Genes required for formation of the apoMoFe protein of Klebsiella pneumoniae nitrogenase in Escherichia coli.
A binary plasmid system was used to produce nitrogenase components in Escherichia coli and subsequently to define a minimum set of nitrogen fixation (nif) genes required for the production of the iron-molybdenum cofactor (FeMoco) reactivatable apomolybdenum-iron (apoMoFe) protein of nitrogenase. The active MoFe protein is an alpha 2 beta 2 tetramer containing two FeMoco clusters and 4 Fe4S4 P centers (for review see, Orme-Johnson, W.H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 419-459). The plasmid pVL15, carrying a tac-promoted nifA activator gene, was coharbored in E. coli with the plasmid pGH1 which contained nifHDKTYENXUSVWZMF' derived from the chromosome of the nitrogen fixing bacterium Klebsiella pneumoniae. The apoMoFe protein produced in E. coli by pGH1 + VL15 was identical to the apoprotein in derepressed cells of the nifB- mutant of K. pneumoniae (UN106) in its electrophoretic properties on nondenaturing polyacrylamide gels as well as in its ability to be activated by FeMoco. The constituent peptides migrated identically to those from purified MoFe protein during electrophoresis on denaturing gels. The concentrations of apoMoFe protein produced in nif-transformed strains of E. coli were greater than 50% of the levels of MoFe protein observed in derepressed wild-type K. pneumoniae. Systematic deletion of individual nif genes carried by pGH1 has established the requirements for the maximal production of the FeMoco-reactivatable apoMoFe protein to be the following gene products, NifHDKTYUSWZM+A. It appears that several of the genes (nifT, Y, U, W, and Z) are only required for maximal production of the apoMoFe protein, while others (nifH, D, K, and S) are absolutely required for synthesis of this protein in E. coli. One curious result is that the nifH gene product, the peptide of the Fe protein, but not active Fe protein itself, is required for formation of the apoMoFe protein. This suggests the possibility of a ternary complex of the NifH, D, and K peptides as the substrate for the processing to form the apoMoFe protein. We also find that nifM, the gene which processes the nifH protein into Fe protein (Howard, K.S., McLean, P.A., Hansen, F. B., Lemley, P.V., Kobla, K.S. & Orme-Johnson, W.H. (1986) J. Biol. Chem. 261, 772-778) can, under certain circumstances, partially replace other processing genes (i.e. nifTYU and/or WZ) although it is not essential for apoMoFe protein formation. It also appears that nifS and nifU, reported to play a role in Fe protein production in Azotobacter vinelandii, play no such role in K. pneumoniae, although these genes are involved in apoMoFe formation.(ABSTRACT TRUNCATED AT 400 WORDS)
Harris
G S
GS
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
White
T C
TC
Flory
J E
JE
Orme-Johnson
W H
WH
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins; D002872:Chromosome Deletion; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005288:Ferredoxins / Q000235:genetics*; D005798:Genes, Bacterial; D005838:Genotype; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008983:Molybdoferredoxin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D001059:Apoproteins; D001426:Bacterial Proteins; D005288:Ferredoxins; D008983:Molybdoferredoxin; D011994:Recombinant Proteins; C065636:nitrogenase apomolybdenum-iron protein, Bacteria; D009591:Nitrogenase