COMPLETE DNA-SEQUENCE, SPECIFIC TN5 INSERTION MAP, AND GENE ASSIGNMENT OF THE CAROTENOID BIOSYNTHESIS PATHWAY OF RHODOBACTER-SPHAEROIDES

The carotenoid biosynthesis genes form a cluster, within the genome of Rhodobacter sphaeroides, lying in the middle of a larger cluster and 45 kb in length, which contains genes for bacteriochlorophyll biosynth esis and for the reaction center and light-harvesting apoproteins. The positions and approximate limits of the carotenoid genes were determi ned previously by localized transposon Tn5 mutagenesis and by comparis on with the closely related Rhodobacter capsulatus carotenoid gene clu ster. In this report, analysis of the DNA and deduced amino acid seque nces of the carotenoid genes in R. sphaeroides are presented. Twenty-f ive Tn5 insertion mutants were used to produce a base-specific Tn5 ins ertion map of this region, and carotenoid gene assignment was supporte d by spectroscopic, ultrastructural, and high-pressure liquid chromato graphy analyses of these mutants. A region in the 3' end of crtD which affects bacteriochlorophyll biosynthesis was discovered, and CrtA was found to possess a proline-rich C-terminal region containing a repeat ed (Ale-Pro)(n) motif. CrtF also showed a high degree of sequence cons ervation with eukaryotic O-methyltransferases. This study provides gen e sequences and assignments based upon a comprehensive structural, spe ctroscopic, and biochemical analysis of a range of carotenoid biosynth etic mutants; in each mutation, the point of Tn5 insertion is determin ed accurate to 1 hp on the gene cluster.