Molecular cloning and nucleotide sequence analysis of the gene coding for heterocyst ferredoxin from the cyanobacterium Anabaena sp. strain PCC 7120

Abstract

In heterocysts of the filamentous cyanobacterium Anabaena 7120 a specific [2Fe-2S] ferredoxin is synthesized, serving as immediate electron donor to nitrogenase. The structural gene for this heterocyst ferredoxin, fdxH, was isolated from a recombinant lambda library, using an oligonucleotide probe derived from a unique segment of the N-terminal amino acid sequence of the purified protein. The sequence of the entire fdxH coding region was determined including 3' and 5' flanking sequences. Assuming proteolytic cleavage of the first methionine residue, the molecular weight of Anabaena 7120 heterocyst ferredoxin is 10,806. Compared with the ferredoxin from vegetative cells, 47 out of 98 amino acid residues are different, including conversions within a highly conserved region responsible for binding of the iron-sulfur cluster. Northern hybridization with a 0.64 kb EcoRI DNA fragment containing the entire fdxH gene indicated two major transcripts of 0.59 and 1.85 kb, which are expressed at a late stage of heterocyst differentiation. By S1 nuclease digestion and primer extension a possible start site of transcription was mapped, 132 bp upstream of fdxH; however, neither a typical Escherichia coli nor nif-type promoter sequence was apparent. Southern hybridization detected only one copy of the fdxH gene in the Anabaena 7120 genome. FdxH is located approximately 7 kb downstream from the nifHDK gene cluster.