The reaction center H subunit is not required for high levels of light-harvesting complex 1 in Rhodospirillum rubrum mutants.
The gene (puhA) encoding the H subunit of the reaction center (RC) was deleted by site-directed interposon mutagenesis by using a kanamycin resistance cassette lacking transcriptional terminators to eliminate polar effects in both the wild-type strain Rhodospirillum rubrum S1 and the carotenoid-less strain R. rubrum G9. The puhA interposon mutants were incapable of photoheterotrophic growth but grew normally under aerobic chemoheterotrophic conditions. Absorption spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the RCs were absent. In minimal medium and also in modified medium containing succinate and fructose, the light-harvesting 1 complex (LH1) levels of the S1-derived mutants were about 70 to 100% of the wild-type levels in the same media. The correct assembly of LH1 in the membrane and the pigment-pigment interaction were confirmed by near-infrared circular dichroism spectroscopy. LH1 formation was almost absent when the carotenoid-less G9-derived puhA mutants were grown in standard minimal medium, suggesting that carotenoids may stabilize LH1. In the fructose-containing medium, however, the LH1 levels of the G9 mutants were 70 to 100% of the parental strain levels. Electron micrographs of thin sections of R. rubrum revealed photosynthetic membranes in all mutants grown in succinate-fructose medium. These studies indicate that the H subunit of the RC is necessary neither for maximal formation of LH1 nor for photosynthetic membrane formation but is essential for functional RC assembly.
Lupo
Domenico
D
Department of Bioenergetics, Institute of Biology, University of Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany.
Ghosh
Robin
R
D000332:Aerobiosis; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism*; D002338:Carotenoids / Q000502:physiology; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004734:Energy Metabolism / Q000235:genetics* / Q000502:physiology; D005632:Fructose / Q000378:metabolism; D017353:Gene Deletion; D045342:Light-Harvesting Protein Complexes / Q000032:analysis*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D016254:Mutagenesis, Insertional; D045322:Photosynthetic Reaction Center Complex Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D012247:Rhodospirillum rubrum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D013057:Spectrum Analysis; D019802:Succinic Acid / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D045342:Light-Harvesting Protein Complexes; D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; C098850:PuhA protein, Rhodobacter; C094744:light-harvesting complex 1, Rhodospirillum; D005632:Fructose; D002338:Carotenoids; D019802:Succinic Acid