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.

Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120

“…Cultures used in the preparation of RNA were grown in Kratz and Myer's liquid medium (28) as described before (2). The E. coli strains used in this study are listed in Table 1 and were grown in L broth (31).…”

“…DNA and RNA were prepared from Anabaena sp. strain PCC 7120 as described previously (2,16). Large-scale preparations of purified plasmid DNA were made by the method of Kahn et al (26).…”

“…strain PCC 7120 chromosomal DNA (2 pRg) were separated by electrophoresis and transferred to a nylon membrane (Gene-Screen Plus; Du Pont) as described by Maniatis et al (31). For Northern blots, RNA (20 pLg) was glyoxalated, separated by electrophoresis, and transferred to Gene-Screen Plus as described before (2). RNA size standards were purchased from Bethesda Research Laboratories.…”

“…RNA size standards were purchased from Bethesda Research Laboratories. End labeling of oligonucleotides and labeling of isolated DNA fragments were carried out as described before (2). All blots were hybridized overnight under conditions described before (2).…”

“…End labeling of oligonucleotides and labeling of isolated DNA fragments were carried out as described before (2). All blots were hybridized overnight under conditions described before (2). Hybridizations with the oligonucleotide as probe were done at 50°C, while all others were done at 65°C.…”