Martin Drummond

The function of isolated domains and chimaeric proteins constructed from the transcriptional activators NifA and NtrC of Klebsiella pneumoniae

A model for the domain structure of σ54‐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, Ser 169 to Ala, also abolished activator function. Exchanging the N‐terminal domains of Klebsiella pneumoniae NtrC, NifA and Escherichia co/r OmpR, did not produce any hybrid activity, suggesting that N‐terminal domains in the native proteins specifically recognize the rest of the molecule.

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.

Sequence of nifL from Klebsiella pneumoniae: mode of action and relationship to two families of regulatory proteins

We present the nucleotide sequence of K. pneumoniae nifL, which negatively regulates nif transcription in response to oxygen and fixed nitrogen. It shows partial sequence homology to the general nitrogen regulatory proteins NtrB of K. pneumoniae and Bradyrhizobium parasponiae. This homology is weaker than that shown between the NifA and NtrC activator components of the nif and general nitrogen control systems. The N‐terminal section of the NifL protein includes a structural duplication sharing sequence homology with part of NtrB, and a region containing a cysteine pair which might be implicated in redox control. Unlike NtrB, NifL appears to lack a DNA‐binding motif, consistent with evidence that NifL represses by interacting directiy with NifA. The C‐terminal section of NifL shows clear homology to NtrB and to a family of proteins involved in transcriptional control or chemotaxis, each of which probably interacts with a member of the family of regulatory proteins showing homology to NtrC.

The use of cloned nif regulatory elements from Klebsiella pneumoniae to examine nif regulation in Azotobacter vinelandii

Summary: Regulatory genes controlling nif expression and also nif promoters fused to lacZ from Klebsiella pneumoniae were cloned on wide-host-range plasmids and introduced into Azotobacter vinelandii to compare regulation of nif expression in the two organisms. A low-copy-number plasmid carrying K. pneumoniae nif A corrected an A. vinelandii Nif-regulatory mutation, whereas a plasmid carrying ntrC did not. A high-copy-number plasmid carrying K. pneumoniae nifL eliminated nif expression in K. pneumoniae but not in A. vinelandii. K. pneumoniae nifL- and nifF-lacZ fusions were expressed strongly in A. vinelandii and were not repressed by ammonium. A nifH-lacZ fusion was not expressed in any conditions in this background except very weakly when K. pneumoniae nifA was also present. The implications of these findings are discussed.

Redundancy of the conserved His residue in Azotobacter vinelandii NifL, a histidine autokinase homologue which regulates transcription of nitrogen fixation genes.

The NifL protein of Azotobacter vinelandii inhibits NifA, the activator of nif (nitrogen fixation) transcription, in response to oxygen and fixed nitrogen. NifL shows strong homology in its C‐terminal domain to the histidine autokinase domains of the canonical two‐component sensor proteins, including the region around His‐304, which corresponds to the residue known to be phosphorylated in other systems. To examine the mechanism of sensory transduction by NifL, mutations encoding 10 substitutions for His‐304 were introduced into the A. vinelandii chromosome. Regulation of nif transcription was measured using acetylene reduction and RNA blots. The substitutions His‐304 → Arg and His‐304 → Pro impaired regulation by both fixed nitrogen and oxygen, but substitution of Ala, Phe, Ile, Lys, Asn, Ser, Thr, Val had no effect. None of the mutants, including His‐304 → Arg and His‐304 → Pro, excreted ammonium during diazotrophy, a phenotype of nifL deletion mutants, suggesting that the molecular basis of this effect differs from that responsible for the inhibition of nif transcription. The data show conclusively that phosphorylation of His‐304 is not essential for any of the known functions of A. vinelandii NifL. Homology to the family of histidine autokinases is therefore inadequate evidence for a mechanism of sensory transduction involving phosphorylation of the conserved histidine residue.

A counterselectable pACYC184-based lacZα-complementing plasmid vector with novel multiple cloning sites; construction of chromosomal deletions in Klebsiella pneumoniae

We have constructed a series of small, chloramphenicol-resistance-encoding, lacZ alpha-complementing vectors with novel multiple cloning sites, based on the pACYC184 replicon. The sacB gene of Bacillus subtilis, which is lethal to Gram- organisms in the presence of sucrose, was cloned into one of these, giving the counterselectable vector pSG335. This was used to substitute a streptomycin-resistance-encoding cassette for the ntrBC genes in the Klebsiella pneumoniae chromosome.

Positional requirements for the function of nif-specific upstream activator sequences.

SummaryThe upstream activator sequence (UAS) found in Klebsiella pneumoniae nif promoters and required for the activation of transcription by nifA, is absent from the nifF-nifL intergenic region, but is present downstream from the nifLA transcription start at+59. To determine whether nif upstream activator sequences can function in a 3′ position, the nifH UAS was cloned downstream from the NifH transcription start, but no activation of transcription by nifA dependent upon the UAS in its 3′ location could be detected. A mild repressive effect was detectable when the nifH UAS was placed downstream of the nifH promoter, but not when the cat promoter was substituted for the nifLA promoter upstream from the motif at+59 described above. However, deletion analysis showed that the UAS motif located downstream of the nifLA promoter has a role in transcription from the nifF promoter, although it is situated at position-263 with respect to the nifF transcription start, about 100 bp further upstream than previously described occurrences of the activator sequence.

Construction of chimeric proteins from the σ;N‐associated transcriptional activators VnfA and AnfA of Azotobacter vinelndii shows that the determinants of promoter specificity lie outside the‘recognition’helix of the HTH motif in the C‐terminal domain

Functional chimeras have been generated from the transcriptional activators VnfA and AnfA, which control expression of the alternative nitrogenases in Azotobacter vinelandii. The activation profiles of the native and chimeric proteins have been determined using lacZ fusions to A. vinelandii anf and vnf promoters in Klebsiella pneumoniae. Repiacing the C‐terminal domain of AnfA with that of VnfA gives a protein with the promoter specificity of VnfA, confirming that the C‐terminal domain contains the determinants of promoter specificity. However, substituting the VnfA sequence from the turn in the helix‐turn‐helix motif to the C‐terminus does not alter the promoter specificity of AnfA. These changes in promoter specificity were reflected in changes in affinity for a VnfA‐binding site, as measured by an in vivo repression assay using a lacZ fusion to a synthetic promoter. This supports the assumption that promoter recognition is determined by activator binding to enhancer‐like sequences, and shows that the principal determinants of specific DNA‐binding lie outside the‘recognition’helix. This may be a general feature of transcriptional activators dependent on σ;N (σ54). The chimera with the promoter specificity of VnfA retained the dependence on nitrogenase Fe protein characteristic of AnfA, indicating that this property is not related to particular promoter sequences, but is a function of the central or N‐terminal domains of AnfA.

Cys184 and Cys187 of NifL protein of Klebsiella pneumoniae are not absolutely required for inhibition of NifA activity

The sequence Cys184-Ala-Asp-Cys187 in the NifL protein of Klebsiella pneumoniae, for which a role in oxygen sensing and/or metal binding has been proposed, was altered by introducing two mutations, Cys184----Ala and Cys187----Ala, using oligodeoxyribonucleotide-directed mutagenesis. Neither mutation abolished ammonium or oxygen control of nif transcription, although some impairment of function was apparent. The two Cys residues are therefore unlikely to have a direct role in oxygen sensing or metal binding, but probably make some contribution to protein folding or stability.

Transcriptional Control of the Nif Regulon in Klebsiella Pneumoniae

Expression of the seventeen nitrogen fixation (nif) genes in Klebsiella pneumoniae is regulated in response to both nitrogen source and oxygen tension, so that nitrogenase synthesis is severely repressed by the presence of ammonia, certain amino acids or by dissolved oxygen in the growth medium. Nitrogen control of nif transcription is maintained at two levels; firstly by the ntr system which exerts a general control on nitrogen metabolism and secondly by regulatory proteins encoded by two nif genes, nifA and nifL. The nifA product is required for transcriptional activation of the nif operons (Dixon et al, 1980; Buchanan-Wollaston et al, 1981a) whereas the nifL product has been implicated in repression of nif transcription in response to fixed nitrogen and oxygen (Buchanan-Wollaston et al, 1981b; Hill et al, 1981; Merrick et al, 1982).