PHOTOSYNTHETIC DEFICIENCY OF A PUFX DELETION MUTANT OF RHODOBACTER-SPHAEROIDES IS SUPPRESSED BY POINT MUTATIONS IN THE LIGHT-HARVESTING COMPLEX GENES PUFB OR PUFA

The pufX gene of the facultative phototroph Rhodobacter sphaeroides en codes a membrane protein that is required for photoheterotrophic growt h. Deletion of pufX impairs the photosynthetic generation of a transme mbrane potential, suggesting a role for the PufX protein in light-driv en cyclic electron transfer [Farchaus, J.W., et al. (1992) EMBO J. 11, 2779-2788]. Here we describe the isolation and characterization of 65 spontaneous suppressor mutants in which photosynthetic competence was restored by secondary mutations. Genetic analysis revealed the occurr ence of single point mutations altering highly conserved residues with in the light-harvesting complex, B875. One of three tryptophan codons was changed to stop or arginine codons in 89% of these suppressor muta nts. Spectral characterization and Western blot analysis were used to examine the B875 assembly and the stable expression of the altered lig ht-harvesting polypeptides. Three different groups of suppressor mutan ts were found: (1) No stable expression of altered B875 polypeptides w as detected for the alpha 43W--> and beta 44W-->* mutants. (2) There was expression of the mutated B875-beta chain, but no stable B875 asse mbly in the beta 47W-->R mutants. (3) Intact B875 complexes were found for the alpha 47S-->F or beta 20H-->R mutants. These results provide evidence that the differently altered B875 polypeptides do not substit ute directly for the PufX protein but lead to structural rearrangement s in the macromolecular membrane organization, thus restoring a suffic iently high capacity for light-driven cyclic electron transfer.