Effects of Mannose on the Growth of N(2)-Fixing Azotobacter vinelandii

Mannose is not a suitable substrate for N(2)-fixing Azotobacter vinelandii. However, when H(2) gas is provided, A. vinelandii can grow mixotrophically with H(2) as the energy source and mannose as the carbon source (T.-Y. Wong and R. J. Maier, J. Bacteriol. 163:528-533, 1985). In this report, seven sugars were used to determine whether A. vinelandii could derive energy from these sugars for mannose utilization. Supplementation of fructose- or galactose-limited medium with mannose did not influence the biomass produced by N(2)-fixing A. vinelandii. The presence of mannose in glucose- or maltose-limited cultures increased cell yield slightly. The addition of mannose decreased the total biomass in the melibiose-limited culture slightly. Mannose was a potent inhibitor of growth when sucrose or turanose was used as the primary sugar. The inhibitory effect of mannose on utilization of sucrose and turanose seems to be related to the energy requirement of the N(2)-fixing processes.     

Growth of Azotobacter vinelandii on Soil Nutrients

Azotobacter vinelandii cells grew well in a medium made from soil and distilled water which contained little or no carbohydrate. They utilized p-hydroxybenzoic acid and other phenolic acids, soil nitrogen, and water-soluble mineral substances. Seventeen soils which supported excellent growth of A. vinelandii contained 11 to 18 different phenolic acids each, including p-hydroxybenzoic, m-hydroxybenzoic, vanillic, p-coumeric, syringic, cis- and trans-ferrulic, and other unidentified aromatic acids. Three white, chalky “caliche” soils which were taken from areas where no plants grew failed to support the growth of A. vinelandii, and these contained no, two, and three phenolic acids, respectively. A. vinelandii did not fix nitrogen when growing in dialysates of soils which contained numerous phenolic acids. Growth was ample and rapid in most of the soils tested, but cell morphology was different from that usually seen in chemically defined, nitrogen-free media which contain glucose.     

N2O Reduction by Azotobacter vinelandii with Emphasis on Kinetic Nitrogen Isotope Effects

A nitrogen-fixing bacterium Azotobacter vinelandii was successfullygrown in a specially designed system with constant partial pressuresof N₂O (0.2 atm) and O₂ (0.2 atm) in a nitrogen-free liquidmedium. Reduction of N₂O proceeded with the evolution of N₂in the gas phage. Large nitrogen isotope fractionation was found for both processes,reduction of N₂O to N₂ and N₂O-fixation. The kinetic isotopefractionation factors of these reactions were at most 1.039and 1.034, respectively. Furthermore, an unexpected inverseisotope effect (organic-N, the end-product, is more enrichedin ¹⁵N than N₂, the intermediate) strongly suggested that N₂Owas directly assimilated within the bacterial cells. Simultaneousassimilation of N₂O and N₂ was also confirmed by using a ¹⁵Ntracer technique. Three independent pathways were demonstrated for the nitrogenfixing system investigated in this study: (1) a direct reductionof N₂O to ammonium (apparently 8-electron reduction), (2) reductionof N₂ to ammonium (6-electron reduction) and (3) N₂O reductionto N₂ (2-electron reduction). ³ Present address: Department of Environmental Sciences, Facultyof Integrated Arts and Sciences, Hiroshima University, Hiroshima730, Japan ⁴ Present address: Department of Earth Sciences, Faculty ofScience, Toyama University, Gofuku, Toyama 930, Japan (Received June 18, 1986; Accepted December 16, 1986)     

H2-dependent mixotrophic growth of N2-fixing Azotobacter vinelandii.

Azotobacter vinelandii can grow with a variety of organic carbon sources and fix N2 without the need for added H2. However, due to an active H2-oxidizing system, H2-dependent mixotrophic growth in an N-free medium was demonstrated when mannose was provided as the carbon source. There was no appreciable growth with either H2 or mannose alone. Both the growth rate and the cell yield were dependent on the concentrations of both substrates, H2 and mannose. Cultures growing mixotrophically with H2 and mannose consumed approximately 4.8 mmol of O2 and produced 4.6 mmol of CO2 per mmol of mannose consumed. In the absence of H2, less CO2 was produced, less O2 was consumed, and cell growth was negligible. The rate of acetylene reduction in mixotrophic cultures was comparable to the rate in cultures grown in N-free sucrose medium. The rate of [14C]mannose uptake of cultures with H2 was greater than with argon, whereas [14C]sucrose uptake was unaffected by the addition of H2; therefore, the role of H2 in mixotrophic metabolism may be to provide energy for mannose uptake. A. vinelandii is not an autotroph, as attempts to grow the organism chemoautotrophically with H2 or to detect ribulose bisphosphate carboxylase activity were unsuccessful.     

Effects of Calcium on Sugar Transport in Azotobacter vinelandii

A fast and environmentally safe procedure was used to study sugar uptake by Azotobacter vinelandii. Transport experiments were performed in a 24-well plate and aerated by rapid oscillatory vibration. Samples were washed by centrifugation and dissolved in biodegradable scintillation cocktail for counting. At cell concentrations up to 6 × 10⁸ cells per ml, the uptake of sucrose was a function of time and was proportional to the cell concentration. This modified uptake assay was used to test the effect of cations on sugar uptake in A. vinelandii. Results showed that Ca²⁺ at 1 to 2 mM stimulated sucrose uptake by decreasing the apparent K_m of sucrose transport. Higher Ca²⁺ concentrations inhibited sucrose uptake in this organism.     

Cytochrome and Ubiquinone Patterns During Growth of Azotobacter vinelandii

After synthesis during the early log phase, the concentrations of ubiquinone and cytochromes did not vary during the growth cycle of Azotobacter vinelandii, when grown with either high or low aeration on nitrogen-free or urea-containing media. The level of aeration had no effect on the concentrations of the electron carriers synthesized, but affected the growth rate. On urea-containing medium, the concentration of cytochrome a(2) was low, but it was synthesized at a linear rate when the bacteria were transferred to nitrogen-free medium. A. vinelandii was shown to utilize sufficient medium urea to account for all of the cell nitrogen. Growth on urea-containing medium with an oxygen atmosphere resulted in low growth yields, and cytochromes c(4) + c(5) were not synthesized; the concentrations of ubiquinone and cytochromes b(1), a(1), and a(2) were only 12 to 18% of the values for growth on nitrogen-free medium with high aeration.     

Stimulation of Agrobacterium tumefaciens Growth by Azotobacter vinelandii Ferrisiderophores

Azotobacter vinelandii stimulated the growth of Agrobacterium tumefaciens H2, H23, H24, H27, and ATCC 15955 on media containing insoluble iron sources. The Azotobacter vinelandii siderophores appeared to promote Agrobacterium tumefaciens growth by solubilizing mineral iron, and the ferrisiderophores so formed then acted as iron sources for Agrobacterium tumefaciens. Agrobactin, the Agrobacterium siderophore, appeared to be inefficient in solubilizing mineral iron directly.     

Growth and nitrogenase activity of Azotobacter vinelandii on soil phenolic acids

M oreno , J., de la R ubia , T., R amos -C ormenzana , A. & V ela , G.R. 1990. Growth and nitrogenase activity of Azotobacter vinelandii on soil phenolic acids. Journal of Applied Bacteriology 69 , 850–855.
Growth and nitrogenase activity (acetylene reduction) of Azotobacter vinelandii were studied in soil suspensions supplemented with p -hydroxybenzoic, vanillic, p -coumaric and ferulic acids. Nitrogenase activity was detected only when the microorganism was cultured on p -hydroxybenzoic acid, suggesting that this compound could be utilized as a carbon source by A. vinelandii for the maintenance of its biological activities under certain environmental conditions.     

Phospholipids of Azotobacter vinelandii

Analyses of resting cells of Azotobacter vinelandii revealed that numerous phospholipids were present that did not concentrate in the membranous R(3) fraction which carried out electron transport function.     

Ultrastructure of Azotobacter vinelandii

Vegetative cells and cysts of Azotobacter vinelandii 12837 were prepared for electron microscopy by several methods assumed to preserve structural details destroyed by techniques previously reported in the literature. Examination of large numbers of cells and cysts by these methods revealed four structural details not reported previously: intine fibrils, intine vesicles, intine membrane, and microtubules. The intine fibrils form a network in the gel-like homogeneous matrix of the CC2 layer. Intine vesicles which seem to originate in the cell wall complex of the central body are seen in the intine and exine of cysts. Analogous structures are found on vegetative cells. The intine is divided into two chemically distinct areas by the two-layered intine membrane. Microtubules, previously reported only in vegetative cells, were found in cysts.     

Hyperproduction of Poly-beta-Hydroxybutyrate during Exponential Growth of Azotobacter vinelandii UWD

The transformation of Azotobacter vinelandii UW with A. vinelandii 113 DNA resulted in the formation of rifampin-resistant colonies, 13% of which also inherited a previously unrecognized mutation in the respiratory NADH oxidase. These transformants produced colonies with a white-sectored phenotype after prolonged incubation. Cells from these sectors were separated and purified by streaking and were named UWD. The dense white phenotype was due to the production of a large amount of poly-beta-hydroxybutyrate during the exponential growth of strain UWD. The polymer accounted for 65 or 75% of the cell dry weight after 24 h of incubation of cultures containing glucose and either ammonium acetate or N(2), respectively, as the nitrogen source. Under the same conditions, strain UW cells contained 22 to 25% poly-beta-hydroxybutyrate, but O(2)-limited growth was required for these optimal production values. Polymer production was not dependent on O(2) limitation in strain UWD, but the efficiency of conversion of glucose to poly-beta-hydroxybutyrate was enhanced in O(2)-limited cultures. Conversion efficiencies were >0.25 and 0.33 mg of poly-beta-hydroxybutyrate per mg of glucose consumed under vigorous- and low-aeration conditions, respectively, compared with an efficiency of 0.05 achieved by strain UW. Strain UWD, therefore, appeared to from poly-beta-hydroxybutyrate under novel conditions, which may be useful in designing new methods for the industrial production of biodegradable plastics.     

Possible mechanism of mannose inhibition of sucrose-supported growth in N2-fixing Azotobacter vinelandii

When mannose was added to a sucrose-supported culture of Azotobacter vinelandii under N2-fixing conditions, cell growth was inhibited. The degree of inhibition was proportional to the amount of mannose and to the aeration rate (T.-Y. Wong, Appl. Environ. Microbiol. 54:473-475, 1988). In this report, we demonstrate that once inside the cell, mannose was phosphorylated to mannose 6-phosphate. It was then isomerized to fructose 6-phosphate and to glucose 6-phosphate. Mannose inhibited sucrose uptake noncompetitively. The decrease in sucrose uptake after mannose addition coincided with a lower rate of respiration and a decrease in nitrogenase activity. The decrease in sucrose uptake and in the ATP pool may decrease the electron flow and reduce protection of the nitrogenase from O2. Cells became very sensitive to O2, and therefore, cell growth was inhibited under high aeration conditions.     

Possible mechanism of mannose inhibition of sucrose-supported growth in N2-fixing Azotobacter vinelandii.

When mannose was added to a sucrose-supported culture of Azotobacter vinelandii under N2-fixing conditions, cell growth was inhibited. The degree of inhibition was proportional to the amount of mannose and to the aeration rate (T.-Y. Wong, Appl. Environ. Microbiol. 54:473-475, 1988). In this report, we demonstrate that once inside the cell, mannose was phosphorylated to mannose 6-phosphate. It was then isomerized to fructose 6-phosphate and to glucose 6-phosphate. Mannose inhibited sucrose uptake noncompetitively. The decrease in sucrose uptake after mannose addition coincided with a lower rate of respiration and a decrease in nitrogenase activity. The decrease in sucrose uptake and in the ATP pool may decrease the electron flow and reduce protection of the nitrogenase from O2. Cells became very sensitive to O2, and therefore, cell growth was inhibited under high aeration conditions.     

Characteristics of N2 fixation in Mo-limited batch and continuous cultures of Azotobacter vinelandii.

Steady-state chemostat cultures of Azotobacter vinelandii were established in a simple defined medium that had been chemically purified to minimize Mo and that contained no utilizable combined N source. Growth was dependent on N2 fixation, the limiting nutrient being the Mo contaminating the system. The Mo content of the organisms was at least 100-fold lower than that of Mo-sufficient cultures, and they lacked the characteristic g = 3.7 e.p.r. feature of the MoFe-protein of nitrogenase. A characteristic of nitrogenase activity in vivo in Mo-limited populations was a disproportionately low activity for acetylene reduction, which was 0.3 to 0.1 of that expected from the rate of N2 reduction. Acetylene was also a poor substrate in comparison with protons as a substrate for nitrogenase, and did not markedly inhibit H2 evolution, in contrast with Mo-sufficient populations. In batch cultures in similar medium or 'spent' chemostat medium inoculated with Mo-limited organisms, the addition of Mo elicited a biphasic increased growth response at concentrations as low as 2.5 nM, provided that sufficient Fe was supplied. In this system V did not substitute for Mo, and Mo-deficient cultures ceased growth at a 25-fold lower population density compared with cultures supplemented with Mo. Nitrogenase component proteins could not be unequivocally detected by visual inspection of fractionated crude extracts of Mo-limited organisms. 35SO42-pulse-labelling studies also showed that the rate of synthesis of the MoFe-protein component of nitrogenase was too low to be quantified. However, the Fe-protein of nitrogenase was apparently synthesized at high rates. The discussion includes an evaluation of the possibility that A. vinelandii possesses an Mo-independent N2-fixation system.     

Effects of monovalent cations on AMP nucleosidase from Azotobacter vinelandii.

The effect of monovalent cations on the purified AMP nucleosidase (AMP phosphoribohydrolase, EC 3.2.2.4) from Azotobacter vinelandii was investigated. All the monovalent cations were activators of the enzyme: Rb+ and Cs+ were the most effective, followed by K+, Na+, NH4+ and Li+ in that order. The apparent Ka for MgATP and nH values (Hill's interaction coefficient) decreased from 0.9 to 0.1 mM, and from 4 to 1, respectively, with the increase in K+ concentration, suggesting that the cation effects are on MgATP binding rather than catalysis. Gel filtration studies have revealed that the enzyme forms a non-dissociable enzyme species with a Stokes radius of 6.0--6.2 nm in the presence of saturating concentrations of monovalent cations, which can be distinguished from the 5.5-nm enzyme species showing temperature-dependent dissociation of the molecule in sulfate or phosphate. These results suggest that these ligands affect the association of the subunits through changes in the environment of the hydrophobic side chains of the enzyme molecules.     

Effect of Inoculation with N2-Fixing Spirilla and Azotobacter on Nitrogenase Activity on Roots of Maize Grown Under Subtropical Conditions

Inoculated and non-inoculated seedlings of maize were grown in fertile clayloam soils of Egypt and Belgium under subtropical conditions provided in a greenhouse. Acetylene-reducing activity and microbial counts were determined during a period ranging from 6 to 12 weeks after sowing. Irrespective of soil origin, N₂-fixing spirilla and Azotobacter were common under maize cultivation. Inoculation resulted in a transitional increase in their numbers at early stages of growth. Nitrogenase activity was not detected in the rhizosphere of young plants. The maximum activities measured (81 to 1,436 nmol of C₂H₄ g⁻¹ h⁻¹) occurred close to the 50 to 70% silking stage. Inoculation with N₂-fixing spirilla, particularly in Nile Delta soil, doubled the amount of N₂ fixed in a late period of growth (12 weeks), whereas inoculation with Azotobacter had no noticeable effect.     

Growth of Azotobacter vinelandii in a solid-state fermentation of technical lignin

Azotobacter vinelandii was cultured on technical lignin, derived from Kraft pulping processes, for biofertilizer production in solid-state fermentation. The effects of the ratio of technical lignin to corn straw, initial water content, and material bed depth on the microorganisms were studied in detail. At 30 degrees C, technical lignin to corn straw at the ratio of 1:0.75, the bed depth of 5 cm, and 67% moisture content, A. vinelandii was grown and reached 4.2 x 10(10) cfu g(-1) dry rot after 36 h.