Summary
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.
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References
Alam J, Whitaker RA, Krogman DW, Curtis SE (1986) Isolation and sequence of the gene for ferredoxin I from the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 168:1265–1271
Biggin MD, Gibson TJ, Hong GF (1983) Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci USA 80:3963–3965
Böhme H, Böger P (1982) Ferredoxin. In: Zaborksi OR, Mitsui A, Black CC (eds) CRC handbook ser of biosolar resources, vol 1: Basic prinicples. CRC Press Inc., Boca Raton, Fla, pp 147–152
Böhme H, Schrautemeier B (1987a) Comparative characterization of ferredoxins from heterocysts and vegetative cells of Anabaena variabilis. Biochim Biophys Acta 891:1–7
Böhme H, Schrautemeier B (1987b) Electron donation to nitrogenase in a cell free system from heterocysts of Anabaena variabilis. Biochim Biophys Acta 891:115–120
Cammack R, Rao KK, Bargeron CP, Hutson KG, Andrew PW, Rogers LJ (1977) Midpoint redox potentials of plant and algal ferredoxins. Biochem J 168:205–209
Feinberg AP, Vogelstein B (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 139:266–267
Golden JW, Robinson SJ, Haselkorn R (1985) Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena. Nature 314:419–423
Golden SS, Brusslan J, Haselkorn R (1986) Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2. EMBO J 5:2789–2798
Golden SS, Brusslan J, Haselkorn R (1987) Genetic engineering of the cynaobacterial chromosome. Methods Enzymol 153:215–231
Hase R, Matsubara H, Hutber GN, Rogers LJ (1982) Amino acid sequences of Nostoc strain MAC ferredoxins I and II. J Biochem 92:1347–1355
Haselkorn R (1978) Heterocysts. Annu Rev Plant Physiol 29:319–344
Haselkorn R (1986) Organisation of the genes for nitrogen fixation in photosynthetic bacteria and cyanobacteria. Annu Rev Microbiol 40:525–547
Kratz WA, Myers J (1955) Nutrition and growth of several bluegreen algae. Am J Bot 42:282–287
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Matsubara H, Hase T (1983) Phylogenetic consideration of ferredoxin sequences in plants, particularly agae. In: Jensen U, Faibrothers DE (eds) Proteins and nucleic acids in plant systematics. Springer, Berlin Heidelberg New York, pp 168–181
Maxam AM, Gilbert W (1980) Sequencing of end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560
Mazur BJ, Rice D, Haselkorn R (1980) Identification of blue-green algal nitrogen fixation genes using heterologous DNA hybridization probes. Proc Natl Acad Sci USA 77:186–190
McMaster GK, Carmichael GG (1977) Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci USA 74:4835–4838
Murray N, Brammar W, Murray K (1977) Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet 150:53–61
Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106
Reed KC, Mann DA (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13:7207–7221
Reith ME, Laudenbach DE, Straus NA (1986) Isolation and nucleotide sequence analysis of the ferredoxin I gene from the cyanobacterium Anacystis nidulans R2. J Bacteriol 168:1319–1324
Rice D, Mazur BJ, Haselkorn R (1982) Isolation and physical mapping of nitrogen fixation genes from the cyanobacterium Anabaena 7120. J Biol Chem 257:13157–13163
Rosenberg M, Court D (1979) Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet 13:319–353
Sanger F, Nicklen S, Coulson AR (1977) DNA sequences with chain termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schrautemeier B, Böhme H (1985) A distinct ferredoxin for nitrogen fixation isolated from heterocysts of the cyanobacterium Anabaena variabilis. FEBS Lett 184:304–308
Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346
Smeekens S, Binsbergen JV, Weisbeek P (1985) The plant ferredoxin precursor: nucleotide sequence of a full length cDNA clone. Nucleic Acids Res 13:3179–3194
Smith BE, Campell F, Eady RR, Eldrige M, Ford CM, Hill S, Kavanagh EP, Lowe DJ, Miller DW, Richardson TH, Robson RL, Thorneley RNF, Yates MG (1987) Biochemistry of nitrogenase and the physiology of related metabolism. Philos Trans R Soc Lond [Biol] 317:131–146
Smith DR, Calvo JM (1980) Nucleotide sequence of the E. coli gene coding for dihydrofolate reductase. Nucleic Acids Res 8:2255–2275
Stern DB, Gruissem W (1987) Control of plastid gene expression: 3′ inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription. Cell 51:1145–1157
Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771
Thomas PS (1983) Hybridisation of denatured DNA transferred or dotted to nitrocellulose paper. Methods Enzymol 100:255–266
Tomioka N, Sugiura m (1983) The complete nucleotid sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans. Mol Gen Genet 191:46–50
Tsukihara T, Fukuyama K, Nakamura M, Katsube Y, Tanaka N, Kakudo M, Wada K, Hase T, Matsubara H (1981) X-ray analysis of a [2Fe-2S] ferredoxin from Spirulina platensis. Main chain fold and location of side chains at 2.5 Å resolution. J Biochem 90:1763–1773
Tumer NE, Robinson SJ, Haselkorn R (1983) Different promoters for the Anabaena glutamine synthetase gene during growth using molecular or fixed nitrogen. Nature 306:337–342
Van der Plas J, de Groot RP, Weisbeek PJ, van Arkel GA (1986a) Coding sequence of a ferredoxin gene from Anabaena variabilis ATCC 29413. Nucleic Acids Res 14:7803
Van der Plas J, de Groot RP, Woortman MR, Weisbeek PJ, van Arkel GA (1986b) Coding sequence of a ferredoxin from Anacystis nidulans R2 (Synechococcus PCC 7942). Nucleic Acids Res 14:7804
Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119
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Böhme, H., Haselkorn, R. Molecular cloning and nucleotide sequence analysis of the gene coding for heterocyst ferredoxin from the cyanobacterium Anabaena sp. strain PCC 7120. Mol Gen Genet 214, 278–285 (1988). https://doi.org/10.1007/BF00337722
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DOI: https://doi.org/10.1007/BF00337722