Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Oct;174(19):6025–6032. doi: 10.1128/jb.174.19.6025-6032.1992

Synthesis of nitrogenase in mutants of the cyanobacterium Anabaena sp. strain PCC 7120 affected in heterocyst development or metabolism.

A Ernst 1, T Black 1, Y Cai 1, J M Panoff 1, D N Tiwari 1, C P Wolk 1
PMCID: PMC207667  PMID: 1328150

Abstract

Mutants of Anabaena sp. strain PCC 7120 that are incapable of sustained growth with air as the sole source of nitrogen were generated by using Tn5-derived transposons. Nitrogenase was expressed only in mutants that showed obvious morphological signs of heterocyst differentiation. Even under rigorously anaerobic conditions, nitrogenase was not synthesized in filaments that were unable to develop heterocysts. These results suggest that competence to synthesize nitrogenase requires a process that leads to an early stage of visible heterocyst development and are consistent with the idea that synthesis of nitrogenase is under developmental control (J. Elhai and C. P. Wolk, EMBO J. 9:3379-3388, 1990). We isolated mutants in which differentiation was arrested at an intermediate stage of heterocyst formation, suggesting that differentiation proceeds in stages; those mutants, as well as mutants with aberrant heterocyst envelopes and a mutant with defective respiration, expressed active nitrogenase under anaerobic conditions only. These results support the idea that the heterocyst envelope and heterocyst respiration are required for protection of nitrogenase from inactivation by oxygen. In the presence of air, such mutants contained less nitrogenase than under anaerobic conditions, and the Fe-protein was present in a posttranslationally modified inactive form. We conclude that internal partial oxygen pressure sufficient to inactivate nitrogenase is insufficient to repress synthesis of the enzyme completely. Among mutants with an apparently intact heterocyst envelope and normal respiration, three had virtually undetectable levels of dinitrogenase reductase under all conditions employed. However, three others expressed oxygen-sensitive nitrogenase activity, suggesting that respiration and barrier to diffusion of gases may not suffice for oxygen protection of nitrogenase in these mutants; two of these mutants reduced acetylene to ethylene and ethane.

Full text

PDF
6025

Images in this article

6030Image
on p.6030
6030Image
on p.6030

Selected References

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

  1. Bancroft I., Wolk C. P., Oren E. V. Physical and genetic maps of the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol. 1989 Nov;171(11):5940–5948. doi: 10.1128/jb.171.11.5940-5948.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  3. Borthakur D., Haselkorn R. Tn5 mutagenesis of Anabaena sp. strain PCC 7120: isolation of a new mutant unable to grow without combined nitrogen. J Bacteriol. 1989 Oct;171(10):5759–5761. doi: 10.1128/jb.171.10.5759-5761.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buikema W. J., Haselkorn R. Characterization of a gene controlling heterocyst differentiation in the cyanobacterium Anabaena 7120. Genes Dev. 1991 Feb;5(2):321–330. doi: 10.1101/gad.5.2.321. [DOI] [PubMed] [Google Scholar]
  5. Buikema W. J., Haselkorn R. Isolation and complementation of nitrogen fixation mutants of the cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol. 1991 Mar;173(6):1879–1885. doi: 10.1128/jb.173.6.1879-1885.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cai Y. P., Wolk C. P. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J Bacteriol. 1990 Jun;172(6):3138–3145. doi: 10.1128/jb.172.6.3138-3145.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elhai J., Wolk C. P. Developmental regulation and spatial pattern of expression of the structural genes for nitrogenase in the cyanobacterium Anabaena. EMBO J. 1990 Oct;9(10):3379–3388. doi: 10.1002/j.1460-2075.1990.tb07539.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ernst A., Reich S., Böger P. Modification of dinitrogenase reductase in the cyanobacterium Anabaena variabilis due to C starvation and ammonia. J Bacteriol. 1990 Feb;172(2):748–755. doi: 10.1128/jb.172.2.748-755.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Golden J. W., Robinson S. J., Haselkorn R. Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena. Nature. 1985 Apr 4;314(6010):419–423. doi: 10.1038/314419a0. [DOI] [PubMed] [Google Scholar]
  10. Helber J. T., Johnson T. R., Yarbrough L. R., Hirschberg R. Regulation of nitrogenase gene expression in anaerobic cultures of Anabaena variabilis. J Bacteriol. 1988 Feb;170(2):552–557. doi: 10.1128/jb.170.2.552-557.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heusterspreute M., Ha Thi V., Emery S., Tournis-Gamble S., Kennedy N., Davison J. Vectors with restriction site banks. IV. pJRD184, a 3793-bp plasmid vector with 49 unique restriction sites. Gene. 1985;39(2-3):299–304. doi: 10.1016/0378-1119(85)90327-0. [DOI] [PubMed] [Google Scholar]
  12. Holland D., Wolk C. P. Identification and characterization of hetA, a gene that acts early in the process of morphological differentiation of heterocysts. J Bacteriol. 1990 Jun;172(6):3131–3137. doi: 10.1128/jb.172.6.3131-3137.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Houchins J. P., Hind G. Concentration and function of membrane-bound cytochromes in cyanobacterial heterocysts. Plant Physiol. 1984 Oct;76(2):456–460. doi: 10.1104/pp.76.2.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hu N. T., Thiel T., Giddings T. H., Jr, Wolk C. P. New Anabaena and Nostoc cyanophages from sewage settling ponds. Virology. 1981 Oct 15;114(1):236–246. doi: 10.1016/0042-6822(81)90269-5. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lambein F., Wolk C. P. Structural studies on the glycolipids from the envelope of the heterocyst of Anabaena cylindrica. Biochemistry. 1973 Feb 27;12(5):791–798. doi: 10.1021/bi00729a002. [DOI] [PubMed] [Google Scholar]
  17. Maldener I., Lockau W., Cai Y. P., Wolk C. P. Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis. Mol Gen Genet. 1991 Jan;225(1):113–120. doi: 10.1007/BF00282649. [DOI] [PubMed] [Google Scholar]
  18. Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  19. Mazur B. J., Rice D., Haselkorn R. Identification of blue-green algal nitrogen fixation genes by using heterologous DNA hybridization probes. Proc Natl Acad Sci U S A. 1980 Jan;77(1):186–190. doi: 10.1073/pnas.77.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mevarech M., Rice D., Haselkorn R. Nucleotide sequence of a cyanobacterial nifH gene coding for nitrogenase reductase. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6476–6480. doi: 10.1073/pnas.77.11.6476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mulligan M. E., Haselkorn R. Nitrogen fixation (nif) genes of the cyanobacterium Anabaena species strain PCC 7120. The nifB-fdxN-nifS-nifU operon. J Biol Chem. 1989 Nov 15;264(32):19200–19207. [PubMed] [Google Scholar]
  22. Stragier P., Losick R. Cascades of sigma factors revisited. Mol Microbiol. 1990 Nov;4(11):1801–1806. doi: 10.1111/j.1365-2958.1990.tb02028.x. [DOI] [PubMed] [Google Scholar]
  23. Wolk C. P., Cai Y., Cardemil L., Flores E., Hohn B., Murry M., Schmetterer G., Schrautemeier B., Wilson R. Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen. J Bacteriol. 1988 Mar;170(3):1239–1244. doi: 10.1128/jb.170.3.1239-1244.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wolk C. P., Cai Y., Panoff J. M. Use of a transposon with luciferase as a reporter to identify environmentally responsive genes in a cyanobacterium. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5355–5359. doi: 10.1073/pnas.88.12.5355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wolk C. P., Vonshak A., Kehoe P., Elhai J. Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1561–1565. doi: 10.1073/pnas.81.5.1561. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES