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
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
Atkinson M, Ninfa A (1992) Characterization ofE. coli glnL mutations affecting nitrogen regulation. J Bacteriol 174:4538–4548
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
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
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
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
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
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
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
Bueno R, Pahel G, Magasanik B (1985) Role ofglnB andglnD gene products in regulation of theglnALG operon ofE. coli. J Bacteriol 164:816–822
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
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
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
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
Drummond M, Clements J, Merrick M, Dixon R (1983) Positive control and autogenous regulation of thenifLA promoter inKlebsiella pneumoniae. Nature 301:302–307
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Prentki P, Krisch H (1984)In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313
Reitzer LJ, Magasanik B (1985) Expression ofglnA in Escherichia coli is regulated at tandem promoters. Proc Natl Acad Sci USA 82:1979–1983
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
Son H, Rhee S (1987) Cascade control ofE. coli glutamine synthetase. J Biol Chem 262:8690–8695
Stock J, Ninfa A, Stock A (1989) Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53:450–490
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
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
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
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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