Active NDH-1 complexes from the cyanobacterium Synechocystis sp. strain PCC 6803

We identified eight bands by staining native gels for NADPH-nitrobluetetrazolium oxidoreductase activity after electrophoresis ofn-dodecyl-ß-D-maltoside-treated membranes of Synechocystissp. strain PCC 6803. Among them, bands A, C, D and E were attributedto the activity of NADPH dehydrogenase (NDH-1). Band A is ahighly active supercomplex of NDH-1 (about 1,000 kDa) that wasabsent in the     

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b₆/f complex, plastocyanin, and cytochrome c₆, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700⁺) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700⁺ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700⁺ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

     

Properties of mutants of Synechocystis sp. strain PCC 6803 lacking inorganic carbon sequestration systems

A mutant (Delta5) of Synechocystis sp. strain PCC 6803 constructed by inactivating five inorganic carbon sequestration systems did not take up CO(2) or HCO(3)(-) and was unable to grow in air with or without glucose. The Delta4 mutant in which BicA is the only active inorganic carbon sequestration system showed low activity of HCO(3)(-) uptake and grew under these conditions but more slowly than the wild-type strain. The Delta5 mutant required 1.7% CO(2) to attain half the maximal growth rate. Electron transport activity of the mutants was strongly inhibited under high light intensities, with the Delta5 mutant more susceptible to high light than the Delta4 mutant. The results implicated the significance of carbon sequestration in dissipating excess light energy.     

Identification of NdhL and Ssl1690 (NdhO) in NDH-1L and NDH-1M complexes of Synechocystis sp. PCC 6803

The subunit compositions of two types of NAD(P)H dehydrogenase complexes of Synechocystis sp. PCC 6803, NDH-1L and NDH-1M, were studied by two-dimensional blue-native/SDS-PAGE followed by electrospray tandem mass spectrometry. Fifteen proteins were observed in NDH-1L including hydrophilic subunits (NdhH, -K, -I, -J, -M, and -N) and hydrophobic subunits (NdhA, -B, -E, -G, -D1, and -F1). In addition, NdhL and a novel subunit, Ssl1690 (designated NdhO), were shown to be components of this complex. All subunits mentioned above were present in the NDH-1M complex except NdhD1 and NdhF1. NdhL and Ssl1690 (NdhO) were homologous to hypothetical proteins encoded by genomic DNA in higher plants, suggesting that chloroplast NDH-1 complexes contain related subunits. Diagnostic sequence motifs were found for both NdhL and NdhO homologous proteins. Analysis of ndhL deletion mutant (M9) revealed the presence of assembled NDH-1L and NDH-1M complexes, but these complexes appear to be functionally impaired in the absence of NdhL. Both NDH-1 complexes were absent in the ndhB deletion mutant (M55).     

Ultrafast primary processes in PS I from Synechocystis sp. PCC 6803: roles of P700 and A(0)

The excitation transport and trapping kinetics of core antenna-reaction center complexes from photosystem I of wild-type Synechocystis sp. PCC 6803 were investigated under annihilation-free conditions in complexes with open and closed reaction centers. For closed reaction centers, the long-component decay-associated spectrum (DAS) from global analysis of absorption difference spectra excited at 660 nm is essentially flat (maximum amplitude <10(-5) absorbance units). For open reaction centers, the long-time spectrum (which exhibits photobleaching maxima at approximately 680 and 700 nm, and an absorbance feature near 690 nm) resembles one previously attributed to (P700(+) - P700). For photosystem I complexes excited at 660 nm with open reaction centers, the equilibration between the bulk antenna and far-red chlorophylls absorbing at wavelengths >700 nm is well described by a single DAS component with lifetime 2.3 ps. For closed reaction centers, two DAS components (2.0 and 6.5 ps) are required to fit the kinetics. The overall trapping time at P700 ( approximately 24 ps) is very nearly the same in either case. Our results support a scenario in which the time constant for the P700 --> A(0) electron transfer is 9-10 ps, whereas the kinetics of the subsequent A(0) --> A(1) electron transfer are still unknown.     

Evidence for hydrogen bond formation to the PsaB chlorophyll of P700 in photosystem I mutants of Synechocystis sp. PCC 6803

P700, the primary electron donor of photosystem I, is an asymmetric dimer made of one molecule of chlorophyll a' (P(A)) and one of chlorophyll a (P(B)) that are bound to the homologous PsaA and PsaB polypeptides. While the carbonyl groups of P(A) are involved in hydrogen-bonding interactions with several surrounding amino acid side chains and a water molecule, P(B) does not engage hydrogen bonds with the protein. Notably, the residue Thr A739 is donating a strong hydrogen bond to the 9-keto C=O group of P(A) and the homologous residue Tyr B718 is free from interaction with P(B). Light-induced FTIR difference spectroscopy of the photooxidation of P700 has been combined with a site-directed mutagenesis attempt to introduce hydrogen bonds to the carbonyl groups of P(B) in Synechocystis sp. PCC 6803. The FTIR study of the Y(B718)T mutant provides evidence that the 9-keto C=O group of P(B) and P(B)(+) engages a relatively strong hydrogen-bonding interaction with the surroundings in a significant fraction (40 +/-10%) of the reaction centers. Additional mutations on the two PsaB residues homologous to those involved in the main interactions between the PsaA polypeptide and the 10a-carbomethoxy groups of P(A) affect only marginally the vibrational frequency of the 10a-ester C=O group of P(B). The FTIR data on single, double, and triple mutants at these PsaB sites indicate a plasticity of the interactions of the carbonyl groups of P(B) with the surrounding protein. However, these mutations do not perturb the hydrogen-bonding interactions assumed by the 9-keto and 10a-ester C=O groups of P(A) and P(A)(+) with the protein and have only a limited effect on the relative charge distribution between P(A)(+) and P(B)(+).     

集胞藻PCC6803中基因slr1761突变体的构建

PCR扩增了集胞藻PCC6803的slr1761基因,进一步以PGEM-T为载体将其克隆到大肠杆菌中,构建了P1761质粒。通过DNA体外重组,以卡那霉素抗性基因插入目的基因片段,构建了既含目的基因上游及下游序列、又携带选择性标记卡那霉素抗性的PK1761质粒。该质粒转化野生型集胞藻PCC6803细胞,利用同源重组原理获得了能在含卡那霉素的培养基上正常生长的基因敲除突变株。对该突变株基因组DNA进行PCR扩增,验证了其基因结构的正确性。     

Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803

The biological process for phosphate (P(i)) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P(i) regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P(i) from the medium than the wild-type strain removed.     

Mutational and kinetic analysis of basic amino acid transport in the cyanobacterium Synechocystis sp. PCC 6803

Two nitrogen-deregulated mutants of Phanerochaete chrysosporium, der8-2 and der8-5, were isolated by subjecting wild type conidia to gamma irradiation, plating on Poly-R medium containing high levels of nitrogen, and identifying colonies that are able to decolorize Poly-R. The mutants showed high levels of ligninolytic activity (¹⁴C-synthetic lignin ¹⁴CO₂), and lignin peroxidase, manganese peroxidase and glucose oxidase activities in both low nitrogen (2.4 mM) and high nitrogen (24 mM) media. The wild type on the otherhand displayed these activities in low nitrogen medium but showed little or no activities in high nitrogen medium. Fast protein liquid chromatographic analyses showed that the wild type as well as the der mutants produce three major lignin peroxidase peaks (designated L1, L2 and L3) with lignin peroxidase activity in low nitrogen medium. Furthermore, in low nitrogen medium, mutant der8-5 produced up to fourfold greater lignin peroxidase activity than that produced by the wild type. In high nitrogen medium, the wild type produced no detectable lignin peroxidase peaks whereas the mutants produced peaks L1 and L2, but not L3, and a new lignin peroxidase protein peak designated LN. Mutants der8-2 and der8-5 also produced high levels of glucose oxidase, an enzyme known to be associated with secondary metabolism and an important source of H₂O₂ in ligninolytic cultures, both in low and high nitrogen media. In contrast, the wild type produced high levels of glucose oxidase in low nitrogen medium and only trace amounts of this enzyme in high nitrogen medium. The results of this study indicate that the der mutants are nitrogen-deregulated for the production of a set of secondary metabolic activities associated with lignin degradation such as lignin peroxidases, manganese peroxidases and glucose oxidase.     

Changes in cyclic and respiratory electron transport by the movement of phycobilisomes in the cyanobacterium Synechocystis sp. strain PCC 6803

Phycobilisomes (PBS) are the major accessory light-harvesting complexes in cyanobacteria and their mobility affects the light energy distribution between the two photosystems. We investigated the effect of PBS mobility on state transitions, photosynthetic and respiratory electron transport, and various fluorescence parameters in Synechocystis sp. strain PCC 6803, using glycinebetaine to immobilize and couple PBS to photosystem II (PSII) or photosystem I (PSI) by applying under far-red or green light, respectively. The immobilization of PBS at PSII inhibited the increase in cyclic electron flow, photochemical and non-photochemical quenching, and decrease in respiration that occurred during the movement of PBS from PSII to PSI. In contrast, the immobilization of PBS at PSI inhibited the increase in respiration and photochemical quenching and decrease in cyclic electron flow and non-photochemical quenching that occurred when PBS moved from PSI to PSII. Linear electron transport did not change during PBS movement but increased or decreased significantly during longer illumination with far-red or green light, respectively. This implies that PBS movement is completed in a short time but it takes longer for the overall photosynthetic reactions to be tuned to a new state.     

Cobalt chloride induced stimulation of Photosystem II electron transport in Synechocystis sp. PCC 6803 cells

Synechocystis sp. PCC 6803 when grown in the presence of sublethal (M) levels of cobalt chloride shows an enhancement of Photosystem II (PS II) catalyzed Hill reaction. This stimulation seems to be induced by cobalt ions as other metal ions inhibit para-benzoquinone catalyzed Hill reaction. At saturating white light intensity, this enhancement is two times over that of the control cells on unit chlorophyll basis. Analysis of the PS II electron transport rate at varying intensities of white, blue or yellow light suggests an increased maximal rates but no change in the quantum yield or effective antenna size of CoCl₂-grown cells. There were no structural and functional changes in the phycobilisome as judged by the absence of changes in the phycocyanin/allophycocyanin ratio, fluorescence emission spectra, second derivative absorption spectra at 77 K and SDS-PAGE analysis of isolated phycobilisomes. The 77 K fluorescence emission spectra of the cells showed a decrease in the ratio of Photosystem I emission (F725) to Photosystem II emission (F685) in CoCl₂-grown cells compared to the control cells. These observations indicate three possibilities: (1) there is an increase in the number of Photosystem II units; (2) a faster turnover of Photosystem II centers; or (3) an alteration in energy redistribution between PS II and PS I in CoCl₂-grown cells which causes stimulation of Photosystem II electron transport rate.Abbreviations APC allophycocyanin - Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - EDTA ethylene diamine tetraacetic acid - PBS phycobilisome - PC phycocyanin - PSI Photosystem I - PS II Photosystem II - pBQ p-benzoquinone - PMSF phenyl methyl sulfonyl fluoride     

Expression of a cyanobacterial sucrose-phosphate synthase from Synechocystis sp. PCC 6803 in transgenic plants

Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp. PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation. The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate. To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters. The Synechocystis SPS protein was expressed at a high level in the plants, which should have been sufficient to increase overall SPS activity 2-8-fold in the leaves. However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not significantly different. The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants. Tomato plants expressing the maize SPS showed 2-3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis. To investigate the apparent inactivity of the Synechocystis SPS the enzyme was purified from transgenic tobacco and rice plants. Surprisingly, the purified enzyme was found to have full catalytic activity. It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.     

Remodeling of photosynthetic electron transport in Synechocystis sp. PCC 6803 for future hydrogen production from water

Photosynthetic microorganisms such as the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) can be exploited for the light-driven synthesis of valuable compounds. Thermodynamically, it is most beneficial to branch-off photosynthetic electrons at ferredoxin (Fd), which provides electrons for a variety of fundamental metabolic pathways in the cell, with the ferredoxin-NADP⁺ Oxido-Reductase (FNR, PetH) being the main target. In order to re-direct electrons from Fd to another consumer, the high electron transport rate between Fd and FNR has to be reduced. Based on our previous in vitro experiments, corresponding FNR-mutants at position FNR_K190 (Wiegand, K., et al.: “Rational redesign of the ferredoxin-NADP-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H₂-production”. Biochim Biophys Acta, 2018) have been generated in Synechocystis cells to study their impact on the cellular metabolism and their potential for a future hydrogen-producing design cell. Out of two promising candidates, mutation FNR_K190D proved to be lethal due to oxidative stress, while FNR_K190A was successfully generated and characterized: The light induced NADPH formation is clearly impaired in this mutant and it shows also major metabolic adaptations like a higher glucose metabolism as evidenced by quantitative mass spectrometric analysis. These results indicate a high potential for the future use of photosynthetic electrons in engineered design cells – for instance for hydrogen production. They also show substantial differences of interacting proteins in an in vitro environment vs. physiological conditions in whole cells.     

Localization of NAD(P)H dehydrogenase in the cyanobacterium Synechocystis sp. strain PCC 6803

Antibodies raised against NdhH and NdhB detected these proteins in the thylakoid membrane of Synechocystis sp. strain PCC 6803, but not in a purified cytoplasmic membrane. We conclude that NAD(P)H dehydrogenase is largely, if not exclusively, confined to the thylakoid membrane.     

Distinct roles of CpcG1 and CpcG2 in phycobilisome assembly in the cyanobacterium Synechocystis sp. PCC 6803

Structural role of the second copy of the rod–core linker CpcG, which was found by genome analysis, was studied in Synechocystis sp. PCC 6803 by gene disruption and fractionation of phycobilisome (sub)complexes. Disruption of cpcG2 (sll1471) resulted in a marked decrease in phycocyanin content both in the background of wild-type and cpcG1 (slr2051)-disruptant. The unique phycocyanin rod–CpcG2 complex without the major allophycocyanin components was isolated from the cpcG1-disruptant. By fluorescence analysis, it was proposed that CpcG2 protein connects the rods with a minor allophycocyanin component, to support energy transfer to Photosystem I.     

Identification of thylakoid membrane thermal transitions in Synechocystis sp. PCC6803 photosynthetic mutants

We used differential scanning calorimetry (DSC) as a technique capable of identifying photosynthetic complexes on the basis of their calorimetric transitions. Annotation of thermal transitions was carried out with thylakoid membranes isolated from various photosynthetic mutants of Synechocystis sp. PCC6803. The thylakoid membranes exhibited seven major DSC bands between 40 and 85°C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherms and by a subsequent annealing procedure. The successive annealing procedure proved to be more reliable technique than mathematical deconvolution in assigning thermal transitions. The main DSC band, around 47°C, resulting from the high enthalpy change that corresponds to non-interacting complex of PSII, was assigned using the PSI-less/apcE(-) mutant cells. Another band around 68-70°C relates to the denaturation of PSII surrounded by other proteins of the photosynthetic complexes in wild type and PSI-less/apcE(-) cells. A further major transition found at 82-84°C corresponds to the PSI core complex of wild type and PSII-deficient BE cells. Other transition bands between 50-67 and 65-75°C are believed to relate to ATP synthase and cytochrome b(6)f, respectively. These thermal transitions were obtained with thylakoids isolated from PSI(-)/PSII(-) mutant cells. Some minor bands determined at 59 and 83-84°C correspond to an unknown complex and NADH dehydrogenase, respectively. These annotations were done by PSI-less/apcE(-) and PSI(-)/PSII(-) mutants.     

Towards functional proteomics of membrane protein complexes in Synechocystis sp. PCC 6803

The composition and dynamics of membrane protein complexes were studied in the cyanobacterium Synechocystis sp. PCC 6803 by two-dimensional blue native/SDS-PAGE followed by matrix-assisted laser-desorption ionization time of flight mass spectrometry. Approximately 20 distinct membrane protein complexes could be resolved from photoautotrophically grown wild-type cells. Besides the protein complexes involved in linear photosynthetic electron flow and ATP synthesis (photosystem [PS] I, PSII, cytochrome b6f, and ATP synthase), four distinct complexes containing type I NAD(P)H dehydrogenase (NDH-1) subunits were identified, as well as several novel, still uncharacterized protein complexes. The dynamics of the protein complexes was studied by culturing the wild type and several mutant strains under various growth modes (photoautotrophic, mixotrophic, or photoheterotrophic) or in the presence of different concentrations of CO2, iron, or salt. The most distinct modulation observed in PSs occurred in iron-depleted conditions, which induced an accumulation of CP43' protein associated with PSI trimers. The NDH-1 complexes, on the other hand, responded readily to changes in the CO2 concentration and the growth mode of the cells and represented an extremely dynamic group of membrane protein complexes. Our results give the first direct evidence, to our knowledge, that the NdhF3, NdhD3, and CupA proteins assemble together to form a small low CO2-induced protein complex and further demonstrate the presence of a fourth subunit, Sll1735, in this complex. The two bigger NDH-1 complexes contained a different set of NDH-1 polypeptides and are likely to function in respiratory and cyclic electron transfer. Pulse labeling experiments demonstrated the requirement of PSII activity for de novo synthesis of the NDH-1 complexes.     

Ssr2998 of Synechocystis sp. PCC 6803 is involved in regulation of cyanobacterial electron transport and associated with the cytochrome b6f complex

To analyze the function of a protein encoded by the open reading frame ssr2998 in Synechocystis sp. PCC 6803, the corresponding gene was disrupted, and the generated mutant strain was analyzed. Loss of the 7.2-kDa protein severely reduced the growth of Synechocystis, especially under high light conditions, and appeared to impair the function of the cytochrome b6 f complex. This resulted in slower electron donation to cytochrome f and photosystem 1 and, concomitantly, over-reduction of the plastoquinone pool, which in turn had an impact on the photosystem 1 to photosystem 2 stoichiometry and state transition. Furthermore, a 7.2-kDa protein, encoded by the open reading frame ssr2998, was co-isolated with the cytochrome b6 f complex from the cyanobacterium Synechocystis sp. PCC 6803. ssr2998 seems to be structurally and functionally associated with the cytochrome b6 f complex from Synechocystis, and the protein could be involved in regulation of electron transfer processes in Synechocystis sp. PCC 6803.