Na+-stimulated phosphate uptake system in Synechocystis sp. PCC 6803 with Pst1 as a main transporter

<p>Abstract</p> <p>Background</p> <p>Most living cells uptake phosphate, an indispensable nutrient for growth from their natural environment. In <it>Synechocystis </it>sp. PCC 6803, the cells lack phosphate-inorganic transport (Pit) system but contain two phosphate-specific transport (Pst) systems, Pst1 and Pst2. We investigated the kinetics of Pi uptake of these two Pst systems by constructing the two mutants, ΔPst1 and ΔPst2, and comparing their kinetic properties with those of the wild-type cells under both Pi-sufficient and deficient conditions. The effects of pH and Na⁺on the uptake of phosphate in <it>Synechocystis </it>were also studied.</p> <p>Results</p> <p>Growth rates of the two mutants and wild type were similar either under phosphate-sufficient or deficient condition. The <it>K_m</it>for phosphate uptake was 6.09 μM in wild type and this was reduced to 0.13 μM in ΔPst1 cells and 5.16 μM in the ΔPst2 strain. The <it>V_max</it>values of 2.48, 0.22, and 2.17 μmol • (min • mg of chlorophyll <it>a</it>)^-1were obtained for wild type, the ΔPst1 and ΔPst2 strains, respectively. A monophasic phosphate uptake was observed in wild-type cells. The uptake of phosphate was energy and pH-dependent with a broad pH optimum between pH 7-10. Osmolality imposed by NaCl stimulated phosphate uptake whereas that imposed by sorbitol decreased uptake, suggesting stimulation of uptake was dependent upon ionic effects.</p> <p>Conclusion</p> <p>The data demonstrate that Pst2 system of <it>Synechocystis </it>has higher affinity toward phosphate with lower <it>V_max</it>than Pst1 system. The Pst1 system had similar <it>K_m</it>and <it>V_max</it>values to those of the wild type suggesting that Pst1 is the main phosphate transporter in <it>Synechocystis </it>sp. PCC 6803. The <it>K_m</it>of Pst1 of <it>Synechocystis </it>is closer to that of Pit system than to that of the Pst system of <it>E. coli</it>, suggesting that <it>Synechocystis </it>Pst1 is rather a medium/low affinity transporter whereas Pst2 is a high affinity transporter.</p

The importance of the hydrophilic region of PsbL for the plastoquinone electron acceptor complex of Photosystem II

AbstractThe PsbL protein is a 4.5kDa subunit at the monomer–monomer interface of Photosystem II (PS II) consisting of a single membrane-spanning domain and a hydrophilic stretch of ~15 residues facing the cytosolic (or stromal) side of the photosystem. Deletion of conserved residues in the N-terminal region has been used to investigate the importance of this hydrophilic extension. Using Synechocystis sp. PCC 6803, three deletion strains: ∆(N6–N8), ∆(P11–V12) and ∆(E13–N15), have been created. The ∆(N6–N8) and ∆(P11–V12) strains remained photoautotrophic but were more susceptible to photodamage than the wild type; however, the ∆(E13–N15) cells had the most severe phenotype. The Δ(E13–N15) mutant showed decreased photoautotrophic growth, a reduced number of PS II centers, impaired oxygen evolution in the presence of PS II-specific electron acceptors, and was highly susceptible to photodamage. The decay kinetics of chlorophyll a variable fluorescence after a single turnover saturating flash and the sensitivity to low concentrations of PS II-directed herbicides in the Δ(E13–N15) strain indicate that the binding of plastoquinone to the QB-binding site had been altered such that the affinity of QB is reduced. In addition, the PS II-specific electron acceptor 2,5-dimethyl-p-benzoquinone was found to inhibit electron transfer through the quinone-acceptor complex of the ∆(E13–N15) strain. The PsbL Y20A mutant was also investigated and it exhibited increased susceptibility to photodamage and increased herbicide sensitivity. Our data suggest that the N-terminal hydrophilic region of PsbL influences forward electron transfer from QA through indirect interactions with the D–E loop of the D1 reaction center protein. Our results further indicate that disruption of interactions between the N-terminal region of PsbL and other PS II subunits or lipids destabilizes PS II dimer formation. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy

The extrinsic proteins of Photosystem II

In this review we examine the structure and function of the extrinsic proteins of Photosystem II. These proteins include PsbO, present in all oxygenic organisms, the PsbP and PsbQ proteins, which are found in higher plants and eukaryotic algae, and the PsbU, PsbV, CyanoQ, and CyanoP proteins, which are found in the cyanobacteria. These proteins serve to optimize oxygen evolution at physiological calcium and chloride concentrations. They also shield the Mn 4CaO 5 cluster from exogenous reductants. Numerous biochemical, genetic and structural studies have been used to probe the structure and function of these proteins within the photosystem. We will discuss the most recent proposed functional roles for these components, their structures (as deduced from biochemical and X-ray crystallographic studies) and the locations of their proposed binding domains within the Photosystem II complex. This article is part of a Special Issue entitled: Photosystem II. © 2011 Elsevier B.V. All rights reserved

Environmental pH Affects Photoautotrophic Growth of Synechocystis sp. PCC 6803 Strains Carrying Mutations in the Lumenal Proteins of PSII.

Synechocystis sp. strain PCC 6803 grows photoautotrophically across a broad pH range, but wild-type cultures reach a higher density at elevated pH; however, photoheterotrophic growth is similar at high and neutral pH. A number of PSII mutants each lacking at least one lumenal extrinsic protein, and carrying a second PSII lumenal mutation, are able to grow photoautotrophically in BG-11 medium at pH 10.0, but not pH 7.5. We investigated the basis of this pH effect and observed no pH-specific change in variable fluorescence yield from PSII centers of the wild type or the pH-dependent ΔPsbO:ΔPsbU and ΔPsbV:ΔCyanoQ strains; however, 77 K fluorescence emission spectra indicated increased coupling of the phycobilisome (PBS) antenna at pH 10.0 in all mutants. DNA microarray data showed a cell-wide response to transfer from pH 10.0 to pH 7.5, including decreased mRNA levels of a number of oxidative stress-responsive transcripts. We hypothesize that this transcriptional response led to increased tolerance against reactive oxygen species and in particular singlet oxygen. This response enabled photoautotrophic growth of the PSII mutants at pH 10.0. This hypothesis was supported by increased resistance of all strains to rose bengal at pH 10.0 compared with pH 7.5

Environmental pH and a Glu364 to Gln mutation in the chlorophyll-binding CP47 protein affect redox-active TyrD and charge recombination in Photosystem II

In Photosystem II, loop E of the chlorophyll-binding CP47 protein is located near a redox-active tyrosine, Y-D, forming a symmetrical analog to loop E in CP43, which provides a ligand to the oxygen-evolving complex (OEC). A Glu364 to Gln substitution in CP47, near Y-D, does not affect growth in the cyanobacterium Synechocystis sp. PCC 6803; however, deletion of the extrinsic protein PsbV in this mutant leads to a strain displaying a pH-sensitive phenotype. Using thermoluminescence, chlorophyll fluorescence, and flash-induced oxygen evolution analyses, we demonstrate that Glu364 influences the stability of Y-D and the redox state of the OEC, and highlight the effects of external pH on photosynthetic electron transfer in intact cyanobacterial cells

Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers?

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g. Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides,RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+QA− or P+QB−, respectively; (b) the kinetics of electron transfer from QA− to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (τ = 3.5 ps), followed by reduction of the bacteriopheophytin (τ = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (τ = 410 ± 30 and 47 ± 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type). EPR studies of the isolated RCs showed a characteristic g = 1.82 signal for the QA semiquinone coupled to the iron atom, which was indistinguishable from the wild type. It is concluded that GluM234 is not essential to the normal functioning of the acceptor quinone complex in bacterial RCs and that the role of bicarbonate in PS II is distinct from the role of this residue in bacterial RCs

Ethylene Synthesis and Regulated Expression of Recombinant Protein in Synechocystis sp PCC 6803

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P(trc) resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P(A1lacO-1), induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density

Assembly of the Photosystem II Membrane-Protein Complex of Oxygenic Photosynthesis

Photosystem II is a 700-kDa membrane-protein super-complex responsible for the light-driven splitting of water in oxygenic photosynthesis. The photosystem is comprised of two 350-kDa complexes each made of 20 different polypeptides and over 80 co-factors. While there have been major advances in understanding the mature structure of this photosystem many key protein factors involved in the assembly of the complex do not appear in the holoenzyme. The mechanism for assembling this super-complex is a very active area of research with newly discovered assembly factors and subcomplexes requiring characterization. Additionally the ability to split water is inseparable from light-induced photodamage that arises from radicals and reactive O2 species generated by Photosystem II chemistry. Consequently, to sustain water splitting, a “self repair” cycle has evolved whereby damaged protein is removed and replaced so as to extend the working life of the complex. Understanding how the biogenesis and repair processes are coordinated is among several important questions that remain to be answered. Other questions include: how and when are the inorganic cofactors inserted during the assembly and repair processes and how are the subcomplexes protected from photodamage during assembly? Evidence has also been obtained for Photosystem II biogenesis centers in cyanobacteria but do these also exist in plants? Do the molecular mechanisms associated with Photosystem II assembly shed fresh light on the assembly of other major energy-transducing complexes such as Photosystem I or the cytochrome b6/f complex or indeed other respiratory complexes? The contributions to this Frontiers in Plant Science Research Topic are likely to reveal new details applicable to the assembly of a range of membrane-protein complexes, including aspects of self-assembly and solar energy conversion that may be applied to artificial photosynthetic systems. In addition, a deeper understanding of Photosystem II assembly — particularly in response to changing environmental conditions — will provide new knowledge underpinning photosynthetic yields which may contribute to improved food production and long-term food security