Tetramethyl-p-phenylenediamine oxidase reaction in Azotobacter vinelandii.

It was possible to quantitate the tetramethyl-p-phenylenediamine (TMPD) oxidase reaction in Azotobacter vinelandii strain O using turbidimetrically standarized resting cell suspensions. The Q(O2) value obtained for whole cell oxidation of ascorbate-TMPD appeared to reflect the full measure of the high respiratory oxidative capability usually exhibited by this genera of organisms. The Q(O2) value for the TMPD oxidase reaction ranged from 1,700 to 2,000 and this value was equivalent to that obtained for the oxidation of the growth substrate, e.g., acetate. The kinetic analyses for TMPD oxidation by whole cells was similar to that obtained for the "particulate" A. vinelandii electron transport particle, that fraction which TMPD oxidase activity is exclusively associated with. Under the conditions used, there appeared to be no permeability problems; TMPD (reduced by ascorbate) readily penetrated the cell and oxidized at a rate comparable to that of the growth substrate. This, however, was not true for the oxidation of another electron donor, 2,6-dichloroindophenol, whose whole cell Q(O2) values, under comparable conditions, were twofold lower. The TMPD oxidase activity in A. vinelandii whole cells was found to be affected by the physiological growth conditions, and resting cells obtained from cells grown on sucrose, either under nitrogen-fixing conditions or on nitrate as the combined nitrogen source, exhibited low TMPD oxidase rates. Such low TMPD oxidase rates were also noted for chemically induced pleomorphic A. vinelandii cells, which suggests that modified growth conditions can (i) alter the nature of the intracellular terminal oxidase formed (or induced), or (ii) alter surface permeability, depending upon the growth conditions used. Preliminary studies on the quantitative TMPD oxidation reaction in mutant whole cells of both Azotobacter and a well-known Mucor bacilliformis strain AY1, deficient in cytochrome oxidase activity, showed this assay can be very useful for detecting respiratory deficiencies in the metabolism of whole cells.     

Diauxic Growth of Azotobacter vinelandii on Galactose and Glucose: Regulation of Glucose Transport by Another Hexose

The growth curve of Azotobacter vinelandii was biphasic when the organism was grown in a medium containing a mixture of galactose and glucose. Galactose was the primary carbon source; glucose was also consumed, but the rate at which it was consumed was lower than the rate at which galactose was consumed during the first phase of growth. Metabolic pathways for both sugars were induced. Cell cultures exhibited a second lag period as galactose was depleted. The length of this lag phase varied from 2 to 10 h depending on the pregrowth history of the cells. The second log growth phase occurred at the expense of the remaining glucose in the medium and was accompanied by induction of the high-maximum rate of metabolism glucose-induced glucose permease and increases in the levels of glucose metabolic enzymes. The second lag phase of diauxie may have been due to the time required for induction of the glucose-induced glucose permease.     

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.     

Simultaneous uptake of galactose and glucose by Azotobacter vinelandii.

Azotobacter vinelandii growing on galactosides induced two distinct permeases for glucose and galactose. The apparent Vmax and Km of the galactose permease were 16 nmol galactose/min per 10(10) cells and 0.5 mM, respectively. The apparent Vmax and Km of the glucose permease were 7.8 nmol glucose/min per 10(10) cells and 0.04 mM, respectively. Excess glucose had no effect on the galactose uptake. However, excess galactose inhibited glucose transport. The galactosides-induced glucose permease also exhibited different uptake kinetics from that induced by glucose.     

Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase

The activity of nitrogenase in the nitrogen-fixing bacterium Azotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O(2) by a high respiration rate. In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture. Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture. Depending on the O(2) tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly. Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A. vinelandii. The composition, thickness, and compactness of this alginate capsule also varied significantly. In general, increasing O(2) tension led to the formation of alginate with a higher molecular weight and a greater L-guluronic acid content. The alginate capsule was accordingly thicker and more compact. In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor. Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O(2) transfer into the cell. It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.     

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.     

Effect of dissolved oxygen on nitrogen fixation by A. vinelandii. II. Ionically adsorbed cells

Continuous culture studies of Azotobacter vinelandii cells immobilized by ionic adsorption to Cellex E anion exchange resin were conducted under oxygen-limited conditions for comparison to free-cell cultures. Immobilization had little effect upon the specific respiration and sucrose consumption rates as compared to free cells. However, maxima in specific nitrogen fixation rate and nitrogenase activity as a function of dissolved oxygen occurred at a C(O(2) ) value of approximately 0.005 mM as opposed to 0.02 mM for free cells. Further, in contrast to free-cell culture, most of the fixed nitrogen appeared in the medium rather than within intact cells. There were strong indications that reproduction of bound cells often resulted in cell lysis accounting for the fixed nitrogen content in solution.     

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.     

The DeLey-Doudoroff Pathway of Galactose Metabolism in Azotobacter vinelandii

Azotobacter vinelandii cell extracts reduced NAD and oxidized d-galactose to galactonate that subsequently was converted to 2-keto-3-deoxy-galactonate. Further metabolism of 2-keto-3-deoxy-galactonate required the presence of ATP and resulted in the formation of pyruvate and glyceraldehyde 3-P. Radiorespirometry indicated a preferential release of CO(2) at the first carbon position of the d-galactose molecule. This suggested that Azotobacter vinelandii metabolizes d-galactose via the DeLey-Doudoroff pathway. The first enzyme of this pathway, d-galactose dehydrogenase, was partially characterized. It has a molecular weight of about 74,000 Da and an isoelectric point of 6.15. The pH optimum of the galactose dehydrogenase was about 9. The apparent K(m)s for NAD and d-galactose were 0.125 and 0.56 mM, respectively. Besides d-galactose, the active fraction of this galactose dehydrogenase also oxidized l-arabinose effectively. The electron acceptor for d-galactose or l-arabinose oxidation, NAD, could not be replaced by NADP. These substrate specificities were different from those reported in Pseudomonas saccharophila, Pseudomonas fluorescens, and Rhizobium meliloti.     

Diauxic growth in Azotobacter vinelandii.

Azotobacter vinelandii exhibited diauxie when grown in a medium containing both acetate and glucose as carbon sources. Acetate was used as the primary carbon source during the acetate-glucose diauxie. Uptake of acetate was constitutively expressed during both diauxic phases of growth. Induction of the glucose uptake system was inhibited in the presence of acetate. Acetate was also the preferred growth substrate for A. vinelandii grown in a medium containing either fructose, maltose, xylitol, or mannitol. The tricarboxylic acid cycle intermediates citrate, isocitrate, and 2-oxoglutarate inhibited glucose utilization in cells grown in glucose medium containing these substrates, and diauxic growth was observed under these growth conditions. Temporal expression of isocitrate-lyase, ATPase, and nitrogenase was exhibited during acetate-glucose diauxie.     

Melibiose is hydrolyzed exocellularly by an inducible exo-alpha-galactosidase in Azotobacter vinelandii.

Azotobacter vinelandii hydrolyzed melibiose exocellularly, leading to an accumulation of free glucose and galactose in the medium. This enzyme could also be induced by galactose, raffinose, and stachyose. The alpha-galactosidase activity could be detected quantitatively by using p-nitrophenyl-alpha-galactopyranoside as a substrate for intact cells. Chloramphenicol totally inhibited the induction of this enzyme. However, benzyl alcohol inhibited the secretion of this enzyme but did not inhibit the biosynthesis of the enzyme.     

Melibiose is hydrolyzed exocellularly by an inducible exo-alpha-galactosidase in Azotobacter vinelandii

Azotobacter vinelandii hydrolyzed melibiose exocellularly, leading to an accumulation of free glucose and galactose in the medium. This enzyme could also be induced by galactose, raffinose, and stachyose. The alpha-galactosidase activity could be detected quantitatively by using p-nitrophenyl-alpha-galactopyranoside as a substrate for intact cells. Chloramphenicol totally inhibited the induction of this enzyme. However, benzyl alcohol inhibited the secretion of this enzyme but did not inhibit the biosynthesis of the enzyme.     

Dependence of oxygen-tolerant nitrogenase activity on divalent cations in Azotobacter vinelandii.

Nitrogenase activity of washed Azotobacter vinelandii cells was enhanced by the addition of Ca2+ and Mg2+, and the enhancement increased with the O2 concentration. In assays provided with a level of O2 that was initially supraoptimal and inhibitory to nitrogenase activity, the addition of Ca2+ or Mg2+ affected both the maximum respiration rate (Vmax) of the cells and the apparent affinity [KS(O2)] of cell respiration for O2. Changes in these parameters correlated with changes in nitrogenase activity. Aeration-dependent increases in Vmax and KS(O2) were inhibited by rifampin and chloramphenicol and were also observed in ammonium-grown cultures.     

Cloning and mutagenesis of genes encoding the cytochrome bd terminal oxidase complex in Azotobacter vinelandii: mutants deficient in the cytochrome d complex are unable to fix nitrogen in air.

The genome of Azotobacter vinelandii contains DNA sequences homologous to the structural genes for the Escherichia coli cytochrome bd terminal oxidase complex. Two recombinant clones bearing cydA- and cydB-like sequence were isolated from an A. vinelandii gene library and subcloned into the plasmid vector pACYC184. Physical mapping demonstrated that the cydA- and cydB-like regions in A. vinelandii are contiguous. The cydAB and flanking DNA was mutagenized by the insertion of Tn5-B20. Mutations in the cydB-hybridizing region resulted in the loss of spectral features associated with cytochromes b595 and d. A new locus, cydB, encoding cytochromes b595 and d in A. vinelandii is proposed. A second region adjacent to cydB was also involved in expression of the cytochrome bd complex in A. vinelandii, since mutations in this region resulted in an increase in the levels of both cytochrome b595 and cytochrome d. The regions involved in expression of the cytochrome bd complex and cydB are transcribed in the same direction. Mutants deficient in cytochromes b595 and d were unable to grow on N-deficient medium when incubated in air but could fix nitrogen when the environmental O2 concentration was reduced to 1.5% (vol/vol). It is proposed that the branch of the respiratory chain terminated by the cytochrome bd complex supports the high respiration rates required for the respiratory protection of nitrogenase.     

Role of the Azotobacter vinelandii nitrogenase-protective shethna protein in preventing oxygen-mediated cell death

Azotobacter vinelandii strains lacking the nitrogenase-protective Shethna protein lost viability upon carbon-substrate deprivation in the presence of oxygen. This viability loss was dependent upon the N(2)-fixing status of cultures (N(2)-fixing cells lost viability, while non-N(2)-fixing cells did not) and on the ambient O(2) level. Supra-atmosheric O(2) tensions (40% partial pressure) decreased the viable cell number of the mutant further, and the mutant had a slightly higher spontaneous mutation frequency than the wild type in the high-O(2) conditions. Iron starvation conditions, which resulted in fourfold-reduced superoxide dismutase levels, were also highly detrimental to the viability of the protective protein mutants, but these conditions did not affect the viability of the wild-type strain. Nitrogenase or other powerful reductants associated with N(2) fixation may be sources of damaging partially reduced oxygen species, and the production of such species are perhaps minimized by the Shethna protein.     

Effects of glyphosate on nitrogen fixation of free-living heterotrophic bacteria

The effect of the herbicide glyphosate ( N -(phosphonomethyl)glycine) on the growth, respiration and nitrogen fixation of Azotobacter chroococcum and A. vinelandii was studied. Azotobacter vinelandii was more sensitive to glyphosate toxicity than A. chroococcum. Recommended dosages of glyphosate did not affect growth rates. More than 4 kg ha^-1 is needed to find some inhibitory effect. Specific respiration rates were 19.17 mmol O₂ h^-1 g^-1 dry weight for A. chroococcum and 12.09 mmol h^-1 g^-1 for A. vinelandii. When 20 kg ha^-1 was used with A. vinelandii , respiration rates were inhibited 60%, the similar percentage inhibition A. chroococcum showed at 28 kg ha^-1. Nitrogen fixation dropped drastically 80% with 20 kg ha^-1 in A. vinelandii and 98% with 28 kg ha^-1 in A. chroococcum. Cell size as determined by electron microscopy decreased in the presence of glyphosate, probably because glyphosate induces amino acid depletion and reduces or stops protein synthesis.     

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.     

Internal Membrane Control in Azotobacter vinelandii

Azotobacter vinelandii was grown on N(2), NH(4) (+), or NO(3) (-), and an internal membrane network was observed by electron microscopy of thin sections of cells. Cells obtained in early exponential growth contained less internal membrane than did cells from cultures in late exponential growth. It seems likely that O(2) has a role in regulating the amount of internal membrane structure.     

Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

Background

Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes.

Methodology/Principal Findings

We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels.

Conclusions/Significance

Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.