Isolation and sequence of the gene for ferredoxin I from the cyanobacterium Anabaena sp. strain PCC 7120

Abstract

The structural gene for ferredoxin I, petF, from the cyanobacterium Anabaena sp. strain PCC 7120 has been isolated from a recombinant lambda library. Mixtures of tetradecanucleotides and heptadecanucleotides, each containing all possible DNA sequences corresponding to two separate regions of the ferredoxin amino acid sequence, were synthesized and used as hybridization probes to identify a genomic clone containing the coding sequence for the petF gene. The sequence of the entire petF coding region and portions of the 3'-and 5'-flanking regions was determined. The DNA sequence of petF suggests that, in contrast to the nucleus-encoded plant protein, cyanobacterial apoferredoxin is not synthesized as a higher-molecular-weight precursor. The Anabaena petF gene is a single-copy gene. During growth on complete medium it was transcribed into a monocistronic mRNA species of approximately 500 bases that initiated 100 base pairs upstream from the petF coding region.Ferredoxin is an acidic, nonheme, iron-sulfur protein with a wide distribution in organisms ranging from nonphotosynthetic, anaerobic bacteria to higher plants and animals. In plants and cyanobacteria, ferredoxin is recognized primarily as a component of the photosynthetic electron transport chain (31). In addition to its central role in NADP+ reduction and cyclic photophosphorylation, ferredoxin functions, directly or indirectly, in a variety of biological electron transfer systems. These include such diverse processes as nitrogen fixation (38), sulfate-sulfite reduction (32), nitrate-nitrite reduction (17, 25), and glutamate synthesis (17). Furthermore, ferredoxin is responsible, via the ferredoxinthioredoxin system, for the regulation of enzymes involved in photosynthetic carbon metabolism (3).Two distinct molecular forms of ferredoxin have been identified in some plants and cyanobacteria. The primary structure of ferredoxins I and II from a number of organisms has been determined (for a compilation, see references 12 and 21), and the amino acid sequence data suggest that these two ferredoxins arose via gene duplication (21). At present, however, no functional differences have been attributed to this dimorphism. Aside from ferredoxins I and II, some cyanobacteria apparently contain yet another form of this protein. Cohn et al. (6) have isolated a membrane-associated ferredoxin from the cyanobacterium Aphanizomenon flosaquae that is distinct from ferredoxin I with regard to location within the cell, chromatographic behavior on a DEAE-cellulose column, and electron spin resonance spectra. The limited amino acid sequence data suggest that the membrane-associated ferredoxin is more similar to ferredoxin I than most ferredoxin II molecules are to ferredoxin

Genes encoding the alpha, gamma, delta, and four F0 subunits of ATP synthase constitute an operon in the cyanobacterium Anabaena sp. strain PCC 7120

“…6). S1 nuclease mapping with a probe that included the 5' end of atpI, all of gene 1, and 500 bp 5' to gene 1 (Fig. 1, probe 1) confirmed the single mRNA endpoint identified in the primer extension assay and demonstrated that no additional RNA endpoints map between gene 1 and atpl (data not shown).…”

Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinase-encoding genes (pta and ack) from Methanosarcina thermophila

“…The 5Ј ends of the pta and ack mRNAs were mapped with the following oligonucleotides: pta1 (5Ј-GTTAAGTTTCTTTGCTCTTTCACT-3Ј, nucleotides 52 to 75 relative to the identified transcriptional start site of pta), pta2 (5Ј-CAAGGATCTTGGCAGCTGCCTG-3Ј, nucleotides 118 to 139), ack1 (5Ј-CCTATCCTCTCGCAAAGACCTAC-3Ј, nucleotides 1215 to 1237), and ack2 (5Ј-CCAGTACTTTCATGTGTAAACC-3Ј, nucleotides 1125 to 1146). Primer extension analysis was based on a procedure described by Alam et al (2). Briefly, RNA (10 to 20 g) isolated from acetate-grown M. thermophila cells was pelleted and the pellet was resuspended in a solution consisting of 2 mol of primer and 7.5 M (each) dGTP, dCTP, and dTTP.…”

Cloning, nucleotide sequence, and transcriptional analyses of the gene encoding a ferredoxin from Methanosarcina thermophila

“…The 5' ends of the fdxA-and ORFl-specific mRNAs were mapped as described before (3) except that [a-35S]dATP (1,000 to 1,500 Ci/mmol) was substituted for [a-32P]dATP. The 21-mer oligonucleotide probes II (complementary to nucleotides +85 to +105 offdxA; see ORFl-specific primers (15 ng) were annealed to 10 or 25 ,ug, respectively, of total M. thermophila RNA which had been treated with DNase.…”