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M J Dilworth

Publications and source records attributed to M J Dilworth.

At least 19 recordsLinked to original sources
PubMed1994

Cloning and sequencing show that 4-hydroxybenzoate hydroxylase (PobA) is required for uptake of 4-hydroxybenzoate in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae MNF300 and R. leguminosarum bv. trifolii WU95 unable to accumulate 4-hydroxybenzoate lack 4-hydroxybenzoate hydroxylase. The capacity of these mutants to take up and grow on 4-hydroxybenzoate was restored by a 2.0 kb EcoRI-PstI DNA fragment. This contained only one ORF which had over 60% DNA sequence similarity with the structural gene for 4-hydroxybenzoate hydroxylase (pobA) from Pseudomonas spp. and Acinetobacter. Reported effects of metabolic inhibitors and substrate analogues on the apparent uptake of 4-hydroxybenzoate have now been shown to be due to their direct effect on 4-hydroxybenzoate hydroxylase. We propose that uptake of 4-hydroxybenzoate is via a metabolic 'drag' mechanism dependent on the activity of the pobA gene product.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1992

Correction for creatine interference with the direct indophenol measurement of NH3 in steady-state nitrogenase assays.

Creatine was identified as a major source of interference with the direct phenol/hypochlorite colorimetric determination of ammonia in nitrogenase reaction mixtures. A method is described for removing other compounds which inhibit color development and for compensating for the interference produced by creatine. This method avoids time-consuming microdiffusion and also routinely makes available the efficiency of ATP hydrolysis coupled to substrate reduction (ATP/2e ratio) with N2 as a reducible substrate. Using this method we determined values for this ratio at 30 degrees C of 4.87 +/- 0.03 during the reduction of protons to H2 and 7.16 +/- 0.14 during the reduction of N2 by the vanadium-containing nitrogenase of Azotobacter chroococcum.

Adenosine Triphosphate↗
PubMed1992

Siderophore and organic acid production in root nodule bacteria.

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 microM added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2-5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron. All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 microM added iron). Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 microM added iron) during the stationary phase of growth.

Catechols↗
PubMed1991

Hydrazine is a product of dinitrogen reduction by the vanadium-nitrogenase from Azotobacter chroococcum.

During the enzymic reduction of N2 to NH3 by Mo-nitrogenase, free hydrazine (N2H4) is not detectable, but an enzyme-bound intermediate can be made to yield N2H4 by quenching the enzyme during turnover [Thorneley, Eady & Lowe (1978) Nature (London) 272, 557-558]. In contrast, we show here that the V-nitrogenase of Azotobacter chroococcum produces a small but significant amount of free N2H4 (up to 0.5% of the electron flux resulting in N2 reduction) as a product of the reduction of N2. The amount of N2H4 formed increased 15-fold on increasing the assay temperature from 20 degrees C to 40 degrees C. Activity cross-reactions between nitrogenase components of Mo- and V-nitrogenases showed that the formation of free N2H4 was associated with the VFe protein. These data provide the first direct evidence for an enzyme intermediate at the four-electron-reduced level during the reduction of N2 by V-nitrogenase.

Azotobacter↗
PubMed1990

Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419.

Four Tn5-induced mutants of Rhizobium meliloti WSM419 were unable to grow or maintain intracellular pH at an external pH of 5.6. Restriction analysis of DNA fragments carrying Tn5 and flanking sequences cloned from these mutants indicated that all four cloned mutations are unique and that the two strains (TG1-6 and TG1-11) carry Tn5 insertions which are within 4.4 kilobases of each other on a single EcoRI fragment. Southern analysis of total mutant DNA indicated a single copy of Tn5 in each mutant. A limited cosmid gene bank of wild-type WSM419 DNA was probed for homology to mutant DNA cloned from the acid-sensitive mutants. Dot hybridization experiments identified one cosmid element within this bank carrying wild-type DNA sequences corresponding to DNA implicated in acid tolerance. This cosmid was able to complement defects in growth and intracellular pH maintenance in TG1-11 but not TG1-6.

Cloning, Molecular↗
PubMed1988

The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane.

1. The vanadium (V-) nitrogenase of Azobacter chroococcum transfers up to 7.4% of the electrons used in acetylene (C2H2) reduction for the formation of ethane (C2H6). The apparent Km for C2H2 (6 kPa) is the same for either ethylene (C2H4) or ethane (C2H6) formation and much higher than the reported Km values for C2H2 reduction to C2H4 by molybdenum (Mo-) nitrogenases. Reduction of C2H2 in 2H2O yields predominantly [cis-2H2]ethylene. 2. The ratio of electron flux yielding C2H6 to that yielding C2H4 (the C2H6/C2H4 ratio) is increased by raising the ratio of Fe protein to VFe protein and by increasing the assay temperature up to at least 40 degrees C. pH values above 7.5 decrease the C2H6/C2H4 ratio. 3. C2H4 and C2H6 formation from C2H2 by V-nitrogenase are not inhibited by H2. CO inhibits both processes much less strongly than it inhibits C2H4 formation from C2H2 with Mo-nitrogenase. 4. Although V-nitrogenase also catalyses the slow CO-sensitive reduction of C2H4 to C2H6, free C2H4 is not an intermediate in C2H6 formation from C2H2. 5. Propyne (CH3C identical to CH) is not reduced by the V-nitrogenase. 6. Some implications of these results for the mechanism of C2H6 formation by the V-nitrogenase are discussed.

Acetylene↗
PubMed1987

Klebsiella pneumoniae nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane.

Ethylene (C2H4) inhibited H2 evolution by the Mo-containing nitrogenase of Klebsiella pneumoniae. The extent of inhibition depended on the electron flux determined by the ratio of Fe protein (Kp2) to MoFe protein (Kp1) with KiC2H4 = 409 kPa ([Kp2]/[Kp1] = 22:1) and KC2H4i = 88 kPa ([Kp1]/[Kp2] = 21:1) at 23 degrees C at pH 7.4. At [Kp2]/[Kp1] = 1:1, inhibition was minimal with C2H4 (101 kPa). Extrapolation of data obtained when C2H4 was varied from 60 to 290 kPa indicates that at infinite pressure of C2H4 total inhibition of H2 evolution should occur. C2H4 inhibited concomitant S2O4(2-) oxidation to the same extent that it inhibited H2 evolution. Although other inhibitors of total electron flux such as CN- and CH3NC uncouple MgATP hydrolysis from electron transfer, C2H4 did not affect the ATP/2e ratio. Inhibition of H2 evolution by C2H4 was not relieved by CO. C2H4 was reduced to C2H6 at [Kp2]/[Kp1] ratios greater than or equal to 5:1 in a reaction that accounted for no more than 1% of the total electron flux. These data are discussed in terms of the chemistry of alkyne and alkene reduction on transition-metal centres.

Dithionite↗
PubMed1985

Ammonia movements in rhizobia.

When free-living rhizobia are grown under N-excess conditions they appear to take up ammonia by a diffusive mechanism. Under low or limiting N, they derepress an ammonium permease which serves to scavenge NH4+. Current data suggest that N2-fixing bacteroids lose ammonia by a diffusive movement sustained by the continual removal of ammonia via the plant ammonia assimilatory system(s).

Ammonia↗
PubMed1981

Nitrogenase of Klebsiella pneumoniae. Hydrazine is a product of azide reduction.

Klebsiella pneumoniae nitrogenase reduced azide, at 30 degrees C and pH 6.8-8.2, to yield ammonia (NH3), dinitrogen (N2) and hydrazine (N2H4). Reduction of (15N = 14N = 14N)-followed by mass-spectrometric analysis showed that no new nitrogen-nitrogen bonds were formed. During azide reduction, added 15N2H4 did not contribute 15N to NH3, indicating lack of equilibration between enzyme-bound intermediates giving rise to N2H4 and N2H4 in solution. When azide reduction to N2H4 was partially inhibited by 15N2, label appeared in NH3 but not in N2H4. Product balances combined with the labelling data indicate that azide is reduced according to the following equations: (formula: see text); N2 was a competitive inhibitor and CO a non-competitive inhibitor of azide reduction to N2H4. The percentage of total electron flux used for H2 evolution concomitant with azide reduction fell from 26% at pH 6.8 to 0% at pH 8.2. Pre-steady-state kinetic data suggest that N2H4 is formed by the cleavage of the alpha-beta nitrogen-nitrogen bond to bound azide to leave a nitride (= N) intermediate that subsequently yields NH3.

Ammonia↗
PubMed1976

Synthesis and turnover of leghaemoglobin in lupin root nodules.

1. The problem of whether leghaemoglobin is synthesized on plant of bacterial ribosomes in root nodules of yellow lupin has been examined. 2. Leghaemoglobin, soluble plant protein and soluble bacteroid protein were labelled with 14C administered by uptake of 14CO2. 3. Exposure of roots to 1 mM D-threo-chloramphenicol resulted in inhibition of soluble bacteroid protein synthesis, but leghaemoglobin synthesis and soluble plant protein synthesis were unaffected. This result is consistent with leghaemoglobin being synthesized on plant ribosomes. 4. After nitrogen-fixing plants had been supplied with a pulse of 14CO2, the decay of specific radioactivity of nodule protein fractions was observed. Leghaemoglobin had an apparent half-life of 18 days and is a stable protein in nitrogen-fixing yellow lupin nodules.

Chloramphenicol↗
PubMed1976

Comparative structural and immunochemical properties of leghaemoglobins.

Circular dichroism studies on leghaemoglobins from snake bean, lupin, serradella and other plants show that, in common with soybean (reported earlier) they have a similar overall polypeptide chain conformation and haem environment and orientation. Immunochemical studies, on the other hand, suggest that the antigenic determinants on the surface of the leghaemoglobins vary considerably. Thus, firstly the alpha-helix content of the leghaemoglobins as a class is very similar (60-65%) and approaches that of the myoglobins, secondly, the sign, magnitude and shape of their circular dichroism spectra in the near ultraviolet, Soret and visible regions suggest close similarities in the environment and orientation of a structurally important tryptophan residue and of the haem moiety, and thirdly, there is comparatively weak haem-protein interaction. The extent of immuno cross-reactivity was found to be best deomonstrated using the Farr radioimmunoassay procedure. The results were (a) 5 leghaemoglobins from one plant (soybean) crossreacted completely but with varying affinities. (b) The extent of cross reactivity between leghaemoglobins from different plants was compared to that within a single plant; the reaction of antiserum to a soybean leghaemoglobin with a serradella leghaemoglobin was weak, with a snake bean leghaemoglobin still weaker (and incomplete) while lupin leghaemoglobins showed no cross reactivity at all. (c) The "rapid" attenuation of cross reactivity among different plant leghaemoglobins is explicable in terms of the extensive amino acid substitutions which have been demonstrated in the literature and in the present studies. (d) In view of this rapid divergence it is not surprising that sperm whale and horse heart myoglobins showed no cross reactivity with soybean leghaemoglobins. In summary, amino acid substitutions in the leghaemoglobin family are conformationally but not immunochemically conservative.

Amino Acid Sequence↗
PubMed1975

Ammonia assimilation by rhizobium cultures and bacteroids.

The enzymes involved in the assimilation of ammonia by free-living cultures of Rhizobium spp. are glutamine synthetase (EC. 6.o.I.2), glutamate synthase (L-glutamine:2-oxoglutarate amino transferase) and glutamate dehydrogenase (ED I.4.I.4). Under conditions of ammonia or nitrate limitation in a chemostat the assimilation of ammonia by cultures of R. leguminosarum, R. trifolii and R. japonicum proceeded via glutamine synthetase and glutamate synthase. Under glucose limitation and with an excess of inorganic nitrogen, ammonia was assimilated via glutamate dehydrogenase, neither glutamine synthetase nor glutamate synthase activities being detected in extracts. The coenzyme specificity of glutamate synthase varied according to species, being linked to NADP for the fast-growing R. leguminosarum, R. melitoti, R. phaseoli and R. trifolii but to NAD for the slow-growing R. japonicum and R. lupini. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase activities were assayed in sonicated bacteroid preparations and in the nodule supernatants of Glycine max, Vicia faba, Pisum sativum, Lupinus luteus, Medicago sativa, Phaseolus coccineus and P. vulgaris nodules. All bacteroid preparations, except those from M. sativa and P. coccineus, contained glutamate synthase but substantial activities were found only in Glycine max and Lupinus luteus. The glutamine synthetase activities of bacteroids were low, although high activities were found in all the nodule supernatants. Glutamate dehydrogenase activity was present in all bacteroid samples examined. There was no evidence for the operation of the glutamine synthetase/glutamate synthase system in ammonia assimilation in root nodules, suggesting that ammonia produced by nitrogen fixation in the bacteroid is assimilated by enzymes of the plant system.

Acetylene↗
PubMed1975

Inhibition of protein synthesis by D-threo-chloramphenicol in the laboratory and nodule forms of Rhizobium lupini.

Protein synthesis by both laboratory-grown bacteria and isolated nodule bacteroids of Rhizobium lupini (strain WU8) is inhibited by D-threo-chloramphenicol, the bacteroid form being the more sensitive to the antibiotic. A comparison between the two forms of the uptake of [14C]chloramphenicol showed that the bacteria always attained a lower intracellular chloramphenicol concentration. It is proposed that the sensitivity difference is due to a difference in membrane permeability between the two forms.

Bacterial Proteins↗