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.
Department of Bioenergetics, Institute of Biology, University of Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany.
Domenico
D
Lupo
Robin
R
Ghosh
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