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. 1988 Jan;170(1):258–265. doi: 10.1128/jb.170.1.258-265.1988

Isolation, sequence analysis, and transcriptional studies of the flavodoxin gene from Anacystis nidulans R2.

D E Laudenbach 1, M E Reith 1, N A Straus 1
PMCID: PMC210636  PMID: 3121586

Abstract

The nonheme, iron-sulfur protein ferredoxin is the terminal constituent of the photosynthetic electron transport chain. Under conditions of iron stress, many cyanobacteria and eucaryotic algae replace ferredoxin with the flavoprotein flavodoxin. The gene for flavodoxin was cloned from the cyanobacterium Anacystis nidulans R2 by using three mixed oligonucleotide probes derived from the partial Synechococcus sp. strain PCC 6301 amino acid sequence. Nucleotide sequence analysis revealed a 513-base-pair open reading frame with a deduced amino acid sequence having homology to other long-chain flavodoxins. Assuming proteolytic cleavage of the initial methionine residue, the molecular weight of the A. nidulans R2 flavodoxin is 18,609. Southern blot hybridization under conditions of reduced stringency detected only one copy of the flavodoxin sequence in the A. nidulans R2 genome. Northern (RNA) blot hybridization analyses by using cloned flavodoxin gene probes indicated that no transcripts are detectable under conditions of iron saturation. However, under iron-deficient growth conditions the flavodoxin gene appeared to be transcribed as part of a larger operon. The operon yielded at least three transcripts. The first was of approximately 1,100 bases (designated RNA 1) and terminated immediately upstream from the 5' end of the flavodoxin open reading frame. A second, less abundant transcript of approximately 1,900 bases (designated RNA 2) encoded all of RNA 1 as well as the flavodoxin polypeptide. Analysis indicated that both transcripts initiate in close proximity to each other. A third, minor transcript of approximately 1,100 bases (designated RNA 3) was detectable downstream of the flavodoxin gene sequence. Addition of iron-stressed A. nidulans R2 cells resulted in almost total loss of detectable mRNA transcripts within 60 min of the addition. The ferredoxin gene transcript has previously been characterized as a monocistronic message of approximately 430 bases (M. E. Reith, D. E. Laudenbach, and N. A. Straus, J. Bacteriol. 168: 1319-1324, 1986). Here we show that the ferredoxin message is detectable under all iron regimes tested is quantitatively unaffected by decreases in iron availability to the cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. André E., Kessler M., Briquel M. E., Alexandre P., Hurault de Ligny B., Huriet C. Intérêt pratique du dosage des produits de dégradation de la fibrine urinaire dans la surveillance précoce des transplantés rénaux. Pathol Biol (Paris) 1983 Jan;31(1):23–27. [PubMed] [Google Scholar]
  2. Belasco J. G., Beatty J. T., Adams C. W., von Gabain A., Cohen S. N. Differential expression of photosynthesis genes in R. capsulata results from segmental differences in stability within the polycistronic rxcA transcript. Cell. 1985 Jan;40(1):171–181. doi: 10.1016/0092-8674(85)90320-4. [DOI] [PubMed] [Google Scholar]
  3. Borbely G., Simoncsits A. 3'-Terminal conserved loops of 16S rRNAs from the cyanobacterium Synechococcus AN PCC 6301 and maize chloroplast differ only in two bases. Biochem Biophys Res Commun. 1981 Aug 14;101(3):846–852. doi: 10.1016/0006-291x(81)91827-1. [DOI] [PubMed] [Google Scholar]
  4. Conley P. B., Lemaux P. G., Grossman A. R. Cyanobacterial light-harvesting complex subunits encoded in two red light-induced transcripts. Science. 1985 Nov 1;230(4725):550–553. doi: 10.1126/science.3931221. [DOI] [PubMed] [Google Scholar]
  5. Dale R. M., McClure B. A., Houchins J. P. A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA. Plasmid. 1985 Jan;13(1):31–40. doi: 10.1016/0147-619x(85)90053-8. [DOI] [PubMed] [Google Scholar]
  6. Drummond M. H. The base sequence of the nifF gene of Klebsiella pneumoniae and homology of the predicted amino acid sequence of its protein product to other flavodoxins. Biochem J. 1985 Dec 15;232(3):891–896. doi: 10.1042/bj2320891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  8. Guikema J. A., Sherman L. A. Influence of Iron Deprivation on the Membrane Composition of Anacystis nidulans. Plant Physiol. 1984 Jan;74(1):90–95. doi: 10.1104/pp.74.1.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guikema J. A., Sherman L. A. Organization and Function of Chlorophyll in Membranes of Cyanobacteria during Iron Starvation. Plant Physiol. 1983 Oct;73(2):250–256. doi: 10.1104/pp.73.2.250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hutson K. G., Rogers L. J., Haslett B. G., Boulter D., Cammack R. Comparative studies on two ferredoxins from the cyanobacterium Nostoc strain MAC. Biochem J. 1978 Jun 15;172(3):465–477. doi: 10.1042/bj1720465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lomax T. L., Conley P. B., Schilling J., Grossman A. R. Isolation and characterization of light-regulated phycobilisome linker polypeptide genes and their transcription as a polycistronic mRNA. J Bacteriol. 1987 Jun;169(6):2675–2684. doi: 10.1128/jb.169.6.2675-2684.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Merchant S., Bogorad L. Regulation by copper of the expression of plastocyanin and cytochrome c552 in Chlamydomonas reinhardi. Mol Cell Biol. 1986 Feb;6(2):462–469. doi: 10.1128/mcb.6.2.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nieva-Gómez D., Roberts G. P., Klevickis S., Brill W. J. Electron transport to nitrogenase in Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1980 May;77(5):2555–2558. doi: 10.1073/pnas.77.5.2555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pustell J., Kafatos F. C. A convenient and adaptable package of computer programs for DNA and protein sequence management, analysis and homology determination. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):643–655. doi: 10.1093/nar/12.1part2.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Reith M. E., Laudenbach D. E., Straus N. A. Isolation and nucleotide sequence analysis of the ferredoxin I gene from the cyanobacterium Anacystis nidulans R2. J Bacteriol. 1986 Dec;168(3):1319–1324. doi: 10.1128/jb.168.3.1319-1324.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  17. Sandmann G., Malkin R. Iron-sulfur centers and activities of the photosynthetic electron transport chain in iron-deficient cultures of the blue-green alga aphanocapsa. Plant Physiol. 1983 Nov;73(3):724–728. doi: 10.1104/pp.73.3.724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shah V. K., Stacey G., Brill W. J. Electron transport to nitrogenase. Purification and characterization of pyruvate:flavodoxin oxidoreductase. The nifJ gene product. J Biol Chem. 1983 Oct 10;258(19):12064–12068. [PubMed] [Google Scholar]
  20. Smillie R. M. Isolation of two proteins with chloroplast ferredoxin activity from a blue-green alga. Biochem Biophys Res Commun. 1965 Sep 8;20(5):621–629. doi: 10.1016/0006-291x(65)90445-6. [DOI] [PubMed] [Google Scholar]
  21. Smith W. W., Pattridge K. A., Ludwig M. L., Petsko G. A., Tsernoglou D., Tanaka M., Yasunobu K. T. Structure of oxidized flavodoxin from Anacystis nidulans. J Mol Biol. 1983 Apr 25;165(4):737–753. doi: 10.1016/s0022-2836(83)80277-0. [DOI] [PubMed] [Google Scholar]
  22. Stewart W. D. Some aspects of structure and function in N2-fixing cyanobacteria. Annu Rev Microbiol. 1980;34:497–536. doi: 10.1146/annurev.mi.34.100180.002433. [DOI] [PubMed] [Google Scholar]
  23. Tanaka M., Haniu M., Yasunobu K. T., Yoch D. C. Complete amino acid sequence of azotoflavin, a flavodoxin from Azotobacter vinelandii. Biochemistry. 1977 Aug 9;16(16):3525–3537. doi: 10.1021/bi00635a005. [DOI] [PubMed] [Google Scholar]
  24. Tandeau de Marsac N., Mazel D., Bryant D. A., Houmard J. Molecular cloning and nucleotide sequence of a developmentally regulated gene from the cyanobacterium Calothrix PCC 7601: a gas vesicle protein gene. Nucleic Acids Res. 1985 Oct 25;13(20):7223–7236. doi: 10.1093/nar/13.20.7223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tarr G. E. Determination of the sequence which spans the beginning of the insertion region in Anacystis nidulans flavodoxin. J Mol Biol. 1983 Apr 25;165(4):754–755. doi: 10.1016/s0022-2836(83)80278-2. [DOI] [PubMed] [Google Scholar]
  26. Thomas P. S. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 1983;100:255–266. doi: 10.1016/0076-6879(83)00060-9. [DOI] [PubMed] [Google Scholar]
  27. Tomioka N., Sugiura M. The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans. Mol Gen Genet. 1983;191(1):46–50. doi: 10.1007/BF00330888. [DOI] [PubMed] [Google Scholar]
  28. Wood P. M. Interchangeable copper and iron proteins in algal photosynthesis. Studies on plastocyanin and cytochrome c-552 in Chlamydomonas. Eur J Biochem. 1978 Jun 1;87(1):9–19. doi: 10.1111/j.1432-1033.1978.tb12346.x. [DOI] [PubMed] [Google Scholar]

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