A Puf-strain of Rhodobacter sphaeroides (PUFB1) was constructed by deleting a portion of the proximal region of the puf operon and inserting a kanamycin resistance gene cartridge. Southern blot analysis demonstrated that in PUFB1, the defective copy of the puf operon had replaced, through homologous recombination, the normal chromosomal copy. The Puf phenotype was characterized by the inability of PUFB1 to grow photoheterotrophically (PS-), the lack of detectable puf-specific transcripts, the absence of the light-harvesting I complex and, by inference, the reaction center spectral complex, and greatly reduced levels of the light-harvesting II complex. The PS' phenotype was restored to PUFB1 when a 13-kilobase BamHI restriction endonuclease fragment containing the entire puf operon and flanking regions was cloned into the broad-host-range plasmid vector RK2 derivative pRK404 and introduced by conjugation into PUFB1. In these complemented strains, there was an increased number of copies of the puf operon (four to six copies per defective chromosomal copy) as the result of plasmid copy number. However, there was no concomitant increase in either the specific bacteriochlorophyll content or the level of puf-specific transcripts when these strains were grown photoheterotrophically.Photoheterotrophically grown Rhodobacter sphaeroides contains a specialized intracytoplasmic membrane system (ICM) (5,8,16) which houses all of the protein-pigment complexes responsible for capturing light energy and -catalyzing photosynthetic electron transport and synthesis of chemical bond energy (28,30,37).The photosynthetic unit of R. sphaeroides comprises three distinct bacteriochlorophyll (Bchl) a pigment-protein complexes: the photochemical reaction center (RC) (30) and two light-harvesting complexes (6). These two spectrally distinct light-harvesting complexes (37) contain 95% of the total Bchl in R. sphaeroides and are classified according to their absorption maxima in the near infrared (9). The B800-850 complex transfers exciton energy to the B875 complex, which in turn transfers such energy to the RC (25, 45). Both B875 and B800-850 pigment-protein complexes contain equimolar amounts of two small but unique hydrophobic polypeptides, designated a and P, which exist in the membrane as heterodimers (1,4,36).Although similarities exist between the B800-850 and B875polypeptides, the spectral differences between the two lightharvesting complexes can be attributed to differences in Bchl pigment-protein interactions within each complex. The B875 complex contains two molecules each of Bchl a and carotenoid, while three molecules of Bchl a and one carotenoid have been proposed to be associated with the B800-B850 complex (4, 17, 31). There are three molecules of Bchl a per a and ,B polypeptides in the B800-850 complex (8). Fluorescence studies suggest a topological model whereby the RC is surrounded by B875 pigment-protein complexes and the B875-RC complexes are interconnected in a lake of B800-850 spectral units (26).The stoichiometry of B...