Characterisation of mutations in the Klebsiella pneumoniae nitrogen fixation regulatory gene nifL which impair oxygen regulation

The nifL gene product of Klebsiella pneumoniae inhibits the activity of the positive activator protein NifA in response to increased levels either of fixed nitrogen or of oxygen in the medium. In order to demonstrate that the responses to these two effectors are discrete we have subjected nifL to hydroxylamine mutagenesis and isolated nifL mutants that are impaired in their ability to respond to oxygen but not to fixed nitrogen. Two such mutations were sequenced and shown to be single base pair changes located in different parts of nifL. The amino acid sequence of NifL shows limited homology to the histidine protein kinases which comprise the sensing component of bacterial two-component regulatory systems. In the light of the location of one of the oxygen-insensitive mutations (Leu294Phe) we have reassessed this homology and we suggest that the Gln273-Leu317 region of NifL may facilitate interactions between NifL and NifA.Abbreviations X-gal 5-bromo-4-chloro-3-indolyl--D-galactopy-ranoside - USAs upstream activator sequences     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3-region of the nifM gene, the nifL and nifA genes and the 5-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.Communicated by H. Böhme     

Sequence and molecular analysis of the nifL gene of Azotobacter vinelandii

In both Klebsiella pneumoniae and Azotobacter vinelandii the nifL gene, which encodes a negative regulator of nitrogen fixation, lies immediately upstream of nifA. We have sequenced the A. vinelandii nifL gene and found that it is more homologous in its C-terminal domain to the histidine protein kinases (HPKs) than Is K. pneumoniae NifL. In particular A. vinelandii NifL contains a conserved histidine at a position shown to be phosphorylated in other systems. Both NifL proteins are homologous in their N-termini to a part of the Halobacterium halobium bat gene product; Bat is involved in regulation of bacterio-opsin, the expression of which is oxygen sensitive. The same region showed homology to the haembinding N-terminai domain of the Rhizobium meliloti fixL gene product, an oxygen-sensing protein. Like K. pneumoniae NifL, A. vinelandii NifL is shown here to prevent expression of nif genes in the presence of NH⁺₄ or oxygen. The sequences found homologous in the C-terminal regions of NifL, FixL and Bat might therefore be involved in oxygen binding or sensing. An in-frame deletion mutation in the nifL coding region resulted in loss of repression by NH⁺₄ and the mutant excreted high amounts of ammonia during nitrogen fixation, thus confirming a phenotype reported earlier for an insertion mutation. In addition, nifLA are cotranscribed in A. vinelandii as in K. pneumoniae, but expression from the A. vinelandii promoter requires neither RpoN nor NtrC.     

Regulation of nif gene expression in Enterobacter agglomerans: nucleotide sequence of the nifLA operon and influence of temperature and ammonium on its transcription

The nucleotide sequence of a plasmid-borne 3.9 kb XhoI-SmaI fragment comprising the 3′-region of the nifM gene, the nifL and nifA genes and the 5′-region of nifB gene of Enterobacter agglomerans was determined. The genes were identified by their homology to the corresponding nif genes of Klebsiella pneumoniae. A typical σ⁵⁴-dependent promoter and a consensus NtrC-binding motif were identified upstream of nifL. The predicted amino acid sequence of NifL showed close similarities to NifL of K. pneumoniae and Azotobacter vinelandii. However, no histidine residue was found to correspond to histidine-304 of A. vinelandii NifL, which had been proposed to be required for the repressor activity of NifL. The NifA sequence with a putative DNA binding motif (Q(X₃) A (X₃) G (X₅)I) and an ATP binding site in the C-terminal and central domains, respectively, resembles that of other known NifA proteins. The function of the nifL and nifA genes was demonstrated in vivo using a binary plasmid system by their ability to activate a nifH promoter-lacZ fusion at different temperatures and concentrations of NH ₄ ⁺ . Maximal promoter activity occurred at 25°C, and it appears that the sensitivity of NifA to elevated temperatures is independent of NifL. The expression of nifL inhibited promoter activity in the presence of NifA when the initial NH ₄ ⁺ concentration in the medium exceeded 4 mM.     

The Azotobacter vinelandii nifL-like gene: nucleotide sequence analysis and regulation of expression

Summary The nucleotide sequence of the Azotobacter vinelandii nifL-like gene (Av-nifL) was determined. The 1.9 kb sequence shows an open reading frame (ORF) of 1557 by which encodes a polypeptide of 519 amino acids, with a calculated molecular weight of 57 793. Av-nifL has about 50 % homology with the Klebsiella pneumoniae nifL gene (Kp-nifL) at the nucleotide level and a little more than 52% homology at the amino acid level. The N-terminal regions show more homology than the C-terminal regions. As is the case in K. pneumoniae, Av-nifL is located just upstream of the A. vinelandii nifA gene (Av-nifA) and both genes constitute an operon. The expression of Av-nifL, however, seems to be independent of NtrA and NtrC. Furthermore, Av-nifL expression is not autogenously regulated by NifA, unlike the case in K. pneumoniae. The expression of an Av-nifL: lacZ fusion in A. vinelandii is inhibited by novobiocin and coumermycin A, which are inhibitors of DNA gyrase.     

Transcriptional regulation of nitrogen fixation genes by DNA supercoiling

DNA gyrase (ATP dependent topoisomerase type II, EC 5.99.1.3) was found to be essential for the expression of the Klebsiella pneumoniae nitrogen fixation gene cluster carried by plasmid pRD1 in Escherichia coli. In the absence of DNA gyrase activity, nitrogen fixation activity could be restored by providing a constitutively expressed nifA function in trans. Our results suggest that nif gene regulation by oxygen may be mediated through the alteration of the superhelical status of the promoter of the nifLA regulatory operon, in addition to the action of the nifL gene product.Communicated by J. Schell     

The Klebsiella pneumoniae PII protein (glnB gene product) is not absolutely required for nitrogen regulation and is not involved in NifL-mediated nif gene regulation

Summary The role of theKlebsiella pneumoniae P_II protein (encoded byglnB) in nitrogen regulation has been studied using two classes ofglnB mutants. In Class I mutants P_II appears not to be uridylylated in nitrogen-limiting conditions and in Class II mutants P_II is not synthesised. The effects of these mutations on expression from nitrogen-regulated promoters indicate that P_II is not absolutely required for nitrogen control. Furthermore the uridylylated form of P_II(P_II-UMP) plays a significant role in the response to changes in nitrogen status by counteracting the effect of P_II on NtrB-mediated dephosphorylation of NtrC. P_II is not involved in thenif-specific response to changes in nitrogen status mediated by NifL.     

Cloning and characterisation of nifLA regulatory mutations from Klebsiella pneumoniae

Summary A total of nine regulatory mutations in the nifLA operon of Klebsiella pneumoniae were cloned in the high copy-number plasmid vector pACYC184. The regulatory phenotypes of the resultant clones were then correlated with their restriction maps and their ability to synthesise nifL and nifA polypeptides in vivo. One mutation, nifL2401, was identified as a 400 bp. deletion within the nifL gene. This mutation was non-polar and caused a Nif⁺ phenotype which showed escape from repression by oxygen and low levels of fixed nitrogen. Identification of this deletion allows the first definitive allocation of a mutation with this phenotype to the nifL gene and provides further evidence for the role of the nifL gene product in nif-specific repression.