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 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.