Honoring Bacon Ke at 100: a legend among the many luminaries and a highly collaborative scientist in photosynthesis research.

Bacon Ke, who did pioneering research on the primary photochemistry of photosynthesis, was born in China on July 26, 1920, and currently, he is living in a senior home in San Francisco, California, and is a centenarian. To us, this is a very happy and unique occasion to honor him. After providing a brief account of his life, and a glimpse of his research in photosynthesis, we present here "messages" for Bacon Ke@ 100 from: Robert Alfano (USA), Charles Arntzen (USA), Sandor Demeter (Hungary), Richard A. Dilley (USA), John Golbeck (USA), Isamu Ikegami (Japan), Ting-Yun Kuang (China), Richard Malkin (USA), Hualing Mi (China), Teruo Ogawa (Japan), Yasusi Yamamoto (Japan), and Xin-Guang Zhu (China).

A Cytoplasmic Protein Ssl3829 Is Important for NDH-1 Hydrophilic Arm Assembly in Synechocystis sp. Strain PCC 6803.

Despite significant progress in clarifying the subunit compositions and functions of the multiple NDH-1 complexes in cyanobacteria, the assembly factors and their roles in assembling these NDH-1 complexes remain elusive. Two mutants sensitive to high light for growth and impaired in NDH-1-dependent cyclic electron transport around photosystem I were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-tagged library. Both mutants were tagged in the ssl3829 gene encoding an unknown protein, which shares significant similarity with Arabidopsis (Arabidopsis thaliana) CHLORORESPIRATORY REDUCTION7. The ssl3829 product was localized in the cytoplasm and associates with an NDH-1 hydrophilic arm assembly intermediate (NAI) of about 300 kD (NAI300) and an NdhI maturation factor, Slr1097. Upon deletion of Ssl3829, the NAI300 complex was no longer visible on gels, thereby impeding the assembly of the NDH-1 hydrophilic arm. The deletion also abolished Slr1097 and consequently reduced the amount of mature NdhI in the cytoplasm, which repressed the dynamic assembly process of the NDH-1 hydrophilic arm because mature NdhI was essential to stabilize all functional NAIs. Therefore, Ssl3829 plays an important role in the assembly of the NDH-1 hydrophilic arm by accumulating the NAI300 complex and Slr1097 protein in the cytoplasm.

The NDH-1L-PSI Supercomplex Is Important for Efficient Cyclic Electron Transport in Cyanobacteria.

Two mutants isolated from a tagging library of Synechocystis sp. strain PCC 6803 were sensitive to high light and had a tag in sll1471 encoding CpcG2, a linker protein for photosystem I (PSI)-specific antenna. Both mutants demonstrated strongly impaired NDH-1-dependent cyclic electron transport. Blue native-polyacrylamide gel electrophoresis followed by immunoblotting and mass spectrometry analyses of the wild type and a mutant containing CpcG2 fused with yellow fluorescent protein-histidine6 indicated the presence of a novel NDH-1L-CpcG2-PSI supercomplex, which was absent in the cpcG2 deletion mutant, the PSI-less mutant, and several other strains deficient in NDH-1L and/or NDH-1M. Coimmunoprecipitation and pull-down analyses on CpcG2-yellow fluorescent protein-histidine6, using antibody against green fluorescent protein and nickel column chromatography, confirmed the association of CpcG2 with the supercomplex. Conversely, the use of antibodies against NdhH or NdhK after blue native-polyacrylamide gel electrophoresis and in coimmunoprecipitation experiments verified the necessity of CpcG2 in stabilizing the supercomplex. Furthermore, deletion of CpcG2 destabilized NDH-1L as well as its degradation product NDH-1M and significantly decreased the number of functional PSI centers, consistent with the involvement of CpcG2 in NDH-1-dependent cyclic electron transport. The CpcG2 deletion, however, had no effect on respiration. Thus, we propose that the formation of an NDH-1L-CpcG2-PSI supercomplex in cyanobacteria facilitates PSI cyclic electron transport via NDH-1L.

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-ß-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.

NdhO, a subunit of NADPH dehydrogenase, destabilizes medium size complex of the enzyme in Synechocystis sp. strain PCC 6803.

Two mutants that grew faster than the wild-type (WT) strain under high light conditions were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in ssl1690 encoding NdhO. Deletion of ndhO increased the activity of NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET), while overexpression decreased the activity. Although deletion and overexpression of ndhO did not have significant effects on the amount of other subunits such as NdhH, NdhI, NdhK, and NdhM in the cells, the amount of these subunits in the medium size NDH-1 (NDH-1M) complex was higher in the ndhO-deletion mutant and much lower in the overexpression strain than in the WT. NdhO strongly interacts with NdhI and NdhK but not with other subunits. NdhI interacts with NdhK and the interaction was blocked by NdhO. The blocking may destabilize the NDH-1M complex and repress the NDH-CET activity. When cells were transferred from growth light to high light, the amounts of NdhI and NdhK increased without significant change in the amount of NdhO, thus decreasing the relative amount of NdhO. This might have decreased the blocking, thereby stabilizing the NDH-1M complex and increasing the NDH-CET activity under high light conditions.

NdhP is an exclusive subunit of large complex of NADPH dehydrogenase essential to stabilize the complex 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 and NdhF1, which are absent in a medium size complex (NDH-1M). They play important roles in respiration, cyclic electron transport around photosystem I, and CO2 acquisition. Two mutants sensitive to high light for growth and impaired in NDH-1-mediated cyclic electron transfer were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in sml0013 encoding NdhP, a single transmembrane small subunit of the NDH-1 complex. During prolonged incubation of the wild type thylakoid membrane with n-dodecyl ß-d-maltoside (DM), about half of the NDH-1L was disassembled to NDH-1M and the rest decomposed completely without forming NDH-1M. In the ndhP deletion mutant (ΔndhP), disassembling of NDH-1L to NDH-1M occurred even on ice, and decomposition to a small piece occurred at room temperature much faster than in the wild type. Deletion of the C-terminal tail of NdhP gave the same result. The C terminus of NdhP was tagged by YFP-His6. Blue native gel electrophoresis of the DM-treated thylakoid membrane of this strain and Western analysis using the antibody against GFP revealed that NdhP-YFP-His6 was exclusively confined to NDH-1L. During prolonged incubation of the thylakoid membrane of the tagged strain with DM at room temperature, NDH-1L was partially disassembled to NDH-1M and the 160-kDa band containing NdhP-YFP-His6 and possibly NdhD1 and NdhF1. We therefore conclude that NdhP, especially its C-terminal tail, is essential to assemble NdhD1 and NdhF1 and stabilize the NDH-1L complex.

Oxygenic photosynthesis-specific subunits of cyanobacterial NADPH dehydrogenases.

Cyanobacterial NADPH dehydrogenase (NDH-1) or NDH-1-like complex is localized in the thylakoid membrane and participates in a variety of bioenergetic reactions, including respiration, cyclic electron transport around photosystem I and CO2 uptake. Over the past decade, a significant achievement has been made in identifying seven oxygenic photosynthesis-specific (OPS) subunits of NDH-1 enzyme, NdhL to NdhQ and NdhS, in several cyanobacterial strains. Six of them are also found in higher plants but not in nonphototrophs. This indicates an exclusive existence of these OPS Ndh subunits in oxygenic photosynthetic organisms and suggested certain role of cyanobacterial and chloroplastic NDH-1 in photosynthetic reactions. In this review, we describe these seven OPS subunits of cyanobacterial NDH-1, focusing on their identification, localization, function, and evolution from cyanobacteria to higher plants. A crucial role of these OPS subunits on the function of cyanobacterial NDH-1 is proposed.

Identification of a cyanobacterial CRR6 protein, Slr1097, required for efficient assembly of NDH-1 complexes in Synechocystis sp. PCC 6803.

Despite significant progress in clarifying the subunit compositions and functions of the multiple NADPH dehydrogenase (NDH-1) complexes in cyanobacteria, the subunit maturation and assembly of their NDH-1 complexes are poorly understood. By transformation of wild-type cells with a transposon-tagged library, we isolated three mutants of Synechocystis sp. PCC 6803 defective in NDH-1-mediated cyclic electron transfer and unable to grow under high light conditions. All the mutants were tagged in the same slr1097 gene, encoding an unknown protein that shares significant homology with the Arabidopsis protein chlororespiratory reduction 6 (CRR6). The slr1097 product was localized in the cytoplasm and was required for efficient assembly of NDH-1 complexes. Analysis of the interaction of Slr1097 with 18 subunits of NDH-1 complexes using a yeast two-hybrid system indicated a strong interaction with NdhI but not with other Ndh subunits. Absence of Slr1097 resulted in a significant decrease of NdhI in the cytoplasm, but not of other Ndh subunits including NdhH, NdhK and NdhM; the decrease was more evident in the cytoplasm than in the thylakoid membranes. In the ∆slr1097 mutant, NdhH, NdhI, NdhK and NdhM were hardly detectable in the NDH-1M complex, whereas almost half the wild-type levels of these subunits were present in NDH-1L complex; similar results were observed in the NdhI-less mutant. These results suggest that Slr1097 is involved in the maturation of NdhI, and that assembly of the NDH-1M complex is strongly dependent on this factor. Maturation of NdhI appears not to be crucial to assembly of the NDH-1L complex.

Distinct roles of multiple NDH-1 complexes in the cyanobacterial electron transport network as revealed by kinetic analysis of P700+ reduction in various Ndh-deficient mutants of Synechocystis sp. strain PCC6803.

While methyl viologen had only a small effect on P700(+) rereduction kinetics after far-red pulses in KCN-treated wild-type Synechocystis sp. strain PCC6803 and an NdhF3/NdhF4 (NdhF3/F4)-defective mutant, it involved a rather slow P700(+) rereduction in an NdhF1-defective mutant. This strongly indicates that (i) active electron flow from metabolites to plastoquinone is suppressed upon deletion of ndhF1 and (ii) photosystem 1-mediated cyclic electron transport is dependent on NdhF3/F4-type NDH-1 complexes.

Possibilities of subunit localization with fluorescent protein tags and electron microscopy examplified by a cyanobacterial NDH-1 study.

Cyanobacterial NDH-1 is a multisubunit complex involved in proton translocation, cyclic electron flow around photosystem I and CO2 uptake. The function and location of several of its small subunits are unknown. In this work, the location of the small subunits NdhL, -M, -N, -O and CupS of Synechocystis 6803 NDH-1 was established by electron microscopy (EM) and single particle analysis. To perform this, the subunits were enlarged by fusion with the YFP protein. After classification of projections, the position of the YFP tag was revealed; all five subunits are integrated in the membrane domain. The results on NDH-1 demonstrate that a GFP tag can be revealed after data processing of EM data sets of moderate size, thus showing that this way of labeling is a fast and reliable way for subunit mapping in multisubunit complexes after partial purification.

Cross-talk between photomixotrophic growth and CO(2) -concentrating mechanism in Synechocystis sp. strain PCC 6803.

Simultaneous catabolic and anabolic glucose metabolism occurs in the same compartment during photomixotrophic growth of the model cyanobacterium Synechocystis sp. PCC 6803. The presence of glucose is stressful to the cells; it is reflected in the high frequency of suppression mutations in glucose-sensitive mutants. We show that glucose affects many cellular processes. It stimulates respiration and the rate of photosynthesis and quantum yield in low- but not high-CO(2) -grown cells. Fluorescence and thermoluminescence parameters of photosystem II are also affected but the results did not lend support to sustained glucose driven over reduction in the light. Glucose-sensitive mutants such as ΔpmgA (impaired in photomixotrophic growth) and Δhik31 (lacking histidine kinase 31) are far more susceptible under high than low air level of CO(2) . A glycine to tryptophan mutation in position 354 in NdhF3, involved in the high-affinity CO(2) uptake, rescued ΔpmgA. A rise in the apparent photosynthetic affinity to external inorganic carbon is observed in high-CO(2) -grown wild-type cells after the addition of glucose, but not in mutant ΔpmgA. This is attributed to upregulation of certain low-CO(2) -induced genes, involved in inorganic carbon uptake, in the wild type but not in ΔpmgA. These data uncovered a new level of interaction between CO(2) fixation (and the CO(2) -concentrating mechanism) and photomixotrophic growth in cyanobacteria.

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.

Single particle analysis of thylakoid proteins from Thermosynechococcus elongatus and Synechocystis 6803: localization of the CupA subunit of NDH-1.

The larger protein complexes of the cyanobacterial photosynthetic membrane of Thermosynechoccus elongatus and Synechocystis 6803 were studied by single particle electron microscopy after detergent solubilization, without any purification steps. Besides the "standard" L-shaped NDH-1L complex, related to complex I, large numbers of a U-shaped NDH-1MS complex were found in both cyanobacteria. In membranes from Synechocystis DeltacupA and DeltacupA/cupB mutants the U-shaped complexes were absent, indicating that CupA is responsible for the U-shape by binding at the tip of the membrane-bound arm of NDH-1MS. Comparison of membranes grown under air levels of CO(2) or 3% CO(2) indicates that the number of NDH-1MS particles is 30-fold higher under low-CO(2).

Identification and localization of the CupB protein involved in constitutive CO2 uptake in the cyanobacterium, Synechocystis sp. strain PCC 6803.

Antibody against cMyc cross-reacted strongly with the CupB protein tagged with His6-cMyc (HM) in thylakoid membrane of Synechocystis sp. strain PCC 6803 but only faintly with the cytoplasmic membrane fraction. The protein was not detected in the membranes of the DeltandhD4 and DeltandhF4 mutants in which CupB was tagged with HM. We concluded that a CupB complex containing NdhD4 and NdhF4 is largely, if not exclusively, confined to the thylakoid membrane. Both CupB and NdhH were detected in a fraction containing protein complexes of > 450 kDa, obtained after nickel column and gel filtration chromatography of the membranes solubilized with n-dodecyl-beta-maltoside.

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.

Structural characterization of NDH-1 complexes of Thermosynechococcus elongatus by single particle electron microscopy.

The structure of the multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes from cyanobacteria was investigated by growing the wild type and specific ndh His-tag mutants of Thermosynechococcus elongatus BP-1 under different CO(2) conditions, followed by an electron microscopy (EM) analysis of their purified membrane protein complexes. Single particle averaging showed that the complete NDH-1 complex (NDH-1L) is L-shaped, with a relatively short hydrophilic arm. Two smaller complexes were observed, differing only at the tip of the membrane-embedded arm. The smallest one is considered to be similar to NDH-1M, lacking the NdhD1 and NdhF1 subunits. The other fragment, named NDH-1I, is intermediate between NDH-1L and NDH-1M and only lacks a mass compatible with the size of the NdhF1 subunit. Both smaller complexes were observed under low- and high-CO(2) growth conditions, but were much more abundant under the latter conditions. EM characterization of cyanobacterial NDH-1 further showed small numbers of NDH-1 complexes with additional masses. One type of particle has a much longer peripheral arm, similar to the one of NADH: ubiquinone oxidoreductase (complex I) in E. coli and other organisms. This indicates that Thermosynechococcus elongatus must have protein(s) which are structurally homologous to the E. coli NuoE, -F, and -G subunits. Another low-abundance type of particle (NDH-1U) has a second labile hydrophilic arm at the tip of the membrane-embedded arm. This U-shaped particle has not been observed before by EM in a NDH-I preparation.

Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria.

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).

Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1.

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

Physical separation of chlorophyll-protein complexes.

This minireview focuses on the physical separation of chlorophyll-protein complexes and particles from the two photosystems by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) or differential/sucrose density-gradient centrifugation during the decade of 1960s.

Inorganic carbon acquisition systems in cyanobacteria.

This minireview focuses on the mechanism of inorganic carbon uptake in cyanobacteria and in particular the two CO(2)-uptake systems and two bicarbonate transporters recently identified in Synechocycstis PCC 6803, and their presence in other cyanobacterial strains.