Summary
The complete nucleotide sequence of the regulatory operon nifLA of a nitrogen fixer Klebsiella oxytoca NG13 was determined, and the transcriptional start point was assigned by S1 mapping. The nifL protein (a repressor) was coded by an open reading frame of 1,485 bases, corresponding to a protein of 495 amino acids with a calculated molecular weight of 55,242. The open reading frame (1,572 bases) of the nifA protein (an activator), corresponding to a molecular weight of 58,649, was confirmed by in vitro transcription-translation experiments, using the wild type and artificially deleted nifA genes. The initiation codon (ATG) of nifA overlapped the termination condon(TGA) of nifL, sharing the two bases T and G. A conserved DNA contact point [Gln-(X)3-Ala-(X)3-Gly-(X)5-Val] common in many DNA binding proteins was found in the C-terminal region of the nifA sequence. The promoter sequences of nifLA, nifB and nifF in K. oxytoca coincided exactly with those of K. pneumoniae in the consensus regions at-12 and-26, although the overall homology in the promoter regions was 96%. Changes of four amino acids were found between the nifA coding sequences of K. oxytoca and K. pneumoniae.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alavares-Morales A, Dixon R, Merrick M (1984) Positive and negative control of the glnA ntrBC regulon in Klebsiella pneumoniae. EMBO J 3:501–507
Ausubel FM, Cannon FC (1981) Molecular genetic analysis of Klebsiella pneumoniae nitrogen fixation (nif) gene. Cold Spring Harbor Symp Quant Biol 45:487–491
Berk AJ, Sharp AP (1977) Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12:721–732
Berk AJ, Sharp AP (1978a) Structure of the adenovirus early mRNA. Cell 14:695–711
Berk AJ, Sharp AP (1978b) Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci USA 75:1274–1278
Beynon J, Cannon M, Buchnan-Wollaston V, Cannon FC (1983) The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell 34:665–670
Bolivar F, Rodriquez RL, Greene PJ, Bethach MC, Heyneker HL, Boyer HW (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113
Buchanan-Wollaston V, Cannon MC, Beynon JL, Cannon FC (1981a) Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae. Nature 294:776–778
Buchanan-Wollaston V, Cannon MC, Cannon FC (1981b) The use of cloned nif (nitrogen fixation) DNA to investigate transcriptional regulation of nif expression in Klebsiella pneumoniae. Mol Gen Genet 184:102–106
Buck M, Miller S, Drummond M, Dixon R (1986) Upstream activator sequences are present in the promoters of nitrogen fixation genes. Nature 320:374–378
Buikema WJ, Szeto WW, Lemley PV, Orme-Johnson WH, Ausubel FM (1985) Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with general nitrogen regulatory gene ntrC of K. pneumoniae. Nucleic Acids Res 13:4539–4555
Burton Z, Burgess RR, Lin J, Moore D, Holder S, Gross CA (1981) The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subumit from E. coli K12. Nucleic Acids Res 9:2889–2903
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P12A cryptic miniplasmid. J Bacteriol 134:1141–1156
Dixon RA (1984) The genetic complexity of nitrogen fixation. J Gen Microbiol 130:2745–2755
Dixon RA, Postgate JR (1971) Transfer of nitrogen fixation genes by conjugation in Klebsiella pneumoniae. Nature 234:47–48
Dixon RA, Eady R, Epsin G, Hill S, Iaccarino M, Kahn D, Merrick M (1980) Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions. Nature 286:128–132
Drummond M, Clements J, Merrick M, Dixon R (1983) Positive control and autogenous regulation of nifLA promoter in Klebsiella pneumoniae. Nature 301:302–306
Drummond M, Whitty P, Wootton J (1986) Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: Homologies to other regulatory proteins. EMBO J 5:441–447
Feramisco FR, Helfman DM, Smart JE, Burridge K, Thomas GP (1982) Coexistence of vinculin-like protein of higher molecular weight in smooth muscle. J Biol Chem 257:11024–11031
Hill S, Kennedy C, Kavanagh E (1981) Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in Klebsiella pneumoniae. Nature 290:424–426
Hirschman J, Wong P-K, Sei K, Keener J, Kustu S (1985) Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: Evidence that the ntrA product is a σ factor. Proc Natl Acad Sci USA 82:7525–7529
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
Kuchino Y, Takanami M (1984) Methods of M13 shotgun DNA sequencing. Protein, Nucleic Acid and Enzyme 29:294–306
Matthews BW, Ohlendorf DH, Anderson WF, Fisher RG, Takeda Y (1982) cro repressor protein and its interaction with DNA. Cold Spring Harbor Symp Quant Biol 47:427–433
Maxam A, Gilbert W (1980) Sequencing end-labeled DNA with base specific chemical cleavages. Methods Enzymol 65:499–560
Merrick MJ (1983) Nitrogen control of the nif region in Klebsiella pneumoniae: Involvement of the ntrA gene and analogies between ntrC and nifA. EMBO J 2:39–44
Merrick M, Hill S, Hennecke H, Hahn M, Dixon R, Kennedy C (1982) Repressor properties of nifL gene product in Klebsiella pneumoniae. Mol Gen Genet 185:75–81
Ow DW, Ausubel FM (1983) Regulation of nitrogen metabolism genes by nifA gene product in Klesiella pneumoniae. Nature 301:307–313
Ow DW, Sundaresan V, Rothstein DM, Brown SE, Ausubel FM (1983) Promoters regulated by the glnG(ntrC) and nifA gene products share a heptameric consensus sequence in the-15 region. Proc Natl Acad Sci USA 80:2524–2528
Roberts GP, Brill WJ (1980) Gene-product relationships of the nif regulon of Klebsiella pneumoniae. J Bacteriol 144:210–216
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Sauer RT, Yocum RR, Doolittle RF, Lewis M, Pabo CO (1982) Homology among DNA-binding proteins suggests use of a conserved super-secondary structure. Nature 298:447–451
Soberon X, Covarrubias L, Bolivar F (1980) Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene 9:287–295
Uozumi T, Wang P-L, Tonouchi N, Nam J-H, Kim Y-M, Beppu T (1986) Cloning and expression of nifA gene of Klebsiella oxytoca in K. pneumoniae and Azospirillum lipoferm. Agric Biol Chem 50:1539–1544
Wang P-L, Koh S-K, Chung K-S, Uozumi T, Beppu T (1985) Cloning and expression in E. coli of the whole nif genes of Klebsiella oxytoca, a nitrogen fixer in the rhizosphere of rice. Agric Biol Chem 49:1469–1477
Author information
Authors and Affiliations
Additional information
Communicated by M. Takanami
Rights and permissions
About this article
Cite this article
Kim, YM., Ahn, KJ., Beppu, T. et al. Nucleotide sequence of the nifLA operon of Klebsiella oxytoca NG13 and characterization of the gene products. Molec. Gen. Genet. 205, 253–259 (1986). https://doi.org/10.1007/BF00430436
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00430436