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PubMed1981

Recovery of competence in calcium-limited Azotobacter vinelandii.

Azotobacter vinelandii cells required 0.5 mM calcium in the iron-limited competence induction medium. This requirement also was fulfilled by strontium, but not by magnesium. Cells pregrown in competence medium lacking calcium rapidly recovered competence with the addition of 0.5 mM calcium, provided they were suspended in the growth supernatant. A 60,000-dalton glycoprotein (pI 5.10) present in competent or incompetent culture supernatants participated in calcium-mediated competence recovery. Cells grown in calcium-limited medium appeared to have leaky cell envelopes and released a diverse array of proteins into the culture supernatant and into distilled water washes of the cells, seven of which appeared to be more dominant in competent cells. Two distilled water washes of cells grown in calcium-limited medium did not prevent calcium-mediated recovery of competence in the culture supernatant. Four to six distilled water washes removed a competence-specific protein (pI 5.19) and prevented calcium-mediated recovery of competence in the culture supernatant.

Azotobacter↗

PubMed1981

Characterization of the predominant Azotobacter vinelandii envelope protein.

A protein with a molecular weight of 60,000 (60K) constitutes approximately 20% of the envelope protein of Azotobacter vinelandii. This protein was removed from cells and purified from other proteins by a simple washing procedure that had no effect on cell viability. Anti-60K antiserum blocked azotophage A-22 adsorption and agglutinated both vegetative cells and cysts; ferritin-conjugated antibodies used in indirect labeling studies bound uniformly to the periphery of vegetative cells. We conclude that 60K is present on the outer surface of vegetative cells and cysts. The protein is similar to the surface protein alpha of Acinetobacter ssp. in molecular weight, reassociation characteristics, and high ratio of acidic to basic amino acids. We propose that 60K forms a layer external to the outer membrane of A. vinelandii.

Agglutination Tests↗

PubMed1981

Metabolism of resorcinylic compounds by bacteria: new pathway for resorcinol catabolism in Azotobacter vinelandii.

We present evidence to document a third pathway for the microbial catabolism of resorcinol. Resorcinol is converted to pyrogallol by resorcinol-grown cells of Azotobacter vinelandii. Pyrogallol is the substrate for one of two ring cleavage enzymes induced by growth with resorcinol. Oxalocrotonate, CO2, pyruvate, and acetaldehyde have been identified as products of pyrogallol oxidation catalyzed by extracts of resorcinol-grown cells. The enzymes pyrogallol 1,2-dioxygenase, oxalocrotonate tautomerase (isomerase), oxalocrotonate decarboxylase, and vinylpyruvate hydratase are present in extracts from resorcinol-grown cells but not in succinate-grown cells.

Acetaldehyde↗

PubMed1981

Transport of ammonium and methylammonium ions by Azotobacter vinelandii.

Ammonium and methylammonium are rapidly taken up by cultures of Azotobacter vinelandii respiring in the presence of succinate. The rate of methylamine uptake increased with external pH from 5.5 to 7.5 but increasing the pH further to 8.5 had little effect on activity, indicating that methylammonium cation rather than uncharged methylamine is the permeant species. The kinetics of methylammonium entry followed the Michaelis-Menten relationship, yielding a K(m) of 25 muM and a V(max) of 3.8 nmol/min per mg of cell protein. At saturating concentrations ammonium was taken up at rates 30-fold higher than those for methylammonium. Ammonium was a competitive inhibitor of methylammonium uptake and gave an inhibition constant of 1 muM. Ammonium derivatives were inhibitors of methylammonium entry in order of effectiveness: hydrazine > methylhydrazine > formamidine > guanidine > dimethylamine > ethylamine; amides and amino acids did not block uptake. Likewise, metal cations inhibited in the order Tl(+) > Cs(+) > Rb(+), whereas Na(+), K(+), and Li(+) produced no significant effect. Methylammonium uptake was blocked in cells exposed to an uncoupler, p-trifluorome-thoxycarbonyl cyanide-phenyl hydrazone or gramicidin D, but not with dicyclo-hexylcarbodiimide or arsenate. Valinomycin stimulated methylammonium entry into cells in a K(+)-free medium but prevented entry in the presence of 10 mM K(+). Monensin and nigericin had little effect on transport. These results indicate that methylammonium and ammonium ions enter A. vinelandii electrogenically via a specific transporter.

Azotobacter↗

PubMed1981

Transformation of Azotobacter vinelandii with plasmids RP4 (IncP-1 group) and RSF1010 (IncQ group).

Multicopy plasmid RSF1010 and four of its in vitro-constructed derivatives were mobilized by the self-transmissible RP4 plasmid into Azotobacter vinelandii UW. Modifications of the Escherichia coli transformation procedure of Cohen et al. (Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114, 1972) allowed transformation of A. vinelandii strains UW and ATCC 12837 with purified RP4 or RSF1010 deoxyribonucleic acid.

Azotobacter↗

PubMed1981

Isolation and characterization of several unique lipids from Azotobacter vinelandii cysts.

Unique cyclic compounds were found in the lipid fraction of Azotobacter vinelandii cysts. In addition to two major molecular species which had already been identified, 5-n-alkylresorcinol and its galactoside derivative, five other molecular species (two alkyl side chain homologs of each) were isolated, and their structures were established by infrared, ultraviolet, nuclear magnetic resonance, and mass spectroscopy. These 10 compounds were 6-n-heneicosylresorcylic acid methyl ester and 6-n-tricosylresorcylic acid methyl ester, 5-n-(2-hydroxy)heneicosylresorcinol and 5-n-(2-hydroxy-tricosylresorcinol, 5-n-heneicosyl-4-acetylresorcinol and 5-n-tricosyl-4-acetylresorcinol, 6-n-heneicosyl-4-hydroxypyran-2-one and 6-n-tricosyl-4-hydroxypyran-2-one, and 6-(2-oxotricosyl)-4-hydroxy-pyran-2-one and 6-(2-oxopentacosyl)-4-hydroxypyran-2-one.

Azotobacter↗

PubMed1981

Unique lipids in Azotobacter vinelandii cysts: synthesis, distribution, and fate during germination.

Unique lipids found in Azotobacter vinelandii cysts are derived primarily from beta-hydroxybutyrate used to induce encystment. Tracer studies with beta-[14C]hydroxybutyrate showed that the biosynthesis of these compounds during encystment began at 8 to 12 h after induction and reached maximal levels after 2 days, Seventy percent of these unique lipids were found in the central body of the cysts, and 23% were found in the exine. Pyronic compounds, which are located mostly in the central body, were degraded during germination of the cysts, but little change occurred in the phenolic compounds, which are more uniformly distributed in the cysts.

Azotobacter↗

PubMed1981

Ammonium and methylammonium transport by the nitrogen-fixing bacterium Azotobacter vinelandii.

Azotobacter vinelandii, grown with NH4+ as nitrogen source, was shown to possess an active transport system which can take up NH4+ against a concentration gradient of 58-fold. The properties of the NH4+ uptake system were investigated with the NH4+ analog CH3NH3+. The use of this analog was justified on the basis of the conclusion that the uptake of NH4+ and CH3NH3 involves a common binding site, as shown by the competitive inhibition of CH3NH3+ uptake by NH4+ (Ki approximately 3 microM). A Lineweaver-Burk plot for CH3NH3+ uptake revealed a biphasic curve, suggesting the existence of two CH3NH3+ (NH4+) uptake systems with apparent Km's for CH3NH3+ equal to 61 microM and 661 microM. The uptake of CH3NH3+ was inhibited by arsenate, as well as by cyanide or carbonyl cyanide-m-chlorophenyl hydrazone, indicating that phosphate bond energy is required.

Arsenates↗

PubMed1982

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.

Acetylene↗

PubMed1982

Iron- and molybdenum-repressible outer membrane proteins in competent Azotobacter vinelandii.

Azotobacter vinelandii produced three major proteins of 93,000, 85,000, and 81,000 daltons and a minor 77,000-dalton protein in the outer membrane of Fe-limited cells, and these cells were competent for transformation by DNA. The synthesis of these proteins was repressed in Fe-sufficient medium. Mo limitation of nitrogen-fixing cells resulted in the hyperproduction of a 44,000-dalton protein and the production of a minor 77,000-dalton protein in the outer membrane. Mo limitation enhanced competence in Fe-limited medium and induced competence in Fe-sufficient medium. The 44,000-dalton protein was replaced by a 45,000-dalton protein when Fe-sufficient medium also contained NH4+, but the cells were noncompetent. The synthesis of these proteins was repressed in Mo-sufficient medium and by NH4+ in Fe-limited medium. All of the culture supernatants contained a blue-white fluorescent material (absorbance maximum, 214 nm) which appeared to coordinate Fe3+, Fe2+, MoO4(2-), WO3(2-), and VO3(-).

Azotobacter↗

PubMed1982

Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii.

Mutants of Azotobacter vinelandii ATCC 12837 were isolated which could fix N2 in the presence of high tungsten concentrations. The most studied of these mutants (WD2) grew well in N-free modified Burk broth containing 10 mM W, whereas the wild type would not grow in this medium. WD2 would also grow in Burk N-free broth at about the same rate as the wild type. WD2 in broth containing W exhibited 22% of the whole cell acetylene reduction activity of the wild type in broth containing Mo and showed a lowered affinity for acetylene. Two-dimensional gel electrophoresis experiments showed that N2-fixing cells of WD2 from broth containing W or Mo did not produce significant amounts of component I of native nitrogenase protein. Electron spin resonance spectra of whole cells and cell-free extracts of WD2 from broth containing W lacked any trace of the g = 3.6 resonance associated with FeMoCo.

Acetylene↗

PubMed1983

Heat sensitivity of Azotobacter vinelandii genetic transformation.

Heating competent Azotobacter vinelandii at 37 or 42 degrees C resulted in a total loss of competence with no loss of viability. The transformation process was relatively insensitive to heating at either temperature once DNase-resistant DNA binding was nearly complete. Although competent and 42 degrees C-treated cells bound equivalent amounts of [32P]DNA in a DNase-resistant state, no donor DNA marker (nif) or radioactivity was detected in the envelope-free cell lysate of heated cells, suggesting that DNA transport across the cell envelope was a heat-sensitive event. Competence was reacquired in a 42 degrees C-treated culture after 2 h of incubation at 30 degrees C by a process which required RNA and protein syntheses. The release of a surface glycoprotein, required for competence, from cells treated at 42 degrees C occurred in an insufficient amount to account for the total loss of competence. Recovery of competence in 42 degrees C-treated cells and further transformation of competent cells were prevented by the exposure of cells to saturating amounts of transforming DNA. Further DNase-resistant DNA binding, however, still occurred, suggesting that there were two types of receptors for DNase-resistant DNA binding to competent A. vinelandii. DNase-resistant DNA binding was dependent on magnesium ions, and at least one receptor type did not discriminate against heterologous DNA.

Azotobacter↗

PubMed1983

Role of glutamine synthetase in the uptake and metabolism of methylammonium by Azotobacter vinelandii.

Methylammonium is a substrate for the ammonium transport system of Azotobacter vinelandii. During cellular uptake methylammonium is rapidly converted to a less polar metabolite (E. M. Barnes, Jr., and P. Zimniak, J. Bacteriol. 146:512-516, 1981). This metabolite has been isolated from A. vinelandii and identified as gamma-glutamylmethylamide by mass spectroscopy, 1H nuclear magnetic resonance spectroscopy, and cochromatography with the authentic compound. Escherichia coli also accumulated gamma-glutamylmethylamide during methylammonium uptake. The biosynthesis of gamma-glutamylmethylamide in vitro required methylammonium, ATP, L-glutamate, and a soluble cell extract from A. vinelandii. The enzyme responsible for gamma-glutamylmethylamide synthesis was glutamine synthetase. In a crude extract, L-methionine-DL-sulfoximine was equipotent in inhibiting the activities for gamma-glutamyltransferase and for the synthesis of glutamine and gamma-glutamylmethylamide. Likewise, an antiserum against the glutamine synthetase of E. coli precipitated the transferase and both synthetic activities at similar titers. During repression by growth of cells on ammonium medium, the synthesis of glutamine and gamma-glutamylmethylamide in vitro was also inhibited coordinately. A partially purified preparation of glutamine synthetase from A. vinelandii utilized methylammonium as substrate (Km = 78 mM, Vmax = 0.30 mumol/min per mg), although less efficiently than ammonium (Km = 0.089 mM, Vmax = 1.1 mumol/min per mg). The kinetic properties of glutamine synthetase with methylammonium as substrate as well as the insensitivity of this activity to inhibition by T1+ were strikingly different from methylammonium translocation. Thus, methylammonium (ammonium) translocation and intracellular trapping as glutamylamides are experimentally distinguishable processes.

Azotobacter↗

PubMed1983

Protein turnover in Azotobacter vinelandii during encystment and germination.

Protein turnover occurs during differentiation of Azotobacter vinelandii 12837 to the extent of 50% during encystment and 7% during germination. The addition of rifampin at the initiation of encystment prevents encystment and inhibits turnover. In germinating cysts, protein turnover is essential owing to an apparent lack of certain amino acid biosynthetic enzymes. The capacity to synthesize sulfur-containing amino acids from inorganic precursors is regained about halfway through the germination process.

Acetates↗

PubMed1984

Inhibition of nitrogenase activity by ammonium chloride in Azotobacter vinelandii.

In Azotobacter vinelandii cells, the short-term inhibition of nitrogenase activity by NH4Cl was found to depend on several factors. The first factor is the dissolved oxygen concentration during the assay of nitrogenase. When cells are incubated with low concentrations of oxygen, nitrogenase activity is low and ammonia inhibits strongly. With more oxygen, nitrogenase activity increases. Cells incubated with an optimum amount of oxygen have maximum nitrogenase activity, and the extent of inhibition by ammonia is small. With higher amounts of oxygen, the nitrogenase activity of the cells is decreased and strongly inhibited by ammonia. The second factor found to be important for the inhibition of nitrogenase activity by NH4Cl was the pH of the medium. At a low pH, NH4+ inhibits more strongly than at a higher pH. The third factor that influenced the extent of ammonia inhibition was the respiration rate of the cells. When cells are grown with excess oxygen, the respiration rate of the cells is high and inhibition of nitrogenase activity by ammonia is small. Cells grown under oxygen-limited conditions have a low respiration rate and NH4Cl inhibition of nitrogenase activity is strong. Our results explain the contradictory reports described in the literature for the NH4Cl inhibition of nitrogenase in A. vinelandii.

Ammonium Chloride↗

PubMed1984

Derepression of the Azotobacter vinelandii siderophore system, using iron-containing minerals to limit iron repletion.

Azotobacter vinelandii solubilized iron from certain minerals using only dihydroxybenzoic acid, which appeared to be produced constitutively. Solubilization of iron from other minerals required dihydroxybenzoic acid and the siderophore N,N'-bis-(2,3- dihydroxybenzoyl )-L-lysine ( azotochelin ) or these chelators plus the yellow-green fluorescent siderophore azotobactin . In addition to this sequential production of siderophores, cells also demonstrated partial to hyperproduction relative to the iron-limited control. The iron sources which caused partial derepression of the siderophores caused derepression of all the high-molecular-weight iron-repressible outer membrane proteins except a 77,000-molecular-weight protein, which appeared to be coordinated with azotobactin production. Increased siderophore production correlated with increased production of outer membrane proteins with molecular weights of 93,000, 85,000, and 77,000, but an 81,000-molecular-weight iron-repressible protein appeared at a constant level despite the degree of derepression. When iron was readily available, it appeared to complex with a 60,000-molecular-weight protein believed to form a surface layer on the A. vinelandii cell.

Azotobacter↗

PubMed1984

Incorporation of isotope from specifically labeled glucose into alginates of Pseudomonas aeruginosa and Azotobacter vinelandii.

The incorporation of isotope from [6-14C]glucose into alginate by both Pseudomonas aeruginosa and Azotobacter vinelandii was 10-fold greater than that from either [1-14C]- or [2-14C]glucose, indicating preferential utilization of the bottom half of the glucose molecule for alginate synthesis. These data strongly suggest that the Entner - Doudoroff pathway plays a major role in alginate synthesis in both P. aeruginosa and A. vinelandii.

Alginates↗

PubMed1984

Regular surface layer of Azotobacter vinelandii.

Washing Azotobacter vinelandii UW1 with Burk buffer or heating cells at 42 degrees C exposed a regular surface layer which was effectively visualized by freeze-etch electron microscopy. This layer was composed of tetragonally arranged subunits separated by a center-to-center spacing of approximately 10 nm. Cells washed with distilled water to remove an acidic major outer membrane protein with a molecular weight of 65,000 did not possess the regular surface layer. This protein, designated the S protein, specifically reattached to the surface of distilled-water-washed cells in the presence of the divalent calcium, magnesium, strontium, or beryllium cations. All of these cations except beryllium supported reassembly of the S protein into a regular tetragonal array. Although the surface localization of the S protein has been demonstrated, radioiodination of exposed envelope proteins in whole cells did not confirm this. The labeling behavior of the S protein could be explained on the basis of varying accessibilities of different tyrosine residues to iodination.

Azotobacter↗