Requirement of carotene isomerization for the assembly of photosystem II in Synechocystis sp. PCC 6803

Carotene isomerase mutant (crtH mutant) cells of Synechocystis sp. PCC 6803 can accumulate beta-carotene under light conditions. However, the mutant cells grown under a light-activated heterotrophic growth condition contained detectable levels of neither beta-carotene nor D1 protein of the photosystem (PS) II reaction center, and no oxygen-evolving activity of PSII was detected. beta-Carotene and D1 protein appeared and a high level of PSII activity was detected after the cells were transferred to a continuous light condition. The PSI activities of thylakoid membranes from mutant cells were almost the same as those of thylakoid membranes from wild-type cells, both before and after transfer to the continuous light condition. These results suggest that beta-carotene is required for the assembly of PSII but not for that of PSI.     

Physiological significance of the regulation of photosystem stoichiometry upon high light acclimation of Synechocystis sp. PCC 6803

We characterized the photosynthetic properties of the pmgA mutant of Synechocystis PCC 6803, which cannot change its photosystem stoichiometry under a high-light condition (200 micromol x m(-2) x s(-1)), in order to clarify the physiological significance of the regulation of photosystem stoichiometry. We found that (1) PSII activity was inhibited more in wild-type cells on the first day under the high-light conditions than in mutant cells. (2) The growth of the mutants following the initial imposition of high light was faster than that of wild-type cells. (3) However, growth was severely inhibited in the mutants after the third day of exposure to high light. (4) The growth inhibition in the mutants under the extended high-light conditions was reversed by the addition of sublethal concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which seemed to mimic photoinhibition of PSII. These results suggest that the main role of adjusting the photosystem stoichiometry with respect to light intensity is not to maintain efficient photosynthesis, but to down regulate electron transfer. Failure to down regulate electron flow leads to cell death under prolonged exposure to high light in this cyanobacterium.     

PsaK2 subunit in photosystem I is involved in state transition under high light condition in the cyanobacterium Synechocystis sp. PCC 6803

To avoid the photodamage, cyanobacteria regulate the distribution of light energy absorbed by phycobilisome antenna either to photosystem II or to photosystem I (PSI) upon high light acclimation by the process so-called state transition. We found that an alternative PSI subunit, PsaK2 (sll0629 gene product), is involved in this process in the cyanobacterium Synechocystis sp. PCC 6803. An examination of the subunit composition of the purified PSI reaction center complexes revealed that PsaK2 subunit was absent in the PSI complexes under low light condition, but was incorporated into the complexes during acclimation to high light. The growth of the psaK2 mutant on solid medium was inhibited under high light condition. We determined the photosynthetic characteristics of the wild type strain and the two mutants, the psaK1 (ssr0390) mutant and the psaK2 mutant, using pulse amplitude modulation fluorometer. Non-photochemical quenching, which reflects the energy transfer from phycobilisome to PSI in cyanobacteria, was higher in high light grown cells than in low light grown cells, both in the wild type and the psaK1 mutant. However, this change of non-photochemical quenching during acclimation to high light was not observed in the psaK2 mutant. Thus, PsaK2 subunit is involved in the energy transfer from phycobilisome to PSI under high light condition. The role of PsaK2 in state transition under high light condition was also confirmed by chlorophyll fluorescence emission spectra determined at 77 K. The results suggest that PsaK2-dependent state transition is essential for the growth of this cyanobacterium under high light condition.     

RNA helicase, CrhR is indispensable for the energy redistribution and the regulation of photosystem stoichiometry at low temperature in Synechocystis sp. PCC6803

We investigated the role of a cold-inducible and redox-regulated RNA helicase, CrhR, in the energy redistribution and adjustment of stoichiometry between photosystem I (PSI) and photosystem II (PSII), at low temperature in Synechocystis sp. PCC 6803. The results suggest that during low temperature incubation, i.e., when cells are shifted from 34°C to 24°C, wild type cells exhibited light-induced state transitions, whereas the mutant deficient in CrhR failed to perform the same. At low temperature, wild type cells maintained the plastoquinone (PQ) pool in the reduced state due to enhanced respiratory electron flow to the PQ pool, whereas in ?crhR mutant cells the PQ pool was in the oxidized state. Wild type cells were in state 2 and ?crhR cells were locked in state 1 at low temperature. In both wild type and ?crhR cells, a fraction of PSI trimers were changed to PSI monomers. However, in ?crhR cells, the PSI trimer content was significantly decreased. Expression of photosystem I genes, especially the psaA and psaB, was strongly down-regulated due to oxidation of downstream components of PQ in ?crhR cells at low temperature. We demonstrated that changes in the low temperature-induced energy redistribution and regulation of photosystem stoichiometry are acclimatization responses exerted by Synechocystis cells, essentially regulated by the RNA helicase, CrhR, at low temperature.     

Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803

In cyanobacterial membranes photosynthetic light reaction and respiration are intertwined. It was shown that the single hydrogenase of Synechocystis sp. PCC 6803 is connected to the light reaction. We conducted measurements of hydrogenase activity, fermentative hydrogen evolution and photohydrogen production of deletion mutants of respiratory electron transport complexes. All single, double and triple mutants of the three terminal respiratory oxidases and the ndhB-mutant without a functional complex I were studied. After activating the hydrogenase by applying anaerobic conditions in the dark hydrogen production was measured at the onset of light. Under these conditions respiratory capacity and amount of photohydrogen produced were found to be inversely correlated. Especially the absence of the quinol oxidase induced an increased hydrogenase activity and an increased production of hydrogen in the light compared to wild type cells. Our results support that the hydrogenase as well as the quinol oxidase function as electron valves under low oxygen concentrations. When the activities of photosystem II and I (PSII and PSI) are not in equilibrium or in case that the light reaction is working at a higher pace than the dark reaction, the hydrogenase is necessary to prevent an acceptor side limitation of PSI, and the quinol oxidase to prevent an overreduction of the plastoquinone pool (acceptor side of PSII). Besides oxygen, nitrate assimilation was found to be an important electron sink. Inhibition of nitrate reductase resulted in an increased fermentative hydrogen production as well as higher amounts of photohydrogen.     

Site-directed mutagenesis of the CPa-1 protein of photosystem II: alteration of the basic residue pair 384,385R to 384,385G leads to a defect associated with the oxygen-evolving complex.

The psbB gene encodes the intrinsic chlorophyll-a binding protein CPa-1 (CP-47), a component of photosystem II in higher plants, algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into a segment of the psbB gene encoding the large extrinsic loop region of CPa-1 in the cyanobacterium Synechocystis sp. PCC 6803. Altered psbB genes were introduced into a mutant recipient strain (DEL-1) of Synechocystis in which the genomic psbB gene had been partially deleted. Initial target sites for mutagenesis were absolutely conserved basic residue pairs occurring within the large extrinsic loop. One mutation, RR384385GG, produced a strain with impaired photosystem II activity. This strain exhibited growth characteristics comparable to controls. However, at saturating light intensities this mutant strain evolved oxygen at only 50% of the rate of the control strains. Quantum yield measurements at low light intensities indicated that the mutant had 30% fewer fully functional photosystem II centers than do control strains of Synechocystis. Immunological analysis of a number of photosystem II protein components indicated that the mutant accumulates normal quantities of photosystem II proteins and that the ratio of photosystem II to photosystem I proteins is comparable to that found in control strains. Upon exposure to high light intensities the mutant cells exhibited a markedly increased susceptibility to photoinactivation. However, Tris-treated thylakoid membranes from both the mutant and wild-type exhibited comparable rates of photoinactivation. Thylakoid membranes isolated from RR384385GG exhibited only 15% of the H2O to 2,6-dichlorophenolindophenol electron transport rate observed in wild-type strains.(ABSTRACT TRUNCATED AT 250 WORDS)     

The high light-inducible polypeptides stabilize trimeric photosystem I complex under high light conditions in Synechocystis PCC 6803

The high light-inducible polypeptides (HLIPs) are critical for survival under high light (HL) conditions in Synechocystis PCC 6803. In this article, we determined the localization of all four HLIPs in thylakoid protein complexes and examined effects of hli gene deletion on the photosynthetic protein complexes. The HliA and HliB proteins were found to be associated with trimeric photosystem I (PSI) complexes and the Slr1128 protein, whereas HliC was associated with PsaL and TMP14. The HliD was associated with partially dissociated PSI complexes. The PSI activities of the hli mutants were 3- to 4-fold lower than that of the wild type. The hli single mutants lost more than 30% of the PSI trimers after they were incubated in intermediate HL for 12 h. The reduction of PSI trimers were further augmented in these cells by the increase of light intensity. The quadruple hli deletion mutant contained less than one-half of PSI trimers following 12-h incubation in intermediate HL. It lost essentially all of the PSI trimers upon exposure to HL for 12 h. Furthermore, a mutant lacking both PSI trimers and Slr1128 showed growth defects similar to that of the quadruple hli deletion mutant under different light conditions. These results suggest that the HLIPs stabilize PSI trimers, interact with Slr1128, and protect cells under HL conditions.     

Analysis of photosynthetic complexes from a cyanobacterial ycf37 mutant

The Ycf37 protein has been suggested to be involved in the biogenesis and/or stability of the cyanobacterial photosystem I (PSI). With Ycf37 specific antibodies, we analyzed the localization of Ycf37 within the thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Inspection of a sucrose gradient profile indicated that small amounts of Ycf37 co-fractionated with monomeric photosynthetic complexes, but not with trimeric PSI. Isolating 3xFLAG epitope-tagged Ycf37 by affinity-tag purification rendered several PSI subunits that specifically co-precipitated with this protein. Blue-native PAGE newly revealed two monomeric PSI complexes (PSI and PSI*) in wild-type thylakoids. The lower amount of PsaK present in PSI* may explain its higher electrophoretic mobility. PSI* was more prominent in high-light grown cells and interestingly proved absent in the Deltaycf37 mutant. PSI* appeared again when the mutant was complemented in trans with the wild-type ycf37 gene. In the Deltaycf37 mutant the amount of trimeric PSI complexes was reduced to about 70% of the wild-type level with no significant changes in photochemical activity and subunit composition of the remaining photosystems. Our results indicate that Ycf37 plays a specific role in the preservation of PSI* and the biogenesis of PSI trimers.     

Mutagenesis of hydrogenase accessory genes of Synechocystis sp. PCC 6803. Additional homologues of hypA and hypB are not active in hydrogenase maturation

Genes homologous to hydrogenase accessory genes are scattered over the whole genome in the cyanobacterium Synechocystis sp. PCC 6803. Deletion and insertion mutants of hypA1 (slr1675), hypB1 (sll1432), hypC, hypD, hypE and hypF were constructed and showed no hydrogenase activity. Involvement of the respective genes in maturation of the enzyme was confirmed by complementation. Deletion of the additional homologues hypA2 (sll1078) and hypB2 (sll1079) had no effect on hydrogenase activity. Thus, hypA1 and hypB1 are specific for hydrogenase maturation. We suggest that hypA2 and hypB2 are involved in a different metal insertion process. The hydrogenase activity of DeltahypA1 and DeltahypB1 could be increased by the addition of nickel, suggesting that HypA1 and HypB1 are involved in the insertion of nickel into the active site of the enzyme. The urease activity of all the hypA and hypB single- and double-mutants was the same as in wild-type cells. Therefore, there seems to be no common function for these two hyp genes in hydrogenase and urease maturation in Synechocystis. Similarity searches in the whole genome yielded Slr1876 as the best candidate for the hydrogenase-specific protease. The respective deletion mutant had no hydrogenase activity. Deletion of hupE had no effect on hydrogenase activity but resulted in a mutant unable to grow in a medium containing the metal chelator nitrilotriacetate. Growth was resumed upon the addition of cobalt or methionine. Because the latter is synthesized by a cobalt-requiring enzyme in Synechocystis, HupE is a good candidate for a cobalt transporter in cyanobacteria.     

Insertional inactivation of the gene encoding subunit II of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterium Synechocystis sp. PCC 6803 carries out oxygenic photosynthesis analogous to higher plants. Its photosystem I contains seven different polypeptide subunits. The cartridge mutagenesis technique was used to inactivate the psaD gene which encodes subunit II of photosystem I. A mutant strain lacking subunit II was generated by transforming wild type cells with cloned DNA in which psaD gene was interrupted by a gene conferring kanamycin resistance. The photoautotrophic growth of mutant strain is much slower than that of wild type cells. The membranes prepared from mutant cells lack subunit II of photosystem I. Studies on the purified photosystem I reaction center revealed that the complex lacking subunit II is assembled and is functional in P700 photooxidation but at much reduced rate. Therefore, subunit II of photosystem I is required for efficient function of photosystem I.     

集胞藻PCC6803 EGY同源基因破坏突变体的构建及表型分析

摘要：拟南芥中近来发现的定位于叶绿体的膜嵌合金属蛋白酶EGY1影响叶绿体发育与脂肪酸合成,经生物信息学分析,集胞藻PCC6803 (Synechocystis sp. PCC6803)中slr0643、sll0862基因编码同源蛋白。【目的】为了鉴定这两个基因的功能,【方法】本文通过同源重组插入卡那霉素抗性基因、切断目的基因,分别构建了slr0643::km和sll0862::km两种突变体,检测突变体的生理生化表型。【结果】在30℃,20 μE/m2s自养培养下,slr0643::km与野生型相比,早期     

The PsaE subunit of photosystem I prevents light-induced formation of reduced oxygen species in the cyanobacterium Synechocystis sp. PCC 6803

The PsaE protein is located at the reducing side of photosystem I (PSI) and is involved in docking the soluble electron acceptors, particularly ferredoxin. However, deletion of the psaE gene in the cyanobacterium Synechocystis sp. strain PCC 6803 inhibited neither photoautotrophic growth, nor in vivo linear and cyclic electron flows. Using photoacoustic spectroscopy, we detected an oxygen-dependent, PSI-mediated energy storage activity in the DeltapsaE null mutant, which was not present in the wild type (WT). The expression of the genes encoding catalase (katG) and iron superoxide dismutase (sodB) was upregulated in the DeltapsaE mutant, and the increase in katG expression was correlated with an increase in catalase activity of the cells. When catalases were inhibited by sodium azide, the production of reactive oxygen species was enhanced in DeltapsaE relative to WT. Moreover, sodium azide strongly impaired photoautotrophic growth of the DeltapsaE mutant cells while WT was much less sensitive to this inhibitor. The katG gene was deleted in the DeltapsaE mutant, and the resulting double mutant was more photosensitive than the single mutants, showing cell bleaching and lipid peroxidation in high light. Our results show that the presence of the PsaE polypeptide at the reducing side of PSI has a function in avoidance of electron leakage to oxygen in the light (Mehler reaction) and the resulting formation of toxic oxygen species. PsaE-deficient Synechocystis cells can counteract the chronic photoreduction of oxygen by increasing their capacity to detoxify reactive oxygen species.     

Monitoring Photosynthesis in Individual Cells of Synechocystis sp. PCC 6803 on a Picosecond Timescale

Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I (PSI) in vivo for the first time, and the observed 6-ps equilibration process and 25-ps trapping process are the same as found previously for isolated PSI. No major differences are detected between different cells. The PAL mutant cells, devoid of phycobilisomes, show four lifetimes: ∼20 ps (PSI and PSII), ∼80 ps, ∼440 ps, and 2.8 ns (all due to PSII), but not all cells are identical and variations in the kinetics are traced back to differences in the PSI/PSII ratio. Finally, FLIM measurements on WT cells reveal that in some cells or parts of cells, phycobilisomes are disconnected from PSI/PSII. It is argued that the FLIM setup used can become instrumental in unraveling photosynthetic regulation mechanisms in the future.     

The BtpA protein stabilizes the reaction center proteins of photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 at low temperature

Specific inhibition of photosystem I (PSI) was observed under low-temperature conditions in the cyanobacterium Synechocystis sp. strain PCC 6803. Growth at 20 degrees C caused inhibition of PSI activity and increased degradation of the PSI reaction center proteins PsaA and PsaB, while no significant changes were found in the level and activity of photosystem II (PSII). BtpA, a recently identified extrinsic thylakoid membrane protein, was found to be a necessary regulatory factor for stabilization of the PsaA and PsaB proteins under such low-temperature conditions. At normal growth temperature (30 degrees C), the BtpA protein was present in the cell, and its genetic deletion caused an increase in the degradation of the PSI reaction center proteins. However, growth of Synechocystis cells at 20 degrees C or shifting of cultures grown at 30 degrees C to 20 degrees C led to a rapid accumulation of the BtpA protein, presumably to stabilize the PSI complex, by lowering the rates of degradation of the PsaA and PsaB proteins. A btpA deletion mutant strain could not grow photoautotrophically at low temperature, and exhibited rapid degradation of the PSI complex after transfer of the cells from normal to low temperature.