Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides light-harvesting B800-850-alpha and B800-850-beta genes

Abstract

Two deoxyoligonucleotide probes were synthesized in accordance with the available amino acid sequence of the B800-850-beta polypeptide from Rhodobacter sphaeroides and were used to isolate a 2.6-kilobase PstI fragment from R. sphaeroides 2.4.1 chromosomal DNA. Identification of the B800-850-beta and B800-850-alpha structural genes, pucB and pucA, was confirmed by DNA sequencing. Northern (RNA) blot analysis, using restriction endonuclease fragments from the cloned genes as probes, revealed a single puc-operon-specific, highly stable transcript of approximately 640 bases present in photosynthetically grown cells. In vitro transcription-translation analysis of the puc operon revealed that the maximum synthesis of the puc operon gene products was achieved when the entire 2.6-kilobase PstI fragment was used as the template, although a 537-base-pair XmaIII fragment was sufficient to direct the synthesis of pucB and pucA fusion product.