Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413.
In oxygenic photosynthetic organisms the PSI-C polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] centers, FA and FB, the terminal electron acceptors in the photosystem I (PSI) complex. An insertion mutation introduced in the psaC locus of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 resulted in the creation of a mutant strain, T398-1, that lacks the PSI-C polypeptide. In medium supplemented with 5 mM fructose, the mutant cells grew well in the dark. However, when grown in the same medium under light, the doubling rate of T398-1 cells was significantly decreased. In intact cells of T398-1, bicarbonate-dependent whole-chain electron transport (PSII and PSI) could not be detected, although partial electron transport reactions involving either one of the two photosystems could be measured at significant rates. The low-temperature EPR signals attributed to the [4Fe-4S] centers FA and FB were absent in the mutant cells. Chemical titration measurements indicated that the ratios of chlorophyll to the primary donor P700 were virtually identical in membranes from the wild-type and mutant cells. Moreover, room-temperature optical spectroscopic analysis of the thylakoid membranes isolated from T398-1 showed flash-induced P700 oxidation followed by dark rereduction, indicating primary photochemistry in PSI. Thus stable assembly of the reaction center of PSI can occur in the absence of the Fe-S cluster cofactors FA and FB. These studies demonstrate that Anabaena 29413 offers a useful genetic system for targeted mutagenesis of the PSI complex.
Mannan
R M
RM
Department of Biology, Washington University, St. Louis, MO 63130.
Whitmarsh
J
J
Nyman
P
P
Pakrasi
H B
HB
D017033:Anabaena / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods; D003227:Conjugation, Genetic; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes; D016297:Mutagenesis, Site-Directed; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics* / Q000378:metabolism; D045331:Photosystem I Protein Complex; D010957:Plasmids; D015183:Restriction Mapping; D013053:Spectrophotometry
D007506:Iron-Sulfur Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D004247:DNA