1887

Microbiology

Volume 147, Issue 1

Isolation and characterization of mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Free

Abstract

In , dinitrogenase reductase ADP-ribosyltransferase (DRAT) is responsible for the ADP-ribosylation of dinitrogenase reductase in response to the addition of \(NH_{4}^{+}\) or removal from light, resulting in a decrease in nitrogenase activity. DRAT is itself subject to post-translational regulation; to investigate the mechanism for the regulation of DRAT activity, random PCR mutagenesis of (encoding DRAT) was performed and mutants with altered DRAT regulation were screened. Two mutants (with substitutions of K103E and N248D) were obtained in which DRAT showed activity under conditions where wild-type DRAT (DRAT-WT) did not. These mutants showed lower nitrogenase activity and a higher degree of ADP-ribosylation of dinitrogenase reductase under N-fixing conditions than was seen in a wild-type control strain. DRAT-K103E was overexpressed and purified. DRAT-K103E displayed a much weaker affinity for an Affi-gel Blue matrix than did DRAT-WT, suggestive of a fairly striking biochemical change. However, there was no significant difference in kinetic constants, such as for NAD and , between DRAT-K103E and DRAT-WT. Like DRAT-WT, DRAT-K103E also modified reduced dinitrogenase reductase poorly. The biochemical properties of these variants are rationalized with respect to their behaviour .

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): ADP-ribosylation , DRAG, dinitrogenase reductase activating glycohydrolase , DRAT, dinitrogenase reductase ADP-ribosyltransferase , DRAT-WT, DRAT from wild-type , nitrogen fixation , random PCR mutagenesis and regulation
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-1-193
2001-01-01
2021-03-01
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/1/1470193a.html?itemId=/content/journal/micro/10.1099/00221287-147-1-193&mimeType=html&fmt=ahah

References

  1. Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Liang, X., Finlay, D. R., Guiney, D. & Helinski, D. R. ( 1985; ). Plasmids related to the broad range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13, 149-153.[CrossRef]
    [Google Scholar]
  2. Fitzmaurice, W. P., Saari, L. L., Lowery, R. G., Ludden, P. W. & Roberts, G. P. ( 1989; ). Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum. Mol Gen Genet 218, 340-347.[CrossRef]
    [Google Scholar]
  3. Fu, H. A., Hartmann, A., Lowery, R. G., Fitzmaurice, W. P., Roberts, G. P. & Burris, R. H. ( 1989; ). Posttranslational regulatory system for nitrogenase activity in Azospirillum spp. J Bacteriol 171, 4679-4685.
     [Google Scholar]
  4. Fu, H. A., Fitzmaurice, W. P., Roberts, G. P. & Burris, R. H. ( 1990; ). Cloning and expression of draTG genes from Azospirillum lipoferum. Gene 86, 95-98.[CrossRef]
    [Google Scholar]
  5. Grunwald, S. K. & Ludden, P. W. ( 1997; ). NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum. J Bacteriol 179, 3277-3283.
     [Google Scholar]
  6. Grunwald, S. K., Lies, D. P., Roberts, G. P. & Ludden, P. W. ( 1995; ). Posttranslational regulation of nitrogenase in Rhodospirillum rubrum strains overexpressing the regulatory enzymes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase activating glycohydrolase. J Bacteriol 177, 628-635.
     [Google Scholar]
  7. Halbleib, C. M., Zhang, Y. & Ludden, P. W. ( 2000; ). Regulation of dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase by a redox-dependent conformational change of nitrogenase Fe protein. J Biol Chem 275, 3493-3500.[CrossRef]
    [Google Scholar]
  8. Hartmann, A. & Burris, R. H. ( 1987; ). Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum. J Bacteriol 169, 944-948.
     [Google Scholar]
  9. Hartmann, A., Fu, H. & Burris, R. H. ( 1986; ). Regulation of nitrogenase activity by ammonium chloride in Azospirillum spp. J Bacteriol 165, 864-870.
     [Google Scholar]
  10. Inoue, A., Shigematsu, T., Hidaka, M., Masaki, H. & Uozumi, T. ( 1996; ). Cloning, sequencing and transcriptional regulation of the draT and draG genes of Azospirillum lipoferum FS. Gene 170, 101-106.[CrossRef]
    [Google Scholar]
  11. Jouanneau, Y., Roby, C., Meyer, C. & Vignais, P. M. ( 1989; ). ADP-ribosylation of dinitrogenase reductase in Rhodobacter capsulatus. Biochemistry 28, 6524-6530.[CrossRef]
    [Google Scholar]
  12. Kanemoto, R. H. & Ludden, P. W. ( 1984; ). Effect of ammonia, darkness, and phenazine methosulfate on whole-cell nitrogenase activity and Fe protein modification in Rhodospirillum rubrum. J Bacteriol 158, 713-720.
     [Google Scholar]
  13. Keohavong, P. & Thilly, W. G. ( 1989; ). Fidelity of DNA polymerases in DNA amplification. Proc Natl Acad Sci USA 86, 9253-9257.[CrossRef]
    [Google Scholar]
  14. Kim, K., Zhang, Y. & Roberts, G. P. ( 1999; ). Correlation of activity regulation and substrate recognition of the ADP-ribosyltransferase that regulates nitrogenase activity in Rhodospirillum rubrum. J Bacteriol 181, 1698-1702.
     [Google Scholar]
  15. Lehman, L. J. & Roberts, G. P. ( 1991; ). Identification of an alternative nitrogenase system in Rhodospirillum rubrum. J Bacteriol 173, 5705-5711.
     [Google Scholar]
  16. Leung, D. W., Chen, E. & Goedde, D. V. ( 1989; ). A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1, 11-15.
     [Google Scholar]
  17. Liang, J. H., Nielsen, G. M., Lies, D. P., Burris, R. H., Roberts, G. P. & Ludden, P. W. ( 1991; ). Mutations in the draT and draG genes of Rhodospirillum rubrum result in loss of regulation of nitrogenase by reversible ADP-ribosylation. J Bacteriol 173, 6903-6909.
     [Google Scholar]
  18. Lowery, R. G. & Ludden, P. W. ( 1988; ). Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum. J Biol Chem 263, 16714-16719.
     [Google Scholar]
  19. Lowery, R. G. & Ludden, P. W. ( 1989; ). Effect of nucleotides on the activity of dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum. Biochemistry 28, 4956-4961.[CrossRef]
    [Google Scholar]
  20. Ludden, P. W. & Roberts, G. P. ( 1989; ). Regulation of nitrogenase activity by reversible ADP ribosylation. Curr Top Cell Regul 30, 23-56.
     [Google Scholar]
  21. Lundberg, K. S., Shoemaker, D. D., Adams, M. W. W., Short, J. M., Sorge, J. A. & Mathur, E. J. ( 1991; ). High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1-6.[CrossRef]
    [Google Scholar]
  22. Ma, L. & Li, J. ( 1997; ). Cloning and sequencing of draTG genes and their downstream region of Azospirillum brasilense Yu62. Chin J Biotechnol 13, 143-152.
     [Google Scholar]
  23. Masepohl, B., Krey, R. & Klipp, W. ( 1993; ). The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of both the molybdenum and the alternative nitrogenase. J Gen Microbiol 139, 2667-2675.[CrossRef]
    [Google Scholar]
  24. Norén, A., Soliman, A. & Nordlund, S. ( 1997; ). The role of NAD+ as a signal during nitrogenase switch-off in Rhodospirillum rubrum. Biochem J 322, 829-832.
     [Google Scholar]
  25. Pierrard, J., Ludden, P. W. & Roberts, G. P. ( 1993; ). Posttranslational regulation of nitrogenase in Rhodobacter capsulatus: existence of two independent regulatory effects of ammonium. J Bacteriol 175, 1358-1366.
     [Google Scholar]
  26. Schweizer, H. P. ( 1993; ). Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. BioTechniques 15, 831-833.
     [Google Scholar]
  27. Simon, R., Priefer, U. & Pühler, A. ( 1983; ). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1, 784-791.[CrossRef]
    [Google Scholar]
  28. Soliman, A. & Nordlund, S. ( 1992; ). Studies on the effect of NAD(H) on nitrogenase activity in Rhodospirillum rubrum. Arch Microbiol 157, 431-435.[CrossRef]
    [Google Scholar]
  29. Thompson, S. T., Cass, K. H. & Stellwagen, E. ( 1975; ). Blue Dextran-sepharose: an affinity column for the dinucleotide fold in protein. Proc Natl Acad Sci USA 72, 669-672.[CrossRef]
    [Google Scholar]
  30. Triplett, E. W., Wall, J. D. & Ludden, P. W. ( 1982; ). Expression of the activating enzyme and Fe protein of nitrogenase from Rhodospirillum rubrum. J Bacteriol 152, 786-791.
     [Google Scholar]
  31. Vieira, J. & Messing, J. ( 1982; ). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268.[CrossRef]
    [Google Scholar]
  32. Vogel, A. M. & Das, A. ( 1994; ). Mutational analysis of Agrobacterium tumefaciens pTiA6 virD1: identification of functionally important residues. Mol Microbiol 12, 811-817.[CrossRef]
    [Google Scholar]
  33. Zhang, Y., Burris, R. H. & Roberts, G. P. ( 1992; ). Cloning, sequencing, mutagenesis, and functional characterization of draT and draG genes from Azospirillum brasilense. J Bacteriol 174, 3364-3369.
     [Google Scholar]
  34. Zhang, Y., Burris, R. H., Ludden, P. W. & Roberts, G. P. ( 1993; ). Posttranslational regulation of nitrogenase activity by anaerobiosis and ammonium in Azospirillum brasilense. J Bacteriol 175, 6781-6788.
     [Google Scholar]
  35. Zhang, Y., Burris, R. H., Ludden, P. W. & Roberts, G. P. ( 1994; ). Posttranslational regulation of nitrogenase activity in Azospirillum brasilense ntrBC mutants: ammonium and anaerobic switch-off occurs through independent signal transduction pathways. J Bacteriol 176, 5780-5787.
     [Google Scholar]
  36. Zhang, Y., Burris, R. H., Ludden, P. W. & Roberts, G. P. ( 1995a; ). Comparison studies of dinitrogenase reductase ADP-ribosyl transferase/dinitrogenase reductase activating glycohydrolase regulatory systems in Rhodospirillum rubrum and Azospirillum brasilense. J Bacteriol 177, 2354-2359.
     [Google Scholar]
  37. Zhang, Y., Cummings, A. D., Burris, R. H., Ludden, P. W. & Roberts, G. P. ( 1995b; ). Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. J Bacteriol 177, 5322-5326.
     [Google Scholar]
  38. Zhang, Y., Burris, R. H., Ludden, P. W. & Roberts, G. P. ( 1997; ). Regulation of nitrogen fixation in Azospirillum brasilense. FEMS Microbiol Lett 152, 195-204.[CrossRef]
    [Google Scholar]
  39. Zhang, Y., Pohlmann, E. L., Ludden, P. W. & Roberts, G. P. ( 2000; ). Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum. J Bacteriol 182, 983-992.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-1-193
Loading
Isolation and characterization of draT mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Rhodospirillum rubrum
Microbiology 147, 193 (2001); https://doi.org/10.1099/00221287-147-1-193
/content/journal/micro/10.1099/00221287-147-1-193
/content/journal/micro/10.1099/00221287-147-1-193
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error