Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803.
We describe the first complete segregation of a targeted inactivation of psaA encoding one of the P700-chlorophyll a apoproteins of photosystem (PS) I. A kanamycin resistance gene was used to interrupt the psaA gene in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Selection of a fully segregated mutant, ADK9, was performed under light-activated heterotrophic growth (LAHG) conditions; complete darkness except for 5 min of light every 24 h and 5 mM glucose. Under these conditions, wild-type cells showed a 4-fold decrease in chlorophyll (chl) per cell, primarily due to a decrease of PS I reaction centers. Evidence for the absence of PS I in ADK9 includes: the lack of EPR (electron paramagnetic resonance) signal I, from P700+; undetectable P700-apoprotein; greatly reduced whole-chain photosynthesis rates; and greatly reduced chl per cell, resulting in a turquoise blue phenotype. The PS I peripheral proteins PSA-C and PSA-D were not detected in this mutant. ADK9 does assemble near wild-type levels of functional PS II per cell, evidenced by: EPR signal II from YD+; high rates of oxygen evolution with 2,6-dichloro-p-benzoquinone (DCBQ), an electron acceptor from PS II; and accumulation of D1, a PS II core polypeptide. The success of this transformation indicates that this cyanobacterium may be utilized for site-directed mutagenesis of the PS I core.
Smart
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DOE-Plant Research Laboratory, Michigan State University, East Lansing 48824.
Anderson
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McIntosh
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D001345:Autoradiography; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002734:Chlorophyll / Q000235:genetics; D000458:Cyanobacteria / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000378:metabolism; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D045331:Photosystem I Protein Complex; D012313:RNA / Q000235:genetics; D015183:Restriction Mapping
D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D002734:Chlorophyll; D012313:RNA