Construction, characterization, and complementation of Rhodospirillum rubrum puf region mutants

Abstract

Rhodospirillum rubrum is a facultatively phototrophic bacterium that, under certain growth conditions, forms an intracytoplasmic chromatophore membrane (ICM) housing the photochemical apparatus. The puf operon of R. rubrum encodes protein subunits of the photochemical reaction center and the B880 lightharvesting antenna complex. Mutant strains of R. rubrum were constructed by interposon mutagenesis through which a kanamycin resistance gene cartridge was inserted into restriction sites and in place of restriction fragments of the pufregion. Southern blot analysis demonstrated that the defective copies ofpuf sequences had replaced their normal chromosomal counterparts through homologous recombination. The pheriotypes of the mutant strains were evaluated on the basis of puf gene expression, spectral analysis, pigment content of membranes, and electron-microscopic examination of thin sections of cells grown under semiaerobic and dark anaerobic conditions. Alterations of the puf region affect phototrophic competence and the formation of the ICM. The latter result implies an obligatory role for puf gene products in ICM formation in R. rubrum. One mutant with a deletion in puf structural genes was complemented in trans to the wild-type phenotype. Other mutants could be restored to the wild-type phenotype only by recombination.Rhodospirillum rubrum is a gram-negative, facultatively phototrophic, purple nonsulfur bacterium that grows chemoheterotrophically under aerobic conditions and phototrophically under reduced-oxygen conditions in the light. During the transition from aerobic to reduced-oxygen conditions, photopigments and associated proteins are synthesized and inserted into a differentiated intracytoplasmic chromatophore membrane (ICM). Previous studies have demonstrated that the photosynthetic ICM is physically continuous with (31) and formed by invagination of (16,17,27) the cytoplasmic membrane.Contained within the ICM of R. rubrum are two proteinpigment complexes, the P870 reaction center complex (RC) and the B880 light-harvesting I complex. The RC of R. rubrum consists of three polypeptide subunits, L, M, and H, which are present in a 1:1:1 ratio. Associated with these polypeptides are bacteriochlorophyll (BCHL), bacteriopheophytin, the carotenoid spirilloxanthin (CRT), iron, and ubiquinone (58, 59). R. rubrum has only one antenna complex, the B880 antenna complex, composed of ot and ,B polypeptides, BCHL, CRT, diphosphatidylethanolamine, and diphosphatidylglycerol (50).The genes encoding the B880 ot and polypeptides, and those encoding the reaction center L and M polypeptides, have been cloned and sequenced in Rhodobacter capsulatus (66, 67), Rhodobacter sphaeroides (35, 61, 62), R. rubrum (7, 10), and Rhodopseudomonas viridis (40, 60). These genes are closely linked in a single operon termed puf. The pufL and pufM genes of R. rubrum, Rhodopseudomonas viridis, Rhodobacter sphaeroides, and Rhodobacter capsulatus show a nucleotide sequence similarity ranging from 61 to 78% (7,10,35,60,66,67). R. rubrum puf seq...

Role of the H Protein in Assembly of the Photochemical Reaction Center and Intracytoplasmic Membrane in Rhodospirillum rubrum

“…R. rubrum P5, in which most of the puf genes were deleted, was shown to be incapable of phototrophic growth and ICM formation. P5 was restored to phototrophic growth and ICM formation by complementation with puf in trans (21,26). These results imply that in R. rubrum the puf gene products are required for ICM formation.…”

“…Phototrophic cultures were grown at 25°C in screw-cap tubes on a rotating platform illuminated by four incandescent lamps at 100 W/m 2 . R. rubrum R5 was grown in the presence of rifampin (15 g/ml) to counterselect for donors in conjugations as previously described (21). Kanamycin (15 g/ml for R. rubrum and 50 g/ml for Escherichia coli), tetracy-cline (12.5 g/ml), chloramphenicol (30 g/ml), and spectinomycin (25 g/ml) were added to the medium as appropriate.…”

“…An interposon mutant was generated by the approach used previously (21). The PstI fragment of pH3.6ϩ extending from within G115 through the first 161 bp of puhA ( Fig.…”

“…Under reduced oxygen concentration, this organism forms an intracytoplasmic membrane (ICM) that is the site of the photosynthetic apparatus (15,16,21). This apparatus consists of the light-harvesting antenna (LH) and the photochemical reaction center (RC).…”

“…In previous studies from this laboratory, the puf region was cloned and interposon mutations within this region were constructed (21). R. rubrum P5, in which most of the puf genes were deleted, was shown to be incapable of phototrophic growth and ICM formation.…”