Heterologous hybridization of Frankia DNA to Rhizobium meliloti and Klebsiella pneumoniae nif genes

A nif gene probe from Rhizobium meliloti was used to isolate a recombinant bacteriophage from a Frankia sp. ArI3 gene bank. There is a large homology between nif D and nif H genes of R. meliloti or Klebsiella pneumoniae and Frankia DNA sequences. Approximately 4.5 kb to the right of nif K, we have localized a DNA region hybridizing to a R. meliloti probe containing nif A and nif B genes. The extent of the homology was greater for nif B than for nif A.     

Expression of the nif and ntr genes of Klebsiella pneumoniae in Rhodobacter sphaeroides cells

The genes glnA, ntr, nif or their promoters from Klebsiella pneumoniae cloned on the vectors, based on the plasmid RSF1010, were introduced into Rhodobacter sphaeroides cells. It was found that K. pneumoniae genes glnA, nifB, nifE, nifL and nifH are not expressed in R. sphaeroides. Neither was the glnA gene from cyanobacterium Anabaena 7120 expressed in R. sphaeroides. No functional activity of K. pneumoniae product of ntrA gene which is expressed from its own promoter, and the product of the gene nifA which is expressed from the constitutive promoter of the kanamycin resistance gene of the transposon Tn903, was detected. The implications of these findings are discussed.     

Isolation and characterization of lambda specialized transducing bacteriophages carrying Klebsiella pneumoniae nif genes

Seve lambda dnif specialized transducing bacteriophages were isolated from Escherichia coli strains containing plasmids carrying the his-nif region of Klebsiella pneumoniae. These phages collectively carry deoxyribonucleic acid for all of the genes in the nif regulon and adjacent deoxyribonucleic acid of K. pneumoniae. The phages were isolated by using Mu insertions in the nif region to direct the integration of lambda pMu phages in nif via formation of lambda pMu-Mu dilysogens which, upon induction, yielded lambda dnif phages. This procedure should be generally applicable for isolating lambda specialized transducing phages carrying genes from E. coli or other bacteria.     

Expression of Klebsiella nif and his genes in Salmonella typhimurium.

Derivatives of Salmonella typhimurium carrying F prime or P prime plasmids with Klebsiella nif and his genes had specific nitrogenase activities similar to Klebsiella in selective conditions, even to showing "hyperinduction" under argon. No evidence was obtained for catabolite repression of normal nif expression but dibutyl cyclic AMP often augmented "hyperinduction". In non-selective conditions the Klebsiella his nif determinants were rapidly lost from the plasmids; the low levels of nif expression and temperature-sensitive his expression previously reported were probably due to ready loss of his nif in the test conditions used.     

The ntr genes of Escherichia coli activate the hut and nif operons of Klebsiella pneumoniae

Regulation of the synthesis of glutamine synthetase and of the arginine and glutamine transport systems (Ntr phenotype) in Salmonella have been shown to require two regulatory genes on the C-terminal side of the glnA gene (McFarland et al., 1981). We have cloned a HindIII-EcoRI DNA fragment from Escherichia coli coding for analogous properties with respect to the Ntr phenotype in E. coli. A plasmid containing this E. coli DNA fragment joined to another fragment carrying a cyanobacterial glnA gene (but no functional regulatory genes) was introduced into a Klebsiella pneumoniae mutant with a Gln-Ntr- phenotype, i.e., which could not derepress nitrogenase. The cyanobacterial gene made the Klebsiella strain Gln+ and the E. coli DNA fragment made the strain Ntr+, including the ability to derepress nitrogenase fully. Thus the products of the glnA-linked ntr genes of E. coli can regulate expression of the Ntr-dependent genes of Klebsiella.     

Expression ofKlebsiella pneumoniae nif genes inProteus mirabilis

Self-transmissible plasmids carryinghis andnif genes fromKlebsiella pneumoniae have been introduced into threehis mutants ofProteus mirabilis: strains 5006-1, WR19 and WR20. Expression ofhis by the transconjugants was unequivocal, if slightly temperature-sensitive, but none was Nif⁺ when tested for acetylene reduction in anaerobic glucose medium using inocula from rich or glucose-minimal aerobic agar cultures. Succinate or pyruvate in place of glucose, low glucose, lower temperature or elevated Na₂MoO₄ did not allownif expression and no nitrogenase MoFe-protein peptide was detected immunologically after exposure to conditions in which diazotrophic enterobacteria, normal or genetically constructed, derepressnif.One strain,P. mirabilis WR19, carrying thehis nif Km^r plasmid pMF250 was examined in detail. Thenif activator genenifA was introduced on the plasmid pCK1. Such derivatives remained Nif^- when tested, after aerobic growth on rich agar media, with normal or low glucose, with succinate or with elevated Mo. However, pre-conditioning by aerobic growth on glucose-minimal agar led to subsequent anaerobic expression ofnif in glucose medium from pMF250 in WR19 carrying pCK1. NH ₄ ⁺ or proline could serve as N-source in the glucose-minimal agar. Maximum activity was about 5% of that ofK. pneumoniae in our assay conditions. Material cross-reacting with anti-serum to the nitrogenase MoFe protein was formed. Nitrogenase activity was not switched off by NH ₄ ⁺ .P. mirabilis WR19 (pCK1) showed NH ₄ ⁺ -constitutive temperature-sensitive kanamycin resistance (anif-related phenotype of this plasmid) in aerobic glucose minimal medium. Expression ofnif inP. mirabilis WR19 (pCK1, pMF250) was NH ₄ ⁺ -repressible despite the constitutivenifA character of pCK1 and introduction of thentrA ⁺ plasmid pMM17 did not alter this phenotype. However, pCK1 did not give rise to NH ₄ ⁺ -constitutive diazotrophy in the wild-typeK. pneumoniae M5al. A construct of WR19 carrying pMF250 and constitutiventrC plasmid (pMD45) remained Nif^- even after pre-growth on glucose-minimal media.We conclude (a) thatP. mirabilis forms a gene product functionally equivalent to that ofntrA inK. pneumoniae, (b) that it forms no functional equivalent of thentrC product in our growth conditions. The need for pre-conditioning on aerobic glucose media remains perplexing.Non-common abbreviation NFDM Nitrogen-free-Davis-Mingioli medium     

Bacteriophage mu-induced deletions in a plasmid containing the nif (N2 fixation) genes of Klebsiella pneumoniae.

Five plasmids with insertions of a heat-inducible Mu prophage in a Mu-sensitive and P1-sensitive derivative of plasmid pRD1, a recombinant R factor containing the his-nif region of Klebsiella pneumoniae, were isolated and characterized. In one plasmid containing the Mu prophage integrated at the his-distal end of nif, selection for heat resistance resulted in the generation of deletions extending from the Mu prophage into the nif region. Thirty of these deltions were used to map 26 point mutations in nif.     

Intergeneric mobilization of Rhizobium nif genes to Agrobacterium and Klebsiella.

Summary The P-1 incompatibility group plasmid RP1 transfers itself from Escherichia coli J53 to the clover endosymbiont bacterium Rhizobium trifolii strain T1 at low frequency in agar surface matings. R. trifolii T1 R-plasmid recipients display a phenotype identical to the wild-type parent strain in all respects except RP1 antibiotic resistances, allowing straightforward donor counterselection and differentiation of excojugants in further intergeneric plasmid transfer experiments. Hence RP1 can readily transfer itself intergenerically from R. trifolii T1 to the related plant pathogenic organism Agrobacterium tumefaciens and to a strain of the free-living diazotroph Klebsiella pneumoniae.Using R. trifolii T1 (RP1) as donor and as recipient LBA 4006, an avirulent strain of A. tumefaciens lacking the tumour-inducing (Ti) plasmid, selection was made for intergeneric transfer of the R-plasmid and its potential as vector of nitrogen-fixation genes evaluated by subsequent indirect screening. Exconjugant Agrobacteria were obtained which carried RP1 resistance markers and, given specific physiological conditions, would reduce acetylene under air. This is the first report of expression of nif genes in a hybrid strain of A. tumefaciens and is of interest since the Ti plasmid of this organism has been suggested as a natural vector for the introduction of these genes into plants. Plasmid RP1 also cotransferred Rhizobium nif genes to KP52, a strain of K. pneumoniae M5al, with deletion by phage eduction of the chromosomal genes for histidine biosynthesis, one of the nif regulatory genes (nif A), the gene for molybdenum cofactor (nif B) and for an electron transport protein of the nitrogen-fixation pathway (nif F). KP52 exconjugants carried RP1 drug resistances and reduced acetylene under anaerobic conditions.     

Qualitative evidence for expression of Klebsiella pneumoniae nif in Pseudomonas putida

Pseudomonas putida MT20-3 carrying the Klebsiella pneumoniae nif plasmids pRD1 or pMF250 showed highly O2-sensitive aerobic acetylene reduction on low-N pyruvate or glucose agar. This finding confirms unequivocally that K. pneumoniae nif can be expressed in an obligate aerobe.     

Mutations in nif genes that cause Klebsiella pneumoniae to be derepressed for nitrogenase synthesis in the presence of ammonium.

Four Nif+ revertants from strains with polar insertions in nifL, were insensitive to ammonium and amino acid repression of nitrogenase synthesis. These strains have mutations located in or near the nifL region. The derepressed phenotype was dominant in a merodiploid containing a nif+ plasmid. These nif regulatory mutations also suppressed the Nif- phenotype of Gln- strains. Thus, regulation by fixed nitrogen (possible via glutamine synthetase) occurs on the nifLA operon but not on the other six nif operons.     

Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae.

Two hundred and thirty-five Nif- strains of Klebsiella pneumoniae were characterized by two-dimensional polyacrylamide gel electrophoresis. Forty-two of these strains were tested further by in vitro acetylene reduction assays. By these techniques, nine nif-coded polypeptides were identified, and eight of these were assigned to specific nif genes. Nitrogenase component I required nifK and nifD, which coded for the beta and alpha subunits, and nifB, -E, and -N were required for the iron-molybdenum cofactor, which is a part of the active site of nitrogenase. nifH coded for the structural protein of component II, and nifM and nifS products seemed to be necessary for the synthesis of an active component II. There were two genes, nifF and nifJ, that were required for N2 fixation in vivo but not for N2 fixation in vitro. There were at least two cases (nifE and nifN, nifK and nifD) of two proteins that seemed to require each other for stability in vivo. Regulation of N2 fixation is apparently complex, and this is reflected by the assignment of regulatory functions to the gene products of nifA, nifL, nifK, nifD, nifH, and NIFJ.     

肺炎克氏杆菌nifA基因在巴西固氮螺菌固氮基因表达的铵调节中的作用

固氯螺菌(Azospirillum)是一类仅在限铵和微好氧条件下固氮的微生物,它可与许多禾本科作物联合共生⑴,具有较大的应用潜力。铵作为固氮作用的调节信号,在固氮螺菌的实际应用中是首要的限制因素。在固氯螺菌中,铵不但具有与肺炎克氏杆菌(Klebsiella pneumoniae)相似的阻遏固氮酶合成的作用,而且还对已合成的固氮酶进行活性调节⑵。研究表明,其固氮酶翻译后活性调节的机制类似于深红红螺菌(Rhodospirillum rubrum)⑶,即在有铵条件下其固氮酶铁蛋白的一个亚基被共价修饰而丧失活性,这一过程是可逆的。由于铵在固氮螺菌中双水平地调节固氮作用,使得在野生菌株中研究其固氮基因表达水平上的调节较为困难。Zhang等⑷利用区域定位诱变技术获得了巴西固氮螺菌Sp7（A．Brasilense Sp7)的draT-突变株,在该突变株中铵不再影响固氮酶的活性,这为其固氮基因表达调节的研究提供了一个良好的材料。本文将组成型表达的肺炎克氏杆菌nifA基围引入该突变株中,通过分析讨论铵对巴西固氮螺菌固氮基固表达的调节作用方式。