nitrogenase and methylamine

A series of Rhodobacter capsulatus AmtB variants were created and assessed for effects on ammonia transport, formation of AmtB-GlnK complexes, and regulation of nitrogenase activity and NifH ADP-ribosylation. Confirming previous reports, H193 and H342 were essential for ammonia transport and the replacement of aspartate 185 with glutamate reduced ammonia transport. Several amino acid residues, F131, D334, and D335, predicted to be critical for AmtB activity, are shown here for the first time by mutational analysis to be essential for transport. Alterations of the C-terminal tail reduced methylamine transport, prevented AmtB-GlnK complex formation, and abolished nitrogenase switch-off and NifH ADP-ribosylation. On the other hand, D185E, with a reduced level of transport, was capable of forming an ammonium-induced complex with GlnK and regulating nitrogenase. This reinforces the notions that ammonia transport is not sufficient for nitrogenase regulation and that formation of an AmtB-GlnK complex is necessary for these processes. However, some transport-incompetent AmtB variants, i.e., F131A, H193A, and H342A, form ammonium-induced complexes with GlnK but fail to properly regulate nitrogenase. These results show that formation of an AmtB-GlnK complex is insufficient in itself for nitrogenase regulation and suggest that partial ammonia transport or occupation of the pore by ammonia is essential for this function.

Topics: Ammonia; Bacterial Proteins; Biological Transport; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Bacterial; Immunoblotting; Methylamines; Mutagenesis, Site-Directed; Mutation; Nitrogenase; Oxidoreductases; Rhodobacter capsulatus

Rhodobacter capsulatus possesses two genes potentially coding for ammonia transporters, amtB and amtY. In order to better understand their role in the physiology of this bacterium and their possible significance in nitrogen fixation, we created single-knockout mutants. Strains mutated in either amtB or amtY did not show a growth defect under any condition tested and were still capable of taking up ammonia at nearly wild-type rates, but an amtB mutant was no longer capable of transporting methylamine. The amtB strain but not the amtY strain was also totally defective in carrying out ADP-ribosylation of Fe-protein or the switch-off of in vivo nitrogenase activity in response to NH(4)(+) addition. ADP-ribosylation in response to darkness was unaffected in amtB and amtBY strains, and glutamine synthetase activity was normally regulated in these strains in response to ammonium addition, suggesting that one role of AmtB is to function as an ammonia sensor for the processes that regulate nitrogenase activity.

Topics: Adenosine Diphosphate; Ammonia; Bacterial Proteins; Carrier Proteins; Darkness; Glutamate-Ammonia Ligase; Membrane Proteins; Methylamines; Mutation; Nitrogen Fixation; Nitrogenase; Nonheme Iron Proteins; Rhodobacter capsulatus

Wild-type and three altered Azotobacter vinelandii nitrogenase MoFe proteins, with substitutions either at alpha-195(His) (replaced by alpha-195(Asn) or alpha-195(Gln)) or at alpha-191(Gln) (replaced by alpha-191(Lys)), were used to probe the interactions of HCN and CN(-), both of which are present in NaCN solutions at pH 7.4, with nitrogenase. The first goal was to determine how added C(2)H(2) enhances the rate of CH(4) production from HCN reduction by wild-type nitrogenase. In the absence of C(2)H(2), wild-type Mo-nitrogenase showed a declining total electron flux, which is an overall measure of all products formed, as the NaCN concentration was increased from 1 to 5 mM, whereas the rates of both CH(4) and NH(3) production increased with increasing NaCN concentration. The NH(3) production rate exceeded the CH(4) production rate up to 5 mM NaCN, at which point they became equal. The "excess NH(3)" likely arises from the two-electron reduction of HCN to CH(2)=NH, some of which is released and hydrolyzed to HCHO plus NH(3). With added C(2)H(2), the rate of CH(4) production increased but only until it equaled that of NH(3) production, which remained unchanged. In addition, total electron flux was decreased even more at each NaCN concentration by C(2)H(2). The increased CH(4) production did not arise from the added C(2)H(2). The lowered total electron flux with C(2)H(2) present would decrease the affinity of the enzyme for HCN, making it a poorer competitor for the binding site. Thus, less CH(2)=NH would be displaced, more CH(2)=NH would undergo the full six-electron reduction, and the rate of CH(4) production would be enhanced. A second goal was to gain mechanistic insight into the roles of the amino acid residues in the alpha-subunit of the MoFe protein at positions alpha-191 and alpha-195 in substrate reduction. At 5 mM NaCN and in the presence of excess wild-type Fe protein, the specific activity for CH(4) production by the alpha-195(Asn), alpha-195(Gln), and alpha-191(Lys) MoFe proteins was 59%, 159%, and 6%, respectively, of that of wild type. For the alpha-195(Asn) MoFe protein, total electron flux decreased with increasing NaCN concentration like wild type. However, the rates of both CH(4) and NH(3) production were maximal at 1 mM NaCN, and they remained unequal even at 5 mM NaCN. With the alpha-195(Gln) MoFe protein, the rates of production of both CH(4) and NH(3) were equal at all NaCN concentrations, and total electron flux was hardly affected by

Topics: Acetylene; Amino Acid Substitution; Azotobacter vinelandii; Carbon Monoxide; Cyanides; Enzyme Inhibitors; Ethylenes; Hydrogen Cyanide; Methane; Methylamines; Molybdoferredoxin; Nitrogenase; Oxidation-Reduction; Protons; Sodium Cyanide; Substrate Specificity

Spontaneous mutants resistant to methionine sulfoximine (Msx), methyl alanine (Mal) and methyl ammonium chloride (Mac) were derived from A. chroococcum strain A103. Msx and Mal-resistant mutants expressed 1.73 to 10.98% of the fully derepressed nitrogenase activity when grown in Burk's medium containing ammonium acetate. Mac-resistant mutants did not express nitrogenase activity in ammonium acetate supplemented medium. The mutants excreted ammonia even after 2 days of growth and some mutants excreted more ammonia as compared to the parent. Selected mutants were inoculated on wheat (Triticum aestivum) and barley (Hordeum vulgare) under field conditions. Majority of the derepressed mutants increased grain yield of wheat and barley varying from 1.2 to 33.3%. However, host-dependent effects on grain yield were observed with different mutants. Two mutants, Mal 27 and Mac 19 showed significant increase in grain yields of both the crops. The results suggest that metabolic analogue-resistant mutants of Azotobacter have potential for use as a biofertilizer for cereal crops.

Topics: Alanine; Ammonia; Azotobacter; Drug Resistance, Microbial; Edible Grain; Methionine Sulfoximine; Methylamines; Mutation; Nitrogen Fixation; Nitrogenase

The Rhodobacter sphaeroides mutants Drn12 and Drn21 derepressed for nitrogenase synthesis in the presence of ammonia and impaired in utilization of certain nitrogen sources have been analyzed. Both mutants show a low level of expression of the glnBA operon. The DNA fragment restoring the wild-type phenotype to these mutants contains the 3'-portion of ntrB gene and the entire ntrC gene. Sequence analysis showed that Drn12 bears a missense mutation in the ntrC gene. The mutation results in the replacement of a glycine residue by aspartate within the N-terminal domain of the NtrC protein. Pleiotropic phenotypes of Drn12 and Drn21 appear to be associated with an alteration in the regulation of glnBA expression.

Topics: Bacterial Proteins; Cloning, Molecular; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genes, Bacterial; Glutamate-Ammonia Ligase; Glutamine; Methylamines; Mutation; Nitrogen; Nitrogenase; Open Reading Frames; PII Nitrogen Regulatory Proteins; Plasmids; Quaternary Ammonium Compounds; Rhodobacter sphaeroides; Sequence Analysis, DNA; Trans-Activators; Transcription Factors

A dansyl chloride precolumn derivatization method has been developed for high-performance liquid chromatography analysis of NH3 and/or CH3NH2 produced by nitrogenase-catalyzed reduction of substrates such as N2 (NH3) or diazirine (NH3, CH3NH2). The dansyl chloride reagent can be used immediately after preparation and is stable at 4 degrees C for 1 month. The derivatization products from NH3 and CH3NH2 are prepared by direct treatment of the assay mixture (30 min of incubation) and are then stable in air and ambient light at room temperature for at least 1 day. They are separated isocratically within several minutes on a mu Bondapak C-18 reversed-phase column (Altex) at 2000-2500 psi using an eluent of 7:7:3 H2O:methanol:acetonitrile and are detected fluorometrically (368 nm excitation; 500 nm emission). The NH3 sensitivity is limited by the background NH3 in the reagents and under practical conditions is ca. 0.02 nmol (20-microliters injection volume). Methylamine sensitivity is an order of magnitude greater. The detector response for either product is linear to at least 2.4 nmol. The method is compared to alternative analytical procedures with respect to sensitivity, convenience, and absence of interferences.

Topics: Ammonia; Azotobacter; Chromatography, High Pressure Liquid; Indicators and Reagents; Methylamines; Microchemistry; Nitrogenase; Spectrometry, Fluorescence

(1) Cyanamide (N identical to C-NH2) has been shown to be a substrate for purified Mo-nitrogenases of Klebsiella pneumoniae and Azotobacter chroococcum, with apparent Km values near 0.8 mM. (2) Reduction products were CH4, CH3NH2 and NH3 formed by pathways requiring 6 or 8 electrons: N identical to CNH2 + 6e + 6H+----CH3NH2 + NH3; N identical to CNH2 + 8e + 8H+----CH4 + 2NH3 (3) Acetylene reduction and hydrogen evolution were inhibited more than 75% by cyanamide (10 mM). Cyanamide also inhibited total electron flux at nitrogenase protein component ratios (Fe/MoFe) near 10. (4) Cyanamide was also a substrate for the recently isolated Va-nitrogenase of A. chroococcum, but with an apparent Km of 2.6 mM showed weaker binding and an 8-fold lower Vmax than did either Mo-nitrogenase. (5) The component ratios of nitrogenase proteins favouring CH4 formation was 3.5 Fe/MoFe protein and 1 Fe/VaFe protein.

Topics: Acetylene; Ammonia; Cyanamide; Cyanides; Hydrogen; Kinetics; Methane; Methylamines; Molybdenum; Nitrogenase; Oxidation-Reduction; Substrate Specificity; Vanadium

In the photosynthetic bacterium Rhodopseudomonas capsulata, NH4+ switch-off of nitrogenase activity can be mimicked by its analog, methylamine. Like NH4+, methylamine appeared to require processing by glutamine synthetase (GS) before it was effective; gamma-glutamylmethylamide was shown to be the product of this reaction. Evidence that this glutamine analog functioned directly to initiate nitrogenase inactivation was suggested first by the fact that it was a poor substrate for glutamate synthase (i.e., it was not further metabolized by this pathway) and secondly, azaserine which blocks the transfer of the glutamine amide group had no effect on CH3NH3+ (or NH4+) switch-off. These observations are taken as preliminary evidence to suggest that when NH4+ inhibits nitrogenase activity, inactivation is initiated by glutamine itself, and not a molecule derived from it. Finally, evidence was presented that R. capsulata would use CH3NH3+ as a nitrogen substrate, but lag periods and generation times increased with subsequent passages.

Topics: Ammonia; Glutamates; Methylamines; Nitrogenase; Rhodopseudomonas; Substrate Specificity