Localization of the glnD gene on a revised map of the 200-kilobase region of the Escherichia coli chromosome.
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Chromosome Mapping↗
Biomedical subjects
M Merrick
Publications and source records attributed to M Merrick.
Casecart systems: integrated and computerized.
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Computer Systems↗
The product of the nitrogen fixation regulatory gene nfrX of Azotobacter vinelandii is functionally and structurally homologous to the uridylyltransferase encoded by glnD in enteric bacteria.
We sequenced the nitrogen fixation regulatory gene nfrX from Azotobacter vinelandii, mutations in which cause a Nif- phenotype, and found that it encodes a 105-kDa protein (NfrX), the N terminus of which is highly homologous to that of the uridylyltransferase-uridylyl-removing enzyme encoded by glnD in Escherichia coli. In vivo complementation experiments demonstrate that the glnD and nfrX products are functionally interchangeable. A vinelandii nfrX thus appears to encode a uridylyltransferase-uridylyl-removing enzyme, and in this paper we report the first sequence of such a protein. The Nif- phenotype of nfrX mutants can be suppressed by a second mutation in a recently identified nifL-like gene immediately upstream of nifA in A. vinelandii. NifL mediates nif regulation in response to the N status in A. vinelandii, presumably by inhibiting NifA activator function as occurs in Klebsiella pneumoniae; thus, one role of NfrX is to modify, either directly or indirectly, the activity of the nifL product.
Adenine↗
The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis.
Active Fe protein of nitrogenase was synthesised in a non-nitrogen fixing organism when Escherichia coli was transformed with a plasmid encoding only two nif-specific genes, nifH and nifM of Klebsiella pneumoniae. Hence proteins NifH and NifM are sufficient to produce active Fe protein in E. coli. K. pneumoniae strains carrying chromosomal nifW- and nifZ- mutations were constructed and shown to be significant C2H2-reducing activity and to grow on N-free plates. Nevertheless, derepressing cultures of the mutant strains had reduced levels of MoFe protein activity, and consequently significantly lower levels of nitrogenase activity, than the nif+ parent strain. NifW and NifZ therefore appear to be involved in the formation or accumulation of active MoFe protein, but are not essential for nitrogen fixation in K. pneumoniae under the conditions tested.
Enzyme Repression↗
Identification of the Klebsiella pneumoniae glnB gene: nucleotide sequence of wild-type and mutant alleles.
The glnB gene of Klebsiella pneumoniae, which encodes the nitrogen regulation protein PII, has been cloned and sequenced. The gene encodes a 12429 dalton polypeptide and is highly homologous to the Escherichia coli glnB gene. The sequences of a glnB mutation which causes glutamine auxotrophy and of a Tn5 induced Gln+ suppressor of this mutation were also determined. The glutamine auxotrophy was deduced to be the result of a modification of the uridylylation site of PII, and the suppression was shown to be caused by Tn5 insertion in glnB. The 3' end of an open reading frame of unknown function was identified upstream of glnB and may be part of an operon containing glnB. Potential homologues of glnB encoding polypeptides extremely similar in sequence to PII were identified upstream of published sequences of the glutamine synthetase structural gene (glnA) in Rhizobium leguminosarum, Bradyrhizobium japonicum and Azospirillum brasilense.
Alleles↗
The nucleotide sequence of the nifM gene of Klebsiella pneumoniae and identification of a new nif gene: nifZ.
A 1.4-kb PstI-HpaI DNA fragment carrying the Klebsiella pneumoniae nifM gene has been sequenced; nifM has been shown to encode a 30.6-kDa polypeptide. Two other open-reading frames were identified upstream of nifM. The one immediately upstream of nifM encodes a 16.6-kDa polypeptide which has been identified by in vitro transcription/translation in an Escherichia coli 30,000 x g supernatant system; we propose to designate this gene nifZ. The sequence of the second open reading frame is incomplete but it does not correspond to nifV, the gene previously thought to be immediately upstream of nifM, and may therefore identify another new nif gene. Both nifM and nifZ have functional nif promoters with the characteristic-24, -12 consensus sequence, we find no evidence for a nifM upstream activator sequence. The role of nifZ in nitrogenase biosynthesis is unknown but its identification calls into question previous assertions that only nifM and nifH are required for the synthesis of nitrogenase Fe protein.
Amino Acid Sequence↗
The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins.
The nucleotide sequence of the Azotobacter vinelandii ntrA gene has been determined. It encodes a 56916 Dalton acidic polypeptide (AvNtrA) with substantial homology to NtrA from Klebsiella pneumoniae (KpNtrA) and Rhizobium meliloti (RmNtrA). NtrA has been shown to act as a novel RNA polymerase sigma factor but the predicted sequence of AvNtrA substantiates our previous analysis of KpNtrA in showing no substantial homology to other known sigma factors. Alignment of the predicted amino acid sequences of AvNtrA, KpNtrA and RmNtrA identified three regions; two showing greater than 50% homology and an intervening sequence of less than 10% homology. The predicted protein contains a short sequence near the centre with homology to a conserved region in other sigma factors. The C-terminal region contains a region of homology to the beta' subunit of RNA polymerase (RpoC) and two highly conserved regions one of which is significantly homologous to known DNA-binding motifs. In A. vinelandii, ntrA is followed by another open reading frame (ORF) which is highly homologous to a comparable ORF downstream of ntrA in K. pneumoniae and R. meliloti.
Amino Acid Sequence↗
The nucleotide sequence of the nitrogen regulation gene ntrB and the glnA-ntrBC intergenic region of Klebsiella pneumoniae.
The nucleotide sequence of the Klebsiella pneumoniae ntrB gene and the glnA-ntrBC intergenic region has been determined. NtrB encodes a 38,409 Dalton polypeptide with a potential DNA-binding domain between residues 67 and 86. This N-terminal domain may play a role in the co-operative control of ntr-regulated promoters by the ntrB and ntrC products. Mapping of in vivo transcripts with S1 nuclease identified three transcripts in the glnA-ntrBC intergenic region. Two transcripts originate upstream of glnA; one reading through into ntrBC and one terminating at a sequence resembling a rho-independent terminator between glnA and ntrBC. A third transcript originates from the ntrBC promoter which has a consensus binding site for the ntrC product in the -10 region. Comparison of the glnA-ntrBC intergenic sequences from K. pneumoniae, Escherichia coli and Salmonella typhimurium has identified a number of conserved features and some significant differences.
Cloning, Molecular↗
Interaction of purified NtrC protein with nitrogen regulated promoters from Klebsiella pneumoniae.
The product of the Klebsiella pneumoniae nitrogen regulatory gene ntrC has been purified and shown to be a dimeric protein of subunit molecular weight 54Kd, designated NtrC. In an in vitro coupled transcription-translation system NtrC inhibited expression from both the ntrBC and glnA promoters. NtrC bound to both of these ntr repressible promoters with equal affinity, but did not bind to the activatable nitrogen fixation promoters nifF or nifLA. NtrC makes contact with nucleotides flanking the -10 region of the glnA (RNA2) promoter at sequences homologous with the proposed consensus binding site.
Bacterial Proteins↗
Positive and negative control of the glnA ntrBC regulon in Klebsiella pneumoniae.
The nitrogen regulation system of Klebsiella pneumoniae comprises three genes ntrA, ntrB and ntrC. We have found that the glnA ntrBC regulon in K. pneumoniae has a similar structure, P1 glnA P2 ntrBC, to that in other enterobacteria. We have constructed plasmids with glnA and ntrB translational lacZ fusions and measured expression from P1 and/or P2 in a K. pneumoniae delta (glnA ntrBC) background with different plasmids which provided the ntrB, ntrC or nifA products in trans. These studies demonstrate that, as in other enterobacteria, transcription of ntrBC is from P1 under nitrogen deficiency and from P2 under nitrogen excess. The P1 promoter can be regulated both positively and negatively; activation requires both ntrB and ntrC products but the ntrC product is sufficient to repress. The P2 promoter is negatively controlled by the ntrC product. Comparison of the modes of regulation of P1 and P2 with regulation of the promoter of the nifLA operon leads us to suggest that these may represent three different classes of ntr-regulated promoters. Although previous studies have shown that the nifA product can substitute for the ntrC product as a positive activator of transcription for a number of promoters, we find that nifA product cannot substitute for ntrC product as a negative regulator at P1 or P2.
Gene Expression Regulation↗
Positive control and autogenous regulation of the nifLA promoter in Klebsiella pneumoniae.
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.
Base Sequence↗
Cloning and characterisation of nifLA regulatory mutations from Klebsiella pneumoniae.
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.
Chromosome Mapping↗
Cloning of the glnA, ntrB and ntrC genes of Klebsiella pneumoniae and studies of their role in regulation of the nitrogen fixation (nif) gene cluster.
The glnA, ntrB and ntrC genes of Klebsiella pneumoniae have been cloned, on a 12 kb HindIII fragment, into the plasmid pACYC184. In a coupled in vitro transcription/translation system the resultant plasmid, pGE100, directed synthesis of five polypeptides (molecular weights 73, 53, 51, 39, 36 kd) from the cloned fragment. A number of plasmids were derived from pGE100 and studied by complementation analysis and in vitro transcription/translation in order to locate particular genes and identify their products. On the basis of the results presented here, together with previous genetic and physical characterisation of the glnA gene and its product in other enteric bacteria, we propose that the 53 kd polypeptide is the glnA gene product (glutamine synthetase monomer). Two polypeptides (36 kd and 51 kd) were synthesised from a 3 kb region previously defined as glnR. In E. coli and S. typhimurium this region comprises two genes ntrB and ntrC with products of 36 kd and 54 kd respectively. This analogy supports the idea that the 36 kd and 51 kd polypeptides are the products of the K. pneumoniae ntrB and ntrC genes respectively. Comparison of these assignments with the physical map of the region indicates a gene order glnA, ntrB, ntrC. Assessment of the Nif phenotype of a glnA-ntrC deletion strain carrying various clones from pGE100 demonstrated that glnA is not required for expression of the nif regulon and that of the three genes cloned, ntrC alone is sufficient for nif expression.
Cloning, Molecular↗
Complementation analysis of glnA-linked mutations which affect nitrogen fixation in Klebsiella pneumoniae.
A number of mutants have been isolated which affect regulation of the nitrogen fixation (nif) gene cluster in Klebsiella pneumoniae and all of which are linked to glnA, the structural gene for glutamine synthetase (G.S.). These mutants were classified on the basis of their G.S. and nitrogenase activities in conditions of nitrogen limitation and excess. The plasmid R68.45 was then used to generate a number of R-primes carrying the glnA region of the K. pneumoniae chromosome. One of these R-primes (pGE10) was subsequently used in complementation analysis and by isolation of transposon-induced insertion mutations in pGE10 we have demonstrated the existence of a gene, glnG, closely linked to glnA. Mutations in glnG have a similar phenotype to glnG mutants described in Escherichia coli (Pahel and Tyler 1979) and Salmonella typhimurium (Kustu et al. 1979) in that substantially reduce G.S. activity but are not glutamine auxotrophs. GlnG mutants have very low nitrogenase activity indicating that the glnG product may be involved regulation of the nif gene cluster in K. pneumoniae.
Genes↗
Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions.
Gene fusions in which the lac genes are under the control of each promoter in the Klebsiella pneumoniae, nitrogen fixation (nif) gene cluster have been constructed. These fusions have been used to examine positive control of the cluster and the response of individual genes to repression by ammonia and oxygen. De-repression of nif transcriptional units is coordinate and molybdate is required for maximal expression of the structural gene operon, which is autogenously regulated.
Ammonia↗
The use of translocatable genetic elements to construct a fine-structure map of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster.
The transposons Tn5, Tn7 and Tn10 and bacteriophage Mu have been used to derive insertion mutations in the Klebsiella pneumoniae nif gene cluster. A large number of deletion mutants have been derived by imprecise excision of insertion mutations and these deletions have been used to construct a fine-structure map of the nif cluster. Comparison of this genetic map with a physical map of the nif cluster derived by Reidel et al. (1979) showed a very good correlation between genetic and physical mapping methods. A new complementation group, designated nifU, has been identified and mapped between nifN and nifS. Polarity studies on the 14 nif cistrons now identified suggests that they are organized in at least seven transcriptional units and that all the multicistronic units are transcribed in the same direction.
Chromosome Deletion↗
Polarity of mutations induced by insertion of transposons Tn5, Tn7 and Tn10 into the nif gene cluster of Klebsiella pneumoniae.
Three new genes nifM, nifI and nifN have been mapped in the nif gene cluster of Klebsiella pneumoniae and a fourth gene nifJ has been confirmed as being a separate cistron. Polar nif mutations were obtained by transposition of Tn7 to plasmid pRD1, and of Tn5 and Tn10 to plasmid pMF100, a derivative of pRD1. Complementation analysis of the nif::Tn mutants led to the identification of at least six transcriptional units: nifB; nifA; nifJ; nifH, nifD and nifK; nifE and nifI; nifN, nifM and nifF. Biochemical and genetic evidence suggest that the three genes nifH, nifD and nifK, which are probably the structural genes for nitrogenase, belong to the same operon and are transcribed from nifH to nifK. A polypeptide with a molecular weight of approximately 120,000 is presumed to be the nifJ product.
Chromosome Mapping↗
Complementation analysis of Klebsiella pneumoniae mutants defective in nitrogen fixation.
A series of mutants defective in nitrogen fixation (nif) were isolated in Klebsiella pneumoniae strain M5a1. The nif mutations were either located on plasmid pRD1 or on the K. pneumoniae chromosome. A total of 37 plasmid mutants and 28 chromosomal mutants were employed in complementation tests using the acetylene reduction technique. Most mutants could be assigned to one of seven nif cistrons: nifA, nifB, nifD, nifE, nifF, nifH, and nifK. Complementation analysis of two nif deletion mutants confirmed transductional evidence that these strains carry nifB-A-F deletions. One deletion mutant had, in contrast to previous transductional analysis, a functional nifK cistron and presumably is deleted for nifB-A-F-E. Examination of the biochemical phenotype of several mutants suggests that the nifA product has a regulatory function, and nifK, nifD and nifH are most probably the structural genes for nitrogenase.
Chromosomes, Bacterial↗