Analysis of the promoter and regulatory sequences of an oxygen-regulated bch operon in Rhodobacter capsulatus by site-directed mutagenesis

Abstract

The biosynthesis of pigments (carotenoids and bacteriochlorophylls) in the photosynthetic bacterium Rhodobacter capsulatus is regulated by the oxygen concentration in the environment. However, the mechanism of this regulation has remained obscure. In this study, transcriptional fusions of the bchCXYZ promoter region to lacZ were used to identify the promoter and regulatory sequences governing transcription of these bacteriochlorophyll biosynthesis genes. The promoter region was identified in vivo by making deletions and site-directed mutations. The 50 bp upstream of the promoter region was shown to be required for the oxygen-dependent transcriptional regulation of bchCXYZ. A previously described palindrome sequence is also likely involved in the regulation. A gel mobility shift assay further defined the interaction of transcription regulators with these DNA sequence elements in vitro and demonstrated that a DNA-protein complex is formed at this promoter region. Since the suggested promoter sequence and the palindrome sequence are found upstream of several other bch and crt operons, these sequences may be responsible for regulating oxygen-dependent pigment biosynthesis at the level of transcription in R. capsulatus. In addition, these cis-acting DNA elements are not found upstream of puh and puf operons, which encode the structural polypeptides of the reaction center and light-harvesting I complexes. This observation supports the model of different regulatory mechanism for the pigment biosynthesis enzymes and structural polypeptides required for the production of the photosynthetic apparatus.