Winston J. Brill

Nitrogenase. I. Repression and derepression of the iron-molybdenum and iron proteins of nitrogenase in Azotobacter vinelandii

Abstract Evidence is presented that the dilution effect on nitrogenase from Azotobacter vinelandii can be overcome by the addition of an optimal amount of Component I (iron-molybdenum protein) or Component II (iron protein); and this optimized activity parallels the activity obtained by applying the dilution-factor correction. The synthesis of both of the nitrogenase components, after exhaustion of ammonia from the medium, seems to be coordinate. The degradation of both of the nitrogenase components after repression was found to be coordinate and neither component was found to be in excess at any time after repression. For the initial one-half generation the nitrogenase activity falls at approximately the same rate as the increase in cell mass, suggesting simple dilution. After this point, however, activity falls more rapidly and more than 95 % of the activity is lost in two generations.

Nitrogenase IV. Simple method of purification to homogeneity of nitrogenase components from Azotobacter vinelandii

Extracts of Azotobacter vinelandii have been fractionated by simple techniques to obtain highly purified components of nitrogenase. The yield of each component is greater than 60%. Purified Component I has a specific activity of 1638 nmoles ethylene formed/min per mg protein. The spectrum of Component I exhibits a broad absorption between 300 and 600 nm, with no distinctive peaks or shoulders. Addition of sodium dithionite or exposure to air has no effect on the absorption spectrum. Component I, examined at 4.2 °K has EPR signals at g = 4.2, 3.65 and 2.01. Addition of sodium dithionite does not produce additional resonances nor does it alter the signals already present. Crystals of Component I are dark brown and needle-shaped. Purified Component II has a specific activity of 1815 nmoles ethylene formed/min per mg protein. The absorption spectrum has no peaks or shoulders between 390 and 650 nm. Upon exposure of Component II to air, absorption increases between 400 and 650 nm. Treatment of oxidized Component II with dithionite causes this absorption to fall below that of the native Component II. EPR spectra of Component II has signals at g values of 2.05, 1.94, and 1.88. Upon inactivation by O2, these signals disappear. Neither component by itself has detectable acetylene-reducing or N2-fixing activity. The ratio of acetylene reduced to N2 fixed is 3.86 with different ratios of the components. Both components form aggregated species upon exposure to air. Dithionite does not reverse this effect.

Trifolin: A Rhizobium recognition protein from white clover

A protein agglutinin, trifoliin, was purified from white clover seeds and seedling roots. Trifoliin specifically agglutinates the symbiont of clover, Rhizobium trifolii, at concentrations as low as 0.2 microgram protein/ml, and binds to the surface of encapsulated R. trifolii 0403. This clover protein has a subunit with Mr approximately 50 000, an isoelectric point of 7.3, and contains carbohydrate. Antibody to purified trifoliin binds to the root hair region of 24-h-old clover seedlings, but does not bind to alfalfa, birdsfoot trefoil or joint vetch. The highest concentration of trifoliin on a clover root is present at sites where material in the capsule of R. trifolii binds. 2-Deoxy-D-glucose elutes trifoliin from intact clover-seedling roots, suggesting that this protein is anchored to root cell walls through its carbohydrate binding sites. We propose that trifoliin on the root hair surface plays an important role in the recognition of R. trifolii by clover.

Derepression of nitrogenase synthesis in the presence of excess NH4

Summary Methionine sulfone and methionine sulfoximine, inhibitors of enzymes involved in NH 4 + assimilation, cause nitrogenase to be synthesized in the presence of excess NH 4 + in Azotobacter vinelandii and Klebsiella pneumoniae . These inhibitors also cause NH 4 + excretion by A . vinelandii . These results indicate that NH 4 + alone is not the actual effector of nitrogenase repression in these organisms.

Nitrogenase. III. Nitrogenaseless mutants of Azotobacter vinelandii: Activities, cross-reactions and epr spectra

Abstract Mutant strains of Azotobacter vinelandii that are unable to fix nitrogen were analyzed for their ability to reduce acetylene and oxidize dithionite. The activities of Components I (Fe-Mo-protein) and II (Fe-protein), the presence of antibody cross-reacting material to each of the components and the electron paramagnetic resonance (EPR) intensities at g = 3.65 also were examined in these strains. All mutant strains so far studied that are unable to reduce nitrogen, are also incapable of reducing acetylene or oxidizing dithionite. Representatives of various nitrogenaseless mutants have been characterized. Based on activity measurements they fall into three classes: those lacking both components (I − II − ), those lacking Component I (I − II + ) and those lacking Component II (I + II − ). Many strains have extremely low levels of activity for either component, but in some of these strains, cross-reacting material is made for one or both of the components. The EPR at g = 3.65 correlates well with the activity for Component I in several of these mutant strains, but in four of the mutants there appears to be 10-20-fold higher amounts of paramagnetic center than the nitrogen-fixing activity in in vitro tests would indicate.

Mutant Strains of Rhizobium japonicum with Increased Ability to Fix Nitrogen for Soybean.

A strain of Rhizobium japonicum used in commercial inoculants was mutagenized and screened by a rapid effectiveness assay with soybean plants. Two mutant strains nodulated the roots earlier than the wild type and also expressed greater symbiotic nitrogen-fixing activity than the wild type in the presence and absence of fixed nitrogen. In addition, one of the mutants formed more root nodules than the wild type. Plants inoculated with these strains had increased dry weights (∼60 percent) and nitrogen content (∼100 percent) when grown in growth chambers.

Transient appearance of lectin receptors onRhizobium trifolii

The appearance on the surface ofRhizobium trifolii 0403 of determinants important to both clover lectin (trifoliin) binding and adherence of the bacteria to clover root epithelial surfaces was studied by quantitative agglutination, immunofluorescence, and direct microscopic techniques. These unique determinants were found for only transient periods of time—as cells left lag phase and as they entered stationary phase of growth in broth. When present, these receptors were associated with a fibrillar polyanionic capsule surrounding the cells when grown on solid medium. These studies support earlier proposals that the architecture of the rhizobial cell surface is not constant in composition, but changes with the phase of growth.

Nitrogenase II. Changes in the EPR signal of Component I (iron-molybdenum protein) of Azotobacter vinelandii nitrogenase during repression and derepression

Abstract The purified, native iron-molybdenum protein (Component I) of Azotobacter vinelandii nitrogenase shows EPR signals at g values of 4.32, 3.65, and 2.01, when observed below 40° K. The resonances near g = 4.32 and 3.65 are unobscured when whole nitrogen-fixing cells are observed, and we have compared the appearance and disappearance of these resonances with nitrogenase activity as well as Component I protein determined immunochemically, during derepression and repression of nitrogenase. The characteristic resonances are absent in cells grown on ammonium salts. During derepression the activity, EPR signal, and cross-reacting material appear and pass through a maximum in parallel. During repression the immunochemically-detectable protein decays inversely with cell growth, while the activity and signal are depressed considerably more rapidly. This suggests that a short-term control mechanism, including the substantial alteration of the paramagnetic center of Component I without elimination of the antigenic moiety, is responsible for rapid repression of nitrogenase by ammonia. EPR signals similar to those of Component I in Azotobacter were also observed at low temperatures in nitrogen-fixing cells of Clostridium pasteurianum, Klebsiella pneumoniae, and Bacillus polymyxa, suggesting that in vivo studies of Component I of nitrogenase may be feasible generally.

Nitrogenase: VII. Effect of component ratio, ATP and H2, on the distribution of electrons to alternative substrates

Some kinetic properties of purified component I (Mo-Fe protein) and component II (Fe protein) of nitrogenase (EC 1.7.99.2) from Azotobacter vinelandii have been examined. The apparent Km values for reducible substrates (0.1 atm for N2, 0.01 atm for acetylene) and dithionite (0.5 mM) are similar for osmotically shocked cell lysates and purified components. However, the ATP dependence of acetylene and N2 reduction varies sigmoidally with ATP concentration and as a function of the relative and absolute concentration of components I and II in the assay. Acetylene is reduced in preference to N2 in competitive assays when component I is in relative excess. Acetylene reduction is not as dependent upon ATP concentration as is N2 reduction, so that acetylene is also a preferred substrate at lower ATP levels. Hydrogen specifically inhibits N2 reduction, diverting electrons to acetylene when both substrates are present in the assay. We propose a model of the enzyme activity, in which the substrates for reduction are bound to component I with electrons being activated by component II. ATP may be involved in activating electrons and in maintaining the appropriate conformation or reduction state of components to allow effective reduction of substrates. The relative rate of reduction of alternative substrates is dependent on the concentration of the particular state(s) capable of reacting with each substrate. The concentration of a particular state of component I is a function of components I, II and ATPL

Acetylene reduction by the iron-molybdenum cofactor from nitrogenase

Abstract The iron-molybdenum cofactor isolated from component I of nitrogenase catalyzes the reduction of acetylene to ethylene in the presence of sodium borohydride. Like nitrogenase, this activity is strongly inhibited by carbon monoxide. From the initial rates of the reaction, the specific activity of the iron-molybdenum cofactor is 34 nmoles of ethylene formed per minute per nmole of Mo in the cofactor. This activity is about 8% of the activity of nitrogenase with an equivalent amount of Mo. ATP has no stimulatory effect on the cofactor activity.