Light-regulated expression of the psbD gene family in Synechococcus sp. strain PCC 7942: evidence for the role of duplicated psbD genes in cyanobacteria.
ABSTRACT The genome of the cyanobacterium Synechococcus sp. strain PCC 7942 contains two psbD genes encoding the D2 protein of the photosystem II reaction center: psbDI, which is cotranscribed as a discistronic message with psbC (the gene encoding CP43, a chlorophyll-a binding protein), and psbDII, which is monocistronic. Northern blot analysis of psbD transcripts showed that the two genes responded differently when wild-type cells were shifted from moderate to high light intensity. Whereas psbDII transcripts increased 500% relative to unshifted control cells, psbDI-psbC transcripts remained unchanged. The beta-galactosidase activities expressed from translational fusions between the psbD genes and the Escherichia coli lacZ reporter gene displayed responses similar to those seen in the RNA. D2 protein levels in thylakoid membranes from wild-type cells increased to 250% of those of the unshifted control cells 12 h after a shift to high light intensities. In contrast, in a mutant strain (AMC016) that carries an inactive psbDII gene, D2 levels decreased by 50% under identical conditions. These results suggested that induction of psbDII gene expression by light can serve as a supplementary system for maintaining a functional photosystem II reaction center at high light intensity. This hypothesis was corroborated by mixed-culture experiments, in which AMC016 cells competed poorly with wild-type cells at high light intensity. These data suggest for the first time that differential expression of members of a cyanobacterial gene family serves to maintain a functional PSII reaction center under diverse environmental conditions.
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ABSTRACT: Photosynthetic microorganisms that directly channel solar energy to the production of molecular hydrogen are a potential future biofuel system. Building such a system requires installation of a hydrogenase in the photosynthetic organism that is both tolerant to oxygen and capable of hydrogen production. Toward this end, we have identified the [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "Deep ecotype" that is able to be heterologously expressed in cyanobacteria and has tolerance to partial oxygen. The A. macleodii enzyme shares sequence similarity with the uptake hydrogenases that favor hydrogen uptake activity over hydrogen evolution. To improve hydrogen evolution from the A. macleodii hydrogenase, we examined the three Fe-S clusters found in the small subunit of many [NiFe] uptake hydrogenases that presumably act as a molecular wire to guide electrons to or from the active site of the enzyme. Studies by others altering the medial cluster of a Desulfovibrio fructosovorans hydrogenase from 3Fe-4S to 4Fe-4S resulted in two-fold improved hydrogen evolution activity. We adopted a strategy of screening for improved hydrogenase constructs using an Escherichia coli expression system before testing in slower growing cyanobacteria. From the A. macleodii enzyme, we created a mutation in the gene encoding the hydrogenase small subunit that in other systems is known to convert the 3Fe-4S medial cluster to 4Fe-4S. The medial cluster substitution did not improve the hydrogen evolution activity of our hydrogenase. However, modifying both the medial cluster and the ligation of the distal Fe-S cluster improved in vitro hydrogen evolution activity relative to the wild type hydrogenase by three- to four-fold. Other properties of the enzyme including thermostability and tolerance to partial oxygen did not appear to be affected by the substitutions. Our results show that substitution of amino acids altering the ligation of Fe-S clusters in the A. macleodii [NiFe] uptake hydrogenase resulted in increased hydrogen evolution activity. This activity can be recapitulated in multiple host systems and with purified protein. These results validate the approach of using an E. coli-cyanobacteria shuttle system for enzyme expression and improvement.Journal of Biological Engineering 07/2013; 7(1):17.
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ABSTRACT: Prochlorococcus and Synechococcus, which numerically dominate vast oceanic areas, are the two most abundant oxygenic phototrophs on Earth. Although they require solar energy for photosynthesis, excess light and associated high UV radiations can induce high levels of oxidative stress that may have deleterious effects on their growth and productivity. Here, we compared the photophysiologies of the model strains Prochlorococcus marinus PCC 9511 and Synechococcus sp. WH7803 grown under a bell-shaped light/dark cycle of high visible light supplemented or not with UV. Prochlorococcus exhibited a higher sensitivity to photoinactivation than Synechococcus under both conditions, as shown by a larger drop of photosystem II (PSII) quantum yield at noon and different diel patterns of the D1 protein pool. In the presence of UV, the PSII repair rate was significantly depressed at noon in Prochlorococcus compared to Synechococcus. Additionally, Prochlorococcus was more sensitive than Synechococcus to oxidative stress, as shown by the different degrees of PSII photoinactivation after addition of hydrogen peroxide. A transcriptional analysis also revealed dramatic discrepancies between the two organisms in the diel expression patterns of several genes involved notably in the biosynthesis and/or repair of photosystems, light-harvesting complexes, CO(2) fixation as well as protection mechanisms against light, UV, and oxidative stress, which likely translate profound differences in their light-controlled regulation. Altogether our results suggest that while Synechococcus has developed efficient ways to cope with light and UV stress, Prochlorococcus cells seemingly survive stressful hours of the day by launching a minimal set of protection mechanisms and by temporarily bringing down several key metabolic processes. This study provides unprecedented insights into understanding the distinct depth distributions and dynamics of these two picocyanobacteria in the field.Frontiers in Microbiology 01/2012; 3:285.
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ABSTRACT: Photosynthetic electron transport, chromatic photoacclirnation and expression of the genes encoding the 01, 02, and cytochrome b559 subunits of the Photosystem II complex were studied in the chlorophyll d containing cyanobacterium Acaryochloris marina MBIC11017 under various environmental conditions. During oxygen deprivation and inhibition of photosynthetic electron transport by dibromothymoquinone the psbA1 gene encoding a 01' isoform was induced. All of the three psbA and one of the three psbD (psbD2) genes, encoding two different isoforms of the 01 and the abundant isoform of the 02 proteins, respectively were induced under exposure to UV-B radiation and high intensity visible light. Under far red light the amount of Photosystem II complexes increased, and expression of the psbE2 gene encoding the alpha-subunit of cytochrome b559 was enhanced. However, the psbF and psbE1 genes encoding the beta- and another isoform of alpha-cytochrome b559, respectively remained lowly expressed under all conditions. Far red light also induced the psbD3 gene encoding a 02' isoform whose primary structure is different from the abundant 02 isoform. psbD3 was also induced under low intensity visible light, when chromatic photoacclimation was indicated by a red-shifted absorption of chlorophyll d. Our results show that differential expression of multigene families encoding different isoforms of 01 and 02 plays an important role in the acclimation of A. marina to contrasting environmental conditions. Moreover, the disproportionate quantity of transcripts of the alpha and beta subunits of cytochrome b559 implies the existence of an alpha-alpha homodimer organization of cytochrome b559 in Photosystem II complexes.Biochimica et Biophysica Acta 07/2012; 1817(7):1083-94. · 4.66 Impact Factor