Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803

• Published in: The EMBO journal Vol. 10; no. 11; pp. 3289 - 3296
• Main Authors: Smart, L.B, Anderson, S.L, McIntosh, L
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 01.11.1991
• Subjects: Autoradiography; Biological and medical sciences; Blotting, Northern; Blotting, Southern; Blotting, Western; Chlorophyll - genetics; Cyanobacteria; Cyanobacteria - metabolism; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Intracellular Membranes - metabolism; Light-Harvesting Protein Complexes; Molecular and cellular biology; Molecular genetics; mutagenesis; Mutagenesis. Repair; Photosynthetic Reaction Center Complex Proteins - genetics; photosystem I; Photosystem I Protein Complex; Restriction Mapping; RNA - genetics; site-directed mutagenesis; Synechocystis; Photosystem 1; Reaction center; Growth; Heterotrophy; Selection; Cyanobacteria; Site directed mutagenesis; EPR spectrometry; Bacteria; Synechocystis; Method
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1991.tb04893.x

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 Il per cell, evidenced by: EPR signal 11 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.