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.
"Variants containing a PT7 promoter or PTRC promoters at all four “promoter site”s were also generated. For integration into cyanobacteria, these features are flanked by 1-kb of S. elongatus DNA on either side for homologous recombination into neutral site 1 (NS1) . Amino acid substitutions were generated by PCR of the operon using primers which encode for mutations corresponding to the substitutions, followed by assembly with an empty vector. "
[Show abstract][Hide abstract] 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.
"PCC 7942; Golden et al., 1989), with one co-transcribed with psbC that encodes the internal PSII antenna protein CP43 (Garczarek et al., 2001) and the other isolated in the genome. It is likely that as for psbA, the two copies are differently regulated in response to light and/or UV stress as previously reported in freshwater model cyanobacteria (Bustos and Golden, 1992; Kos et al., 2008), although this was not checked in the present study. Alternatively, this may simply contribute to a higher expression level of this key photosynthetic gene, possibly enabling a higher turnover of the corresponding protein. "
[Show abstract][Hide abstract] 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 08/2012; 3:285. DOI:10.3389/fmicb.2012.00285 · 3.99 Impact Factor
"Synechococcus elongatus strain PCC 7942 was obtained from the University of Toronto Culture Collection as Synechococcus leopoliensis strain UTCC 100. Cyanobacterial cultures were maintained in BGll medium (Bustos and Golden 1992) at 24–28 °C with 12 h light/dark cycles on a rotary shaker or on agar as previously described (Golden et al. 1987). When appropriate the growth medium of S. leopoliensis was supplemented with chloramphenicol at a concentration of 7.5 lg/ mL. "
[Show abstract][Hide abstract] ABSTRACT: We report the transfer of cellulose synthesis genes (acsABΔC) from the heterotropic alpha proteobacterium, Gluconacetobacter xylinus strain ATCC 53582 to a photosynthetic microbe (Synechococcus leopoliensis strain UTCC 100). These genes were functionally expressed in this cyanobacterium, resulting in the production of non-crystalline
cellulose. Although the cellulose lacks the structural integrity of the product synthesized by G. xylinus, the non-crystalline nature of the cyanobacterial cellulose makes it an ideal potential feedstock for biofuel production.
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