Characterization of nif H gene mutants from Klebsiella pneumoniae

The nitrogenase enzyme complex of Klebsiella pneumoniae (Kp) is composed of the Fe protein, encoded by nif (nitrogen fixation) H, and the MoFe protein, encoded by nif D and nif K. The genes nif H, nif D, nif K, and nif Y are located in a single operon which is transcribed from nif H toward nif Y.; Nucleotide changes in the nif H gene were identified by DNA cloning and sequencing from six selected Kp mutant strains. They are UN60, C{dollar}sp{lcub}640{rcub}{dollar} GC {dollar}to{dollar} TGC; UN116, C{dollar}sp{lcub}67{rcub}{dollar}TC {dollar}to{dollar} TTC; UN117, G{dollar}sp{lcub}688{rcub}{dollar}Ag {dollar}to{dollar} AAG; UN1041, CG{dollar}sp{lcub}302{rcub}{dollar}C {dollar}to{dollar} CAC; UN1678, GC{dollar}sp{lcub}713{rcub}{dollar}C {dollar}to{dollar} GTC and UN1795, G{dollar}sp{lcub}439{rcub}{dollar}AG {dollar}to{dollar} AAG. Their corresponding amino acid substitutions are UN60, Arg{dollar}sp{lcub}214{rcub}{dollar} {dollar}to{dollar} Cys; UN116, Leu{dollar}sp{lcub}23{rcub}{dollar} {dollar}to{dollar} Phe; UN 117, Glu{dollar}sp{lcub}230{rcub}{dollar} {dollar}to{dollar} Lys; UN1041, Arg{dollar}sp{lcub}101{rcub}{dollar} {dollar}to{dollar} His; UN1678, Ala{dollar}sp{lcub}238{rcub}{dollar} {dollar}to{dollar} Val; and UN1795, Glu{dollar}sp{lcub}147{rcub}{dollar} {dollar}to{dollar} Lys.; Results from radioimmuno-detection of Western and Northern blots showed significant reductions in both steady-state levels of the accumulated Fe protein and nif H mRNA. The decreased quantities of Fe protein in the mutaint strains as well as their reduced nitrogenase specific activity may explain the lower nitrogenase activity observed in vivo when compared to the wild type.; The relative specific activities of the nitrogenase in UN60, UN1041 and UN1795 were less than 2% of the wild type, whereas those in UN116, UN117 and UN1678 were between 28-40% of the wild type during serine enhanced derepression. The residues of Arg{dollar}sp{lcub}101{rcub}{dollar}, Glu{dollar}sp{lcub}147{rcub}{dollar}, and Arg{dollar}sp{lcub}214{rcub}{dollar} on the Fe protein are invariant in nearly all diazotrophs examined, and changes in these residues almost completely inactivated the nitrogenase activities in UN1041, UN1795 and UN60, respectively. These results demonstrated that the conserved residues of the Fe protein are functionally important.; The Arg{dollar}sp{lcub}101{rcub}{dollar} on the Fe protein had been replaced by His in UN1041. The equivalent arginyl residue of the Fe protein of Rhodospirillum rubrum was found to be ADP-ribosylated when the nitrogenase was inactivated. The Fe protein of UN1041 also had a larger apparent molecular weight than the others, as detected with the rabbit antiserum raised against the C-terminal peptide of Kp Fe protein.; Point-mutations of the nif H genes reduced the steady-state levels of the accumulated nif H mRNA in Kp mutant strains, which suggests that nif H may be involved in self-regulation. Multiple nif H-containing transcripts were detected when a nif H specific probe, CCKp2003, was used in the Northern blot hybridization.