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.     

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.     

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803

The cytochrome b₆f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b₆f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.     

Extended heme promiscuity in the cyanobacterial cytochrome c oxidase: characterization of native complexes containing hemes A, O, and D, respectively.

The cyanobacteria Anacystis nidulans (Synechococcus sp. PCC6301), Synechocystis sp. PCC6803, Anabaena sp. PCC 7120, and Nostoc sp. PCC8009 were grown photoautotrophically under reduced oxygen tension in a medium with sulfate replaced by thiosulfate and nitrate replaced by ammonium as the S- and N-sources, respectively. In addition, Anabaena and Nostoc were grown under dinitrogen-fixing conditions in a medium free of combined nitrogen. Membranes were isolated from late-logarithmic cells (culture density corresponding to approximately 3 microliters packed cells per milliliter); cytoplasmic and thylakoid membranes were separated and purified according to established procedures. Acid-labile hemes were extracted from the membranes and subjected to reversed-phase high-performance liquid chromatography. Separated hemes were analyzed spectroscopically and identified by comparison with authentic standards. In addition to hemes B, A, and O, the latter of which was induced under semianaerobic conditions only, substitution of thiosulfate and ammonium for the oxy-anions sulfate and nitrate led to the appearance of spectrally discernible heme D in the membranes and extracts therefrom. However, spectroscopic and kinetic investigation of the membrane-bound heme D rather disproved any reaction with oxygen or carbon monoxide. Kinetic measurements performed with the membrane-bound respiratory oxidase gave evidence for only two kinetically competent terminal oxidases, a3 and o3, both apparently associated with a single type of apoprotein, viz. subunit I of the known cyanobacterial aa3-type cytochrome c oxidase. The heme D, on the other hand, seems to form a spectrally distinguished, yet kinetically ill-defined hemoprotein complex which does not qualify as a fully functional d-type terminal oxidase on our (wild-type) cyanobacteria even after growth under semianaerobic pseudo-reducing conditions. Also growth (of Anabaena and Nostoc) under dinitrogen-fixing conditions did not change this situation. Thus, we are left with (wild-type) cyanobacteria forming an unbranched respiratory chain with only a single type of terminal oxidase protein, viz. the known aa3-type cytochrome c oxidase. This oxidase, however, may incorporate different prosthetic (heme) groups in the sense of "heme promiscuity." Biosynthesis of the different heme groups thereby seems to respond to the ambient redox environment. In particular, however, conditions for expression of the two quinol oxidases potentially and additionally coded for by the genome of, e. g., Synechocystis sp. PCC6803 (see http://www.kazusa.or.jp/cyano), have not yet been found.     

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.     

NADPH dehydrogenase-mediated respiratory electron transport in thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803 is inactive in the light

Non-photochemical redox changes of the plastoquinone pools in darkness were investigated in the cyanobacterium Synechocystis sp. PCC 6803 by monitoring changes in Chl fluorescence yield during light-to-dark transitions. The inhibitors rotenone and mercury with or without 1 mM succinate fully suppressed the post-illumination increase in Chl fluorescence in both NADPH dehydrogenase-defective (M55) and deltaCtaI cells. The latter cells lack subunit I of cytochrome aa3-type cytochrome c oxidase. These results strongly suggest that NADPH dehydrogenase plays the major role in electron donation in the non-photo-chemical reduction of plastoquinone. The rising phase of post-illumination Chl fluorescence in both wild type pretreated with KCN, and deltaCtaI cells, was significantly slowed by low light illumination. We detected comparable photochemical levels of both photosystem (PS) II and PSI during steady state illumination in wild type and deltaCtaI cells. From these results, we suggest that respiratory electron flow involved in the non-photochemical redox change of plastoquinone is not likely to occur in the light.     

A strain of Synechocystis sp. PCC 6803 without photosynthetic oxygen evolution and respiratory oxygen consumption: implications for the study of cyclic photosynthetic electron transport

Cyclic electron transport around photosystem (PS) I is believed to play a role in generation of ATP required for adaptation to stress in cyanobacteria and plants. However, elucidation of the pathway(s) of cyclic electron flow is difficult because of low rates of this electron flow relative to those of linear photosynthetic and respiratory electron transport. We have constructed a strain of Synechocystis sp. PCC 6803 that lacks both PSII and respiratory oxidases and that, consequently, neither evolves nor consumes oxygen. However, this strain is still capable of cyclic electron flow around PSI. The photoheterotrophic growth rate of this strain increased with light intensity up to an intensity of about 25 mumol photons m-2 s-1, supporting the notion that cyclic electron flow contributes to ATP generation in this strain. Indeed, the ATP-generating ability of PSI is demonstrated by the fact that the PSII-less oxidase-less strain is able to grow at much higher salt concentrations than a strain lacking PSI. A quinone electrode was used to measure the redox state of the plastoquinone pool in vivo in the various strains used in this study. In contrast to what is observed in chloroplasts, the plastoquinone pool was rather reduced in darkness and was oxidized in the light. This is in line with significant electron donation by respiratory pathways (NADPH dehydrogenase and particularly succinate dehydrogenase) in darkness. In the light, the pool becomes oxidized due to the presence of much more PSI than PSII. In the oxidase-less strains, the plastoquinone pool was very much reduced in darkness and was oxidized in the light by PSI. Photosystem II activity did not greatly alter the redox state of the plastoquinone pool. The results suggest that cyclic electron flow around PSI can contribute to generation of ATP, and a strain deficient in linear electron transport pathways provides an excellent model for further investigations of cyclic electron flow.     

Voltammetric measurement of the plastoquinone redox state in isolated thylakoids

The oxidation of plastoquinol by the cytochrome bf complex is commonly believed to be the rate limiting step in photosynthetic electron transport. When input of electrons from PS II exceeds electron flow through the cytochrome bf complex the plastoquinone pool becomes reduced. A voltammetric technique previously used to measure the redox state of the ubiquinone pool in plant mitochondria, was modified to measure the redox state of the plastoquinone pool in thylakoids. The presence or absence of a proton gradient strongly influenced the relationship between the redox state of the plastoquinone pool and other photosynthetic parameters. A linear relationship between the rate of electron transport and the reduction of plastoquinone was found. The slope of this relationship was greater in coupled than in uncoupled thylakoids, indicating that under coupled conditions the plastoquinone pool is more reduced at any given rate of electron flow. A complex relationship was found between QA reduction, calculated as 1 – q_p, and the redox state of the plastoquinone pool. The extent of Q_A reduction was similar in coupled and uncoupled thylakoids, but at any given level of Q_A reduction, PQ was always more reduced in coupled thylakoids. These results suggest that the presence of a proton gradient changes the equilibrium constant between Q_A and PQ.     

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.

     

Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803

Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70-240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803.     

Relationship Between the Growth of Synechocystis sp. PCC 6803 on Medium with Glucose and the Photosynthetic Energy Transformation

Measurements of chlorophyll fluorescence kinetics from dark-starved cells, light-grown cells and mixotrophic cells of Synechocystis sp. PCC 6803 were obtained using a pulse amplitude modulation (PAM) fluorometer. Photosystem Ⅱ photochemical efficiency Ⅱand the extent of reduction of Q－A in the three kinds of cells described above were compared. The millisecond delayed light emission (MDLE) of light-grown cells and mixotrophic cells were also detected. On the basis of the analysis of fluorescence kinetic parameters, comparison of the slow phase of MDLE and the influence of inhibitors of photosynthetic electron transport3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) on the mixotrophic growth of Synechocystis sp. PCC 6803, it was concluded that the reasons for higher growth rate under mixotrophic than that under photoautotrophic might be that glucose promoted the photoautotrophic growth of mixotrephic cells and the donation of eletrons to the plastoquinone pool from the respiratory substance and the transform of energy was promoted by photosynthetic system, which provided the energy needed by anabolism of cells caused by the glucose added to the medium.      

Cytochrome c oxidase of the cyanobacterium Synechocystis sp. PCC 6803 protects photosynthesis from salt stress

We generated cytochrome c oxidase (CtaI)-defective cells of the cyanobacterium Synechocystis sp. PCC 6803 in order to investigate the physiological function of the CtaI-mediated respiratory electron transport pathway. When they were salt stressed, CtaI-defective cells showed a substantial decrease in photosynthesis due to reduction of the photochemical efficiency of Photosystem II and of the chlorophyll in the reaction center of the photo-oxidizable form of Photosystem I. These findings demostrate that CtaI-mediated electron transport is important for resistance to salt stress.     

Type 2 NADH Dehydrogenases in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration

Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool.     

Light inhibition of respiration is due to a dual function of the cytochrome b6-f complex and the plastocyanin/cytochrome c-553 pool in Aphanocapsa

Studies of the respiratory electron transport pathway in the blue-green alga, Aphanocapsa, demonstrated the presence of cytochrome oxidase and a cytochrome complex. The use of antimycin A showed only the occurrence of a plastidal type of cytochrome complex (the cytochrome b6-f complex), which is insensitive to this inhibitor. Determination of the extent of photooxidation of cytochromes c-553 and f-556 under conditions of high and low cytochrome oxidase activities indicated an electron flow through both cytochromes to cytochrome oxidase. Direct evidence for a common segment of photosynthetic and respiratory electron transport from plastoquinone via the cytochrome b6-f complex to the soluble plastocyanin/cytochrome c-553 pool, as well as a competition between cytochrome oxidase and Photosystem I for reductants in this pool in the light, was obtained by measurements of electron transport with suitable electron donors in this alga.     

Acclimation to the growth temperature and thermosensitivity of photosystem II in a mesophilic cyanobacterium, Synechocystis sp. PCC6803

Differences in the temperature dependence and thermosensitivities of PSII activities in Synechocystis sp. PCC6803 grown at 25 and 35 degrees C were studied. Hill reactions in cells, thylakoid membranes and purified PSII core complexes were measured at high temperatures or at their growth temperatures after high-temperature treatments. In the presence of 2,5-dichloro-p-benzoquinone as an electron acceptor, which can accept electrons directly from Q(A), the temperature dependence of the oxygen-evolving activity was almost the same in thylakoid membranes and in the purified PSII complexes from cells grown at 25 or 35 degrees C. When duroquinone, which accepts electrons only through Q(B) plastoquinone, was used as an electron acceptor, the temperature dependence was the same for purified PSII core complexes but was different between thylakoids isolated from the cells grown at 25 and 35 degrees C. No remarkable difference was observed in protein compositions between thylakoids and between purified PSII complexes from cells grown at 25 or 35 degrees C. However, the fluidity of thylakoids, measured by electron flow to P700, was affected by the growth temperature. These results suggest that one of the major factors which cause the changes in the thermosensitivity of PSII is the change in the fluidity of thylakoid membranes. As for the acclimation of PSII in thylakoids to high temperatures, one of the main causes is the decrease in the high-temperature-induced formation of non-Q(B) PSII due to the decreased fluidity in the cells grown at 35 degrees C.     

Respiratory terminal oxidases in the facultative chemoheterotrophic and dinitrogen fixing cyanobacterium Anabaena variabilis strain ATCC 29413: characterization of the cox2 locus

Upon nitrogen step-down, some filamentous cyanobacteria differentiate heterocysts, cells specialized for dinitrogen fixation, a highly oxygen sensitive process. Aerobic respiration is one of the mechanisms responsible for a microaerobic environment in heterocysts and respiratory terminal oxidases are the key enzymes of the respiratory chains. We used Anabaena variabilis strain ATCC 29413, because it is one of the few heterocyst-forming facultatively chemoheterotrophic cyanobacteria amenable to genetic manipulation. Using PCR with degenerate primers, we found four gene loci for respiratory terminal oxidases, three of which code for putative cytochrome c oxidases and one whose genes are homologous to cytochrome bd-type quinol oxidases. One cytochrome c oxidase, Cox2, was the only enzyme whose expression, tested by RT-PCR, was evidently up-regulated in diazotrophy, and therefore cloned, sequenced, and characterized. Up-regulation of Cox2 was corroborated by Northern and primer extension analyses. Strains were constructed lacking Cox1 (a previously characterized cytochrome c oxidase), Cox2, or both, which all grew diazotrophically. In vitro cytochrome c oxidase and respiratory activities were determined in all strains, allowing for the first time to estimate the relative contributions to total respiration of the different respiratory electron transport branches under different external conditions. Especially adding fructose to the growth medium led to a dramatic enhancement of in vitro cytochrome c oxidation and in vivo respiratory activity without significantly influencing gene expression.     

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