Electron transport to nitrogenase in Azotobacter chroococcum: Azotobacter flavodoxin hydroquinone as an electron donor.
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Journal Article↗
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Physiological Study of Azotobacter Chroococcum. I: Influence of Vitamine B (?) and Nucleic Acid on Azotobacter.
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Journal Article↗
Homologous structural genes and similar induction patterns in Azotobacter spp. and Pseudomonas spp.
Intergeneric comparison of the three enzymes that initiate metabolism of protocatechuate in Azotobacter and Pseudomonas species revealed close immunological relatedness of isofunctional proteins. Furthermore, beta-ketoadipate induces all of the enzymes of the protocatechuate pathway (except protocatechuate oxygenase) in Azotobacter and in Pseudomonas species of the "fluorescent" and "cepacia" groups. This regulatory property sets the organisms apart from other bacteria. Protocatechuate oxygenase from Pseudomonas cepacia, like the enzyme from fluorescent Pseudomonas species, cross-reacts strongly with antiserum prepared against protocatechuate oxygenase from Azotobacter vinelandii. Double-diffusion experiments conducted with the antiserum revealed relatedness of Azotobacter spp. Protocatechuate oxygenases in the following order: A. vinelandii = Azotobacter miscellum greater than Azotobacter chroococcum greater than Azotobacter beijerinkii. The antiserum also revealed serological heterogeneity among Pseudomonas spp. protocatechuate oxygenases which were serologically indistinguishable in earlier studies using Pseudomonas aeruginosa protocatechuate oxygenase as reference protein.
Azotobacter↗
Batch biological treatment of nitrogen deficient synthetic wastewater using Azotobacter supplemented activated sludge.
Biological treatment of nitrogen deficient wastewaters are usually accomplished by external addition of nitrogen sources to the wastewater which is an extra cost item. As an alternative for effective biological treatment of nitrogen deficient wastewaters, the nitrogen fixing bacterium, Azotobacter vinelandii, was used in activated sludge and also in pure culture. Total organic carbon (TOC) removal performances of Azotobacter-added and free activated sludge cultures were compared at different initial TN/TOC ratios. The rate and extent of TOC removal were comparable for all cultures when initial TN/TOC ratio was larger than 0.12; however, both the rate and extent of TOC removal from nitrogen deficient (TN/TOC<12%) synthetic wastewater were improved by using Azotobacter-added activated sludge as compared to the Azotobacter-free activated sludge culture. More than 90% TOC removal was obtained with pure Azotobacter or Azotobacter-added activated sludge culture from a nitrogen deficient synthetic wastewater.
Azotobacter↗
In vitro nitrogen fixation, phosphate solubilization, survival and nutrient release by Azotobacter strains in an aquatic system.
In vitro nitrogen fixation, phosphate solubilization, survival and nutrient release by the three strains of Azotobacter (Mac-27, nitrogen fixer; PS-21, phosphate solubilizer and MSX-9, a phytohormone producer) in an aquatic system using three different organic substrates (cow-dung, piggery and poultry waste/excreta) were studied. It was shown that Azotobacter can grow and multiply in an aquatic system when fertilized either with cow-dung (100 mg 1(-1)), poultry (50 mg 1(-1)) or piggery (25 mg 1(-1)) waste/ excreta. Mac-27 and MSX-9 strains of Azotobacter fixed higher quantities of nitrogen (0.03-0.12 mg 1(-1) d(-1)) in aquaria water treated with cow-dung, while with poultry and piggery excreta, the three strains of Azotobacter fixed 0.03-0.09 mg 1(-1)d(-1) of nitrogen. O-PO4 concentration was also high in microbial-inoculated aquaria as compared to the controls which decreased with respect to time. Similarly, a decrease in total nitrogen concentration was also observed in the aquaria water after the maximum levels of fixation had been reached. Concomitant with the decrease in total nitrogen concentration, a simultaneous increase in NO3-N, NO2-N and NH4-N was observed. Maximum phytoplankton population was observed in MSX-9, while the population of zooplankton was maximum in Mac-27 inoculated aquaria. These studies indicated that Azotobacter can be used as a biofertilizer for enhancing nitrogen input and phosphate solubilization in fish ponds. Microbial inoculation along with organic substrate, however, has to be repeated at least at biweekly intervals.
Animals↗
Bacterial, azotobacter, actinomycetes, and fungal population in soil after diazinon, imidacloprid, and lindane treatments in groundnut (Arachis hypogaea L.) fields.
Bacterial, azotobacter, actinomycetes, and fungal populations were determined in groundnut (Arachis hypogaea L.) fields between July and November for three consecutive years (1997-1999) after insecticide treatments. Diazinon was applied for both seed and soil treatments. However, imidacloprid and lindane were used for seed treatments. An average half-life (t1/2) of diazinon in seed- and soil-treated fields was found to be 29.32 and 34.87 days, respectively. Its residues were found for 60 days in both cases. In diazinon seed treatment, an increase in azotobacter, fungi, and actinomycetes populations was observed in samples from the 15th and 30th days, and this trend continued until crop harvest. However, the bacterial population had not been affected by this treatment. The diazinon soil treatment had indicated some significant adverse effects on fungi and actinomycetes population, which recovered after 30 days. The population of bacteria and azotobacter increased significantly in this treatment. The residues of imidacloprid and lindane were found for 90 and 120 days with an average half-life of 40.9 and 53.3 days, respectively. Imidacloprid had no significant effect on fungi and actinomycetes populations up to 15 days, and between 15 to 60 days some adverse effects were indicated. However, some significant increases in bacterial and azotobacter population were observed. Lindane had no effect on bacterial and fungal population. However, its adverse effects were observed in actinomycetes and azotobacter populations between 30 to 60 days.
Actinobacteria↗
Interactions between Azotobacter and "phosphobacteria" and their establishment in the rhizosphere as affected by soil fertility.
The effects on plant growth of "bacterial fertilizers" prepared from Azotobacter spp. and phosphate-solubilizing bacteria ("phosphobacteria") have been the subject of much controversy. Cases where no plant-growth stimulation occurred may often be accounted for by the failure to establish the bacterial inocula in the rhizosphere. Three factors that may influence inocula establishment, i.e. soil fertility, manuring, and interactions between Azotobacter and "phosphobacteria," were examined in pot experiments, designed for statistical analysis, in two neutral-alkaline soils, using lavender plants (Lavandula spica L.). During the experiments the numbers of Azotobacter and "phosphobacteria" were counted. Dry weights of roots and shoots were recorded after 16 weeks of growth. At the end of the experiments there were always more Azotobacter and "phosphobacteria" in the rhizospheres when plants were inoculated with both groups of organisms together than when they were inoculated singly. Addition of 2% farmyard manure to the richer soil enhanced this effect. Plant growth was greatest when seedlings were inoculated with both Azotobacter and the "phosphobacteria".
Azotobacter↗
Transformation of Azotobacter vinelandii strains unable to fix nitrogen with Rhizobium spp. DNA.
The phenotypes of Azotobacter vinelandii ATCC 12837 strains defective in nitrogen fixation (Nif-) were characterized by intrageneric transformation with known Nif- strains of A. vinelandii OP. These former mutant strains were used as recipients for intergeneric transformation by deoxyribonucleic acid (DNA) prepared from Rhizobium spp. to determine if the rhizobia would transform the Azotobacter Nif- phenotypes to Nif+. The frequency of Nif+ transformants using Rhizobium DNA was always less than the frequency using Azotobacter wild-type DNA but was greater than the spontaneous reversion frequency. The Azotobacter Nif+ recombinants also were stable. DNA from all of the Rhizobium spp. transformed to Nif+ Azotobacter mutants defective in the nitrogenase component I (molybdoferredoxin); however, some recombinants had a lower nitrogenase activity and a delayed nitrogenase depression time. Mutants defective in the pleiotrophic transcriptional control of both nitrogenase components were transformed to Nif+ by the asymbiotic nitrogen fixing Rhizobium sp. 32H1 and 41A1, but not the symbiotic nitrogen-fixing species. The significance of these results and the possible future applications of this system are discussed.
Azotobacter↗
PATTERNS OF OXIDATIVE ASSIMILATION IN STRAINS OF ACETOBACTER AND AZOTOBACTER.
Tomlinson, Geraldine A. (The University of British Columbia, Vancouver, B.C., Canada), and J. J. R. Campbell. Patterns of oxidative assimilation in strains of Acetobacter and Azotobacter. J. Bacteriol. 86:1165-1172. 1963.-Oxidative assimilation of glucose-U-C(14) was studied with washed-cell suspensions of Acetobacter aceti, A. xylinum, Azotobacter vinelandii, and A. agilis. The suggestion that oxidative assimilation is largely the incorporation of endogenously produced ammonia is tenable. A. aceti did not exhibit oxidative assimilation and it did not incorporate ammonia in the presence of glucose, alpha-ketoglutarate, or pyruvate. A. xylinum, A. vinelandii, and A. agilis incorporated C(14) into the nitrogenous fractions of the cell. The level of assimilation into A. xylinum was low due to the accumulation of extracellular cellulose, and the level of assimilation into the Azotobacter was low presumably because of the requirement of energy for nitrogen fixation. The Azotobacter were characterized by the presence of a high level of radioactivity in the cold trichloroacetic acid-soluble pool. None of the organisms accumulated compounds in the supernatant fluid that might be considered pacemakers in glucose oxidation, and this could be a contributing factor in the low level of assimilation.
Acetobacter↗
Regulated expression of the nifM of Azotobacter vinelandii in response to molybdenum and vanadium supplements in Burk's nitrogen-free growth medium.
Azotobacter is a diazotrophic bacterium that harbors three genetically distinct nitrogenases referred to as nif, vnf, and anf systems. The nifM is an accessory gene located in the nif gene cluster and is transcriptionally regulated by the NifA. However, Azotobacter mutants that lack NifA are known to synthesize functional NifM and this accessory protein is known to be needed for the activity of nitrogenase-2 and nitrogenase-3. To determine how the transcription of nifM is regulated when Azotobacter is grown under conditions in which nitrogenase-2 or nitrogenase-3 is expressed, we generated an Azotobacter vinelandii strain that carries a nifM:lacZ-kanamycin resistance gene cassette in its chromosome. In this strain the nifM open reading frame was disrupted by the presence of a lacZ-kanamycin resistance gene cassette so that it could not produce active NifM. Moreover, the lacZ gene was placed under the transcriptional control elements of the nifM gene so that the lacZ expression could be used as a marker to determine the extent of expression of the nifM gene under different growth conditions. Our results show that this strain was unable to grow in Burk's nitrogen-free medium supplemented with either molybdenum or vanadium or lacking both metals suggesting that in the absence of functional NifM none of the nitrogenases were active. It was also found that the nifM expression was differentially regulated when the A. vinelandii cells were grown under conditions that activate nitrogenase-2 and nitrogenase-3, as determined by liquid beta-galactosidase activity measurements. These results suggest that the transcriptional activators, VnfA and AnfA, may regulate the nifM expression.
Azotobacter vinelandii↗
Effect of Azotobacter inoculation on plant growth and soil nitrogen.
The validity of seed bacterization with Azotobacter chroococcum in soils of variable densities of naturally present azotobacters was studied. Inoculation of barley grains had no effect on counts of total microflora, neither in rhizosphere nor in root-free soil, but significantly increased Azotobacter population, especially in the rhizosphere. The rate of colonization in the root region was much higher when soil initially harboured low Azotobacter densities. Bacterization improved plant growth and increased soil nitrogen through nitrogen fixation. Nitrogen balance in soils showed higher gains in the inoculated treatments over the uninoculated analogues of 30--98 ppm.
Azotobacter↗
Ecological studies on Azotobacter in Egyptian soils.
The present survey includes 156 representative soil samples. Results obtained confirm the richness of Egyptian soils, particularly the Nile Valley soils, in Azotobacter (60% of the samples contained greater than 10(3) colonies/g soil). Colony counts were lower than MPN estimations. Glucose is recommended for use in plating medium. Among the environmental factors affecting Azotobacter densities in soils of Egypt are: organic carbon content, total soluble salt content, pH and type of the soil, depth, cultivation, and standing crop. Pot experiments performed indicated that high incubation temperature (30, 37 degrees C) markedly enhanced development of Azotobacter as well as the gains of total nitrogen, particularly when soils are amended with 1% maize straw and incubated for 12 months. A. chrococcum was the most prevalent speciee; A. vinelandii was encountered as well. The analysis of some samples, representing soils of near Middle Eastern as well as North African countries, indicated the common occurrence of Azotobacter in these soils. Beijerinckia could not be detected in any of Egyptian or non-Egyptian samples examined.
Azotobacter↗
Growth of Azotobacter vinelandii in dialysed soil medium: studies upon the life cycle.
Azotobacter vinelandii ATCC 12837 cultured in dialysed soil medium with addition of 0.5% glucose showed four distinct morphological cell types: large cells, precyst forms, mature cysts and filterable corpuscles (0.3 micron in diameter). These results indicate that Azotobacter is a bacterium with a complex life cycle under certain culture conditions. Intracellular levels of RNA and poly-beta-hydroxybutyric acid were significantly affected when cells grown in dialysed soil were compared with those obtained after growth on defined medium (N-free). Further studies showed that the chemical composition of filterable corpuscles obtained from dialysed soil medium were different from the composition of normal Azotobacter cells produced in both culture media (dialysed soil and defined media). We suggest that filterable corpuscles represent a stage in the life cycle of Azotobacter in their natural environment.
Azotobacter↗
Azotobacter vinelandii flavodoxin: purification and properties of the recombinant, dephospho form expressed in Escherichia coli.
The nifF gene coding for the flavodoxin from the nitrogen-fixing bacterium Azotobacter vinelandii (strain OP) was cloned into the plasmid vector pUC7 [Bennett, L. T., Jacobsen, M. R., & Dean, D. R. (1988) J. Biol. Chem. 263 1364-1369] and the resulting plasmid transformed and expressed in Escherichia coli strain DH5. Recombinant Azotobacter flavodoxin is expressed at levels 5-6-fold higher in E. coli than in comparable yields of Azotobacter cultures grown under nitrogen-fixing conditions. Even higher levels were observed with flavodoxin expressed in E. coli under control of a tac promoter. Electron spin resonance spectroscopy on whole cells and in cell-free extracts showed the flavodoxin to be largely in the semiquinone form. The flavodoxin purified from E. coli exhibited the same molecular weight, isoelectric point, flavin mononucleotide (FMN) content, N-terminal sequence, and carboxyl-terminal amino acids as for the wild-type Azotobacter protein. The recombinant flavodoxin differed from native flavodoxin in that it exhibited an increased antigenicity to flavodoxin antibody and did not contain a covalently bound phosphate. Small differences are also observed in circular dichroism spectral properties in the visible and ultraviolet spectral regions. The recombinant, dephospho flavodoxin exhibits an oxidized/semiquinone potential (pH 8.0) of -224 mV and a semiquinone/hydroquinone couple (pH 8.0) of -458 mV. This latter couple is 50-60 mV higher than that exhibited by the native flavodoxin. Resolution of recombinant dephospho flavodoxin resulted in an apoflavodoxin that was much less stable than that prepared from the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Intergeneric evolutionary homology revealed by the study of protocatechuate 3,4-dioxygenase from Azotobacter vinelandii.
Protocatechuate 3,4-dioxygenase (EC 1.13.1.3) was purified to homogeneity from extracts of Azotobacter vinelandii. The molecular weight of the oligomeric protein was estimated to be 510 000 by gel filtration and 480 000 by ultracentrifugation. The oligomer appears to be formed by association of equal amounts of nonidentical subunits which were estimated by sodium dodecyl sulfate gel electrophoresis to have respective molecular weights of 23 300 and 25 250. Ten gram-atoms of iron was associated with each mol of oligomer. Therefore, the enzyme appears to be a decamer with the structure 10(alpha beta Fe). T-HE AMINO ACID COMPOSITION OF Azotobacter protocatechuate oxygenase closely resembles the amino acid compositions of protocatechuate 3,4-dioxygenases from Pseudomonas aeruginosa and Thiobacillus sp. These proteins from P. aeruginosa and P. putida are known to be formed by association of nonidentical subunits of a physical size similar to the subunits of the Azotobacter enzyme. Furthermore, antisera prepared against the Azotobacter oxygenase cross-reacted strongly with the isofunctional enzymes from the two fluorescent Pseudomonas species. A weak immunological cross-reaction was observed when the antisera were tested against protocatechuate 3,4-dioxygenase from Acinetobacter calcoaceticus. The results favor the conclusion that the bacterial protocatechuate 3,4-dioxygenases were derived from a common ancestral protein.
Amino Acids↗
The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum.
The structural genes (hupSL) of the membrane-bound NiFe-containing H2-uptake hydrogenase (Hup) of Azotobacter chroococcum were identified by oligonucleotide screening and sequenced. The small subunit gene (hupS) encodes a signal sequence of 34 amino acids followed by a 310-amino-acid, 34156D protein containing 12 cysteine residues. The large subunit gene (hupL) overlaps hupS by one base and codes for a predicted 601-amino-acid, 66433D protein. There are two regions of strong homology with other Ni hydrogenases: a Cys-Thr-Cys-Cys-Ser motif near the N-terminus of HupS and an Asp-Pro-Cys-Leu-Ala-Cys motif near the carboxy-terminus of HupL. Strong overall homology exists between Azotobacter, Bradyrhizobium japonicum and Rhodobacter capsulatus Hup proteins but less exists between the Azotobacter proteins and hydrogenases from Desulfovibrio strains. Mutagenesis of either hupS or hupL genes of A. chroococcum yielded Hup- phenotypes but some of these mutants retained a partial H2-evolving activity. Hybridization experiments at different stages of gene segregation confirmed the multicopy nature of the Azotobacter genome.
Amino Acid Sequence↗
Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii.
The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions. It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given.
Azotobacter↗
Cytochrome c oxidation by the electron transport fraction of Azotobacter vinelandii.
The spectrophotometric oxidation of horse heart ferrocytochrome c was examined by use of the particulate electron transport fraction (R(3)) of Azotobacter vinelandii strain O. Unlike cytochrome c, purified preparations of native Azotobacter cytochromes c(4) + c(5) were oxidized only slowly by the electron transport fraction. The oxidation of mammalian cytochrome c proceeded at an appreciable rate and displayed "apparent" first-order kinetics at a pH optimum of 9.0 with tris(hydroxymethyl)aminomethane-chloride buffer. The calculated V(max) value was 0.22 mumole of cytochrome c oxidized per min per mg of protein (25 C) and a K(m) value for cytochrome c of 2.3 x 10(-5)m was obtained. Ferricytochrome c was a "strict" competitive inhibitor for this oxidation. Cytochrome c oxidation by the Azotobacter electron transport system was markedly sensitive to cyanide, azide, and hydroxylamine, although carbon monoxide inhibition could not be demonstrated. It was sensitive also to high concentrations of phosphate, ethylenediaminetetraacetate, and some metal cations. "Aging" or prolonged storage of the Azotobacter R(3) fraction, at 4 C for 10 days, resulted in a threefold increase in specific activity. The cytochrome c peroxidase type of reaction did not occur with the R(3) electron transport fraction.
Animals↗