nifA Gene Product Research Articles

Nitrogen control of the nif regulon in Klebsiella pneumoniae: involvement of the ntrA gene and analogies between ntrC and nifA.

The ntrC and nifA gene products of Klebsiella pneumoniae are transcriptional activators involved in general nitrogen control and nif-specific regulation, respectively. Multicopy plasmids expressing either ntrC or nifA from a foreign promoter were used to study the relationship between these two genes and ntrA. The nifA product substituted for ntrC product in activation of a number of genes including nifLA, hutUH and genes for arginine and proline utilisation. NtrC could not substitute for nifA in transcriptional activation of the nifHDKY operon. In ntrA- strains, neither the ntrC nor the nifA product functioned to activate transcription of nif promoters. In vitro transcription/translation studies with plasmid clones demonstrated similar levels of expression of ntrC and nifA in ntr+ and ntrA- S-30 extracts. Hence, lack of activator function in an ntrA mutant indicates that both the ntrC and nifA products require a functional ntrA for activity. When expressed from foreign promoters, both the ntrC and nifA products were active in conditions which would normally repress nif expression. Hence, the ntrA product was apparently not limiting in these conditions.

Cloning and expression of a DNA fragment carrying a his nifA fusion and the nifBQ operon from a nif constitutive mutant of Klebsiella pneumoniae

From the nifc mutant plasmid pPC868, previously shown to carry a DNA duplication responsible for the Nifc phenotype, a 10 kb HindIII fragment was cloned into the multicopy vector pBR325. Restriction analysis of the resulting plasmids and in vitro deleted derivatives confirmed that the mutation was a fusion between his and nifLA. The order was hisG-hisD'-'nifL-nifA so that nifA was transcribed under the control of the his promoter and the nifL gene was altered. In addition the cloned fragment contained the adjacent nifBQ operon, and complementation data revealed that the nifA, nifB and hisG genes were expressed. Synthesis of nifA product under the transcription control of the his (or cat [CmR]) promoter enabled complementation of nifA and nifB mutations either in the absence or the presence of ammonia, but did not restore nitrogen fixation in a glnF mutant. Therefore, the nifA gene product requires glnF for its positive control function in a manner analogous to ntrC. Protein content analysis of minicells containing various multicopy nif plasmids confirmed the genetic organization mentioned above. A new polypeptide of 51,500 daltons was found whose synthesis was observed at 30 degrees C but not at 37 degrees C. According to the physical map, this protein could be the nifB gene product. Our results are in agreement with nifB transcription being under the control of a thermolabile nifA product. Moreover we obtained results suggesting that the presence of multiple copies of a functional nifB gene inhibited nitrogen fixation.

Regulation of nitrogenase in the photosynthetic bacterium Rhodopseudomonas capsulata as studied by two-dimensional gel electrophoresis.

By using two-dimensional electrophoresis, five putative soluble nif gene products were identified, and the regulation of nif gene expression in Rhodopseudomonas capsulata was investigated. Expression of nif was repressed by ammonia and atmospheric concentrations of oxygen. Deprivation of molybdenum caused an interesting pattern of partial repression of nif gene expression that was not relieved by tungsten. These results are discussed in relation to the better understood system of nif regulation in Klebsiella pneumoniae.

Effect of nifA gene product on expression of lacZ under nifH promoter in Escherichia coli.

Gene expression of the nitrogen fixation system from Klebsiello pneumonice was studied in Escherichia coli by using compatible plasmids as vectors. One constructed plasmid carried the nifH promoter fused to the structural gene for beta-galactosidase, lac Z. Another plasmid carried the promoter of a tetracycline-resistance gene fused to nifA. We found that anaerobic synthesis of beta-galactosidase was greatly enhanced by the presence of an active nifA gene, indicating that its product is a positive control factor for transcription of nifH. In addition, anaerobic expression of lacZ was repressed by ammonium or serine in the presence of nifA. Thus the regulatory mechanism under study is of physiological relevance.

Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae.

The nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae is linked to his, consists of at least 17 genes arranged in seven or eight operons1–4 (Fig. 1), and its expression is inhibited by oxygen (O2) and fixed nitrogen (N) including ammonia, nitrate and amino acids (see ref. 5 for review). Previous studies have suggested that the nifA and nifL gene products respectively activate6–8 and repress9,10 nif expression. There has also been evidence, from mutants that express nif constitutively in the presence of fixed N, that regulation by fixed N involves the gln (glutamine synthetase) genes11,12, and that their products act at the promoter of the nifLA operon13,14. We have recently shown by direct measurement of transcription in vivo that the nifA and nifL gene products are, respectively, an activator and represser of nif transcription intiation from all nif promoters except that of the nifLA operon15. We now report experiments aimed at clarifying the role of the nifA product and the interaction of the nifLA operon and gln genes in controlling nif expression. Having constructed plasmids that constitutively express the nifA gene and investigated nif expression in a variety of K. neumoniae strains transformed by such plasmids, we show that, in the absence of nifL protein, nifA expression is sufficient to activate nif expression, even in the presence of fixed N and O2. gln-mediated regulation of nif by fixed N exerts its effect by controlling nifLA expression. We have also confirmed that the nif A protein is temperature sensitive, thus explaining the absence of nif expression at 37 ° C. Our results provide a strategy by which the constitutive expression of nif in the presence of fixed N and O2 is stabilized and suggest a model for nif transcriptional regulation.

The use of cloned nif (nitrogen fixation) DNA to investigate transcriptional regulation of nif expression in Klebsiella pneumoniae.

Some restriction endonuclease fragments of nif DNA, when carried on small multicopy plasmids, inhibited nif expression in Klebsiella pneumoniae. A study of this inhibitory effect revealed, (1) that overproduction of the nifL gene product inhibited transcription of two nif operons examined, nifJ and nifHDKY and, (2) that when transcription was initiated from the promoter of the nifHDKY operon on multicopy plasmids there was a corresponding decrease in the transcription rates of the chromosomally located nifJ and nifHDKY but not the nifLA operon. Studies of transcription in vivo also showed that the nifA gene product was essential for transcription initiation from the nifHDKY and nifBQ promoters. These results, taken with earlier observations (see Discussion) provide evidence that the nifL and nifA gene products are respectively a repressor and activator of nif transcription initiation from all nif promoters except that of the nifLA operon.

Kinetic study of the expression of Klebsiella pneumoniae nitrogen fixation ( nif) genes under conditions of inhibited transcription

In vivo acetylene-reducing activity and the translation of nif mRNAs were examined in a N 2-fixing culture of Klebsiella pnuemoniae, following addition of NH 4 + ions, rifampicin, or both. The de novo biosynthesis of six [ 14C] pulse-labelled nif gene products was followed by gel electrophoresis (one and two dimensional). The amount of label incorporation into the nif-encoded polypeptides, which belong to four out of the seven nif transcriptional units, reflects the extent of translation of the corresponding mRNAs. A comparison of the results obtained under the three sets of conditions suggests : i) that the nif mRNAs examined appear to be more stable than the bulk cellular mRNAs ; ii) that the presence of NH 4 + ions does not affect the translation or most of the already pre-formed nif mRNAs.

Gene-product relationships of the nif regulon of Klebsiella pneumoniae.

Products of nifV, M, A, and F of Klebsiella pneumoniae have been identified, and suggestive evidence exists for the assignment of products for nifS, U, and L. These assignments confirm previous descriptions of transcriptional polarity and suggest that existence of several more nif genes whose products are apparently not essential for nitrogen fixation under standard laboratory conditions of growth. Although the nifA product is essential for expression of other nif transcripts, it is not involved in the regulation of its own transcript, nifRLA. The stability of nif-encoded proteins under anaerobic conditions was examined in a variety of mutants, confirming previously reported protein-protein interactions and suggesting several new ones. The stability of nif-encoded proteins in ammonia was found to be comparable to that in ammonia-free conditions, whereas all nif-gene products appeared to be labile in the presence of oxygen.

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

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.