Summary
The glanA, ntrB and ntrC genes of Klebsiella pneumoniae have been cloned, on a 12 kb HindIII fragment, into the plasmid pACYC 184. In a coupled in vitro transcription/translation system the resultant plasmid, pGE100, directed synthesis of five polypeptides (molecular weights 73, 53, 51, 39, 36 kd) from the cloned fragment. A number of plasmids were derived from pGE100 and studied by complementation analysis and in vitro transcription/translation in order to locate particular genes and identify their products.
On the basis of the results presented here, together with previous genetic and physical characterisation of the glnA gene and its product in other enteric bacteria, we propose that the 53 kd polypeptide is the glnA gene product (glutamine synthetase monomer).
Two polypeptides (36 kd and 51 kd) were synthesised from a 3 kb region previously defined as glnR. In E. coli and S. typhimurium this region comprises two genes ntrB and ntrC with products of 36 kd and 54 kd respectively. This analogy supports the idea that the 36 kd and 51 kd polypeptides are the products of the K. pneumoniae ntrB and ntrC genes respectively. Comparison of these assignments with the physical map of the region indicates a gene order glnA, ntrB, ntrC.
Assessment of the Nif phenotype of a glnA-ntrC deletion strain carrying various clones from pGE100 demonstrated that glnA is not required for expression of the nif regulon and that of the three genes cloned, ntrC alone is sufficient for nif expression.
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References
Backman K, Chen Y-M, Magasanik B (1981) Physical and genetic characterisation of the glnA-glnG region of the Escherichia coli chromosome. Proc Natl Acad Sci USA 78:3473–3747
Buchanan-Wollaston V, Cannon MC, Beynon JL, Cannon FC (1981) Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae. Nature 294:776–778
Chang ACY, Cohen SN (1978) Construction and characterisation of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmids. J Bacteriol 134:1141–1156
Cohen SN, Chang ACY, Hsu L (1972) Non-chromosomal antibiotic resistance in bacteria: Genetic transformation of E. coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114
Collins J (1979) Cell-free synthesis of proteins coding for mobilisation functions of ColEl and transposition functions of Tn3. Gene 6:29–42
Covarrubias AA, Rocha M, Bolivar F, Bastarrachea F (1980) Cloning and physical mapping of the glnA gene of Escherichia coli K-12. Gene 11:239–251
de Bruijn FJ, Ausubel FM (1981) The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: Identification of glnR, a gene involved in the regulation of the nif and hut operons. Mol Gen Genet 183:289–297
Dixon R, Kennedy C, Kondorosi A, Krishnapillai V, Merrick M (1977) Complementation analysis of Klebsiella pneumoniae mutants defective in nitrogen fixation. Mol Gen Genet 157:189–198
Dixon R, Eady RR, Espin G, Hill S, Isccarino M, Kahn D, Merrick M (1980) Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions. Nature 286:128–132
Eady RR, Issack R, Kennedy C, Postgate JR, Ratcliffe HD (1978) Nitrogenase synthesis in Klebsiella pneumoniae: Comparison of ammonium and oxygen regulation. J Gen Microbiol 104:277–285
Espin G, Merrick M (1980) Glutamine synthetase mutations which affect the expression of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. In: Gibson AH, Newton WE (eds) Current perspectives in nitrogen fixation. Australian Academy of Sciences, Canberra, p 399
Espin G, Alvarez-Morales A, Merrick M (1981) Complementation analysis of glnA-linked mutations which affect nitrogen fixation in Klebsiella pneumoniae. Mol Gen Genet 184:213–217
Ginsburg A, Stadtman ER (1973) Regulation of glutamine synthetase in Escherichia coli. In: Prusiner S, Stadtman ER (eds) The enzymes of glutamine metabolism. Academic Press, New York pp 9–43
Humphreys GO, Willshaw GA, Anderson ES (1975) A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta 383:457–463
Kahn M, Kolter R, Thomas C, Figurski D, Meyer R, Remant E, Helinski D (1979) Plasmid cloning vehicles derived from plasmids ColEl, F, R6K and RK2. Methods Enzymol 68:268–280
Kennedy C (1977) Linkage map of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. Mol Gen Genet 157:199–204
Koduri RK, Bedwell DM, Brenchley JE (1980) Characterisation of a HindII-generated DNA fragment carrying the glutamine synthetase gene of Salmonella typhimurium. Gene 11:227–237
Kustu SG, Burton D, Garcia E, McCarter L, McFarland N (1979) Nitrogen control in Salmonella: Regulation by the glnR and glnF gene products. Proc Natl Acad Sci USA 76:4576–4580
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Leemans J, Villarroel R, Silva B, Van Montagu M, Schell J (1980) Direet repetition of a 1.2 Md DNA sequence is involved in site-specific recombination by the P1 plasmid R68. Gene 10:319–328
Leonard JM, Goldberg RB (1980) Regulation of nitrogen metabolism in glutamine auxotrophs of Klebsiella pneumoniae. J Bacteriol 142:99–110
MacNeil D (1981) General method, using Mu-Mudl dilysogens, to determine the direction of transcription of and generate deletions in the glnA region of Escherichia coli. J Bacteriol 146:260–268
MacNeil D, Brill WJ (1980) Mutations in nif genes that cause Klebsiella pneumoniae to be derepressed for nitrogenase synthesis in the presence of ammonium. J Bacteriol 144:744–751
MacNeil D, Zhu J, Brill WJ (1981) Regulation of nitrogen fixation in Klebsiella pneumoniae: Isolation and characterisation of strains with nif-lac fusions. J Bacteriol 145:348–357
Magasanik B, Prival MJ, Brenchley JE, Tyler BM, Deleo AB, Streicher SL, Bender RA, Paris CG (1974) Glutamine synthetase as a regulator of enzyme synthesis. Curr Top Cell Regul 8:119–138
McFarland N, McCarter L, Artz S, Kustu S (1981) Nitrogen regulatory locus ‘glnR’ of enteric bacteria is composed of cistrons ntrB and ntrC: Identification of their protein products. Proc Natl Acad Sci USA 78:2135–2139
O'Farrell PH (1975) High resolution two-dimensional electrophoesis of proteins. J Biol Chem 250:4007–4021
Pahel G, Tyler B (1979) A new glnA-linked regulatory gene for glutamine synthetase in Escherichia coli. Proc Natl Acad Sci USA 76:4544–4548
Rothstein DM, Pahel G, Tyler B, Magasanik B (1980) Regulation of expression from the glnA promoter of Escherichia coli in the absence of glutamine synthetase. Proc Natl Acad Sci USA 77:4372–4376
Shanmugam KT, Morandi C (1976) Amino acids as repressors of nitrogenase biosynthesis in Klebsiella pneumoniae. Biochim Biophys Acta 408:101–111
Sibold L, Melck D, Elmerich C (1981) A nif mutant of Klebsiella pneumoniae fixing nitrogen in the presence of ammonia. FEMS Microbiol Lett 10:37–41
Steicher SL, Shanmugam KT, Ausubel F, Morandi C, Goldberg RB (1976) Regulation of nitrogen fixation in Klebsiella pneumoniae: evidence for a role of glutamine synthetase as a regulator of nitrogenase synthesis. J Bacteriol 120:815–821
Tubb RS (1974) Glutamine synthetase and ammonium regulation of nitrogenase synthesis in Klebsiella. Nature 251:481–485
Tubb RS, Postgate JR (1973) Control of nitrogenase synthesis in Klebsiella pneumoniae. J Gen Microbiol 79:103–117
Tuli R, Thomas J (1981) Recombinant DNA analysis has enabled the identification of a regulatory gene for the enzymes of nitrogen metabolism in Klebsiella pneumoniae. In: Proceedings of the Society of Biological Chemists (India). 50th Annual Meeting, University of Baroda, Baroda, India, p 181
Tyler B (1978) Regulation of the assimilation of nitrogen compounds. Annu Rev Biochem 47:1127–1162
Tyler B, Deleo AB, Magasanik B (1974) Activation of transcription of hut DNA by glutamine synthetase. Proc Natl Acad Sci USA 71:225–229
Wei GR, Kustu S (1981) Glutamine auxotrophs with mutations in a nitrogen regulatory gene, ntrC, that is near glnA. Mol Gen Genet 183:392–399
Willetts NS, Crowther C, Holloway BW (1981) The insertion sequence IS21 of R68.45 and the molecular basis for mobilisation of the bacterial chromosome. Plasmid 6:30–52
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Espin, G., Alvarez-Morales, A., Cannon, F. et al. Cloning of the glnA, ntrB and ntrC genes of Klebsiella pneumoniae and studies of their role in regulation of the nitrogen fixation (nif) gene cluster. Mol Gen Genet 186, 518–524 (1982). https://doi.org/10.1007/BF00337959
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DOI: https://doi.org/10.1007/BF00337959