Mike Merrick

Complementation analysis of klebsiella pneumoniae mutants defective in nitrogen fixation

SummaryA series of mutants defective in nitrogen fixation (nif) were isolated in Klebsiella pneunoniae 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.

Repressor properties of the nifL gene product in Klebsiella pneumoniae

SummaryCertain mutations in the nifL gene of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster resulted in altered nif regulaiton such that nitrogenase synthesis was no longer repressed by low levels of exogenous fixed nitrogen, by oxygen or by high temperature. Introduction of a plasmid with a nifL+ allele restored fixed nitrogen and oxygen repression. We therefore conclude that the nifL product acts as a nif-specific repressor in response to these effectors.Hence nif-specific regulation is controlled by the products of two regulatory genes, nifLA, which comprise a single operon. As previously reported (Dixon et al. 1980; Buchanan-Wollaston et al. 1981), the nifA product is necessary for transcription from other nif promoters but not from that of its own operon. We find that the nifL gene product also acts at other nif promoters but does not repress its own synthesis. Transcription of nifLA is in turn controlled by a general nitrogen-regulatory system in the cell, mediated by the products of the ntrA (glnF), ntrB (glnL) and ntrC (glnG) genes.

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

SummaryThe glanA, ntrB and ntrC genes of Klebsiella pneumoniae have been cloned, on a 12 kb HindIII fragment, into the plasmid pACYC 184. 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.

Identification of the Klebsiella pneumoniae glnB gene: Nucleotide sequence of wild-type and mutant alleles

SummaryThe 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.

Polarity of mutations induced by insertion of transposons Tn5, Tn7 and Tn10 into the nif gene cluster of Klebsiella pneumoniae

SummaryThree 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.

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.

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

The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: Analysis of conserved sequences in NtrA proteins

SummaryThe 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>50% homology and an intervening sequence of <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 β′ 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.

Complementation analysis of glnA-linked mutations which affect nitrogen fixation in Klebsiella pneumoniae

SummaryA 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 they 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 in regulation of the nif gene cluster in K. pneumoniae.

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae and Escherichia coli

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae M5a1 and Escherichia coli K12 has been developed. The method, which uses a freeze-thaw cycle in the presence of CaCl2 to facilitate DNA uptake, is substantially more efficient for K. pneumoniae M5a1 than the conventional transformation procedure for E. coli. The simplicity and speed of the method makes it very attractive for routine transformation of K. pneumoniae M5a1 and E. coli K12.

The role of uridylyltransferase in the control ofKlebsiella pneumoniae nif gene regulation

TheglnD gene in enteric bacteria encodes a uridylyltransferase/uridylyl-removing enzyme which acts as the primary nitrogen sensor in the nitrogen regulation (Ntr) system. We have investigated the role of this enzyme in transcriptional regulation of nitrogen fixation genes inKlebsiella pneumoniae by cloningglnD from this organism and constructing a null mutant by insertional inactivation of the chromosomal gene using the Ω interposon.K. pneumoniae glnD encodes a 102.3 kDa polypeptide which is highly homologous to the predicted products of bothEscherichia coli glnD andAzotobacter vinelandii nfrX. TheglnD-Ω mutant was unable to uridylylate PII and was altered in adenylylation/deadenylylation of glutamine synthetase. Uridylyltransferase was required for derepression ofntr-regulated promoters such asglnAp2 and pnifL but was not involved in thenifspecific response to changes in nitrogen status mediated by thenifL product. We conclude that a separate, as yet uncharacterised, nitrogen control system may be responsible for nitrogen sensing by NifL.