Nucleotide sequence of the gene coding for the nitrogenase iron protein from Klebsiella pneumoniae

We report the complete DNA sequence of the Klebsiella pneumoniae nifH gene, the gene which codes for component 2 (Fe protein or nitrogenase reductase) of the nitrogenase enzyme complex. The amino acid sequence of the K. pneumoniae nitrogenase Fe protein is deduced from the DNA sequence. The K. pneumoniae Fe protein contains 292 amino acids, has a Mr = 31,753, and contains 9 cysteine residues. We compare the amino acid sequence of the K. pneumoniae protein with available amino acid sequence data on nitrogenase Fe proteins from two other species, Clostridium pasteurianum and Azotobacter vinelandii. The C. pasteurianum Fe protein, for which the complete sequence is known, shows 67% homology with the K. pneumoniae Fe protein. Extensive regions of strong conservation (90-95%) are found, while other regions show relatively poor conservation (30-35%). It is suggested that these strongly conserved regions are of special importance to the function of this enzyme, and the findings are discussed in the light of evolutionary theories on the origin of nif genes.     

The dependence on iron availability of allocation of iron to nitrogenase components in Klebsiella pneumoniae and Escherichia coli.

Nitrogenase contains approximately 38 iron ions/complete unit. Therefore, we sought to identify steps and genes involved in nitrogenase production that are responsive to iron availability. We have characterized nitrogenase production in Klebsiella pneumoniae grown in a range of different iron concentrations. We find significant accumulation (50-75%) and normal synthesis rates of the structural polypeptides, even under conditions in which the observed nitrogenase activities are only 14-28% of those observed in iron-sufficient conditions. Thus, maturation instead of synthesis of the structural polypeptides is primarily responsible for the iron dependence of nitrogenase activity. We have also used a binary plasmid system in Escherichia coli to investigate the contributions of various nitrogen fixation (nif) genes to the iron dependence of nitrogenase production. At least one of the nif genes DKTYENXUSVW can modulate synthesis of the structural polypeptide NIF H in response to iron availability. We speculate that an iron-deficient complex of the product(s) of at least one of these genes may repress structural polypeptide synthesis in iron-depleted K. pneumoniae. Such a system would compensate for the inactivity of NIF L in iron-depleted cultures and ensure balanced production of the structural polypeptides of nitrogenase in accordance with the iron available for their maturation.     

Microcin-mediated interactions between Klebsiella pneumoniae and Escherichia coli strains

Amensal indirect interactions between a Klebsiella pneumoniae microcin-producing strain and several Escherichia coli strains, all of intestinal origin, were studied. Mixed batch cultures of both microcin-producing and microcin-sensitive strains showed that microcin production and excretion into the medium allowed the producer strain to prevail over sensitive strains, even when initial competition conditions were highly unfavourable for the producer. Mixed cultures also showed the production of a microcin-antagonist by the same microcin-producing strain when the nutrients in the medium had been depleted. The antagonist apparently promoted the viability of sensitive cells already damaged by microcin. These results have likely ecological implications.     

Electron transport to nitrogenase in Klebsiella pneumoniae: purification and properties of the nifJ protein

In Klebsiella pneumoniae, the physiological electron flow to nitrogenase involves specifically, in addition to nitrogenase reductase, the products of the nifF and nifJ genes. The J protein was purified to homogeneity and was found to be an iron-sulfur protein devoid of molybdenum. In its native state, the J protein is a dimer of Mr about 245 000, made up of two subunits of the same molecular weight. It contains about 30 mol iron and 24 mol labile sulfur/mol protein. The addition of J protein to crude extracts of a nifJ mutant reestablishes pyruvate-supported acetylene-reducing activity. This activity is further enhanced by addition of pure nitrogenase (Kp1). Based on its physical properties, the J protein is probably an oxidoreductase whose physiological role might be to transfer electrons from a metabolic donor to the F protein. In addition, another protein whose activity is also dependent on the nifJ gene seems to be required for the formation of a fully active Kp1.     

Cloning of Klebsiella pneumoniae pqq genes and PQQ biosynthesis in Escherichia coli

We have cloned genes from Klebsiella pneumoniae which are required for pyrroloquinoline quinone (PQQ) biosynthesis. The cloned 6.7 kb fragment can complement several chromosomal pqq mutants. Escherichia coli strains are unable to synthesize PQQ but E. coli strains containing the cloned 6.7 kb K. pneumoniae fragment can synthesize PQQ in large amounts and E. coli pts mutants can be complemented on minimal glucose medium by this clone.     

The molybdenum--iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ''mapping''.

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide 'maps' after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.     

The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation.

A comprehensive model for the mechanism of nitrogenase action is used to simulate pre-steady-state kinetic data for H2 evolution in the presence and in the absence of N2, obtained by using a rapid-quench technique with nitrogenase from Klebsiella pneumoniae. These simulations use independently determined rate constants that define the model in terms of the following partial reactions: component protein association and dissociation, electron transfer from Fe protein to MoFe protein coupled to the hydrolysis of MgATP, reduction of oxidized Fe protein by Na2S2O4, reversible N2 binding by H2 displacement and H2 evolution. Two rate-limiting dissociations of oxidized Fe protein from reduced MoFe protein precede H2 evolution, which occurs from the free MoFe protein. Thus Fe protein suppresses H2 evolution by binding to the MoFe protein. This is a necessary condition for efficient N2 binding to reduced MoFe protein.     

Klebsiella pneumoniae nitrogenase: formation and stability of putative beryllium fluoride-ADP transition state complexes.

Incubation of the MoFe protein (Kp1) and Fe protein (Kp2), the component proteins of Klebsiella pneumoniae nitrogenase, with BeF(3)(-) and MgADP resulted in a progressive inhibition of nitrogenase activity. We have shown that at high Kp2 to Kp1 molar ratios this inhibition is due to the formation of an inactive complex with a stoichiometry corresponding to Kp1.{Kp2.(MgADP.BeFx)2}2. At lower Kp2:Kp1 ratios, an equilibrium between this 2:1 complex, the partially active 1:1 Kp1.Kp2.(MgADP. BeFx)2 complex, and active nitrogenase components was demonstrated. The inhibition was reversible since incubation of the 1:1 complex in the absence of MgADP and beryllium resulted in complete restoration of activity over 30 h. Under pseudo-first-order conditions with regard to nitrogenase components and MgADP, the kinetics of the rate of inhibition with increasing concentrations of BeF(3)(-) showed a square dependence on [BeF(3)(-)], consistent with the binding of two Be atoms by Kp2 in the complex. Analytical fplc gel filtration profiles of Kp1.Kp2 incubation mixtures at equilibrium resolved the 2:1 complex and the 1:1 complex from free Kp1. Deconvolution of the equilibrium profiles gave concentrations of the components allowing constants for their formation of 2.1 x 10(6) and 5.6 x 10(5) M(-1) to be calculated for the 1:1 and 2:1 complexes, respectively. When the active site concentration of the different species was taken into account, values for the two constants were the same, indicating the two binding sites for Kp2 are the same for Kp1 with one or both sites unoccupied. The value for K(1) we obtain from this study is comparable with the value derived from pre-steady-state studies of nitrogenase. Analysis of the elution profile obtained on gel filtration of a 1:1 ratio incubation mixture containing 20 microM nitrogenase components showed 97% of the Kp2 present initially to be complexed. These data provide the first unequivocal demonstration that Fe protein preparations which may contain up to 50% of a species of Fe protein defective in electron transfer is nevertheless fully competent in complex formation with MoFe protein.     

Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae

Yoch, D. C. (South Dakota State University, Brookings), and R. M. Pengra. Effect of amino acids on the nitrogenase system of Klebsiella pneumoniae. J. Bacteriol. 92:618-622. 1966.-The effect of exogenous amino acids and the free amino acid pool on the synthesis of the nitrogenase system of Klebsiella pneumoniae M5al (formerly Aerobacter aerogenes M5al) was investigated. When an actively N(2)-fixing culture was used to inoculate a medium containing a limiting concentration of NH(4) (+), an induction lag period was observed. When either a single amino acid or a mixture of amino acids was substituted at the same nitrogen concentration, growth was uninterrupted by the induction period. It appears that a step or steps in the formation of the nitrogenase system are repressed by NH(4) (+) and are not affected by amino acid N. The amino acids, far from repressing formation of nitrogenase as does NH(4) (+), actually stimulate its formation. It appears that both free and amino nitrogen are used simultaneously. The amino acids that served concomitantly with N(2) as a source of nitrogen were: aspartic acid, serine, threonine, leucine, and histidine. Of these amino acids, it was shown that aspartic acid is readily taken up by the cells. Of the amino acids not serving as an immediate nitrogen source, isoleucine is not taken up by the cells. The free amino acid pool of the cells was measured at the onset and termination of the induction period. Ninhydrin-positive material in the amino acid pool was depleted by 35% during the induction period.     

P1 bacteriophage and tellurite sensitivity in Klebsiella pneumoniae and Escherichia coli

Klebsiella pneumoniae and Escherichia coli respond inversely toward P1 bacteriophage or TeO3(-2). Klebsiella pneumoniae is resistant to both antagonists and E. coli is sensitive. However, P1 cmts lysogens (P1 cmts resistant) of K. pneumoniae became sensitive to tellurite and when cured from P1 cmts regained resistance. Escherichia coli spontaneous mutants selected for resistance to either P1 or TeO3(-2) were collaterally resistant to the other. As well, TeO3(-3) enhanced the adsorption of P1 vir to both E. coli and K. pneumoniae. Several outer membrane proteins were enhanced in the K. pneumoniae lysogens and were reduced in E. coli lysogens; one of which was the same molecular weight (77 000) in both bacteria. When partially purified it enhanced the plaque efficiency of P1 vir. Lipopolysaccharide (LPS) from E. coli C600 inactivated P1 vir, but neither the P1 lysogens nor LPS derived from the lysogens inactivated P1 vir. Escherichia coli P1 lysogens produced only heptose-deficient LPS. It is suggested that both LPS and outer membrane protein(s) comprise the P1 receptor. TeO3(-2) may interact with one or both components.     

Citrate substitutes for homocitrate in nitrogenase of a nifV mutant of Klebsiella pneumoniae

An organic acid extracted from purified dinitrogenase isolated from a nifV mutant of Klebsiella pneumoniae has been identified as citric acid. H2 evolution by the citrate-containing dinitrogenase is partially inhibited by CO, and by some substrates for nitrogenase. The response of maximum velocities to changes in pH for both the wild-type and the NifV- dinitrogenase was compared. No substantial differences between the enzymes were observed, but there are minor differences. Both enzymes are stable in the pH range 4.8-10, but the enzyme activities dropped dramatically below pH 6.2.     

Amino acids as repressors of nitrogenase biosynthesis in Klebsiella pneumoniae.

Nitrogenase biosynthesis in Klebsiella pneumoniae including mutant strains, which produce nitrogenase in the presence of NH+4 (Shanmugam, K.T., Chan, Irene, and Morandi, C. (1975) Biochim. Biophys. Acta 408, 101--111) is repressed by a mixture of L-amino acids. Biochemical analysis shows that glutamine synthetase activity in strains SK-24, SK-28, and SK-29 is also repressed by amino acids, with no detectable effect on glutamate dehydrogenase. Among the various amino acids, L-glutamine in combination with L-aspartate was found to repress nitrogenase biosynthesis completely. In the presence of high concentrations of glutamine (1 mg/ml) even NH+4 repressed nitrogenase biosynthesis in the strains SK-27, SK-37, SK-55 and SK-56. Under these conditions, increased glutamate dehydrogenase activity was also detected. Physiological studies show that nitrogenase derepressed strains are unable to utilize NH+4 as sole source of nitrogen for biosynthesis of glutamate for biosynthesis of glutamate, whereas back mutations leading to NH+4 utilization results in sensitivity to repression by NH+4. These findings suggest that amino acids play an important role as regulators of nitrogen fixation.