Construction of insertion mutants of Synechocystis sp. PCC 6803: evidence for an essential function of subunit IV of the cytochrome b6/f complex

The gene encoding subunit IV of the cytochrome b₆/f complex (petD) has been isolated from a genomic library of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 480 nucleotides and can code for a polypeptide with a molecular weight of 17.5 kDa. The deduced amino acid sequence shows high identity with the corresponding sequences of both the photoautotrophic prokaryote Nostos sp. PCC 7906 as well as of lower and higher photoautotrophic eukaryotes (e.g. Chlorella protothecoides, Nicotiana tabacum). Transformation of Synechocystis sp. PCC 6803 with a plasmid containing the cloned petD gene in which the coding sequence is interrupted by the aminoglycoside 3-phosphotransferase gene (aph) from Tn903 resulted in the formation of km resistant transformants. The molecular analysis of independent transformants revealed that all clones were merodiploid containing both uninterrupted wild-type as well as interrupted mutant petD copies. Approaches to segregate these two genomes were unsuccessful implying an essential function of the petD gene product in Synechocystis sp. PCC 6803.Abbreviations aph aminoglycoside 3-phosphotransferase - cpDNA chloroplast DNA - km kanamycin - PSI photosystem I - PSII photosystem II     

Inactivation of the water-oxidizing enzyme in manganese stabilizing protein-free mutant cells of the cyanobacteria Synechococcus PCC7942 and Synechocystic PCC6803 during dark incubation and conditions leading to photoactivation

The previously constructed MSP (manganese stabilizing protein-psbO gene product)-free mutant of Synechococcus PCC7942 (Bockholt R, Masepohl B and Pistorius E K (1991) FEBS Lett 294: 59–63) and a newly constructed MSP-free mutant of Synechocystis PCC6803 were investigated with respect to the inactivation of the water-oxidizing enzyme during dark incubation. O₂ evolution in the MSP-free mutant cells, when measured with a sequence of short saturating light flashes, was practically zero after an extended dark adaptation, while O₂ evolution in the corresponding wild type cells remained nearly constant. It could be shown that this inactivation could be reversed by photoactivation. With isolated thylakoid membranes from the MSP-free mutant of PCC7942, it could be demonstrated that photoactivation required illumination in the presence of Mn²⁺ and Ca²⁺, while Cl^– addition was not required under our experimental conditions. Moreover, an extended analysis of the kinetic properties of the water-oxidizing enzyme (kinetics of the S₃(S₄)S₀ transition, S-state distribution, deactivation kinetics) in wild type and mutant cells of Synechococcus PCC7942 and Synechocystis PCC6803 was performed, and the events possibly leading to the reversible inactivation of the water-oxidizing enzyme in the mutant cells are discussed. We could also show that the water-oxidizing enzyme in the MSP-free mutant cells is more sensitive to inhibition by added NH₄Cl-suggesting that NH₃ might be a physiological inhibitor of the water oxidizing enzyme in the absence of MSP.Abbreviations Chl chlorophyll - DCBQ 2,6-Dichloro-p-benzoquinone - MSP manganese stabilizing protein (psbO gene product) - PS II Photosystem II - WOE water oxidizing enzyme - WT wild type This paper is dedicated to Prof. Dr. Bernard Axelrod on the occasion of his 80th birthday     

Isolation and characterization of the ccmM gene required by the cyanobacterium Synechocystis PCC6803 for inorganic carbon utilization

A high CO₂-requiring mutant of Synechocystis PCC6803 (G3) capable of C_i transport but unable to utilize the intracellular C_i pool for photosynthesis was constructed. A DNA clone of 6.1 kbp that transforms the G3 mutant to the wild-type phenotype was isolated from a Synechocystis PCC6803 genomic library. Complementation test with subclones allocated the mutation site within a DNA fragment of 674 bp nucleotides. Sequencing analysis of the mutation region elucidated an open reading frame encoding a 534 amino-acid protein with a significant sequence homology to the protein coded by the ccmN gene of Synechococcus PCC7942. The ccmM-like gene product of Synechocystis PCC6803 contains four internal repeats with a week similarity to the rbcS gene product. An open reading frame homologous to the ccmN gene of Synechococcus PCC7942 was found downstream to the ccmM-like gene. As opposed to the Synechococcus PCC7942 ccmM and ccmN genes located 2 kbp upstream to, and oriented in the same direction as, the rbc operon, the ccm-like genes in Synechocystis PCC6803 are not located within 22 kbp upstream to the rbcL gene of the Rubisco operon. Thus, despite the resemblance in clustering of the ccmM and ccmN genes in both cyanobacterial species, the difference in their genomic location relative to the rbc genes demonstrates variability in structural organization of the genes involved in inorganic carbon acquisition.Abbreviations CCM CO₂-concentrating mechanism - C_i inorganic carbon - HCR high CO₂-requiring - kbp kilobase pair - ORF open reading frame - Rubisco ribulose 1,5-bisphosphate carboxylase-oxygenase gene - SSC sodium chloride and sodium citrate - WT wild-type     

Interaction of the photosynthetic and respiratory electron transport chains producing slow O2 signals under flashing light in Synechocystis sp. PCC 6803

We investigated the slow signal of apparent O₂ release under brief light flashes by using mutants of Synechocystis sp. PCC 6803 which lacked CP43 and D1. The slow signal was present at higher amplitudes in the mutants. It was inhibited by starving the mutants of glucose (>90%), by 10 mM NaN₃ (85%) and by boiling samples for 2 min (100%). In the mutants and in the wild-type, the slow signal was 95% inhibited by the combination of DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) and HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide). In the wild type, the addition of DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) or CCCP (carbonylcyanide m-chlorophenylhydrazone) completely inhibited photosynthetic O₂ evolution, yet failed to inhibit the slow signal. We explain the kinetics of the wild-type signal as a positive deflection due to the inhibition of respiration by PS I activity, and a negative deflection due to the stimulation of respiration by electrons originating from PS II. We found no evidence of a meta-stable S₃ in Synechocystis sp. PCC 6803 that could contribute to the slow signal of apparent O₂ release. We present a calculation which involves only averaging, division and subtraction, that can remove the contribution of the slow signal from the true photosynthetic O₂ signal and provide up to a 10-fold improved accuracy of the S-state models.Abbreviations ADRY Acceleration of the Deactivation Reactions of the water-splitting enzyme system Y - Ant-2-p 2-(3-chloro-4-trifluoromethyl)-anilino-3,5-dinitrothiophene - CCCP carbonylcyanide m-chlorophenylhydrazone - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a.k.a. Dibromothymoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - S. 6803 Synechocystis sp. PCC 6803     

Characterisation of CO2 and HCO3 − uptake in the cyanobacterium Synechocystis sp. PCC6803

The availability of a complete genome database for the cyanobacterium Synechocystissp. PCC6803 (glucose-tolerant strain) has raised expectations that this organism would become a reference strain for work aimed at understanding the CO₂-concentrating mechanism (CCM) in cyanobacteria. However, the amount of physiological data available has been relatively limited. In this report we provide data on the relative contributions of net HCO₃ ⁻ uptake and CO₂ uptake under steady state photosynthetic conditions. Cells were compared after growth at high CO₂ (2% v/v in air) or limiting CO₂ conditions (20 ppm CO₂). Synechocystishas a very high dependence on net HCO₃ ⁻ uptake at low to medium concentrations of inorganic carbon (Ci). At high Ci concentrations net CO₂ uptake became more important but did not contribute more than 40% to the rate of photosynthetic O₂ evolution. The data also confirm that high Ci cells of Synechocystissp. PCC6803 possess a strong capacity for net HCO₃ ⁻ uptake under steady state photosynthetic conditions. Time course experiments show that induction of maximal Ci uptake capacity on a shift from high CO₂ to low CO₂ conditions was near completion by four hours. By contrast, relaxation of the induced state on return of cells to high CO₂, takes in excess of 230 h. Experiments were conducted to determine if Synechocystissp. PCC6803 is able to exhibit a `fast induction' response under severe Ci limitation and whether glucose was capable of causing a rapid inactivation in Ci uptake capacity. Clear evidence for either response was not found. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification of highly active oxygen-evolving photosystem II from Chlamydomonas reinhardtii

A method is described for the isolation and purification of active oxygen-evolving photosystem II (PS II) membranes from the green alga Chlamydomonas reinhardtii. The isolation procedure is a modification of methods evolved for spinach (Berthold et al. 1981). The purity and integrity of the PS II preparations have been assesssed on the bases of the polypeptide pattern in SDS-PAGE, the rate of oxygen evolution, the EPR multiline signal of the S₂ state, the room temperature chlorophyll a fluorescence yield, the 77 K emission spectra, and the P700 EPR signal at 300 K. These data show that the PS II characteristics are increased by a factor of two in PS II preparations as compared to thylakoid samples, and the PS I concentration is reduced by approximately a factor ten compared to that in thylakoids.Abbreviations BSA bovine serum albumin - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU (diuron) 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMQ 2,5-dimethyl-p-benzoquinone - EDTA ethylenediamine tetraacetic acid - EPR electron paramagnetic resonance - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-[N-Morpholino]ethanesulfonic acid - OEE oxygen evolving enhancer - PS II photosystem II - SDS-PAGE sodium dedocyl sulfate polyacrylamide gel electrophoresis     

Chimaeric CP47 mutants of the cyanobacterium Synechocystis sp. PCC 6803 carrying spinach sequences: Construction and function

Chimaeric mutants of the cyanobacterium Synechocystis sp. PCC 6803 have been generated carrying part or all of the spinach psbB gene, encoding CP47 (one of the chlorophyll-binding core antenna proteins in Photosystem II). The mutant in which the entire psbB gene had been replaced by the homologous gene from spinach was an obligate photoheterotroph and lacked Photosystem II complexes in its thylakoid membranes. However, this strain could be transformed with plasmids carrying selected regions of Synechocystis psbB to give rise to photoautotrophs with a chimaeric spinach/cyanobacterial CP47 protein. This process involved heterologous recombination in the cyanobacterium between psbB sequences from spinach and Synechocystis 6803; which was found to be reasonably effective in Synechocystis. Also other approaches were used that can produce a broad spectrum of chimaeric mutants in a single experiment. Functional characterization of the chimaeric photoautotrophic mutants indicated that if a decrease in the photoautotrophic growth rates was observed, this was correlated with a decrease in the number of Photosystem II reaction centers (on a chlorophyll basis) in the thylakoid membrane and with a decrease in oxygen evolution rates. Remaining Photosystem II reaction centers in these chimaeric mutants appeared to function rather normally, but thermoluminescence and chlorophyll a fluorescence measurements provided evidence for a destabilization of Q_B ^–. This illustrates the sensitivity of the functional properties of the PS II reaction center to mild perturbations in a neighboring protein.Abbreviations diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F_v variable chlorophyll a fluorescence - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - (k)bp (kilo)base pairs - PS II Photosystem II - Q_A primary electron-accepting plastoquinone in Photosystem II - Q_B secondary electron-accepting plastoquinone in Photosystem II - SDS sodium dodecyl sulfate     

Deletion of cytochrome c oxidase genes from the cyanobacterium Synechocystis sp. PCC6803: Evidence for alternative respiratory pathways

An oligonucleotide directed against a highly conserved region of aa₃-type cytochrome c oxidases was used to clone the cox genes from the cyanobacterium Synechocystis sp. PCC6803. Several overlapping clones were obtained that contained the coxB, coxA, and coxC genes, transcribed in the same direction in that order, coding for subunits II, I, and III, respectively. The deduced protein sequences of the three subunits showed high sequence similarity with the corresponding subunits of all known aa₃-type cytochrome c oxidases. A 1.94-kb HindII fragment containing most of coxA and about half of coxC was deleted and replaced by a cassette coding for kanamycin resistance. Mutant cells that were homozygous for the deleted cox locus were obtained. They were viable under photoautotrophic and photoheterotrophic conditions, but contained no cytochrome c oxidase activity. Nevertheless, these mutant cells showed almost normal respiration, defined as cyanide-inhibitable O₂ uptake by whole cells in the dark. It is concluded, therefore, that aa₃-type cytochrome c oxidase is not the only terminal respiratory oxidase in Synechocystis sp. PCC6803.Abbreviations CM cytoplasmic membrane - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HQNO 2-heptyl-4-hydroxyquinoline N-oxide - ICM intracytoplasmic membranes - SU subunit - TES (N-tris(hydroxymethyl)methyl)-2-aminoethane sulfonic acid     

Oxidizing side of the cyanobacterial Photosystem I: Mutational analysis of the luminal H loop of the PsaB subunit

PS I core proteins are expected to interact with the electron donor proteins plastocyanin or cytochrome c ₆. To investigate the role of the luminal H loop of PsaB in the assembly and function of the PS I complex, we generated 15 deletion and repetition mutations in the H loop of the PsaB protein from Synechocystis sp. PCC 6803. The mutant strains differed in their photoautotrophic growth. The PS I proteins could not be detected in the membranes of mutants in which the N438–E448, I453–T464, or S500–G512 region was deleted from the PsaB protein, indicating the essential role of these segments in proper folding of the PsaB protein. Mutants with partial or complete deletion of the L469–D496 segment contained the PS I proteins. These results indicate that the regions near the transmembrane helices are more important for the assembly of PsaB than the middle region of the H loop. The L469-D496 segment in the H loop of PsaB is dispensable in the interaction between the PS I complex and the soluble donor proteins. These results suggested that sections of the H loop of PsaB are crucial for the structural integrity of the PsaB protein.     

Absence of the psbH gene product destabilizes the Photosystem II complex and prevents association of the Photosystem II-X protein in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

PS II-H is a small hydrophobic protein that is universally present in the PS II core complex of cyanobacteria and plants. The role of PS II-H was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The psbH disruptant could grow photoautotrophically; however, its growth was much slower than that of the wild type cell. Chromatography enabled the isolation of active oxygen-evolving PS II complexes from both the mutant and the wild type. The mutant yielded a relatively large amount of inactive PS II complex that lacked the following extrinsic proteins: the 33-kDa protein, the 12-kDa protein, and cytochrome c ₅₅₀ . There were differences between the psbH disruptant and the wild type in terms of the oxygen evolution activities of the cells, thylakoids, and PS II complexes. At high concentrations of 2,6-DCBQ, the activity was much lower in the mutant than in the wild type. Gel filtration chromatography of the PS II complexes showed that both active and inactive PS II complexes isolated from the mutant were mostly in the monomeric form, while the active PS II complex from the wild type was in the dimeric form. The polypeptide composition of both active and inactive PS II complexes from the mutant showed the absence of another small polypeptide, PS II-X. These results suggest that the PS II-H protein is essential for stable assembly of native dimeric PS II complex containing PS II-X.     

Oxygen evolving membranes and particles from the transformable cyanobacterium Synechocystis sp. PCC6803

Membranes and PS II particles retaining high rates of O₂-evolving activity have been isolated from the transformable cyanobacterium, Synechocystis sp. PCC6803. Membranes from cells grown under red light exhibit rates of O₂-evolution ranging from 500–700 mole O₂/mg chl/h. PS II particles are prepared by a simple procedure involving DEAE column chromatography of detergent extracts obtained by simultaneous treatment of membranes with octylglucoside and dodecylmaltoside. The isolated PS II fraction is enriched in polypeptides immunologically cross-reactive with polypeptides present in core reaction center preparations of spinach, exhibits 77 K fluorescence emission maxima at 685 and 696 nm, but not emission and absorption due to phycobilines and is capable of rates of O₂-evolution exceeding 1000 mole O₂/mg chl/h.Abbreviations DM dodecyl--D-maltoside - OG octyl--D-glucoside     

Changes in composition of membrane proteins accompanying the regulation of PS I/PS II stoichiometry observed with Synechocystis PCC 6803

Changes in composition of membrane proteins in Synechocystis PCC 6803 induced by the shift of light regime for photosynthetic growth were studied in relation to the regulation of PS I/PS II stoichiometry. Special attention was paid to the changes in abundance of proteins of PS I and PS II complexes. Composition was examined using a LDS-PAGE and a quantitative enzyme immunoassay. Abundance of PsaA/B polypeptides and the PsaC polypeptide of the PS I complex, on a per cell basis, increased under the light regime exciting preferentially PS II and decreased under the light regime exciting mainly PS I. Similar changes were observed with polypeptides of 18.5, 10 and 8.5 kDa. The abundance of other proteins associated with membranes, including PsbA polypeptide of the PS II complex, was fairly constant irrespective of light regime. These results are consistent with our previous observations with other strains of cyanophytes (Anabaena variabilis M2 and Synechocystis PCC 6714) that PS I is the variable component in changes in PS I/PS II stoichiometry in response to changing light regimes for photosynthesis.Abbreviations CBB Coomassie brilliant blue - Chl chlorophyll - EIA enzyme immunoassay - LDS lithium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - PS photosystem - PVDF polyvinylidene difluoride     

Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.     

Resonance Raman spectroscopy of carotenoids in Photosystem II core complexes

Resonance Raman (RR) spectroscopy has been used to examine the configuration of the carotenoids bound to Synechocystis PCC 6803 Photosystem II (PS II) core complexes. The excitation wavelengths used (514.5, 488.0, 476.5 and 457.9 nm) span the absorption bands of all of the ~12–17 neutral carotenoids in the PS II core complex. The RR spectra of the two carotenoids associated with the D1–D2 polypeptides (Car₅₀₇ and Car₄₈₉) of the reaction center are extracted via light versus dark difference experiments measured at 20 K. The RR results are consistent with all-trans configurations for both Car₅₀₇ and Car₄₈₉ and indicate that majority of the other carotenoids in the PS II core complex must also be in the all-trans configuration. The configuration of β-carotene is relevant to its proposed function as a molecular wire in the secondary electron-transfer reactions of PS II.     

Evaluation of central metabolism based on a genomic database of<Emphasis Type="Italic">Synechocystis</Emphasis> PCC6803

Cyanobacteria produce industrially important secondary metabolites such as lipopeptide, oligosaccharide, fatty acid (esp. sulfolipid),etc. Among them,Synechocystis PCC6803 is the first strain with a publicly available full genome sequence, as of 1996, and is one of the most extensively studied photosynthetic microorganisms. Using this genomic information, the central metabolism ofSynechocystis PCC6803 was reconstructed, including photosynthesis, oxidative phosphorylation, glycolysis, pyruvate metabolism, TCA cycle, carbon fixation, and transport system. Each biochemical reaction was carefully incorporated into the model, taking into consideration the metabolite formula, stoichiometry, charge balance, and thermodynamic properties using information from genomic and metabolic databases as well as biochemical literature. The metabolic flux of the model was calculated using flux balance analysis according to its cultivation with various carbon sources. The results of simulation were in accordance with experimental data, which suggests that the central metabolism model can properly estimate the behavior ofSynechocystis PCC6803. This model would aid in the understanding of the whole cell metabolism ofSynechocystis PCC6803, the first effort of its kind for photosynthetic bacteria.     

Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803

The sll1418 gene encodes a PsbP-like protein in Synechocystis sp. PCC 6803. Expression of sll1418 was similar in BG-11 and in Cl⁻- or Ca²⁺-limiting media, and inactivation of sll1418 did not prevent photoautotrophic growth in normal or nutrient-limiting conditions. Also the wild-type and ΔPsbP strains exhibited similar oxygen evolution and assembly of Photosystem II (PS II) centers. Inactivation of sll1418 in the ΔPsbO: ΔPsbP, ΔPsbQ:ΔPsbP, ΔPsbU:ΔPsbP and ΔPsbV:ΔPsbP mutants did not prevent photoautotrophy or alter PS II assembly and oxygen evolution in these strains. Moreover, the absence of PsbP did not affect the ability of alkaline pH to restore photoautotrophic growth in the ΔPsbO:ΔPsbU strain. The PsbO, PsbU and PsbV proteins are required for thermostability of PS II and thermal acclimation in Synechocystis sp. PCC 6803 [Kimura et al. (2002) Plant Cell Physiol 43: 932–938]. However, thermostability and thermal acclimation in ΔPsbP cells were similar to wild type. These results are consistent with the conclusion that PsbP is associated with ∼3 of PS II centers, and may play a regulatory role in PS II [Thornton et al. (2004) Plant Cell 16: 2164–2175].     

Influence of Histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus

The influence of the histidine axial ligand to the P_D1 chlorophyll of photosystem II on the redox potential and spectroscopic properties of the primary electron donor, P_680, was investigated in mutant oxygen-evolving photosystem II (PSII) complexes purified from the thermophilic cyanobacterium Thermosynechococcus elongatus. To achieve this aim, a mutagenesis system was developed in which the psbA₁ and psbA₂ genes encoding D1 were deleted from a His-tagged CP43 strain (to generate strain WT^?) and mutations D1-H198A and D1-H198Q were introduced into the remaining psbA₃ gene. The O₂-evolving activity of His-tagged PSII isolated from WT^? was found to be significantly higher than that measured from His-tagged PSII isolated from WT in which psbA₁ is expected to be the dominantly expressed form. PSII purified from both the D1-H198A and D1-H198Q mutants exhibited oxygen-evolving activity as high as that from WT^?. Surprisingly, a variety of kinetic and spectroscopic measurements revealed that the D1-H198A and D1-H198Q mutations had little effect on the redox and spectroscopic properties of P₆₈₀, in contrast to the earlier results from the analysis of the equivalent mutants constructed in Synechocystis sp. PCC 6803 [B.A. Diner, E. Schlodder, P.J. Nixon, W.J. Coleman, F. Rappaport, J. Lavergne, W.F. Vermaas, D.A. Chisholm, Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization, Biochemistry 40 (2001) 9265-9281]. We conclude that the nature of the axial ligand to P_D1 is not an important determinant of the redox and spectroscopic properties of P₆₈₀ in T. elongatus.     

Lumenal proteins involved in respiratory electron transport in the cyanobacterium Synechocystis sp. PCC6803

Cyanobacterial thylakoids catalyze both photosynthetic and respiratory activities. In a photosystem I-less Synechocystis sp. PCC 6803 strain, electrons generated by photosystem II appear to be utilized by cytochrome oxidase. To identify the lumenal electron carriers (plastocyanin and/or cytochromes c ₅₅₃, c ₅₅₀, and possibly c _M) that are involved in transfer of photosystem II-generated electrons to the terminal oxidase, deletion constructs for genes coding for these components were introduced into a photosystem I-less Synechocystis sp. PCC 6803 strain, and electron flow out of photosystem II was monitored in resulting strains through chlorophyll fluorescence yields. Loss of cytochrome c ₅₅₃ or plastocyanin, but not of cytochrome c ₅₅₀, decreased the rate of electron flow out of photosystem II. Surprisingly, cytochrome c _M could not be deleted in a photosystem I-less background strain, and also a double-deletion mutant lacking both plastocyanin and cytochromec ₅₅₃ could not be obtained. Cytochrome c _M has some homology with the cytochrome c-binding regions of the cytochromecaa₃ -type cytochrome oxidase from Bacillus spp. and Thermus thermophilus. We suggest that cytochrome c _M is a component of cytochrome oxidase in cyanobacteria that serves as redox intermediate between soluble electron carriers and the cytochromeaa₃ complex, and that either plastocyanin or cytochrome c ₅₅₃ can shuttle electrons from the cytochrome b_6f complex to cytochrome c _M.     

Cloning, nucleotide sequence and mutational analysis of the gene encoding the Photosystem II manganese-stabilizing polypeptide of Synechocystis 6803

Summary Affinity purified, polyclonal antibodies raised against the Photosystem II 33 kDa manganese-stabilizing polypeptide of the spinach oxygen-evolving complex were used to isolate the gene encoding the homologous protein from Synechocystis 6803. Comparison of the amino acid sequence deduced from the Synechocystis psb1 nucleotide sequence with recently published sequences of spinach and pea confirms the homology indicated by antigenic crossreactivity and shows that the cyanobacterial and higher plant sequences are 43% identical and 63% conserved. Regions of identity, varying in length from 1 to 10 consecutive residues, are distributed throughout the protein. The 28 residues at the amino terminus of the psb1 gene product, characteristic of prokaryotic signal peptides, show homology with the carboxyl-terminal third of the transit sequences of pea and spinach and are most likely needed for the transport of the manganese-stabilizing protein across the thylakoid membrane to its destination of the lumen. Synechocystis mutants which contain a kanamycin resistance gene cassette inserted into the coding region for the 32 kDa polypeptide were constructed. These mutants contain no detectable 32 kDa polypeptide, do not evolve oxygen, and are incapable of photoautotrophic growth.     

Expression of the CMV‐CP Gene in Synechocystis 6803 Affects Cyanobacterial Photosynthesis

Previous work has shown that the presence of excess coat protein (CP) of cucumber mosaic virus (CMV) in the chloroplasts was related with mosaic symptoms. However, whether these mosaic symptoms are directly induced by the interaction between CP and chloroplasts is unknown. To directly demonstrate the interaction between CP and the chloroplast, Synechocystis sp. PCC 6803 was used as the chloroplast model. The cDNA encoding the CMV‐CP was cloned in a cyanobacterial shuttle vector (pKT‐CP) and transferred to Synechocystis sp. PCC 6803. The CP was expressed in the cyanobacterium with the psbA promoter. The expression of CMV‐CP hindered the growth of transgenic cyanobacterium cells and decreased its photosynthetic rate and the PS II activity. The transgenic cells showed increased fluorescence (F) from the phycobilisome terminal emitters and increased fluorescence (F) from PS II. The absorption spectra at room temperature showed the Chl and the phycocyanin absorption peak of the mutant strain significantly decreased. These results showed that CP may directly affect the cyanobacterium cells and decreased its photosynthesis, especially the PS II activity. These data might provide new evidence for mosaic symptoms being directly induced by the interaction between CP and chloroplasts.