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. 1994 Aug;176(16):5077–5085. doi: 10.1128/jb.176.16.5077-5085.1994

Identification and structure of the nasR gene encoding a nitrate- and nitrite-responsive positive regulator of nasFEDCBA (nitrate assimilation) operon expression in Klebsiella pneumoniae M5al.

B S Goldman 1, J T Lin 1, V Stewart 1
PMCID: PMC196347  PMID: 8051020

Abstract

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources through the nitrate assimilatory pathway. The structural genes for assimilatory nitrate and nitrite reductases together with genes necessary for nitrate transport form an operon, nasFEDCBA. Expression of the nasF operon is regulated both by general nitrogen control and also by nitrate or nitrite induction. We have identified a gene, nasR, that is necessary for nitrate and nitrite induction. The nasR gene, located immediately upstream of the nasFEDCBA operon, encodes a 44-kDa protein. The NasR protein shares carboxyl-terminal sequence similarity with the AmiR protein of Pseudomonas aeruginosa, the positive regulator of amiE (aliphatic amidase) gene expression. In addition, we present evidence that the nasF operon is not autogenously regulated.

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Selected References

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  1. 't Riet J van, Stouthamer A. H., Planta R. J. Regulation of nitrate assimilation and nitrate respiration in Aerobacter aerogenes. J Bacteriol. 1968 Nov;96(5):1455–1464. doi: 10.1128/jb.96.5.1455-1464.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bender R. A., Friedrich B. Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70. J Bacteriol. 1990 Dec;172(12):7256–7259. doi: 10.1128/jb.172.12.7256-7259.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bender R. A. The role of the NAC protein in the nitrogen regulation of Klebsiella aerogenes. Mol Microbiol. 1991 Nov;5(11):2575–2580. doi: 10.1111/j.1365-2958.1991.tb01965.x. [DOI] [PubMed] [Google Scholar]
  6. Berg B. L., Li J., Heider J., Stewart V. Nitrate-inducible formate dehydrogenase in Escherichia coli K-12. I. Nucleotide sequence of the fdnGHI operon and evidence that opal (UGA) encodes selenocysteine. J Biol Chem. 1991 Nov 25;266(33):22380–22385. [PubMed] [Google Scholar]
  7. Berg B. L., Stewart V. Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12. Genetics. 1990 Aug;125(4):691–702. doi: 10.1093/genetics/125.4.691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bruske A. K., Anton M., Heller K. J. Cloning and sequencing of the Klebsiella pneumoniae tonB gene and characterization of Escherichia coli-K. pneumoniae TonB hybrid proteins. Gene. 1993 Sep 6;131(1):9–16. doi: 10.1016/0378-1119(93)90663-n. [DOI] [PubMed] [Google Scholar]
  10. Cali B. M., Micca J. L., Stewart V. Genetic regulation of nitrate assimilation in Klebsiella pneumoniae M5al. J Bacteriol. 1989 May;171(5):2666–2672. doi: 10.1128/jb.171.5.2666-2672.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Castilho B. A., Olfson P., Casadaban M. J. Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons. J Bacteriol. 1984 May;158(2):488–495. doi: 10.1128/jb.158.2.488-495.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Crawford N. M., Arst H. N., Jr The molecular genetics of nitrate assimilation in fungi and plants. Annu Rev Genet. 1993;27:115–146. doi: 10.1146/annurev.ge.27.120193.000555. [DOI] [PubMed] [Google Scholar]
  14. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Drew R., Lowe N. Positive control of Pseudomonas aeruginosa amidase synthesis is mediated by a transcription anti-termination mechanism. J Gen Microbiol. 1989 Apr;135(4):817–823. doi: 10.1099/00221287-135-4-817. [DOI] [PubMed] [Google Scholar]
  16. Fellay R., Frey J., Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene. 1987;52(2-3):147–154. doi: 10.1016/0378-1119(87)90041-2. [DOI] [PubMed] [Google Scholar]
  17. Garzón A., Li J., Flores A., Casadesus J., Stewart V. Molybdenum cofactor (chlorate-resistant) mutants of Klebsiella pneumoniae M5al can use hypoxanthine as the sole nitrogen source. J Bacteriol. 1992 Oct;174(19):6298–6302. doi: 10.1128/jb.174.19.6298-6302.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hughes K. T., Roth J. R. Directed formation of deletions and duplications using Mud(Ap, lac). Genetics. 1985 Feb;109(2):263–282. doi: 10.1093/genetics/109.2.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Iuchi S., Lin E. C. Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli. J Bacteriol. 1987 Aug;169(8):3720–3725. doi: 10.1128/jb.169.8.3720-3725.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ivey D. M., Guffanti A. A., Zemsky J., Pinner E., Karpel R., Padan E., Schuldiner S., Krulwich T. A. Cloning and characterization of a putative Ca2+/H+ antiporter gene from Escherichia coli upon functional complementation of Na+/H+ antiporter-deficient strains by the overexpressed gene. J Biol Chem. 1993 May 25;268(15):11296–11303. [PubMed] [Google Scholar]
  21. Jeter R. M., Sias S. R., Ingraham J. L. Chromosomal location and function of genes affecting Pseudomonas aeruginosa nitrate assimilation. J Bacteriol. 1984 Feb;157(2):673–677. doi: 10.1128/jb.157.2.673-677.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kleckner N., Bender J., Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991;204:139–180. doi: 10.1016/0076-6879(91)04009-d. [DOI] [PubMed] [Google Scholar]
  23. Kraft R., Tardiff J., Krauter K. S., Leinwand L. A. Using mini-prep plasmid DNA for sequencing double stranded templates with Sequenase. Biotechniques. 1988 Jun;6(6):544-6, 549. [PubMed] [Google Scholar]
  24. Lin J. T., Goldman B. S., Stewart V. Structures of genes nasA and nasB, encoding assimilatory nitrate and nitrite reductases in Klebsiella pneumoniae M5al. J Bacteriol. 1993 Apr;175(8):2370–2378. doi: 10.1128/jb.175.8.2370-2378.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lin J. T., Goldman B. S., Stewart V. The nasFEDCBA operon for nitrate and nitrite assimilation in Klebsiella pneumoniae M5al. J Bacteriol. 1994 May;176(9):2551–2559. doi: 10.1128/jb.176.9.2551-2559.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lowe N., Rice P. M., Drew R. E. Nucleotide sequence of the aliphatic amidase regulator gene (amiR) of Pseudomonas aeruginosa. FEBS Lett. 1989 Mar 27;246(1-2):39–43. doi: 10.1016/0014-5793(89)80249-2. [DOI] [PubMed] [Google Scholar]
  27. Luque I., Herrero A., Flores E., Madueño F. Clustering of genes involved in nitrate assimilation in the cyanobacterium Synechococcus. Mol Gen Genet. 1992 Mar;232(1):7–11. doi: 10.1007/BF00299130. [DOI] [PubMed] [Google Scholar]
  28. MacNeil D., Zhu J., Brill W. J. Regulation of nitrogen fixation in Klebsiella pneumoniae: isolation and characterization of strains with nif-lac fusions. J Bacteriol. 1981 Jan;145(1):348–357. doi: 10.1128/jb.145.1.348-357.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Macaluso A., Best E. A., Bender R. A. Role of the nac gene product in the nitrogen regulation of some NTR-regulated operons of Klebsiella aerogenes. J Bacteriol. 1990 Dec;172(12):7249–7255. doi: 10.1128/jb.172.12.7249-7255.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Maloy S., Stewart V. Autogenous regulation of gene expression. J Bacteriol. 1993 Jan;175(2):307–316. doi: 10.1128/jb.175.2.307-316.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Marzluf G. A. Regulation of sulfur and nitrogen metabolism in filamentous fungi. Annu Rev Microbiol. 1993;47:31–55. doi: 10.1146/annurev.mi.47.100193.000335. [DOI] [PubMed] [Google Scholar]
  32. Merrick M. J. In a class of its own--the RNA polymerase sigma factor sigma 54 (sigma N). Mol Microbiol. 1993 Dec;10(5):903–909. doi: 10.1111/j.1365-2958.1993.tb00961.x. [DOI] [PubMed] [Google Scholar]
  33. Nagatani H., Shimizu M., Valentine R. C. The mechanism of ammonia assimilation in nitrogen fixing Bacteria. Arch Mikrobiol. 1971;79(2):164–175. doi: 10.1007/BF00424923. [DOI] [PubMed] [Google Scholar]
  34. North A. K., Klose K. E., Stedman K. M., Kustu S. Prokaryotic enhancer-binding proteins reflect eukaryote-like modularity: the puzzle of nitrogen regulatory protein C. J Bacteriol. 1993 Jul;175(14):4267–4273. doi: 10.1128/jb.175.14.4267-4273.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Omata T., Andriesse X., Hirano A. Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC7942. Mol Gen Genet. 1993 Jan;236(2-3):193–202. doi: 10.1007/BF00277112. [DOI] [PubMed] [Google Scholar]
  36. Penfold R. J., Pemberton J. M. An improved suicide vector for construction of chromosomal insertion mutations in bacteria. Gene. 1992 Sep 1;118(1):145–146. doi: 10.1016/0378-1119(92)90263-o. [DOI] [PubMed] [Google Scholar]
  37. Ramos F., Blanco G., Gutiérrez J. C., Luque F., Tortolero M. Identification of an operon involved in the assimilatory nitrate-reducing system of Azotobacter vinelandii. Mol Microbiol. 1993 Jun;8(6):1145–1153. doi: 10.1111/j.1365-2958.1993.tb01659.x. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  40. Stewart G. S., Lubinsky-Mink S., Jackson C. G., Cassel A., Kuhn J. pHG165: a pBR322 copy number derivative of pUC8 for cloning and expression. Plasmid. 1986 May;15(3):172–181. doi: 10.1016/0147-619x(86)90035-1. [DOI] [PubMed] [Google Scholar]
  41. Stewart V., Cali B. M. Genetic evidence that NarL function is not required for nitrate regulation of nitrate assimilation in Klebsiella pneumoniae M5al. J Bacteriol. 1990 Aug;172(8):4482–4488. doi: 10.1128/jb.172.8.4482-4488.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stewart V. Nitrate regulation of anaerobic respiratory gene expression in Escherichia coli. Mol Microbiol. 1993 Aug;9(3):425–434. doi: 10.1111/j.1365-2958.1993.tb01704.x. [DOI] [PubMed] [Google Scholar]
  43. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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