Summary
A 15 kilobase HindIII fragment of Klebsiella pneumoniae DNA containing the glnA gene was cloned into the plasmid vector pACYC184. The resulting plasmid, pFB51, complements glnA - mutations in Escherichia coli and K. pneumoniae. pFB51 also complements the GlnR phenotype of a Klebsiella pneumoniae gln regulatory mutant (KP5060) defined by the restoration of Hut+ and Nif+ (histidine utilization and nitrogen fixation) phenotypes to this strain. Three recombinant plasmids containing subsegments of the 15 kb HindIII fragment were derived from pFB51. Plasmid pFB514 which contains a spontaneous 4 kb delection of K. pneumoniae DNA from pFB51 is more stable than pFB51 and is still able to complement glnA - mutations and the GlnR- phenotype of KP5060. Plasmids pFB53 and pFB54, which contain a 6.5 kb SalI DNA fragment from pFB51 recloned into pACYC184 in opposite orientations, complement glnA - mutations but not the GlnR- phenotype of KP5060. Plasmids pFB514 and pFB53 were mutagenized by transposon Tn5 resulting in a total of 92 Tn5 insertions in the cloned fragments. Utilizing these insertion mutations, a correlated physical and genetic map was constructed by determining the physical location of each Tn5 insertion and by analyzing the ability of each Tn5 mutated plasmid to complement a glnA - strain of E. coli and a glnA - GlnR- strain of K. pneumoniae. Two classes of Tn5 insertions with an altered Gln phenotpye were obtained. One cluster of insertions spanning a 1.3 kb region abolished complementation of the glnA - mutations. A second 2 kb cluster of Tn5 insertions, immediately adjacent to the first cluster, abolished the ability of pFB514 plasmid to complement the GlnR- phenotype, while glnA - complementation was unaffected. We propose that the second cluster of Tn5 insertions define a DNA region coding for a positive regulatory factor for nitrogen fixation (nif) and histidine utilization (hut) genes (glnR).
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ausubel FM, Bird SC, Durbin KJ, Janssen KA, Margolskee RF, Peskin AP (1979) Glutamine synthetase mutations which affect expression of nitrogen fixation genes in Klebsiella pneumoniae. J Bacteriol 140:597–606
Berg DE (1977) Insertion and excision of the transposable kanamycinresistance determinant Tn5. In: Bukhari AI, Shapiro JA, Adhya SL (eds) DNA insertion elements, plasmids and episomes. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, p 205
Berg DE, Weiss A, Crossland L (1980) Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol 142:439–446
Berg D, Davies J, Allet B, Rochaix J-D (1975) Transposition of R factor genes to bacteriophage lambda. Proc Natl Acad Sci USA 72:3628–3632
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heynecker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A mulipurpose cloning system. Gene 2:95–113
Brenchley JE, Baker CA, Patil LG (1975) Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium. J Bacteriol 124:182–189
Brenchley JE, Prival MJ, Magasanik B (1973) Regulation of the synthesis of enzymes responsible for glutamate formation in Klebsiella aerogenes. J Biol Chem 248:6122–6128
Cannon FC, Riedel GE, Ausubel FM (1979) Overlapping sequences of K. pneumoniae nif DNA cloned and characterized. Mol Gen Genet 174:59–66
Chaconas G, de Bruijn FJ, Casabadan MJ, Lupski JR, Kwoh TJ, Harshey RM, Du Bow MS, Bukhari AI (1981) In vitro and in vivo manipulations of bacteriophage Mu DNA: Cloning of Mu ends and construction of mini-Mu's carrying selectable markers. Gene 13:37–46
Chang ACY, Cohen SN (1978) Construction and characterization 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
Clewell DB, Helinski DR (1969) Supercoiled circular DNA-protein complex in Escherichia coli: Purification and induced conversion to an open circular DNA form. Proc Natl Acad Sci USA 62:1159–1166
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
Dugaiczyk A, Boyer HW, Goodman HM (1976) Ligation of EcoRI fragments into linear and circular structures. J Mol Biol 96:171–184
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, p 9
Goldberg RB, Bender RA, Streicher SL (1974) Direct selection for P1-sensitive mutants of enteric bacteria. J Bacteriol 118:810–814
Hayward GS, Smith MH (1972) The chromosome of bacteriophage T5: 1. Analysis of the single-stranded DNA fragments by agarose gel electrophoresis. J Mol Bil 63:383–395
Jorgensen RA, Berg DE, Allet B, Rezinikoff WS (1979) Restriction enzyme cleavage map of Tn10, a transposon which encodes tetracycline resistance. J Bacteriol 137:681–685
Kelley WS, Chalmers K, Murray ME (1977) Isolation and characterization of λpolA transducing phage. Proc Natl Acad Sci USA 74:5632–5637
Klein RD, Selsing E, Wells RD (1980) A rapid micro-scale technique for isolation of recombinant plasmid DNA suitable for restriction enzyme analysis. Plasmid 3:88–91
Koduri RK, Bedwell DM, Brenchley JE (1980) Characterization of a HindIII-generated DNA fragment carrying the glutamine synthetase gene of Salmonella typhimurium. Gene 11:227–237
Kustu S, 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
Kustu SG, McFarland NG, Hui SP, Esmon B, Ferro-Luzzi Ames G (1979) Nitrogen control in Salmonella typhimurium: co-regulation of synthesis of glutamine synthetase and amino acid transport systems. J Bacteriol 138:218–234
Leonardo JM, Goldberg RB (1980) Regulation of nitrogen metabolism in glutamine auxotrophs of Klebsiella pneumoniae. J Bacteriol 142:99–110
MacNeil T, MacNeil D, Roberts GP, Supiano MA, Brill WJ (1978) Finestructure mapping and complementation analysis of nif (nitrogen fixation) genes in Klebsiella pneumoniae. J Bacteriol 136:253–266
Maganasik B, Prival MJ, Brenchley JE, Tyler BM, DeLeo AB, Streicher SL, Bender RA and Paris CG (1974) Glutamine synthetase as a regulator of enzyme synthesis. Curr Top Cell Regul 8:119–138
Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218
Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Morrison DA (1979) Transformation and preservation of competent bacterial cells by freezing. In: Wu R (ed) Methods in enzymology, vol 86. Academic Press, New York, p 326
Murray NE, Kelley WS (1979) Characterization of λpolA transducing phages: Effective expression of the E. coli polA gene. Mol Gen Genet 175:77–87
Nakamura Y, Yura T (1975) Evidence for a positive regulation of RNA polymerase synthesis in Escherichia coli. J Mol Biol 97:621–642
Ow DW, Ausubel FM (1980) Recombinant P4 bacteriophages propagate as viable lytic phages or as automonous plasmids in Klebsiella pneumoniae. Mol Gen Genet 180:165–175
Pahel G, Bloom FR, Tyler B (1979) Deletion mapping of the polA-metB region of the E. coli chromosome. J Bacteriol 138:653–656
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
Pahel G, Zelenetz AD, Tyler BM (1978) gltB gene and regulation of nitrigen metabolism by glutamine synthetase in Escherichia coli. J Bacteriol 133:139–148
Prival MJ, Brenchley JE, Magasanik B (1973) Glutamine synthetase and the regulation of histidase formation in Klebsiella aerogenes. J Biol Chem 248:4334–4344
Prival MJ, Magasanik B (1971) Resistance to catabolite repression of histidase and proline oxidase during nitrogen limited growth of Klebsiella aerogens. J Biol Chem 246:6288–6296
Riedel GE, Ausubel FM, Cannon FC (1979) Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae. Proc Natl Acad Sci USA 76:2866–2870
Ruvkun GB, Ausubel FM (1981) A general method for site-directed mutagenesis in prokaryotes. Nature 289:85–88
Sancar A, Rupp WD (1979) Physical map of the recA gene. Proc Natl Acad Sci USA 76:3144–3148
Shaw KJ, Berg CM (1979) Escherichia coli K-12 auxotrophs induced by the insertion of the transposable element Tn5. Genetics 92:741–747
Streicher SL, Shanmugam KT, Ausubel FM, Morandi C, Goldberg RB (1974) Regulation of nitrogen fixation in Klebsiella pneumoniae: Evidence for glutamine synthetase as a regulator of nitrogenase synthesis. J Bacteriol 120:815–821
Tait RC, Rodriguez RL, West RW (1980) The rapid purification of T4 DNA ligase from a λT4 lig lysogen. J Biol Chem 255:813–815
Tyler B (1978) Regulation of the assimilation of nitrogen compounds. Annu Rev Biochem 47:1127–1162
Widera G, Gautier F, Lindenmaier W, Collins J (1978) The expression of tetracycline resistance after insertion of foreign DNA fragments between the EcoRI and HindIII sites of the plasmid cloning vector pBR322. Mol Gen Genet 163:301–305
Wilson GG, Murray NE (1979) Molecular cloning of the DNA ligase gene from phage T4. 1. Characterization of the recombinants. J Mol Biol 132:471–491
Author information
Authors and Affiliations
Additional information
Communicated by D. Sherratt
Rights and permissions
About this article
Cite this article
de Bruijn, F.J., Ausubel, F.M. 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. Molec. Gen. Genet. 183, 289–297 (1981). https://doi.org/10.1007/BF00270631
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00270631