Rakefet Schwarz

Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria.

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).

Thylakoid membrane perforations and connectivity enable intracellular traffic in cyanobacteria

Cyanobacteria, the progenitors of plant and algal chloroplasts, enabled aerobic life on earth by introducing oxygenic photosynthesis. In most cyanobacteria, the photosynthetic membranes are arranged in multiple, seemingly disconnected, concentric shells. In such an arrangement, it is unclear how intracellular trafficking proceeds and how different layers of the photosynthetic membranes communicate with each other to maintain photosynthetic homeostasis. Using electron microscope tomography, we show that the photosynthetic membranes of two distantly related cyanobacterial species contain multiple perforations. These perforations, which are filled with particles of different sizes including ribosomes, glycogen granules and lipid bodies, allow for traffic throughout the cell. In addition, different layers of the photosynthetic membranes are joined together by internal bridges formed by branching and fusion of the membranes. The result is a highly connected network, similar to that of higher‐plant chloroplasts, allowing water‐soluble and lipid‐soluble molecules to diffuse through the entire membrane network. Notably, we observed intracellular membrane‐bounded vesicles, which were frequently fused to the photosynthetic membranes and may play a role in transport to these membranes.

Physiological and Molecular Studies on the Response of Cyanobacteria to Changes in the Ambient Inorganic Carbon Concentration

The ability of cyanobacteria to adapt to a wide range of ambient CO2 concentrations involves modulation of the activity of an inorganic carbon-concentrating mechanism (CCM), as well as other changes at various cellular levels including the biosynthetic pathway of purines. Studies of high-CO2-requiring mutants have identified several of the genes involved in the operation of the CCM and in the ability to grow under changing ambient CO2 concentration. In the case of Synechococcus sp. strain PCC 7942 most of these genes have been mapped in the genomic region of the rbcLS operon. Higher levels of detectable transcripts originating from some of these genes have been observed after exposure of the cells to low CO2 concentration. Studies of mutants have confirmed quantitative models postulating crucial roles for carboxysomes and carboxysome-located carbonic anhydrase (CA) in cyanobacterial photosynthesis. A central role is also proposed for cytoplasmic-membrane-associated CA activity: CA may function to scavenge escaping CO2 by intracellular conversion to bicarbonate against the chemical potential.

Molecular Responses to Environmental Stress

Cyanobacteria are remarkable for their ability to flourish in environments with widely fluctuating chemical and physical parameters, such as nutrient availability, light quality and intensity, temperature and osmotic conditions. This chapter focuses on the responses of cyanobacteria to changes in light intensity, light quality and macronutrient availability. Suboptimal light and nutrient conditions result in a number of specific and general responses that strongly influence the physiology of the cell. Some of the changes can be striking, while others are subtle; they can occur in minutes or take place over several days. In recent years knowledge of the molecular responses of cyanobacteria to stress conditions has increased considerably. Capacity to acclimate to suboptimal light levels for photosynthetic carbon fixation is unique among the bacteria. In contrast, an understanding of acclimation to nutrient deprivation has gained much from the information available from analyses of enteric bacteria, although cyanobacteria have a number of interesting deviations in their responses. Perhaps most provocative in studies of acclimation processes in cyanobacteria is the overlap among the responses elicited by different environmental stimuli, which suggests the existence of a hierarchy of responses and the involvement of global regulatory circuits.

Oxidative Stress in Synechococcus sp. Strain PCC 7942: Various Mechanisms for H2O2 Detoxification with Different Physiological Roles

This study focuses on the mechanisms for hydrogen peroxide detoxification in Synechococcus sp. strain PCC 7942. To gain better understanding of the role of different routes of hydrogen peroxide detoxification, we inactivated TplA (thioredoxin-peroxidase-like), which we recently identified. In addition, we inactivated the gene encoding catalase-peroxidase and examined the ability to detoxify H(2)O(2) and to survive oxidative stress in both of the single mutants and in the double mutant. Surprisingly, we observed that the double mutant survived H(2)O(2) concentrations that the single catalase-peroxidase mutant could not tolerate. This phenotype correlated with an increased ability of the double mutant to detoxify externally added H(2)O(2) compared to the catalase-peroxidase mutant. Therefore, our studies suggested the existence of a hydrogen peroxide detoxification activity in addition to catalase-peroxidase and thioredoxin-peroxidase. The rate of detoxification of externally added H(2)O(2) was similar in the wild-type and the TplA mutant cells, suggesting that, under these conditions, catalase-peroxidase activity was essential for this process and TplA was dispensable. However, during excessive radiation, conditions under which the cell might experience oxidative stress, TplA appears to be essential for growth, and cells lacking it cannot compete with the wild-type strain. Overall, these studies suggested different physiological roles for various cellular hydrogen peroxide detoxification mechanisms in Synechococcus sp. strain PCC 7942.

High CO2 concentration alleviates the block in photosynthetic electron transport in an ndhB-inactivated mutant of Synechococcus sp. PCC 7942.

The high-concentration CO2-requiring mutant N5 of Synechococcus sp. PCC 7942 was obtained by the insertion of a kanamycin-resistant gene at the EcoRI site, 12.4 kb upstream of rbc. The mutant is unable to accumulate inorganic carbon internally and exhibits very low apparent photosynthetic affinity for inorganic carbon but a photosynthetic Vmax similar to that of the wild type. Sequence and northern analyses showed that the insertion inactivated a gene highly homologous to ndhB, encoding subunit II of NADH dehydrogenase in Synechocystis sp. PCC 6803 (T. Ogawa [1991] Proc Natl Acad Sci USA 88: 4275-4279). When the mutant and the wild-type cells were exposed to 5% CO2 in air, their photosynthetic electron transfer capabilities, as revealed by fluorescence and thermoluminescence measurements, were similar. On the other hand, a significant decrease in variable fluorescence was observed when the mutant (but not the wild-type) cells were exposed to low CO2 under continuous light. The same treatment also resulted in a shift (from 38-27[deg]C) in the temperature at which the maximal thermoluminescence emission signal was obtained in the mutant but not in the wild type. These results may indicate that subunit II of NADH dehydrogenase is essential for the functional operation of the photosynthetic electron transport in Synechococcus under low but not high levels of CO2. We suggest that the inability to accumulate inorganic carbon under air conditions stems from disrupture of electron transport in this mutant.

Low Activation State of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Carboxysome-Defective Synechococcus Mutants

The high-CO2-requiring mutant of Synechococcus sp. PCC 7942, EK6, was obtained after extension of the C terminus of the small subunit of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). The carboxysomes in EK6 were much larger than in the wild type, but the cellular distribution of the large and small sub-units of Rubisco was not affected. The kinetic parameters of in vitro-activated Rubisco were similar in EK6 and in the wild type. On the other hand, Rubisco appeared to be in a low state of activation in situ in EK6 cells pretreated with an air level of CO2. This was deduced from the appearance of a lag phase when carboxylation was followed with time in cells permeabilized by detergent and subsequently supplied with saturating CO2 and RuBP. Pretreatment of the cells with high CO2 virtually abolished the lag. After low-CO2 treatment, the internal RuBP pool was much higher in mutant cells than in the wild-type cells; pretreatment with high CO2 reduced the pool in mutant cells. We suggest that the high-CO2-requiring phenotype in mutants that possess aberrant carboxysomes arises from the inactivated state of Rubisco when the cells are exposed to low CO2.

Inactivation of the extrinsic subunit of photosystem II, PsbU, in Synechococcus PCC 7942 results in elevated resistance to oxidative stress

PsbU is a subunit of the extrinsic complex attached to the core of photosystem II. A PsbU‐mutant of Synechococcus PCC 7942 was isolated based on its elevated resistance to externally applied oxidative stress. PsbU‐mutant exhibits fast rates of degradation of the photosystem II core protein, D1, under sub‐saturating as well as high‐light conditions. While forward electron transfer is not affected, back electron flow is severely impaired in the mutant. We suggest that impairment of psbU results in production of reactive‐oxygen‐species, which trigger antioxidative mechanisms even under standard growth conditions. Accordingly, when challenged with external oxidative stress, these cells are more resistant than wild type cells.

NblC, a novel component required for pigment degradation during starvation in Synechococcus PCC 7942

Adjustment of photosynthetic light harvesting to ambient conditions is essential to allow efficient energy capturing and to prevent surplus excitation and the cellular damage resulting from it. Degradation of the cyanobacterial light harvesting complex, the phycobilisome, is a general acclimation response occurring under various stress conditions. This study identifies a novel component, NblC, which mediates phycobilisome degradation under nitrogen, sulphur and phosphorus starvation. Our study indicates the requirement of NblC for efficient expression of nblA, an essential component of the degradation pathway; accumulation of nblA transcripts upon nutrient starvation was impaired in the NblC‐mutant. Furthermore, expression of NblC under the control of a foreign promoter resulted in accumulation of nblA transcripts and degradation of the light harvesting complex. Transcription of nblC is induced upon nutrient starvation, suggesting the requirement of elevated levels of NblC under these conditions. Importantly, NblC could not exert its positive effect on nblA expression in the absence of the response regulator NblR. Sequence alignment suggests kinase motifs as well as homology of NblC to anti‐sigma factors. Accordingly, we suggest a mode of action for this newly identified modulator, which provides new insights into regulation of gene expression in response to environmental stimuli.

Structural, Functional, and Mutational Analysis of the NblA Protein Provides Insight into Possible Modes of Interaction with the Phycobilisome

The enormous macromolecular phycobilisome antenna complex (>4 MDa) in cyanobacteria and red algae undergoes controlled degradation during certain forms of nutrient starvation. The NblA protein (∼6 kDa) has been identified as an essential component in this process. We have used structural, biochemical, and genetic methods to obtain molecular details on the mode of action of the NblA protein. We have determined the three-dimensional structure of the NblA protein from both the thermophilic cyanobacterium Thermosynechococcus vulcanus and the mesophilic cyanobacterium Synechococcus elongatus sp. PCC 7942. The NblA monomer has a helix-loop-helix motif which dimerizes into an open, four-helical bundle, identical to the previously determined NblA structure from Anabaena. Previous studies indicated that mutations to NblA residues near the C terminus impaired its binding to phycobilisome proteins in vitro, whereas the only mutation known to affect NblA function in vivo is located near the protein N terminus. We performed random mutagenesis of the S. elongatus nblA gene which enabled the identification of four additional amino acids crucial for NblA function in vivo. This data shows that essential amino acids are not confined to the protein termini. We also show that expression of the Anabaena nblA gene complements phycobilisome degradation in an S. elongatus NblA-null mutant despite the low homology between NblAs of these cyanobacteria. We propose that the NblA interacts with the phycobilisome via “structural mimicry” due to similarity in structural motifs found in all phycobiliproteins. This suggestion leads to a new model for the mode of NblA action which involves the entire NblA protein.