decade, new subunits of NDH-1 complexes specific to oxygenic photosynthesis have been identified in several cyanobacterial strains. They are NdhM to NdhQ and NdhS (Prommeenate et al., 2004; Battchikova et al., 2005, 2011b; Nowaczyk et al., 2011; Wulfhorst et al., 2014; Zhang et al., 2014; Zhao et al., 2014b, 2015), in addition to NdhL first identified in the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803) about 20 years ago (Ogawa, 1992). Among them, NdhS possesses a ferredoxin (Fd)-binding motif and was shown to bind Fd, which suggested that Fd is one of the electron donors to NDH-1 complexes (Mi et al., 1995; Battchikova et al., 2011b; Ma and Ogawa, 2015). Deletion of NdhS strongly reduced the activity of NDH-CET but had no effect on respiration and CO₂ uptake (Battchikova et al., 2011b; Ma and Ogawa, 2015). The NDH-CET plays an important role in coping with various environmental stresses regardless of its elusive mechanism. For example, this function can greatly alleviate heat-sensitive growth phenotypes (Wang et al., 2006a; Zhao et al., 2014a). Thus, heat treatment strategy can help in identifying the proteins essential to NDH-CET.Here, a new oxygenic photosynthesis-specific (OPS) subunit NdhV was identified in Synechocystis 6803 with the help of heat treatment strategy, and its deletion did not influence the assembly of NDH-1L and NDH-1MS complexes and the activities of CO₂ uptake and respiration but impaired the NDH-CET activity. We give evidence that NdhV interacts with NdhS and is another component of Fd-binding domain of cyanobacterial NDH-1 complex. A possible role of NdhV on the NDH-CET activity is discussed.     

Subunit Q Is Required to Stabilize the Large Complex of NADPH Dehydrogenase in Synechocystis sp. Strain PCC 6803

Two major complexes of NADPH dehydrogenase (NDH-1) have been identified in cyanobacteria. A large complex (NDH-1L) contains NdhD1, NdhF1, and NdhP, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, NDH-1-dependent cyclic electron transport around photosystem I, and CO₂ uptake. Two mutants sensitive to high light for growth and impaired in cyclic electron transport around photosystem I were isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in an open reading frame encoding a product highly homologous to NdhQ, a single-transmembrane small subunit of the NDH-1L complex, identified in Thermosynechococcus elongatus by proteomics strategy. Deletion of ndhQ disassembled about one-half of the NDH-1L to NDH-1M and consequently impaired respiration, but not CO₂ uptake. During prolonged incubation of the thylakoid membrane with n-dodecyl-β-d-maltoside at room temperature, the rest of the NDH-1L in ΔndhQ was disassembled completely to NDH-1M and was much faster than in the wild type. In the ndhP-deletion mutant (ΔndhP) background, absence of NdhQ almost completely disassembled the NDH-1L to NDH-1M, similar to the results observed in the ΔndhD1/ΔndhD2 mutant. We therefore conclude that both NdhQ and NdhP are essential to stabilize the NDH-1L complex.Cyanobacterial NADPH dehydrogenase (NDH-1) complexes are localized in the thylakoid membrane (Ohkawa et al., 2001, 2002; Zhang et al., 2004; Xu et al., 2008; Battchikova et al., 2011a) and participate in a variety of bioenergetic reactions, such as respiration, cyclic electron transport around PSI, and CO₂ uptake (Ogawa, 1991; Mi et al., 1992; Ohkawa et al., 2000). Structurally, the cyanobacterial NDH-1 complexes closely resemble energy-converting complex I in eubacteria and the mitochondrial respiratory chain, regardless of the absence of homologs of three subunits in cyanobacterial genomes that constitute the catalytically active core of complex I (Friedrich et al., 1995; Friedrich and Scheide, 2000; Arteni et al., 2006). Over the past few years, significant achievements have been made in resolving the subunit compositions and functions of the multiple NDH-1 complexes in several cyanobacterial strains (for review, see Battchikova and Aro, 2007; Ogawa and Mi, 2007; Ma, 2009; Battchikova et al., 2011b; Ma and Ogawa, 2015). Four types of NDH-1 have been identified in the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter, Synechocystis 6803), and all four types of NDH-1 are involved in NDH-1-dependent cyclic electron transport (CET) around PSI (NDH-CET; Bernát et al., 2011). The NDH-CET plays an important role in coping with various environmental stresses, regardless of its elusive mechanism. For example, this function can greatly alleviate high light-sensitive growth phenotypes (Endo et al., 1999; Battchikova et al., 2011a; Dai et al., 2013; Zhang et al., 2014; Zhao et al., 2014). Therefore, high light strategy can help in identifying the proteins essential to NDH-CET.Proteomics studies revealed the presence of three major NDH-1 complexes in cyanobacteria: a large complex (NDH-1L), a medium size complex (NDH-1M), and a small complex (NDH-1S) with molecular masses of about 460, 350, and 200 kD, respectively (Herranen et al., 2004). NDH-1M consists of 14 subunits (i.e. NdhA–NdhC, NdhE, NdhG–NdhO, and NdhS). In addition to these subunits, the NDH-1L complex contains NdhD1, NdhF1, NdhP, and NdhQ (Prommeenate et al., 2004; Battchikova et al., 2005, 2011b; Zhang et al., 2005, 2014; Nowaczyk et al., 2011; Wulfhorst et al., 2014; Ma and Ogawa, 2015) and is involved in respiration (Zhang et al., 2004). NDH-1S is composed of NdhD3, NdhF3, CO₂ uptake A (CupA), and CupS (Ogawa and Mi, 2007) and is considered to be associated with NDH-1M in the cells as a functional complex NDH-1MS (Zhang et al., 2004, 2005) participating in CO₂ uptake. Among the several copies of ndhD and ndhF genes found in cyanobacterial genomes, ndhD1 and ndhF1 show the highest homology to chloroplast ndhD and ndhF genes, respectively, and CupA and CupS subunits of the cyanobacteria have no counterparts in higher plants. These facts suggest that the structure and composition of NDH-1L, but not the NDH-1MS complex, are similar to those of the chloroplast NDH-1 complex (Battchikova and Aro, 2007; Ogawa and Mi, 2007; Shikanai, 2007; Ma, 2009; Suorsa et al., 2009; Battchikova et al., 2011b; Ifuku et al., 2011; Peng et al., 2011a; Ma and Ogawa, 2015). Despite their similarity, a large number of subunits that constitute the chloroplast NDH-1 complex, including ferredoxin-binding subcomplex subunits NdhT and NdhU and all the subunits of subcomplex B and lumen subcomplex, are absent in the cyanobacterial NDH-1L complex (Battchikova et al., 2011b; Ifuku et al., 2011; Peng et al., 2011a). This implies that the stabilization strategies for the cyanobacterial NDH-1L complex and chloroplastic NDH-1 complex might be significantly different.Recently, a new oxygenic photosynthesis-specific small subunit NdhQ was identified in the NDH-1L complex purified by Ni²⁺ affinity chromatography from Thermosynechococcus elongatus (Nowaczyk et al., 2011). NdhQ is extensively present in cyanobacteria, but its homolog is absent in higher plants (Nowaczyk et al., 2011). In this study, we demonstrate that deletion of NdhQ disassembled the NDH-1L into NDH-1M, but not NDH-1MS, in Synechocystis 6803 and consequently impaired respiration, but not CO₂ uptake. NdhQ and NdhP stabilize the NDH-1L complex. Thus, the stabilization strategy of cyanobacterial NDH-1L is distinctly different from that of the chloroplastic NDH-1 complex.     

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.     

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.     

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.     

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.     

Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803

Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. strain PCC6803 the two metabolic pathways are linked by coregulated genes that are involved in arsenic transport, sulfide oxidation, and probably in sulfide-based alternative photosynthesis. Although Synechocystis sp. strain PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis, we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. suoS (sll5036) encodes a light-dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200-fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as an arsenite importer at permissive concentrations. We propose that the proteins encoded by the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and that the operon was acquired by the cyanobacterium via horizontal gene transfer.     

Polyhydroxybutyrate particles in Synechocystis sp. PCC 6803: facts and fiction

Transmission electron microscopy has been used to identify poly-3-hydroxybutyrate (PHB) granules in cyanobacteria for over 40 years. Spherical inclusions inside the cell that are electron-transparent and/or slightly electron-dense and that are found in transmission electron micrographs of cyanobacteria are generally assumed to be PHB granules. The aim of this study was to test this assumption in different strains of the cyanobacterium Synechocystis sp. PCC 6803. Inclusions that resemble PHB granules were present in strains lacking a pair of genes essential for PHB synthesis and in wild-type cells under conditions that no PHB granules could be detected by fluorescence staining of PHB. Indeed, in these cells PHB could not be demonstrated chemically by GC/MS either. Based on the results gathered, it is concluded that not all the slightly electron-dense spherical inclusions are PHB granules in Synechocystis sp. PCC 6803. This result is potentially applicable to other cyanobacteria. Alternate assignments for these inclusions are discussed.     

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.