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. 1996 Jul;178(14):4143–4149. doi: 10.1128/jb.178.14.4143-4149.1996

Coexistence of two structurally similar but functionally different PII proteins in Azospirillum brasilense.

M de Zamaroczy 1, A Paquelin 1, G Peltre 1, K Forchhammer 1, C Elmerich 1
PMCID: PMC178171  PMID: 8763942

Abstract

The coexistence of two different PII, proteins in Azospirillum brasilense was established by comparing proteins synthesized by the wild-type strain and two null mutants of the characterized glnB gene (encoding PII) adjacent to glnA. Strains were grown under conditions of nitrogen limitation or nitrogen excess. The proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or isoelectric focusing gel electrophoresis and revealed either by [32P]phosphate or [3H]uracil labeling or by cross-reaction with an anti-A. brasilense PII-antiserum. After SDS-PAGE, a single band of 12.5 kDa revealed by the antiserum in all conditions tested was resolved by isoelectric focusing electrophoresis into two bands in the wild-type strain, one of which was absent in the glnB null mutant strains. The second PII protein, named Pz, was uridylylated under conditions of nitrogen limitation. The amino acid sequence deduced from the nucleotide sequence of the corresponding structural gene, called glnZ, is very similar to that of PII. Null mutants in glnB were impaired in regulation of nitrogen fixation and in their swarming properties but not in glutamine synthetase adenylylation. No glnZ mutant is yet available, but it is clear that PII and Pz are not functionally equivalent, since glnB null mutant strains exhibit phenotypic characters. The two proteins are probably involved in different regulatory steps of the nitrogen metabolism in A. brasilense.

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Selected References

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  1. Adler S. P., Purich D., Stadtman E. R. Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme. J Biol Chem. 1975 Aug 25;250(16):6264–6272. [PubMed] [Google Scholar]
  2. Allikmets R., Gerrard B., Court D., Dean M. Cloning and organization of the abc and mdl genes of Escherichia coli: relationship to eukaryotic multidrug resistance. Gene. 1993 Dec 22;136(1-2):231–236. doi: 10.1016/0378-1119(93)90470-n. [DOI] [PubMed] [Google Scholar]
  3. Amar M., Patriarca E. J., Manco G., Bernard P., Riccio A., Lamberti A., Defez R., Iaccarino M. Regulation of nitrogen metabolism is altered in a glnB mutant strain of Rhizobium leguminosarum. Mol Microbiol. 1994 Feb;11(4):685–693. doi: 10.1111/j.1365-2958.1994.tb00346.x. [DOI] [PubMed] [Google Scholar]
  4. Bender R. A., Janssen K. A., Resnick A. D., Blumenberg M., Foor F., Magasanik B. Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. J Bacteriol. 1977 Feb;129(2):1001–1009. doi: 10.1128/jb.129.2.1001-1009.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bozouklian H., Fogher C., Elmerich C. Cloning and characterization of the glnA gene of Azospirillum brasilense Sp7. Ann Inst Pasteur Microbiol. 1986 Jul-Aug;137B(1):3–18. doi: 10.1016/s0769-2609(86)80089-8. [DOI] [PubMed] [Google Scholar]
  6. Bueno R., Pahel G., Magasanik B. Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli. J Bacteriol. 1985 Nov;164(2):816–822. doi: 10.1128/jb.164.2.816-822.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cheah E., Carr P. D., Suffolk P. M., Vasudevan S. G., Dixon N. E., Ollis D. L. Structure of the Escherichia coli signal transducing protein PII. Structure. 1994 Oct 15;2(10):981–990. doi: 10.1016/s0969-2126(94)00100-6. [DOI] [PubMed] [Google Scholar]
  8. Colonna-Romano S., Patriarca E. J., Amar M., Bernard P., Manco G., Lamberti A., Iaccarino M., Defez R. Uridylylation of the PII protein in Rhizobium leguminosarum. FEBS Lett. 1993 Sep 6;330(1):95–98. doi: 10.1016/0014-5793(93)80927-m. [DOI] [PubMed] [Google Scholar]
  9. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  10. Forchhammer K., Tandeau de Marsac N. Phosphorylation of the PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942: analysis of in vitro kinase activity. J Bacteriol. 1995 Oct;177(20):5812–5817. doi: 10.1128/jb.177.20.5812-5817.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Forchhammer K., Tandeau de Marsac N. The PII protein in the cyanobacterium Synechococcus sp. strain PCC 7942 is modified by serine phosphorylation and signals the cellular N-status. J Bacteriol. 1994 Jan;176(1):84–91. doi: 10.1128/jb.176.1.84-91.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Foster-Hartnett D., Kranz R. G. The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters. J Bacteriol. 1994 Aug;176(16):5171–5176. doi: 10.1128/jb.176.16.5171-5176.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fraser C. M., Gocayne J. D., White O., Adams M. D., Clayton R. A., Fleischmann R. D., Bult C. J., Kerlavage A. R., Sutton G., Kelley J. M. The minimal gene complement of Mycoplasma genitalium. Science. 1995 Oct 20;270(5235):397–403. doi: 10.1126/science.270.5235.397. [DOI] [PubMed] [Google Scholar]
  14. Holtel A., Merrick M. Identification of the Klebsiella pneumoniae glnB gene: nucleotide sequence of wild-type and mutant alleles. Mol Gen Genet. 1988 Dec;215(1):134–138. doi: 10.1007/BF00331314. [DOI] [PubMed] [Google Scholar]
  15. Kamberov E. S., Atkinson M. R., Ninfa A. J. The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP. J Biol Chem. 1995 Jul 28;270(30):17797–17807. doi: 10.1074/jbc.270.30.17797. [DOI] [PubMed] [Google Scholar]
  16. Liang Y. Y., Arsène F., Elmerich C. Characterization of the ntrBC genes of Azospirillum brasilense Sp7: their involvement in the regulation of nitrogenase synthesis and activity. Mol Gen Genet. 1993 Aug;240(2):188–196. doi: 10.1007/BF00277056. [DOI] [PubMed] [Google Scholar]
  17. Liang Y. Y., de Zamaroczy M., Arsène F., Paquelin A., Elmerich C. Regulation of nitrogen fixation in Azospirillum brasilense Sp7: involvement of nifA, glnA and glnB gene products. FEMS Microbiol Lett. 1992 Dec 15;100(1-3):113–119. doi: 10.1111/j.1574-6968.1992.tb14028.x. [DOI] [PubMed] [Google Scholar]
  18. Liu J., Magasanik B. The glnB region of the Escherichia coli chromosome. J Bacteriol. 1993 Nov;175(22):7441–7449. doi: 10.1128/jb.175.22.7441-7449.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Magasanik B. Reversible phosphorylation of an enhancer binding protein regulates the transcription of bacterial nitrogen utilization genes. Trends Biochem Sci. 1988 Dec;13(12):475–479. doi: 10.1016/0968-0004(88)90234-4. [DOI] [PubMed] [Google Scholar]
  20. Moens S., Michiels K., Keijers V., Van Leuven F., Vanderleyden J. Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7. J Bacteriol. 1995 Oct;177(19):5419–5426. doi: 10.1128/jb.177.19.5419-5426.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sibold L., Henriquet M., Possot O., Aubert J. P. Nucleotide sequence of nifH regions from Methanobacterium ivanovii and Methanosarcina barkeri 227 and characterization of glnB-like genes. Res Microbiol. 1991 Jan;142(1):5–12. doi: 10.1016/0923-2508(91)90091-n. [DOI] [PubMed] [Google Scholar]
  22. Son H. S., Rhee S. G. Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII protein and nucleotide sequence of its structural gene. J Biol Chem. 1987 Jun 25;262(18):8690–8695. [PubMed] [Google Scholar]
  23. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tarrand J. J., Krieg N. R., Döbereiner J. A taxonomic study of the Spirillum lipoferum group, with descriptions of a new genus, Azospirillum gen. nov. and two species, Azospirillum lipoferum (Beijerinck) comb. nov. and Azospirillum brasilense sp. nov. Can J Microbiol. 1978 Aug;24(8):967–980. doi: 10.1139/m78-160. [DOI] [PubMed] [Google Scholar]
  25. Vasudevan S. G., Gedye C., Dixon N. E., Cheah E., Carr P. D., Suffolk P. M., Jeffrey P. D., Ollis D. L. Escherichia coli PII protein: purification, crystallization and oligomeric structure. FEBS Lett. 1994 Jan 17;337(3):255–258. doi: 10.1016/0014-5793(94)80203-3. [DOI] [PubMed] [Google Scholar]
  26. Wray L. V., Jr, Atkinson M. R., Fisher S. H. The nitrogen-regulated Bacillus subtilis nrgAB operon encodes a membrane protein and a protein highly similar to the Escherichia coli glnB-encoded PII protein. J Bacteriol. 1994 Jan;176(1):108–114. doi: 10.1128/jb.176.1.108-114.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. de Zamaroczy M., Delorme F., Elmerich C. Characterization of three different nitrogen-regulated promoter regions for the expression of glnB and glnA in Azospirillum brasilense. Mol Gen Genet. 1990 Dec;224(3):421–430. doi: 10.1007/BF00262437. [DOI] [PubMed] [Google Scholar]
  28. de Zamaroczy M., Paquelin A., Elmerich C. Functional organization of the glnB-glnA cluster of Azospirillum brasilense. J Bacteriol. 1993 May;175(9):2507–2515. doi: 10.1128/jb.175.9.2507-2515.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. van Heeswijk W. C., Rabenberg M., Westerhoff H. V., Kahn D. The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen in Escherichia coli. Mol Microbiol. 1993 Aug;9(3):443–457. doi: 10.1111/j.1365-2958.1993.tb01706.x. [DOI] [PubMed] [Google Scholar]
  30. van Heeswijk W. C., Stegeman B., Hoving S., Molenaar D., Kahn D., Westerhoff H. V. An additional PII in Escherichia coli: a new regulatory protein in the glutamine synthetase cascade. FEMS Microbiol Lett. 1995 Oct 1;132(1-2):153–157. doi: 10.1111/j.1574-6968.1995.tb07825.x. [DOI] [PubMed] [Google Scholar]

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