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We have determined the nucleotide sequences of two genes from Klebsiella pneumoniae, nifA, the nif‐specific activator of transcription and ntrC, the bifunctional regulatory protein involved in ‘nitrogen control’. These sequences differ significantly from those previously published. In particular, nifA extends 40 codons beyond the stop codon reported earlier. This extension encodes a putative DNA‐binding domain strongly homologous to the Rhizobium meliloti nifA protein and to some extent to the ntrC protein. In all three proteins this domain is linked by a segment of variable length to a strongly conserved central domain of 240 residues. A short segment having the properties of an interdomain linker joins the central region to an N‐terminal domain, which is weakly related in the case of the two nifA proteins. This homology is not shared by the N‐terminal domain of ntrC, which is clearly but unexpectedly related to the N‐terminal domains of a diverse set of procaryotic pleiotropic control proteins, including ompR, dye and nusA from Escherichia coli and spoOA and spoOF from Bacillus subtilis.

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Evidence is presented that establishes a novel class of interdomain linkers, named Q-linkers, as a defined element of protein structure. Q-linkers occur at the boundaries of functionally distinct domains in a widespread set of bacterial regulatory and sensory transduction proteins, typified by the nitrogen regulatory proteins, NtrB, NtrC, NifA and NifL. Q-linkers are not strongly conserved in sequence in otherwise homologous proteins, are approximately 15-25 residues long and relatively rich in glutamine, arginine, glutamate, serine and proline, and are assigned as 'coil', with a very low probability of alpha or beta structure, by eight secondary structure prediction methods. Hydrophobic amino acids are spaced with a periodicity of approximately 4-5 residues in the C-terminal 15 residues of these sequences. A pattern discriminator is presented that incorporates these properties and is used to predict segments resembling Q-linkers in sequence databases. Insertions of four and eight amino acids, constructed in the Q-linker sequences of NtrC and NifA, were found to have no effect on the function of the proteins in signal transduction and transcriptional activation. However, when NtrC was expressed as two separate polypeptides, consisting of the domains normally joined by the Q-linker, the construct failed to function. These results suggest that the Q-linker serves a simple but essential role in tethering the structurally-distinct but interacting domains of the protein. Q-linkers are therefore potentially applicable as domain fusion junctions for engineered chimaeric multidomain proteins expressed in enteric bacterial systems.

A model for the domain structure of sigma 54-dependent transcriptional activators, based on sequence data, has been tested by examining the function of truncated and chimaeric proteins. Removal of the N-terminal domain of NtrC abolishes transcriptional activation, indicating that this domain is positively required for activator function. Over-expression of this domain as a separate peptide appears to titrate out the phosphorylating activity of NtrB. Removal of the N-terminal domain of NifA reduces activation 3-4-fold. The residual activity is particularly sensitive to inhibition by NifL, suggesting that the role of the N-terminal domain is to block the action of NifL in derepressing conditions. The C-terminal domain of NtrC showed repressor activity when expressed as a separate peptide. This domain is necessary for activator function even when NtrC binding sites are deleted from promoters. A point mutation in the ATP-binding motif of the NtrC central domain, Ser169 to Ala, also abolished activator function. Exchanging the N-terminal domains of Klebsiella pneumoniae NtrC, NifA and Escherichia coli OmpR, did not produce any hybrid activity, suggesting that N-terminal domains in the native proteins specifically recognize the rest of the molecule.

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 haem-binding N-terminal 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.

The nitrogen fixation (nif) genes of Klebsiella pneumoniae are specifically regulated by the products of the nifLA operon. We have located the promoter of this operon, and identified sequences required for nifLA transcription. Transcription from this promoter is shown to be positively regulated by the ntrC gene product (which coordinates the expression of many operons required for nitrogen assimilation) and also autogenously by the product of the nifA gene.

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