Circadian rhythms are endogenous cellular programs that time metabolic and behavioral events to occur at optimal times in the daily cycle. Light and dark cycles synchronize the endogenous clock with the external environment through a process called entrainment. Previously, we identified the bacteriophytochrome-like circadian input kinase CikA as a key factor for entraining the clock in the cyanobacterium Synechococcus elongatus PCC 7942. Here, we present evidence that CikA senses not light but rather the redox state of the plastoquinone pool, which, in photosynthetic organisms, varies as a function of the light environment. Furthermore, CikA associates with the Kai proteins of the circadian oscillator, and it influences the phosphorylation state of KaiC during resetting of circadian phase by a dark pulse. The abundance of CikA varies inversely with light intensity, and its stability decreases in the presence of the quinone analog 2,5-dibromo-3-methyl-6-isopropylp-benzoquinone (DBMIB). The pseudo-receiver domain of CikA is crucial for sensitivity to DBMIB, and it binds the quinone directly, a demonstration of a previously unrecognized ligand-binding role for the receiver fold. Our results suggest that resetting the clock in S. elongatus is metabolism-dependent and that it is accomplished through the interaction of the circadian oscillator with CikA.biological rhythms ͉ photosynthetic electron transport ͉ pseudo-receiver ͉ redox ͉ Synechococcus elongatus

The endogenous 24-h (circadian) rhythms exhibited by the cyanobacterium Synechococcus elongatus PCC 7942 and other organisms are entrained by a variety of environmental factors. In cyanobacteria, the mechanism that transduces environmental input signals to the central oscillator of the clock is not known. An earlier study identified ldpA as a gene involved in light-dependent modulation of the circadian period, and a candidate member of a clock-entraining input pathway. Here, we report that the LdpA protein is sensitive to the redox state of the cell and exhibits electron paramagnetic resonance spectra consistent with the presence of two Fe4S4 clusters. Moreover, LdpA copurifies with proteins previously shown to be integral parts of the circadian mechanism. We also demonstrate that LdpA affects both the absolute level and light-dependent variation in abundance of CikA, a key input pathway component. The data suggest a novel input pathway to the circadian oscillator in which LdpA is a component of the clock protein complex that senses the redox state of a cell

Nitrogen availability is crucial for crop yield with nitrogen fertilizer accounting for a large percentage of farmers’ expenses. However, an untimely or excessive application of fertilizer can increase risks of negative environmental effects. These factors, along with the environmental and energy costs of synthesizing nitrogen fertilizer, led us to seek out novel biotechnology-driven approaches to supply nitrogen to plants. The strategy we focused on involves transgenic expression of nitrogenase, a bacterial multi-subunit enzyme that can capture atmospheric nitrogen. Here we report expression of the active Fe subunit of nitrogenase via integration into the tobacco plastid genome of bacterial gene sequences modified for expression in plastid. Our study suggests that it will be possible to engineer plants that are able to produce their own nitrogen fertilizer by expressing nitrogenase genes in plant plastids.

The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q A Ϫ (more than a 100-fold in the tobacco ⌬psbJ mutant). The decay of the oxidized S 2,3 states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q B Ϫ /S 2,3 recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q A Ϫ to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process. The photosystem II complex (PSII)1 of the thylakoid membrane is involved in the photochemical process resulting in water oxidation, oxygen evolution, and reduction of plastoquinone. PSII comprises a core complex formed by a few proteins binding the ligands required for primary photochemistry and electron transfer and the chlorophyll-binding antennae as well as a number of low molecular weight proteins whose functions have not yet been identified (1, 2). The process of light-induced charge separation and reduction of a quinone acceptor, similar to that performed by PSII, is also carried out by photosynthetic bacteria that do not evolve oxygen and perform cyclic electron flow around the bacterial photochemical reaction center (RC) (3). While in both cases a small number of proteins binding the appropriate ligands are capable of light-driven charge separation and electron transfer, the linear electron flow performed by PSII using water as a source of electrons is more complex and may require regulatory steps that are not demanded by the cyclic electron flow of the bacterial RC. Thus it is plausible that proteins of PSII, besides those acting as energy-transferring antennae (4), may play some role in the regulation of the forward and backward electron flow within the PSII core complex. Recombination of the primary charge-separated pair due to back electron flow may lead to the generation of singlet oxygen considered to be the cause of PSII oxidative stress and turnover of its core proteins (5, 6). Indeed cytochrome b 559 , an essential component of the PSII core complex is considered to play such a regulatory role by diverting electrons from the reducing side of PSII and channeling them to the oxidized donor side of the complex (7). This protein is missing from the RC complex of purple anoxygenic photosynthetic bacteria. The psbE and psbF genes encoding the ␣ and ␤ subunits of cytochrome b 559 , respectiv...

(N.B.I., S.V.S.)The D1 protein is an integral component of the photosystem II reaction center complex. In the cyanobacterium Synechocystis sp. PCC 6803, D1 is synthesized with a short 16-amino acids-long carboxyl-terminal extension. Removal of this extension is necessary to form active oxygen-evolving photosystem II centers. Our earlier studies have shown that this extension is cleaved by CtpA, a specific carboxyl-terminal processing protease. The amino acid sequence of the carboxyl-terminal extension is conserved among D1 proteins from different organisms, although at a level lower than that of the mature protein.In the present study we have analyzed a mutant strain of Synechocystis sp. PCC 6803 with a duplicated extension, and a second mutant that lacks the extension, to investigate the effects of these alterations on the function of the D1 protein in vivo. No significant difference in the growth rates, photosynthetic pigment composition, fluorescence induction, and oxygen evolution rates was observed between the mutants and the control strain. However, using long-term mixed culture growth analysis, we detected significant decreases in the fitness of these mutant strains. The presented data demonstrate that the carboxyl-terminal extension of the precursor D1 protein is required for optimal photosynthetic performance.