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Targeted Genetic Inactivation Of The Photosystem I Reaction Center In The Cyanobacterium Synechocystis Sp. PCC 6803.
Published 1991 · Biology, Medicine
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.