Construction and characterization of cyanobacterial mutants lacking the manganese-stabilizing polypeptide of photosystem II.
Mutants of the cyanobacterium Synechocystis sp. Pasteur Culture Collection (PCC) 6803 that specifically lack the extrinsic 33-kDa manganese-stabilizing polypeptide of the photosystem II oxygen-evolving complex have been constructed by two independent methods. Cartridge mutagenesis was used to insertionally inactivate the psbO gene of one mutant and completely delete the psbO gene of the other mutant. These mutants have no detectable manganese-stabilizing polypeptide, but they do accumulate steady-state levels of the intrinsic photosystem II polypeptides D1, D2, and CP-43 that are comparable to wild-type, as determined by immunoblot analysis. Measurement of the evolution of the relative quantum yields of chlorophyll fluorescence following actinic flash excitation indicates that though the concentration of reaction centers in mutant cells is comparable to that of wild-type cells, approximately 40% of these centers harbor a fluorescence-quenching species other than P680+. The mutants are capable of photoautotrophic growth at a slower rate than that of wild-type. Under conditions of Ca2+ depletion where wild-type growth is unaffected, the mutants are unable to grow at all. The manganese-stabilizing protein, therefore, enhances the binding of Ca2+ or protects the reaction center at low Ca2+ concentrations. The mutant evolve oxygen at approximately 70% of the wild-type rate, but are completely photoinactivated by high light intensities. Our results indicate that the manganese-stabilizing polypeptide is not absolutely required for photosystem II assembly or function in cyanobacteria, but its absence does lead to an enhanced sensitivity to photoinhibition.
Philbrick
J B
JB
Department of Biochemistry and Microbiology, Cook College, Rutgers, State University of New Jersey, New Brunswick 08903.
Diner
B A
BA
Zilinskas
B A
BA
D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D008345:Manganese / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009693:Nucleic Acid Hybridization; D010100:Oxygen / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D045332:Photosystem II Protein Complex; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D014170:Transformation, Genetic
D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D008345:Manganese; D012313:RNA; D004247:DNA; D010100:Oxygen