Molecular cloning and sequence of the B880 holochrome gene from Rhodospirillum rubrum.

Abstract

Restriction fragments of genomic Rhodospirillum rubrum DNA were selected according to size by electrophoresis followed by hybridization with [32P]mRNA encoding the two B880 holochrome polypeptides. The fragments were cloned into Escherchia coli C600 with plasmid pBR327 as a vector. The clones were selected by colony hybridization with 32P-holochrome-mRNA and counterselected by hybridization with Rs. rubrum ribosomal RNA, a minor contaminant of the mRNA preparation. Chimeric plasmid pRR22 was shown to contain the B880 genes by hybrid selection of B880 holochrome-mRNA. We report a restriction map of its 2.2-kilobase insert and the sequence of a 430 base pair fragment thereof. Genes alpha and beta are nearly contiguous, indicating that they are transcribed as a single operon. The predicted amino acid sequences coincide with the sequences of the alpha and beta polypeptides established in other laboratories, except for additional C-terminal tails of 10 and 13 amino acid residues, respectively. We suggest that these tail sequences may serve, during membrane assembly, to give these intrinsic membrane proteins their peculiar orientation with their C-terminus facing the periplasm and their N terminus facing the cytoplasm. Intraspecific sequence homology between the alpha and beta genes of R. rubrum is low, showing no evolutionary relatedness. This is in contrast to the high interspecific homology between the corresponding sequences of Rs. rubrum and Rhodopseudomonas capsulata B880 genes.