Nucleotide sequence of the nifLA operon of Klebsiella oxytoca NG13 and characterization of the gene products

Abstract

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 codon(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.

Sequence and molecular analysis of the nifL gene of Azotobacter vinelandii

“…The UAS contains the conserved sequence TGT-N,o-ACA, While all Gram-negative diazotrophs examined contain nifA. a nifL-\ike gene has been identified only in species of Kiebsielia and Azotobacter (Kennedy, 1977;Kim et al. 1986;Drummond and Wootton.…”

A Haemoprotein Is Not Involved in the Control by Oxygen of Enteric Nitrogenase Synthesis

“…The sequence of nzjL (Kim et al, 1986;Drummond & Wootton, 1987) revealed a -Cys-X-XCys-region that is homologous with redox-sensitive sites in disulphide reductases such as thioredoxin, and with metal-binding sites in redox centres of rubredoxins and cupredoxins. Drummond & Wootton (1987) also found a similarity between the amino acid sequences flanking the Cys-containing site and regions of cytochrome c in which the two cysteines are involved in covalent binding of haem; the amino acids responsible for the 5th and 6th ligands to the iron of the haem in cytochrome c (histidine and methionine) are, however, missing.…”

Oxygen Sensitivity of NifA Protein of Azospirillum lipoferum FS as Suggested by Gene Cloning and Expression in Escherichia coli

“…The SalIdigested genomic DNA of A. /ipoferum FS 2 ) was analyzed by Southern hybridization with a 32P-labeled 850-bp KpnI-PvuII fragment of pNOA 1 4 ) containing the central region of the K. oxytoca NG 13 nifA gene. Hybridizations were done by the standard methods 8 ) with a modification; washing temperature was altered to 50°e.…”

“…pTVAI and pTVKI were constructed as follows: Ncol sites were introduced at the initiation co dons of the cloned A. /ipoferum FS n[fA gene and the K. oxytoca nifA gene 4 ) by site-directed mutagenesis with a change of the second encoded amino acid; from Pro to Ala in the A. /ipoferwn nifA gene and from Ile to Val in the K. oxytoca nifA gene.…”