Abstract
A gene coding for the photosynthetic reaction center-bound cytochrome subunit, pufC, of Blastochloris viridis, which belongs to the alpha-purple bacteria, was introduced into Rubrivivax gelatinosus, which belongs to the beta-purple bacteria. The cytochrome subunit of B. viridis was synthesized in the R. gelatinosus cells, in which the native pufC gene was knocked out, and formed a chimeric reaction center (RC) complex together with other subunits of R. gelatinosus. The transformant was able to grow photosynthetically. Rapid photo-oxidization of the hemes in the cytochrome subunit was observed in the membrane of the transformant. The soluble electron carrier, cytochrome c(2), isolated from B. viridis was a good electron donor to the chimeric RC. The redox midpoint potentials and the redox difference spectra of four hemes in the cytochrome subunit of the chimeric RC were almost identical with those in the B. viridis RC. The cytochrome subunit of B. viridis seems to retain its structure and function in the R. gelatinosus cell. The chimeric RC and its mutagenesis system should be useful for further studies about the cytochrome subunit of B. viridis.
Highlights
Photosynthetic energy conversion begins in pigment-protein complexes called photosynthetic reaction centers
Measurements of spectra and flash-induced redox changes of the cytochromes c showed that the B. viridis cytochrome subunit was synthesized in R. gelatinosus and functioned as the rapid electron donor to the photooxidized special pair
This means that the B. viridis cytochrome subunit can form a functional “chimeric” reaction center (RC) complex together with the R. gelatinosus LM core polypeptides
Summary
Bacteria and Growth Media—R. gelatinosus strain IL144RL2, which is a spontaneous mutant of the wild-type strain IL144 and shows greatly depressed production of the light-harvesting 2 complex, was used as a host strain for gene manipulations in this study. A 2.5-kb DNA fragment flanked by BamHI restriction sites and containing the 3Ј-region of pufM and the entire pufC of B. viridis was cloned from the genomic library of B. viridis DNA using a pUC119 plasmid as a cloning vector. This plasmid was named p9VP3 (Fig. 1B). The nucleotide sequence of pufC in the p9VP3 plasmid was confirmed to be identical to that previously reported for the B. viridis pufC [5] Both the genes for the M subunits of R. gelatinosus and B. viridis have a common SalI restriction site at the conserved 3Ј-regions. The redox potential was raised stepwise by additions of a solution of potassium ferricyanide
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