Characterization and kinetics of the biosynthesis of some nitrogen fixation ( nif) gene products in Klebsiella pneumoniae

Abstract

Analysis of 14C pulse-labelled proteins, synthesized by a Nif Klebsiella pneumoniae strain and by a number of genetically mapped nif::Mu and nif deletion mutants, was performed by two-dimensional gel electrophoresis. By comparison of the autoradiograms, six nif-specific polypeptides were identified. In addition to the previously characterized nifK, nifD, nifH and nifl products, the product of nifF was identified as a polypeptide of 10,000 daltons and pI about 4.5 and the product of nifU as a polypeptide of 22,000 daltons and pI 5. Moreover, the biosynthesis of nifF and nifU polypeptides was shown to be prevented in mutants affecting the regulatory gene nifA, which is known to control the biosynthesis of the other nit genes products so far identified. In all cases, the biochemical phenotypes of the different polar mutants were in good agreement with those expected from the transcriptional organization of the nif cluster previously established by genetic analysis. Kinetic studies of both nitrogenase activity and of the biosynthesis of the six nif-specific polypeptides were performed with the Nif' strain, incubated either under conditions of derepression or under conditions of repression by NH4+ ions. Upon derepression, the biosynthesis of the six nif polypeptides, which belong to four different transcriptional units, seems to be coordinated since they appear simultaneously after a lag of 45 minutes. Under those conditions, both in vivo and in vitro nitrogenase activities were detectable only 30 minutes later. Upon addition of NH4+ ions, the biosynthesis of the six nif polypeptides was rapidly abolished. However, the kinetics of residual biosynthesis, probably due to the transcription of preexisting mRNAs, was not similar for the six nif products. The nifU product was no longer detectable after 5 minutes, the nifF, K, D and J products were not detectable after 30 minutes, whereas some nifH product was still slightly detectable after 60 minutes.