Molecular cloning and sequence analysis of the structural gene of ferredoxin I from the photosynthetic bacterium Rhodobacter capsulatus

Abstract

The structural gene (fdxN) encoding ferredoxin I (FdI) in the photosynthetic bacterium Rhodobacter capsulatus was isolated from a cosmid library by using an oligonucleotide probe corresponding to the N-terminal amino acid sequence of FdI. The nucleotide sequences of the gene and of the 3'- and 5'-flanking regions were determined. The gene fdxN codes for a polypeptide of 64 mino acids having a calculated molecular weight of 6,728. Amino acid sequencing of the N- and C-terminal ends of FdI allowed the determination of 86% of the primary structure and confirmed that FdI is the fdxN gene product. Sequence comparisons indicate that FdI shares common structural features with ferredoxins containing two [4Fe-4S] clusters, including eight conserved cysteines. Maximal homology was found with a ferredoxin from Rhodo-pseudomonas palustris. Northern (RNA) hybridization using a 158-base-pair DNA fragment internal to the fdxN coding region revealed the existence of two mRNA transcripts of approximately 330 and 750 nucleotides. Neither of those transcripts was present under nif-repressing growth conditions. The 5' end of the smaller transcript was mapped by S1 nuclease protection and primer extension experiments. On the basis of Southern hybridization experiments, by using probes homologous to fdxN, nifE, and a fragment complementing a nif point mutation, fdxN was localized inside a cluster of nif genes.