Ray Dixon

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.

The xylABC promoter from the Pseudomonas putida TOL plamid is activated by nitrogen regulatory genes in Escherichia coli

SummaryThe xylABC promoter (OP1), located on the TOL plasmid of Pseudomonas putida contains sequences homologous to the conserved regions found in nitrogen fixation (nif) promoters and in other promoters subject to nitrogen control. XylA-lac fusions were constructed in order to monitor expression from the OP1 promoter in Escherichia coli. Transcription was activated in the presence of the heterologous regulatory genes ntrC or nifA from Klebsiella pneumoniae as well as by the homologous P. putida regulatory gene xylR. In all cases activation was also dependent on the ntrA gene, whose product has been implicated as a specific sigma factor for ntr activatable operons. The 5′ ends of xylA mRNA, detected by S1 nuclease mapping of in vivo transcripts, were identical in strains containing xylR, ntrC or nifA as transcriptional activators. However, activation of the K. pneumoniae nifL or nifH promoters by xylR was not detected.

Role of metal ions in negative regulation of nitrogen fixation by the nifL gene product from Klebsiella pneumoniae

SummaryThe ability of the Klebsiella pneumoniae nifL gene product to antagonise NIFA mediated transcriptional activation from the nifH promoter in vivo was inhibited either by metal deprivation, or by the presence of the iron chelators EDDA or Desferal in the growth medium. This inhibition of the repressive activity of NIFL was reversed by the addition of ferrous or manganous ions to the medium but was unaffected by other transition metals. The dependence on metal ions for NIFL activity was observed when NIFL was overexpressed and when cultures were exposed to oxygen or high levels of fixed nitrogen. Immunochemical evidence suggests that NIFL and NIFA associate to form a functional protein complex. Metal ions are apparently not required for the formation of this complex.

Ammonia assimilation and nitrogen fixation in rhizobium meliloti

SummaryThe enzymes involved in ammonia assimilation by Rhizobium meliloti 4l and their role in the regulation of nitrogen metabolism were studied. Glutamine synthetase (GS) and glutamate synthase (GOGAT) were present at relatively high levels in cells grown in media containing either low or high concentrations of ammonia. NADP-linked glutamate dehydrogenase could not be detected.GOGAT and GS mutants were isolated and characterised. A mutant lacking GOGAT activity did not grow even on high concentrations of ammonia, it was a glutamate auxotroph and was effective in symbiotic nitrogen fixation. The GS and assimilatory nitrate reductase activities of this mutant were not repressible by ammonia but still repressible by casamino acids. A mutant with low GS activity required glutamine for optimal growth. It was ineffective and its nitrate reductase was not inducible.These findings indicate that ammonia is assimilated via the GS/GOGAT pathway in free-living R. meliloti and bacterial GOGAT is not important in symbiosis. Furthermore, GS is suggested to be a controlling element in the nitrogen metabolism of R. meliloti.

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.

Plant viral leaders influence expression of a reporter gene in tobacco

In order to optimise expression of a foreign protein in transgenic plants we investigated the potential benefits of including a viral untranslated leader sequence within a plant transformation vector. A variety of 5 leaders, including the tobacco mosaic virus (TMV) leader sequence and 31 nucleotides of the cauliflower mosaic virus (CaMV) 35S RNA leader, were compared. Viral leader constructs employing the 35S promoter and the reporter β-glucuronidase (GUS) were tested by electroporation into tobacco mesophyll protoplasts and against a cointroduced chloramphenicol acetyl transferase (CAT) gene in transgenic tobacco leaves. In the transient assay system, GUS activities from the viral leaders were compared with those from either a short, random leader or a translational fusion of the CaMV 19S RNA ORF VI to GUS. A two-to-three-fold enhanced level of expression resulted when these leaders were substituted with either the 35S RNA or the TMV leader sequences. This enhancement was further increased, to four-to five-fold, by inclusion of four or seven of the bases from the 35S transcription initiation site adjacent to the TMV leader. In transgenic tobacco the improved GUS levels were maintained from constructs including either the TMV leader (eight-fold) or this sequence with the addition of the 35S transcription initiation site bases (ten-fold). A comparison of GUS enzyme amounts with GUS mRNA amounts, using the CAT gene as an internal standard, revealed that TMV leader-bearing mRNA was translated from four-to six-fold more efficiently than the random leader control.

The role of activator binding sites in transcriptional control of the divergently transcribed nifF and nif LA promoters from Klebsiella pneumoniae

The regulatory region spanning the divergently transcribed nif F and nifLA promoters contains a NIFA‐specific upstream activator sequence (UAS) located around +59, and two NTRC binding sites centred at −142 and −163 with respect to the nifLA transcription start site. We have constructed mutations in each of these binding sites and examined their role in transcriptional activation of the divergently transcribed promoters. Analysis of a mutation at +60 confirms that the UAS is required for efficient NIFA‐mediated activation of nifF transcription. This sequence is also required for maximal activation of the nifLA promoter. Mutations at −169 and −148, within the two NTRC binding sites, reduce activation of the nifLA promoter by NTRC in vivo and lower the affinity of the activator for these sites in vitro. Phosphorylation of NTRC by NTRB is required for efficient binding of NTRC to these sites.

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.

Requirements for transcriptional activation in vitro of the nitrogen‐regulated gInA and nifLA promoters from Klebsiella pneumoniae: dependence on activator concentration

Three proteins involved in nitrogen regulation In Klebsiella pneumoniae, NTRA, NTRB and NTRC, have been purified. In a defined in vitro system all three NTR proteins are required for initiation of transcription at the ntr activatable glnA and nifLA promoters. However, in crude S‐30 extracts, transcription from the glnA promoter, but not the nifLA promoter, can be activated in the absence of NTRB. A higher concentration of NTRC is required for activation of nifLA transcription than for glnA transcription. Sequences located between ‐227 and ‐158 with respect to the nifL transcription start site are required for efficient activation of the nifLA promoter in vitro.