Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants

A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori _fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described.     

Steady-State Phosphorylation of Light-Harvesting Complex II Proteins Preserves Photosystem I under Fluctuating White Light

According to the “state transitions” theory, the light-harvesting complex II (LHCII) phosphorylation in plant chloroplasts is essential to adjust the relative absorption cross section of photosystem II (PSII) and PSI upon changes in light quality. The role of LHCII phosphorylation upon changes in light intensity is less thoroughly investigated, particularly when changes in light intensity are too fast to allow the phosphorylation/dephosphorylation processes to occur. Here, we demonstrate that the Arabidopsis (Arabidopsis thaliana) stn7 (for state transition7) mutant, devoid of the STN7 kinase and LHCII phosphorylation, shows a growth penalty only under fluctuating white light due to a low amount of PSI. Under constant growth light conditions, stn7 acquires chloroplast redox homeostasis by increasing the relative amount of PSI centers. Thus, in plant chloroplasts, the steady-state LHCII phosphorylation plays a major role in preserving PSI upon rapid fluctuations in white light intensity. Such protection of PSI results from LHCII phosphorylation-dependent equal distribution of excitation energy to both PSII and PSI from the shared LHCII antenna and occurs in cooperation with nonphotochemical quenching and the proton gradient regulation5-dependent control of electron flow, which are likewise strictly regulated by white light intensity. LHCII phosphorylation is concluded to function both as a stabilizer (in time scales of seconds to minutes) and a dynamic regulator (in time scales from tens of minutes to hours and days) of redox homeostasis in chloroplasts, subject to modifications by both environmental and metabolic cues. Exceeding the capacity of LHCII phosphorylation/dephosphorylation to balance the distribution of excitation energy between PSII and PSI results in readjustment of photosystem stoichiometry.Plant acclimation to different quantities and qualities of light has been extensively investigated. The light quality experiments have usually concerned the red/blue and far-red light acclimation strategies, which have been closely related to the state transitions and the phosphorylation of the light-harvesting complex II (LHCII) proteins, Lhcb1 and Lhcb2, by the state transition7 (STN7) kinase (Allen, 2003; Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006; Rochaix, 2007). Such studies on acclimation to different qualities of light have uncovered key mechanisms required for the maintenance of photosynthetic efficiency in dense populations and canopies (Dietzel et al., 2008). However, the role of LHCII phosphorylation under fluctuations in the quantity of white light has been scarcely investigated. Light conditions in natural environments may be very complex with respect to the quantity of white light, which constantly fluctuates both in short- and long-term durations (Smith, 1982; Külheim et al., 2002). Thus, the acclimation strategies to natural environments must concomitantly meet the challenges of both high- and low-light acclimation. Changing cloudiness, for example, would initiate both the high-light and low-light acclimation signals in the time scale of minutes and hours, whereas the movements of leaves in the wind or the rapid movement of clouds would initiate even more frequent light acclimation signals. The kinetics of reversible LHCII phosphorylation is far too slow to cope with rapid environmental changes.The phosphorylation level of LHCII proteins in the thylakoid membrane is regulated by both the STN7 kinase and the counteracting PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010). No definite results are available about regulation of the PPH1/TAP38 phosphatase, but the STN7 kinase is strongly under redox regulation (Lemeille et al., 2009) and controls the phosphorylation level of LHCII proteins under varying white light intensities as well as according to chloroplast metabolic cues, as described already decades ago (Fernyhough et al., 1983; Rintamäki et al., 2000; Hou et al., 2003). So far, research on the role of the STN7 kinase and LHCII phosphorylation in the light acclimation of higher plants has heavily focused on reversible LHCII phosphorylation and concomitant state transitions. The state 1-to-state 2 transition, by definition, means the phosphorylation of LHCII proteins, their detachment from PSII in grana membranes, and migration to the stroma membranes to serve in the collection of excitation energy to PSI (Fork and Satoh, 1986; Williams and Allen, 1987; Wollman, 2001; Rochaix, 2007; Kargul and Barber, 2008; Murata, 2009; Lemeille et al., 2010; Minagawa, 2011). Concomitantly, the absorption cross section of PSII decreases and that of PSI increases (Canaani and Malkin, 1984; Malkin et al., 1986; Ruban and Johnson, 2009). Indeed, state transitions have been well documented when different qualities (blue/red and far red) of light, preferentially exciting either PSII or PSI, have been applied.Different from state transitions, the white light intensity-dependent reversible LHCII phosphorylation does not result in differential excitation of the two photosystems (Tikkanen et al., 2010). Instead, both photosystems remain nearly equally excited independently whether the LHCII proteins are heavily phosphorylated or strongly dephosphorylated. Moreover, it is worth noting that the different qualities of light generally used to induce reversible LHCII phosphorylation and state transitions (blue/red and far-red lights) have usually been of very low intensity (for review, see Haldrup et al., 2001), and apparently, minimal protonation of the lumen takes place under such illumination conditions. Yet another difference between induction of LHCII protein phosphorylation by different qualities of light or different quantities of white light concerns the concomitant induction of PSII core protein phosphorylation. In the former case, the level of PSII core protein phosphorylation follows the phosphorylation pattern of LHCII proteins, whereas under different quantities of white light, the phosphorylation behavior of PSII core and LHCII proteins is the opposite (Tikkanen et al., 2008b).To gain a more comprehensive understanding of the physiological role of white light-induced changes in LHCII protein phosphorylation, we have integrated Arabidopsis (Arabidopsis thaliana) LHCII phosphorylation with other light-dependent regulatory modifications of light harvesting and electron transfer in the thylakoid membrane, which include the nonphotochemical quenching of excitation energy (for review, see Niyogi, 1999; Horton and Ruban, 2005; Barros and Kühlbrandt, 2009; de Bianchi et al., 2010; Jahns and Holzwarth, 2012; Ruban et al., 2012) and the photosynthetic control of electron transfer by the cytochrome b₆f (Cytb6f) complex (Rumberg and Siggel, 1969; Witt, 1979; Tikhonov et al., 1981; Bendall, 1982; Nishio and Whitmarsh, 1993; Joliot and Johnson, 2011; Suorsa et al., 2012; for review, see Foyer et al., 1990, 2012), both strongly dependent on lumenal protonation.It is demonstrated that the steady-state LHCII phosphorylation is particularly important under rapidly fluctuating light (FL) conditions. This ensures equal energy distribution to both photosystems, prevents the accumulation of electrons in the intersystem electron transfer chain (ETC), eliminates perturbations in chloroplast redox balance, and maintains PSI functionality upon rapid fluctuations in white light intensity.     

Plasma membrane of Synechocystis PCC 6803: a heterogeneous distribution of membrane proteins

Proteomic studies carried out previously on the plasma membrane of Synechocystis have identified several peripheral and integral proteins. The distribution of these proteins along the membrane still remains obscure. In this study, the distribution of proteins along the plasma membrane of Synechocystis was carried out using subfractions, the right-side-out (RSO) and inside-out (ISO) vesicles, fractionated from a pure and specific fraction of the plasma membrane. These subfractions were analyzed and quantified for several proteins by immunoblotting. It was found that the ISO fraction contained higher quantities of preD1, D1 and PsaD, the integral proteins of photosystem I and II known to be present also in the plasma membrane. Lower amounts of peripheral vesicle inducing protein Vipp1 and nitrate/nitrite binding protein NrtA were present in the ISO compared to the RSO fraction. On the contrary, the distribution of two integral transporter proteins, SbtA and PxcA, was found equal in both fractions. Our studies clearly establish that the plasma membrane of Synechocystis has a heterogeneous composition with respect to protein distribution. The accumulation of photosynthesis-associated proteins in the ISO fraction provides evidence that the discrete regions of the plasma membrane harbor sites for biogenesis of photosystems.     

Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme

Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes. We could identify a significant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled Photosystem II complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.     

Role of Thylakoid ATP/ADP Carrier in Photoinhibition and Photoprotection of Photosystem II in Arabidopsis

The chloroplast thylakoid ATP/ADP carrier (TAAC) belongs to the mitochondrial carrier superfamily and supplies the thylakoid lumen with stromal ATP in exchange for ADP. Here, we investigate the physiological consequences of TAAC depletion in Arabidopsis (Arabidopsis thaliana). We show that the deficiency of TAAC in two T-DNA insertion lines does not modify the chloroplast ultrastructure, the relative amounts of photosynthetic proteins, the pigment composition, and the photosynthetic activity. Under growth light conditions, the mutants initially displayed similar shoot weight, but lower when reaching full development, and were less tolerant to high light conditions in comparison with the wild type. These observations prompted us to study in more detail the effects of TAAC depletion on photoinhibition and photoprotection of the photosystem II (PSII) complex. The steady-state phosphorylation levels of PSII proteins were not affected, but the degradation of the reaction center II D1 protein was blocked, and decreased amounts of CP43-less PSII monomers were detected in the mutants. Besides this, the mutant leaves displayed a transiently higher nonphotochemical quenching of chlorophyll fluorescence than the wild-type leaves, especially at low light. This may be attributed to the accumulation in the absence of TAAC of a higher electrochemical H⁺ gradient in the first minutes of illumination, which more efficiently activates photoprotective xanthophyll cycle-dependent and independent mechanisms. Based on these results, we propose that TAAC plays a critical role in the disassembly steps during PSII repair and in addition may balance the trans-thylakoid electrochemical H⁺ gradient storage.In plants, the chloroplast thylakoid membrane is the site of light-driven photosynthetic reactions coupled to ATP synthesis. There are four major protein complexes involved in these reactions, namely, PSI, PSII, the cytochrome b₆f, and the H⁺-translocating ATP synthase (for review, see Nelson and Ben-Shem, 2004). The photosystems and the cytochrome b₆f complex also contain redox components and pigments bound to protein subunits. Their synthesis, assembly, optimal function, and repair during normal development and stress require a number of transport and regulatory mechanisms. In this context, the water-oxidizing PSII complex composed of more than 25 integral and peripheral proteins attracts special attention since its reaction center D1 subunit is degraded and replaced much faster than the other subunits under excess and even growth light conditions (for review, see Aro et al., 2005). Thus, the D1 protein turnover is the major event in the repair cycle of the PSII complex and occurs subsequently to the inactivation of PSII electron transport. D1 degradation is most likely performed by thylakoid FtsH and Deg proteases, operating on both sides of the thylakoid membrane (Lindahl et al., 2000; Haussühl et al., 2001; Silva et al., 2003; Kapri-Pardes et al., 2007). The PSII repair cycle is regulated by reversible phosphorylation of several core subunits (Tikkanen et al., 2008).ATP is produced as a result of the light-driven photosynthetic reactions in the thylakoid membrane and mainly is utilized in the carbon fixation reactions occurring in the soluble stroma. Besides this, ATP also drives several energy-dependent processes occurring on the stromal side of the thylakoid membrane, including phosphorylation, folding, import, and degradation of proteins. Furthermore, experimental evidence for ATP transport across the thylakoid membrane and nucleotide metabolism inside the lumenal space has been reported (Spetea et al., 2004; for review, see Spetea and Thuswaldner, 2008; Spetea and Schoefs, 2010). The protein responsible for the thylakoid ATP transport activity has been identified in Arabidopsis (Arabidopsis thaliana) as the product of the At5g01500 gene and functionally characterized in Escherichia coli as an ATP/ADP exchanger (Thuswaldner et al., 2007). This protein is homologous to the extensively studied bovine mitochondrial ADP/ATP carrier and therefore has been named thylakoid ATP/ADP carrier (TAAC). In the same report, it has been demonstrated that TAAC transports ATP from stroma to lumen in exchange for ADP, as based on radioactive assays using thylakoids isolated from Arabidopsis wild-type plants and a T-DNA insertion knockout line (named taac). Furthermore, TAAC was shown to be mainly expressed in photosynthetic tissues with an up-regulation during greening, senescence, and stress (e.g. high light) conditions, implying a physiological role during thylakoid biogenesis and turnover.The ATP translocated by TAAC across the thylakoid membrane is converted to GTP by the lumenal nucleoside diphosphate kinase III; GTP can then be bound and hydrolyzed to GDP and inorganic phosphate by the PsbO protein, a lumenal extrinsic subunit of the PSII complex (Spetea et al., 2004; Lundin et al., 2007a). The anion transporter 1 from Arabidopsis has been proposed to export to the stroma the phosphate generated during nucleotide metabolism in the thylakoid lumen (Ruiz Pavón et al., 2008). Between the two PsbO isoforms in Arabidopsis, it has recently been reported that PsbO2 plays an essential role in D1 protein turnover during high light stress and that it has a higher GTPase activity than PsbO1 (Lundin et al., 2007b, 2008; Allahverdiyeva et al., 2009). The precise mechanism of PsbO2-mediated PSII repair is not known. Nevertheless, the requirement of GTP for efficient proteolytic removal of the D1 protein during repair of photoinactivated PSII was previously reported (Spetea et al., 1999). Furthermore, it has been proposed that the PsbO2 type of PSII complexes undergo more efficient repair. This has been attributed to the PsbO2-mediated GTPase activity that induces PsbO2 release from the complex, thus facilitating the next steps in the repair process, namely, dissociation of the CP43 subunit and proteolysis of the D1 subunit (Lundin et al., 2007b, 2008).TAAC may represent the missing link between ATP synthesis on the stromal side of the thylakoid membrane and nucleotide-dependent reactions in the lumenal space. The taac mutant provides an interesting tool to study whether there are any regulatory networks between the activity of TAAC and PSII repair. Based on phenotypic characterization of two different T-DNA insertion lines of the TAAC gene, we report in this article that the PSII repair cycle is malfunctioning in the absence of TAAC and that the thermal photoprotection is faster activated during light stress.     

Dithiol oxidant and disulfide reductant dynamically regulate the phosphorylation of light-harvesting complex II proteins in thylakoid membranes

Light-induced phosphorylation of light-harvesting chlorophyll a/b complex II (LHCII) proteins in plant thylakoid membranes requires an activation of the LHCII kinase via binding of plastoquinol to cytochrome b(6)f complex. However, a gradual down-regulation of LHCII protein phosphorylation occurs in higher plant leaves in vivo with increasing light intensity. This inhibition is likely to be mediated by increasing concentration of thiol reductants in the chloroplast. Here, we have determined the components involved in thiol redox regulation of the LHCII kinase by studying the restoration of LHCII protein phosphorylation in thylakoid membranes isolated from high-light-illuminated leaves of pumpkin (Cucurbita pepo), spinach (Spinacia oleracea), and Arabidopsis. We demonstrate an experimental separation of two dynamic activities associated with isolated thylakoid membranes and involved in thiol regulation of the LHCII kinase. First, a thioredoxin-like compound, responsible for inhibition of the LHCII kinase, became tightly associated and/or activated within thylakoid membranes upon illumination of leaves at high light intensities. This reducing activity was completely missing from membranes isolated from leaves with active LHCII protein phosphorylation, such as dark-treated and low-light-illuminated leaves. Second, hydrogen peroxide was shown to serve as an oxidant that restored the catalytic activity of the LHCII kinase in thylakoids isolated from leaves with inhibited LHCII kinase. We propose a dynamic mechanism by which counteracting oxidizing and reducing activities exert a stimulatory and inhibitory effect, respectively, on the phosphorylation of LHCII proteins in vivo via a novel membrane-bound thiol component, which itself is controlled by the thiol redox potential in chloroplast stroma.     

Site-specific mutations in the D1 polypeptide affect the susceptibility of Synechocystis 6803 cells to photoinhibition

Photoinhibition of photosystem II in the cyanobacterium Synechocystis 6803 was followed after site-specific mutagenesis of the D1 polypeptide. Mutations were created in the stromal/cytosolic loop connecting helices D and E. Two mutations E243K and CA1, a deletion of the three glutamates 242–244 and a substitution Q241H, were made in the putative cleavage area of the D1 polypeptide. A third mutation E229D was made in the PEST-like sequence. Mutants and control cells were illuminated and F_V/F_M was recorded. Compared to the control, the mutants were less photoinhibited. Fluorescence relaxation after a single flash was delayed in CA1. Restoration of F_V/F_M after photoinhibition in the mutants was totally dependent on protein synthesis while control cells were able to recover partially also when protein synthesis was inhibited. In addition, the protein synthesis-dependent recovery of CA1 was slowed down. Our results indicate a correlation between the mutated amino acids and photoinhibition of photosystem II.     

Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II

The degradation rate of the D1 polypeptide was measured in threeSynechocystis PCC 6803 mutantsin vivo. Mutations were introduced into a putative cleavage area of the D1 polypeptide (QEEET motif) and into the PEST-like area. PEST sequences are often found in proteins with a high turnover rate. The QEEET-motif mutants are CA1 [(E242-E244);Q241H] and E243K, and the third mutation, E229D, was directed to the PEST-like area. During high-light illumination (1500 mol photons m^-2s^-1) that induced photoinhibition of photosystem II (PSII), the half-life time of the D1 polypeptide in mutant E229D (t _1/2=35 min) was about twice as long as in AR (control strain) cells (t _1/2=19 min). In growth light (40 mol photons m^-2s^-1), the degradation rate of the D1 polypeptide in E229D and AR strains was the same (t _1/25 h). In growth light the D1 polypeptide was degraded faster in both QEEET-motif mutants than in the AR strain, but in photoinhibitory light the degradation rates were similar. According to these results, the highly conservative QEEET motif as such is not required for the proteolytic cut of the D1 polypeptide, but it does affect the rate of degradation. No simple correlation existed between the degradation rate of the D1 polypeptide and the susceptibility of PSII to photoinhibition in mutant and AR cells under our experimental conditions.     

A previously found thylakoid membrane protein of 14kDa (TMP14) is a novel subunit of plant photosystem I and is designated PSI-P

We show that the thylakoid membrane phosphoprotein TMP14 is a novel subunit of plant photosystem I (PSI). Blue native/SDS-PAGE and sucrose gradient fractionation demonstrated the association of the protein exclusively with PSI. We designate the protein PSI-P. The presence of PSI-P subunit in Arabidopsis mutants lacking other PSI subunits was analyzed and suggested a location in the proximity of PSI-L, -H and -O subunits. The PSI-P protein was not differentially phosphorylated in state 1 and state 2.