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The role of uridylyltransferase in the control ofKlebsiella pneumoniae nif gene regulation

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Abstract

TheglnD gene in enteric bacteria encodes a uridylyltransferase/uridylyl-removing enzyme which acts as the primary nitrogen sensor in the nitrogen regulation (Ntr) system. We have investigated the role of this enzyme in transcriptional regulation of nitrogen fixation genes inKlebsiella pneumoniae by cloningglnD from this organism and constructing a null mutant by insertional inactivation of the chromosomal gene using the Ω interposon.K. pneumoniae glnD encodes a 102.3 kDa polypeptide which is highly homologous to the predicted products of bothEscherichia coli glnD andAzotobacter vinelandii nfrX. TheglnD-Ω mutant was unable to uridylylate PII and was altered in adenylylation/deadenylylation of glutamine synthetase. Uridylyltransferase was required for derepression ofntr-regulated promoters such asglnAp2 and pnifL but was not involved in thenifspecific response to changes in nitrogen status mediated by thenifL product. We conclude that a separate, as yet uncharacterised, nitrogen control system may be responsible for nitrogen sensing by NifL.

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References

  • Adler S, Purich D, Stadtman E (1975) Cascade control ofE. coli glutamine synthetase. J Biol Chem 250:6264–6272

    Google Scholar 

  • Arnott M, Sidoti C, Hill S, Merrick M (1989) Deletion analysis of the nitrogen fixation regulatory gene,nifL ofKlebsiella pneumoniae. Arch Microbiol 151:180–182

    Google Scholar 

  • Atkinson M, Ninfa A (1992) Characterization ofE. coli glnL mutations affecting nitrogen regulation. J Bacteriol 174:4538–4548

    Google Scholar 

  • Backman K, Chen Y, Magasanik B (1981) Physical and genetic characterisation of theglnA-glnG region of theEscherichia coli chromosome. Proc Natl Acad Sci USA 78:3743–3747

    Google Scholar 

  • Bali A, Blanco G, Hill S, Kennedy C (1992) Excretion of ammonium by anifL mutant ofAzotobacter vinelandii fixing nitrogen. Appl Environ Microbiol 58:1711–1718

    Google Scholar 

  • Bancroft S, Rhee S, Neumann C, Kustu S (1978) Mutations that alter the covalent modification of glutamine synthetase inSalmonella typhimurium. J Bacteriol 134:1046–1055

    Google Scholar 

  • Bloom F, Levin S, Foor F, Tyler B (1978) Regulation of glutamine synthetase formation inE. coli: characterization of mutants lacking the uridylyltransferase. J Bacteriol 134:569–577

    Google Scholar 

  • Brown M, Segal A, Stadtman E (1971) Modulation of glutamine synthetase adenylylation and deadenylylation is mediated by metabolic transformation of the PII- regulatory protein. Proc Natl Acad Sci USA 68:2949–2953

    Google Scholar 

  • Buchanan-Wollaston V, Cannon M, Beynon J, Cannon F (1981) Role of thenifA gene product in the regulation of nif expression inK. pneumoniae. Nature 294:776–778

    Google Scholar 

  • Buck M, Khan H, Dixon R (1985) Site directed mutagenesis of theKlebsiella pnuemoniae nifL andnifH promoters andin vivo analysis of promoter activity. Nucleic Acids Res 13:7621–7638

    Google Scholar 

  • Bueno R, Pahel G, Magasanik B (1985) Role ofglnB andglnD gene products in regulation of theglnALG operon ofE. coli. J Bacteriol 164:816–822

    Google Scholar 

  • Cannon W, Charlton W, Buck M (1991) Organization and function of binding sites for the transcriptional activator NifA in theKlebsiella pneumoniae nifE andnifU promoters. J Mol Biol 220:915–931

    Google Scholar 

  • Colonna-Romano S, Patriarca E, Amar M, Bernard P, Manco G, Lamberti A, Iaccarino M, Defez R (1993) Uridylylation of the PII protein inRhizobium leguminosarum. FEBS Lett 330:95–98

    Google Scholar 

  • Contreras A, Drummond M, Bail A, Blanco G, Garcia E, Bush G, Kennedy C, Merrick M (1991) The product of the nitrogen fixation regulatory genenfrX ofAzotobacter vinelandii is functionally and structurally homologous to the uridylyltransferase encoded byglnD in enteric bacteria. J Bacteriol 173:7741–7749

    Google Scholar 

  • Dixon R, Kennedy C, Kondorosi A, Krishnapillai V, Merrick M (1977) Complementation analysis ofKlebsiella pneumoniae mutants defective in nitrogen fixation. Mol Gen Genet 157:189–198

    Google Scholar 

  • Drummond M, Clements J, Merrick M, Dixon R (1983) Positive control and autogenous regulation of thenifLA promoter inKlebsiella pneumoniae. Nature 301:302–307

    Google Scholar 

  • Engleman E, Francis S (1978) Cascade control ofE. coli glutamine synthetase. II. Metabolite regulation of the enzymes in the cascade. Arch Biochem Biophys 191:602–612

    Google Scholar 

  • Foor F, Janssen K, Magasanik B (1975) Regulation of synthesis of glutamine synthetase by adenylylated glutamine synthetase. Proc Natl Acad Sci USA 72:4844–4848

    Google Scholar 

  • Foor F, Cedergren R, Streicher S, Rhee S, Magasanik B (1978) Glutamine synthetase ofKlebsiella aerogenes: properties ofglnD mutants lacking uridylyltransferase. J Bacteriol 134:562–568

    Google Scholar 

  • Forchhammer K, Tandeau de Marsac N (1994) The PII protein in the cyanobacteriumSynechococcus sp. strain PCC 7942 is modified by serine phosphorylation and signals the cellular N-status. J Bacteriol 176:84–91

    Google Scholar 

  • Francis S, Engleman E (1978) Cascade control ofE. coli glutamine synthetase. I. Studies on the uridylyltransferase and uridylylremoving enzyme(s) fromE. coli. Arch Biochem Biophys 191:590–601

    Google Scholar 

  • Garcia E, Federici M, Rhee S, Berberich M (1980) Glutamine synthetase cascade: enrichment of uridylyltransferase inE. coli carrying hybrid ColEl plasmids. Arch Biochem Biophys 203:181–189

    Google Scholar 

  • Geissler S, Drummond M (1993) A counterselectable, pACYC184basedlacZ α-complementing plasmid with novel polylinkers; use in constructing chromosomal deletions inKlebsiella pneumoniae. Gene 136:253–255

    Google Scholar 

  • Ginsburg A, Stadtman E (1973) Regulation of glutamine synthetase inE. coli. In: Prusiner S, Stadtman E (eds) The enzymes of glutamine metabolism. Academic Press, New York, pp 9–43

    Google Scholar 

  • van Heeswijk W, Kuppinger O, Merrick M, Kahn D (1992) Localization of theglnD gene on a revised map of the 200 kilobase region of theE. coli chromosome. J Bacteriol 174:1702–1703

    Google Scholar 

  • van Heeswijk W, Rabenberg M, Westerhoff H, Kahn D (1993) The genes of the glutamine synthetase adenylylation cascade are not regulated by nitrogen inEscherichia coli. Mol Microbiol 9:443–457

    Google Scholar 

  • Holtel A, Merrick M (1988) Identification of theKlebsiella pneumoniae glnB gene: nucleotide sequence of wild-type and mutant alleles. Mol Gen Genet 215:134–138

    Google Scholar 

  • Holtel A, Merrick M (1989) TheKlebsiella pneumoniae PII, protein (glnB gene product) is not absolutely required for nitrogen regulation and is not involved in NifL mediatednif gene regulation. Mol Gen Genet 217:474–480

    Google Scholar 

  • Keener J, Kustu S (1988) Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NtrB and NtrC of enteric bacteria: roles of the conserved amino-terminal domain of NtrC. Proc Natl Acad Sci USA 85:4976–4980

    Google Scholar 

  • Kleiner D, Paul W, Merrick M (1988) Construction of multicopy expression vectors for regulated overproduction of proteins inK. pneumoniae and other enteric bacteria. J Gen Microbiol 134:1779–1784

    Google Scholar 

  • Kustu S, Hirschman J, Burton D, Jelesko J, Meeks J (1984) Covalent modification of bacterial glutamine synthetase: physiological significance. Mol Gen Genet 197:309–317

    Google Scholar 

  • Mangum J, Magni G, Stadtman E (1973) Regulation of glutamine synthetase adenylylation and deadenylylation by the enzymatic uridylylation and deuridylylation of the PII regulatory protein. Arch Biochem Biophys 158:514–525

    Google Scholar 

  • Merrick M (1992) Regulation of nitrogen fixation genes in freeliving and symbiotic bacteria. In: Stacey G, Burris R, Evans H (eds) Biological nitrogen fixation. Chapman and Hall, New York, pp 835–876

    Google Scholar 

  • Minchin S, Austin S, Dixon R (1989) Transcriptional activation of theKlebsiella pnuemoniae nifLA promoter by NtrC is face-ofthe-helix dependent and the activator stabilizes the interaction of sigma 54-RNA polymerase with the promoter. EMBO J 8:3491–3499

    Google Scholar 

  • Movva N, Semon D, Meyer C, Kawashima E, Wingfield P, Miller J, Miller C (1990) Cloning and nucleotide sequence of theSalmonella typhimurium pepM gene. Mol Gen Genet 223:345–348

    Google Scholar 

  • Prentki P, Krisch H (1984)In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313

    Google Scholar 

  • Reitzer LJ, Magasanik B (1985) Expression ofglnA in Escherichia coli is regulated at tandem promoters. Proc Natl Acad Sci USA 82:1979–1983

    Google Scholar 

  • Sibold L, Henriquet M, Possot O, Aubert J-P (1991) Nucleotide sequence ofnifII regions fromMethanobacterium ivanovii andMethanosarcina barkeri 227 and characterisation ofglnB like genes. Res Microbiol 142:5–12

    Google Scholar 

  • Son H, Rhee S (1987) Cascade control ofE. coli glutamine synthetase. J Biol Chem 262:8690–8695

    Google Scholar 

  • Stock J, Ninfa A, Stock A (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53:450–490

    Google Scholar 

  • Takeshita S, Sato M, Toba M, Masahashi W, Hashimoto-Gotoh T (1987) High-copy-number and low-copy-number plasmid vectors forlacZα complementation and chloramphenicol- or kanamycin-resistance selection. Gene 61:63–74

    Google Scholar 

  • Vasudevan S, Gedye C, Dixon N, Cheah E, Carr P, Suffolk P, Jeffrey P, Ollis D (1994)E. coli PII, protein: purification, crystallization and oligomeric structure. FEBS Lett 337:255–258

    Google Scholar 

  • Wray L, Atkinson M, Fisher S (1994) The nitrogen-regulatedBacillus subtilis nrgAB operon encodes a membrane protein and a protein highly similar to theE. coli glnB-encoded PII. J Bacteriol 176:108–114

    Google Scholar 

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Communicated by A. Kondorosi

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Edwards, R., Merrick, M. The role of uridylyltransferase in the control ofKlebsiella pneumoniae nif gene regulation. Molec. Gen. Genet. 247, 189–198 (1995). https://doi.org/10.1007/BF00705649

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  • DOI: https://doi.org/10.1007/BF00705649

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