1887

Microbiology Society logo

Volume 146, Issue 11

The bv. gene, encoding a uridylyltransferase/uridylyl-removing enzyme, is expressed in the root nodule but is not essential for nitrogen fixation Free

Abstract

The GenBank accession number for the sequence reported in this paper is AF155830.

A bv. VF39 gene () encoding the uridylyltransferase/uridylyl-removing enzyme, which constitutes the sensory component of the nitrogen regulation () system, was identified, cloned and characterized. The deduced amino acid sequence contains the conserved active site motif of the nucleotidyltransferase superfamily and is highly homologous to the gene products of other bacterial species. Downstream of the VF39 resides an open reading frame with similarity to the virulence factor gene . Mutation of the gene abolished the ability to use nitrate as a sole nitrogen source but not glutamine. In addition, neither uridylylation of P nor induction of the -regulated gene (encoding glutamine synthetase II) under ammonium deficiency could be observed in mutant strains. This strongly suggests that mutants harbour a permanently deuridylylated P protein and as a consequence are unable to activate transcription from NtrC-dependent promoters. The gene itself is expressed constitutively, irrespective of the nitrogen content of the medium. A functional GlnD protein is not essential for nitrogen fixation in bv. , but detection of expression in the symbiotic and infection zone of the root nodule and quantitative measurements suggest that at least part of the system functions in symbiosis. The results also indicate that the N-terminal part of GlnD is essential for the cell, as deletions in the 5′-region of the gene appear to be lethal and mutations possibly affecting the expression of the first half of the protein have a significant effect on the vitality of the mutant strain.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): glutamine synthetase , GS, glutamine synthetase , mviN , nitrogen regulation and uridylyltransferase, PII uridylylation
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-11-2987
2000-11-01
2024-04-24
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/11/1462987a.html?itemId=/content/journal/micro/10.1099/00221287-146-11-2987&mimeType=html&fmt=ahah

References

  1. Adler S. P., Purich D., Stadtman E. R. 1975; Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme. J Biol Chem 250:6264–6272
     [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. 1990; Basic local alignment search tool. J Mol Biol 215:403–410 [CrossRef]
     [Google Scholar]
  3. Amar M., Patriarca E. J., Manco G., Bernard P., Riccio A., Lamberti A., Defez R., Iaccarino M. 1994; Regulation of nitrogen metabolism is altered in a glnB mutant strain of Rhizobium leguminosarum. Mol Microbiol 11:685–693 [CrossRef]
     [Google Scholar]
  4. Arcondeguy T., Huez I., Fourment J., Kahn D. 1996; Symbiotic nitrogen fixation does not require adenylylation of glutamine synthetase I in Rhizobium meliloti. FEMS Microbiol Lett 145:33–40 [CrossRef]
     [Google Scholar]
  5. Arcondeguy T., Huez I., Tillard P., Gangneux C., de Billy F., Gojon A., Truchet G., Kahn D. 1997; The Rhizobium meliloti PII protein, which controls bacterial nitrogen metabolism, affects alfalfa nodule development. Genes Dev 11:1194–1206 [CrossRef]
     [Google Scholar]
  6. Ausubel F. M., Brent R., Kingston R.editors 1987 Current Protocols in Molecular Biology New York: Wiley;
     [Google Scholar]
  7. Beringer J. E. 1974; R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198 [CrossRef]
     [Google Scholar]
  8. Bladergroen M. R., Spaink H. P. 1998; Genes and signal molecules involved in the rhizobia-leguminoseae symbiosis. Curr Opin Plant Biol 1:353–359 [CrossRef]
     [Google Scholar]
  9. Bravo A., Mora J. 1988; Ammonium assimilation in Rhizobium phaseoli by the glutamine synthetase-glutamate synthase pathway. J Bacteriol 170:980–984
     [Google Scholar]
  10. Brown C. M., Dilworth M. J. 1975; Ammonia assimilation by Rhizobium cultures and bacteroids. J Gen Microbiol 86:39–48 [CrossRef]
     [Google Scholar]
  11. de Bruijn F. J., Rossbach S., Schneider M., Ratet P., Messmer S., Szeto W. W., Ausubel F. M., Schell J. 1989; Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation. J Bacteriol 171:1673–1682
     [Google Scholar]
  12. Bullock J. C., Fernandez J. M., Short J. M. 1987; XL1Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. BioTechniques 5:376–379
     [Google Scholar]
  13. Chiurazzi M., Iaccarino M. 1990; Transcriptional analysis of the glnB-glnA region of Rhizobium leguminosarum biovar viciae. Mol Microbiol 4:1727–1735 [CrossRef]
     [Google Scholar]
  14. Chiurazzi M., Meza R., Lara M., Lahm A., Defez R., Iaccarino M., Espin G. 1992; The Rhizobium leguminosarum biovar phaseoli glnT gene, encoding glutamine synthetase III. Gene 119:1–8 [CrossRef]
     [Google Scholar]
  15. Colonna-Romano S., Riccio A., Guida M., Defez R., Lamberti A., Iaccarino M., Arnold W., Priefer U., Pühler A. 1987; Tight linkage of glnA and a putative regulatory gene in Rhizobium leguminosarum. Nucleic Acids Res 15:1951–1964 [CrossRef]
     [Google Scholar]
  16. Colonna-Romano S., Arnold W., Schlüter A., Boistard P., Pühler A., Priefer U. B. 1990; An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK. Mol Gen Genet 223:138–147 [CrossRef]
     [Google Scholar]
  17. Colonna-Romano S., Patriarca E. J., Amar M., Bernard P., Manco G., Lamberti A., Iaccarino M., Defez R. 1993; Uridylylation of the PII protein in Rhizobium leguminosarum. FEBS Lett 330:95–98 [CrossRef]
     [Google Scholar]
  18. Contreras A., Drummond M., Bali A., Blanco G., Garcia E., Bush G., Kennedy C., Merrick M. 1991; The product of the nitrogen fixation regulatory gene nfrX of Azotobacter vinelandii is functionally and structurally homologous to the uridylyltransferase encoded by glnD in enteric bacteria. J Bacteriol 173:7741–7749
     [Google Scholar]
  19. Dénarié J., Debellé F. 1996; Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem 65:503–535 [CrossRef]
     [Google Scholar]
  20. Downie J. A. 1994; Signalling strategies for nodulation of legumes by rhizobia. Trends Microbiol 2:318–324 [CrossRef]
     [Google Scholar]
  21. Edwards R., Merrick M. 1995; The role of uridylyltransferase in the control of Klebsiella pneumoniae nif gene regulation. Mol Gen Genet 247:189–198 [CrossRef]
     [Google Scholar]
  22. Engleman E. G., Francis S. H. 1978; Cascade control of E. coli glutamine synthetase. II. Metabolite regulation of the enzymes in the cascade. Arch Biochem Biophys 191:602–612 [CrossRef]
     [Google Scholar]
  23. Espin G., Moreno S., Guzman J. 1994; Molecular genetics of the glutamine synthetases in Rhizobium species. Crit Rev Microbiol 20:117–123 [CrossRef]
     [Google Scholar]
  24. Filser D. M., Moscatelli C., Lamberti A., Vincze E., Guida M., Salzano G., Iaccarino M. 1986; Characterization and cloning of two Rhizobium leguminosarum genes coding for glutamine synthetase activities. J Gen Microbiol 132:2561–2569
     [Google Scholar]
  25. Glenn A. R., Dilworth M. J. 1985; Ammonia movements in rhizobia. Microbiol Sci 2:161–162165–167
     [Google Scholar]
  26. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [CrossRef]
     [Google Scholar]
  27. van Heeswijk W., Kuppinger O., Merrick M., Kahn D. 1992; Localization of the glnD gene on a revised map of the 200 kilobase region of the Escherichia coli chromosome. J Bacteriol 174:1702–1703
     [Google Scholar]
  28. van Heeswijk W. C., Rabenberg M., Westerhoff H. V., Kahn D. 1993; The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli. Mol Microbiol 9:443–457 [CrossRef]
     [Google Scholar]
  29. Holm L., Sander C. 1995; DNA polymerase β belongs to an ancient nucleotidyltransferase superfamily. Trends Biochem Sci 20:345–347 [CrossRef]
     [Google Scholar]
  30. Hooykaas P. J. J., Clapwijk P. M., Nuti M. P., Shilperoort R. A., Roersch A. 1977; Transfer of the Agrobacterium tumefaciens T1 plasmid to avirulent Agrobacteria and to Rhizobium ex-planta. J Gen Microbiol 98:477–484 [CrossRef]
     [Google Scholar]
  31. Jefferson R. A. 1987; Assaying chimeric genes in plants: the GUS fusion system. Plant Mol Biol Reporter 5:387–405 [CrossRef]
     [Google Scholar]
  32. Jiang P., Peliska J. A., Ninfa A. J. 1998a; Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2 . 7 . 7 . 59) of Escherichia coli and its interaction with the PII protein. Biochemistry 37:12782–12794 [CrossRef]
     [Google Scholar]
  33. Jiang P., Peliska J. A., Ninfa A. J. 1998b; Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of ntr gene transcription in Escherichia coli. Biochemistry 37:12795–12801 [CrossRef]
     [Google Scholar]
  34. Jiang P., Peliska J. A., Ninfa A. J. 1998c; The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylation state. Biochemistry 37:12802–12810 [CrossRef]
     [Google Scholar]
  35. Kim I. H., Kwak S. J., Kang J., Park S. C. 1998; Transcriptional control of the glnD gene is not dependent on nitrogen availability in Escherichia coli. Mol Cells 8:483–490
     [Google Scholar]
  36. Lorkiewicz Z. 1997; Nodulation genes in the Rhizobium-plant signal exchange. Acta Biochim Pol 44:1–12
     [Google Scholar]
  37. Magasanik B. 1982; Genetic control in nitrogen assimilation in bacteria. Annu Rev Genet 16:135–168 [CrossRef]
     [Google Scholar]
  38. Mangum J. H., Magni G., Stadtman E. R. 1973; Regulation of the glutamine synthetase adenylylation and deadenylylation by the enzymatic uridylylation and deadenylylation of the PII regulatory protein. Arch Biochem Biophys 158:514–525 [CrossRef]
     [Google Scholar]
  39. Maniatis T., Fritsch E. F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  40. Merrick M. J., Edwards R. A. 1995; Nitrogen control in bacteria. Microbiol Rev 59:604–622
     [Google Scholar]
  41. Miller J. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  42. Moreno S., Patriarca E. J., Chiurazzi M., Meza R., Defez R., Lamberti A., Riccio A., Iaccarino M., Espin G. 1992; Phenotype of a Rhizobium leguminosarum ntrC mutant. Res Microbiol 143:161–171 [CrossRef]
     [Google Scholar]
  43. O’Connell K. P., Raffel S. J., Saville B. J., Handelsman J. 1998; Mutants of Rhizobium tropici strain CIAT899 that do not induce chlorosis in plants. Microbiology 144:2607–2617 [CrossRef]
     [Google Scholar]
  44. Patriarca E. J., Chiurazzi M., Manco G., Riccio A., Lamberti A., De Paolis A., Rossi M., Defez R., Iaccarino M. 1992; Activation of the Rhizobium leguminosarum glnII gene by NtrC is dependent on upstream DNA sequences. Mol Gen Genet 234:337–345 [CrossRef]
     [Google Scholar]
  45. Patriarca E. J., Riccio A., Tate R., Colonna-Romano S., Iaccarino M., Defez R. 1993; The ntrBC genes of Rhizobium leguminosarum are part of a complex operon subject to negative regulation. Mol Microbiol 9:569–577 [CrossRef]
     [Google Scholar]
  46. Patriarca E. J., Tate R., Fedorova E., Riccio A., Defez R., Iaccarino M. 1996; Down-regulation of the Rhizobium ntr system in the determinate nodule of Phaseolus vulgaris identifies a specific developmental zone. Mol Plant–Microbe Interact 9:243–251 [CrossRef]
     [Google Scholar]
  47. Patschkowski T., Schlüter A., Priefer U. B. 1996; Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue. Mol Microbiol 21:267–280 [CrossRef]
     [Google Scholar]
  48. Priefer U. B. 1989; Genes involved in lipopolysaccharide production and symbiosis are clustered on the chromosome of Rhizobium leguminosarum biovar viciae VF39. J Bacteriol 171:6161–6168
     [Google Scholar]
  49. van Rhijn P., Vanderleyden J. 1995; The Rhizobium-plant symbiosis. Microbiol Rev 59:124–142
     [Google Scholar]
  50. Schäfer A., Tauch A., Jäger W., Kalinowski J., Thierbach G., Pühler A. 1994; Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73 [CrossRef]
     [Google Scholar]
  51. Schlüter A., Patschkowski T., Quandt J., Selinger L. B., Weidner S., Krämer M., Zhou L., Hynes M. F., Priefer U. B. 1997; Functional and regulatory analysis of the two copies of the fixNOQP operon of Rhizobium leguminosarum strain VF39. Mol Plant–Microbe Interact 10:605–616 [CrossRef]
     [Google Scholar]
  52. Shatters R. G., Somerville J. E., Kahn M. L. 1989; Regulation of glutamine synthetase II activity in Rhizobium meliloti 104A14. J Bacteriol 171:5087–5094
     [Google Scholar]
  53. Shatters R. G., Liu Y., Kahn M. L. 1993; Isolation and characterization of a novel glutamine synthetase from Rhizobium meliloti. J Biol Chem 268:469–475
     [Google Scholar]
  54. Simon R. 1984; High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon. Mol Gen Genet 196:413–420 [CrossRef]
     [Google Scholar]
  55. 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]
  56. Simon R., O’Connell M., Labes M., Pühler A. 1986; Plasmid vectors for the genetic analysis and manipulation of rhizobia and other gram-negative bacteria. Methods Enzymol 118:640–659
     [Google Scholar]
  57. Staden R. 1986; The current status and portability of our sequence handling software. Nucleic Acids Res 14:217–231 [CrossRef]
     [Google Scholar]
  58. Udvardi M. K., Day D. A. 1997; Metabolite transport across symbiotic membranes of legume nodules. Annu Rev Plant Physiol Plant Mol Biol 48:493–523 [CrossRef]
     [Google Scholar]
  59. 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]
  60. 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 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-11-2987
Loading
The Rhizobium leguminosarum bv. viciae glnD gene, encoding a uridylyltransferase/uridylyl-removing enzyme, is expressed in the root nodule but is not essential for nitrogen fixation
Microbiology 146, 2987 (2000); https://doi.org/10.1099/00221287-146-11-2987
/content/journal/micro/10.1099/00221287-146-11-2987
/content/journal/micro/10.1099/00221287-146-11-2987
Loading

Data & Media loading...

Most cited 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