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.     

Plasmids of Azotobacter vinelandii.

Four laboratory strains and two isolates of Azotobacter vinelandii were found to contain plasmids. Twenty-five laboratory strains which could fix nitrogen did not have free, covalently closed circular plasmid DNA. The plasmids varied in size from 9 to 52 megadaltons, and each strain yielded only one plasmid. No discernible differences in ability to fix nitrogen were found between plasmid-bearing and cured cultures.     

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.     

Ubiquinone in hydrogen metabolism by Azotobacter vinelandii

Extraction with n-heptane abolished over 95% of the NADH oxidase and the hydrogenase activity in membrane preparations from Azotobacter vinelandii. Incorporation of ubiquinone-8 or plastoquinone restored each reaction to about 55% of its original activity.     

Multiple chromosomes of Azotobacter vinelandii.

The number of copies of the genes leuB, nifH, nifD, and nifK per cell of Azotobacter vinelandii has been determined to be about 80. A beta-lactamase gene was integrated into the A. vinelandii chromosome by single-point crossover. Subsequently, we have been able to detect nearly 80 copies of this beta-lactamase gene per cell of A. vinelandii when cultured for a large number of generations in the presence of ampicillin. The multiple copies of the beta-lactamase gene do not seem to be present on a single chromosome, as evident from the fragment obtained by digestion of cellular DNA with the appropriate restriction endonuclease. The kinetics of renaturation of DNA of A. vinelandii is suggestive of complexity similar to that of Escherichia coli. The DNA content of A. vinelandii, however, is 40 times that of E. coli. All these indicate the presence of multiple chromosomes, possibly as many as 80, in A. vinelandii.     

Nitrogenase in synchronized Azotobacter vinelandii OP.

Azotobacter vinelandii OP was synchronized by the continuous phased culture technique. The nitrogenase (nitrogen:(acceptor)oxidoreductase)(EC 1.7.99.2) activity of the culture was determined continuously within the fermentor by acetylene reduction. Addition of NH4+ in excess of 5 x 10(-3)M to the culture lowered nitrogenase activity immediately. Other sources of fixed nitrogen had no immediate effect on nitrogenase activity, but nitrogenase synthesis decreased in the cell cycle following the one in which the fixed nitrogen was added.     

Novel exopolysaccharide production by Azotobacter vinelandii

Summary An isolate of Azotobacter vinelandii from the rhizosphere of the rhizomes of lotus produced a novel exopolysaccharide (EPS) having the composition of glucose: galactose: rhamnose (2.5: 1: 1.2). This composition has not been reported previously. The EPS has excellent viscosifying properties and could possibly be pseudoplastic in nature.     

Metabolism of methylammonium by Azotobacter vinelandii

Azotobacter vinelandii takes up the ammonium analog methylammonium from the external medium and metabolizes it to a less polar compound which has been identified as N-methylglutamine. The enzyme glutamine synthetase appears responsible for methylammonium metabolism in this organism and full activity of the enzyme is required for maximal rates of methylammonium uptake. L-methionine-DL-sulfoximine or L-methionine sulfone, inhibitors of glutamine synthetase activity, were shown to reduce the rate of methylammonium uptake by wild type cultures. A mutant strain with low glutamine synthetase activity was shown to be unable to carry out in vitro N-methylglutamine synthesis or in vivo uptake of methylammonium. Thus, methylammonium uptake assays may prove useful as a method of identifying mutants with altered glutamine synthetase activity.Abbreviations MSX L-methionine-DL-sulfoximine - MSF L-methionine sulfone     

Hydrogen-mediated mannose uptake in Azotobacter vinelandii.

Azotobacter vinelandii can grow mixotrophically with H2 plus mannose under N2-fixing conditions (T. Y. Wong and R. J. Maier, J. Bacteriol. 163:528-533, 1985). Mixotrophically grown cultures incubated in H2 transported mannose with a Vmax fourfold greater than that observed for cultures incubated in argon, but H2 did not change the apparent Km for mannose. Respiratory inhibitors, such as potassium cyanide, hydroxylamine, and p-chloromercuribenzoic acid, as well as the proton conductor carbonyl cyanide m-chlorophenyl-hydrazone inhibited mannose uptake. We suggest that one of the roles of H2 in mixotrophic metabolism is to supply energy that facilitates mannose transport.     

Phosphate-limited culture of Azotobacter vinelandii.

Batch cultures of Azotobacter vinelandii grown in phosphate-deficient media were compared with control cultures grown in phosphate-sufficient media. Phosphate limitation was assessed by total cell yield and by growth kinetics. Although cell protein, nucleic acids, and early growth rate were unaffected by phosphate deficiency, cell wall structure, oxygen uptake, and cell viability were significantly affected. Also, phosphate-limited cells contained much larger amounts of poly-beta-hydroxybutyric acid but lower adenylate nucleotide energy charge than did control cells. The ratio of adenosine 5'-triphosphate to adenosine 5'-diphosphate was much lower in phosphate-deficient cells. The data indicate a substrate saving choice of three metabolic pathways available to this organism under different growth conditions.     

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.     

Expression of an alternative nitrogen fixation system in Azotobacter vinelandii.

Nitrogenase activities were determined from maximum acetylene reduction rates for mutant strains of Azotobacter vinelandii which are unable to fix N2 in the presence of molybdenum (Nif-) but undergo phenotypic reversal to Nif+ under conditions of Mo deficiency. The system responsible for N2 fixation under these conditions is thought to be an alternative N2 fixation system (Bishop et al., Proc. Natl. Acad. Sci. U.S.A. 77:7342-7346, 1980). Phenotypic reversal of Nif- strains to Nif+ strains was also observed in N-free medium without Mo but with either V or Re. Two protein patterns were found on two-dimensional gels of proteins from the extracts of wild-type cells cultured in N-free medium without Mo and with or without V or Re. The expression of each protein pattern in the wild-type strain of A. vinelandii seemed to depend upon the physiological state of the N2-fixing culture. Electron paramagnetic resonance experiments were conducted on whole cells of A. vinelandii grown under conditions of Mo deprivation in the absence of fixed N. No g = 3.65 signal (an electron paramagnetic resonance signal characteristic of the Mo-containing component of nitrogenase) was detectable in these cells, regardless of whether V or Re was present during growth of these cells, These results are discussed from the perspective that the well-known effect of V on N2 fixation by A. vinelandii may involve an alternative N2 fixation system.     

The biosynthesis of alginic acid by Azotobacter vinelandii.

The sequence of reactions by which alginic acid is biosynthesized from sucrose in Azotobacter vinelandii was determined both by feeding radioactive individual enzymes involved. Results indicate that the first polymeric substance formed in the synthesis is polymannuronic acid and that mannuronic acid units are epimerized to guluronic acid at the polymer level. Guluronic acid does not appear to be formed at the monomer level, either free or in combination with GDP.     

Beta-ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single β-ketothiolase activity participating in the formation of acetoacetyl-CoA, a precursor for poly-β-hydroxybutyrate (PHB) synthesis, and in β-oxidation (Manchak, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140, 953–963). We designed a degenerate oligonucleotide from a highly conserved region among bacterial β-ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to β-ketothiolases. Immediately downstream of bktA, we identified a gene called hbdH, which encodes a protein exhibiting similarity to β-hydroxyacyl-CoA and β-hydroxybutyryl-CoA dehydrogenases. Two regions with homology to bktA were also observed. One of these was cloned and allowed the identification of the phbA gene, encoding a second β-ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbdH and phbA mutations, respectively, as well as strain EG1 carrying both bktA and phbA mutations, were constructed. The hbdH mutation had no effect on β-hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilation. The bktA mutation had no effect on β-ketothiolase activity, PHB synthesis or fatty acid assimilation, whereas the phbA mutation significantly reduced β-ketothiolase activity and PHB accumulation, showing that this is the β-ketothiolase involved in PHB biosynthesis. Strain EG1 was found to grow under β-oxidation conditions and to possess β-ketothiolase activity. Taken together, these results demonstrate the presence of three genes coding for β-ketothiolases in A. vinelandii.