A system for site-specific mutagenesis of the photosynthetic reaction center in Rhodopseudomonas viridis.
The purple non-sulfur bacterium Rhodopseudomonas viridis contains a photosynthetic reaction center which has been structurally resolved to 2.3 A providing a unique basis for the study of biological electron transfer processes by the method of site-specific mutagenesis. Here we report the construction of a puf operon deleted mutant strain incapable of photosynthetic growth. The deletion was introduced with the help of a newly constructed suicide vector by electroporation which is with conjugation another gene transfer system for R. viridis. The deletion strain was complemented by conjugational gene transfer with wild-type (WT) and mutated LM genes of the puf operon. The complemented WT and mutations YL162F and HL153F grew photosynthetically, expressed and assembled the four subunits L, M, H and Cyt c of the reaction center correctly. These first mutations already demonstrate the value of the R. viridis system for a detailed structure-function analysis of photosynthetic electron transfer.
Laussermair
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E
Max-Planck Institut für Biochemie, Martinsried, Germany.
Oesterhelt
D
D
D001483:Base Sequence; D002872:Chromosome Deletion; D003227:Conjugation, Genetic; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004558:Electric Stimulation / Q000379:methods; D005821:Genetic Techniques; D005822:Genetic Vectors; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012241:Rhodopseudomonas / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D045322:Photosynthetic Reaction Center Complex Proteins