A quantitative model of nitrogen fixation in the presence of ammonium.

Nitrogen fixation provides bioavailable nitrogen, supporting global ecosystems and influencing global cycles of other elements. It provides an additional source of nitrogen to organisms at a cost of lower growth efficiency, largely due to respiratory control of intra-cellular oxygen. Nitrogen-fixing bacteria can, however, utilize both dinitrogen gas and fixed nitrogen, decreasing energetic costs. Here we present an idealized metabolic model of the heterotrophic nitrogen fixer Azotobacter vinelandii which, constrained by laboratory data, provides quantitative predictions for conditions under which the organism uses either ammonium or nitrogen fixation, or both, as a function of the relative supply rates of carbohydrate, fixed nitrogen as well as the ambient oxygen concentration. The model reveals that the organism respires carbohydrate in excess of energetic requirements even when nitrogen fixation is inhibited and respiratory protection is not essential. The use of multiple nitrogen source expands the potential niche and range for nitrogen fixation. The model provides a quantitative framework which can be employed in ecosystem and biogeochemistry models.

Manipulating the molecular weight of alginate produced by Azotobacter vinelandii in continuous cultures.

Alginate production by Azotobacter vinelandii in chemostat cultures was evaluated at different dilution rates (D) and inlet sucrose concentrations of 5 and 20 g l(-1). At the low inlet sucrose concentration, the molecular weight of alginate increased from 800 to 1800 kDa when D increased from 0.05 to 0.10 h(-1), whereas the opposite trend was observed with the high inlet sucrose concentration. This behaviour can be explained by changes in specific sucrose uptake rate. Thus, a decrease in alginate molecular weight was dependent on the specific sucrose uptake rate when this rate was higher than 0.42 g g(-1) h(-1). The manipulation of the D can be used to select the molecular weight of alginate in continuous culture.

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii.

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.

Mechanistic significance of the preparatory migration of hydrogen atoms around the FeMo-co active site of nitrogenase.

The migration of H atoms over S and Fe atoms in the reaction domain of FeMo-co, the active site of nitrogenase, is described and used to explain mechanistic data on the catalyzed reductions of N(2) and C(2)H(2). After electron transfer to FeMo-co, H atoms are generated by fast proton supply to S3B (atom labels from structure 1M1N) and migrate vectorially via several pathways from S3B to locations on the FeMo-co face, specifically Fe6, S2B, Fe2, and S2A (calculated reaction profiles are reported). The E(n)H(n) reduction levels (n = 1-4) in the Thorneley-Lowe kinetic-mechanistic schemes are each potential sequences of substructures with different distributions of H atoms. The positions of H atoms influence the binding of substrates N(2) and C(2)H(2), and the bound substrate subsequently blocks further migration of H atoms past the binding site. This model provides a consistent structural interpretation of (a) the two-site reactivity of C(2)H(2) and the differentiation of the high- and low-affinity sites as due to different preparatory H migration; (b) the differing mutual inhibitions of N(2) and C(2)H(2) in wild-type protein; (c) the modified reactivity of the Azotobacter vinelandii alpha-(Gly)69(Ser) mutant with N(2) and C(2)H(2); and (d) the basis for the stereoselectivity of hydrogenation of C(2)D(2) and its loss in some mutant proteins. Some structures for initially bound N(2) and C(2)H(2), and their hydrogenated intermediates, are presented. The key new concept is that binding sites and binding states for substrates and intermediates are characterized not only by their locations on the FeMo-co face but also by the structural and temporal status of the distribution of H atoms over the FeMo-co reaction domain.

Contact angle, WAXS, and SAXS analysis of poly(beta-hydroxybutyrate) and poly(ethylene glycol) block copolymers obtained via Azotobacter vinelandii UWD.

This study investigated and correlated physical properties and cell interactions of copolymers obtained by a poly(ethylene glycol) (PEG)-modulated fermentation of Azotobacter vinelandii UWD. PEGs with molecular weights of 400 and 3400 Da and di(ethylene glycol) (DEG) were used to modulate the bacterial synthesis of poly(beta-hydroxybutyrate) (PHB). The PHB crystallinity was determined by wide-angle X-ray scattering (WAXS). Small-angle X-ray scattering (SAXS) showed that lamellar distances decreased between the PHB and the PHB modulated with PEG or DEG. Furthermore, the contact angle of water on the PHB/PEG polymer surfaces decreased when compared to that of PHB. The significant decrease of the contact angle and corresponding increase in surface tension, as well as significant decrease in cell adhesion, suggest the presence of hydrophilic PEG and DEG within the hydrophobic surface.

Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-beta-hydroxybutyrate synthesis.

The lipids poly-beta-hydroxybutyrate (PHB) and alkylresorcinols are the major metabolic products of Azotobacter vinelandii cysts. Cysts are formed in less than 0.01% of late stationary phase cells grown on sucrose. Culturing vegetative cells in n-butanol or beta-hydroxybutyrate induces encystment. After induction of encystment, PHB rapidly accumulates in large granules. Then, the cells begin the synthesis of alkylresorcinols that replace the phospholipids in the membranes and are components of the exine, the outer layer of the cyst envelope. Vegetative cells do not synthesize alkylresorcinols. We report here the effect of mutations in the phbBAC operon, coding for the enzymes of the PHB biosynthetic pathway, on the synthesis of alkylresorcinols and cyst formation. The phb mutations did not impair the capacity to form mature cysts. However, the cysts formed by these strains posses a thicker exine layer and a higher content of alkylresorcinols than the cysts formed by the wild-type strain. A blockage of PHB synthesis caused by phb mutations resulted in the synthesis of alkylresorcinols and encystment even under non-inducing conditions. We propose that, as a consequence of the blockage in the PHB biosynthetic pathway, the acetyl-CoA and reducing power pools are increased causing the shift to lipid metabolism required for the synthesis of alkylresorcinols and cyst formation.

Functional expression of the FeMo-cofactor-specific biosynthetic genes nifEN as a NifE-N fusion protein synthesizing unit in Azotobacter vinelandii.

The nifEN encodes an E2N2 tetrameric metalloprotein complex that serves as scaffold for assembly of the FeMo cofactor of nitrogenase. In most diazotrophs, the NifE and NifN are translated as separate polypeptides and then assembled into tetrameric E2N2 complex. However, in Anabaena variabilis which has two nif clusters that encode two different NifEN complexes, the NifEN2 is encoded by a single nifE-N like gene, which has high homology to the NifE at amino-terminus and to the NifN at the carboxy-terminus. These observations implied that a metalloprotein like NifEN can accommodate large variations in their amino acid composition and also in the way they are synthesized (as two separate proteins or as a single protein) and yet remain functional. In Azotobacter vinelandii NifE and NifN are synthesized separately. To test whether NifEN could retain its functionality when encoded by a single gene, we generated a translational fusion of the nifE and nifN genes of A. vinelandii that could encode a large NifE-N fusion protein. When expressed in the nifEN-minus strain of A. vinelandii, the nifE-N gene fusion could complement the NifEN function. Western blot analysis by using polyclonal NifEN antibodies revealed that the complementing nifEN product is a large NifE-N fusion protein unit. The fact that the gene fusion of nifE-N specifies a functional NifE-N fusion protein reflects that these metalloproteins can accommodate a wide range of flexibility in their gene organization, structure, and assembly.

Stereospecificity of acetylene reduction catalyzed by nitrogenase.

In addition to catalyzing the reduction of dinitrogen to ammonia, the metalloenzyme nitrogenase catalyzes the reduction of a number of alternative substrates, including acetylene (C(2)H(2)) to ethylene (C(2)H(4)) and, in certain cases, to ethane (C(2)H(6)). The stereochemistry of proton addition for C(2)D(2) reduction to C(2)D(2)H(2) catalyzed by the Mo-dependent nitrogenase has been used to probe substrate binding and proton addition mechanisms. In the present work, the C(2)D(2) reduction stereospecificity of altered MoFe proteins having amino acid substitutions within the active site FeMo-cofactor environment was examined by Fourier transform infrared (FTIR) spectroscopy. Altered MoFe proteins examined included those having the alpha-subunit 96(Arg) residue substituted by Gln, Leu, or Ala, the alpha-subunit 69(Gly) residue substituted by Ser, and the alpha-subunit 195(His) residue substituted by Asn. The stereochemistry of proton addition to C(2)D(2) does not correlate with the measured K(m) values for C(2)H(2) reduction, or with the ability of the enzyme to reduce C(2)H(2) by four electrons to yield C(2)H(6). Instead, the electron flux through nitrogenase was observed to significantly influence the ratio of cis- to trans-1,2-C(2)H(2)D(2) formed. Finally, the product distribution observed for reduction of C(2)H(2) in D(2)O is not consistent with an earlier proposed enzyme-bound intermediate. An alternative model that accounts for the stereochemistry of C(2)H(2) reduction by nitrogenase based on a branched reaction pathway and an enzyme-bound eta(2)-vinyl intermediate is proposed.

Mannuronan C-5 epimerases and cellular differentiation of Azotobacter vinelandii.

Differentiation in Azotobacter vinelandii involves the encystment of the vegetative cell under adverse environmental circumstances and the germination of the resting cell into the vegetative state when growth conditions are satisfactory again. Morphologically, the encystment process involves the development of a protective coat around the resting cell. This coat partly consists of multiple layers of alginate, which is a copolymer of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G). Alginate contributes to coat rigidity by virtue of a high content of GG blocks. Such block structures are generated through a family of mannuronan C-5 epimerases that convert M to G after polymerization. Results from immunodetection and light microscopy, using stains that distinguish between different cyst components and types, indicate a correlation between cyst coat organization and the amount and appearance of mannuronan C-5 epimerases in the extracellular medium and attached to the cells. Specific roles of individual members of the epimerase family are indicated. Calcium and magnesium ions appear to have different roles in the structural organization of the cyst coat. Also reported is a new gene sharing strong sequence homology with parts of the epimerase-encoded R-modules. This gene is located within the epimerase gene cluster of Azotobacter vinelandii.

Investigations on the cell volumes of Azotobacter vinelandii by scanning electron microscopy.

Previous experiments by other investigators on the DNA content of Azotobacter vinelandii have demonstrated that the DNA content in these cells is several folds higher than that of E. coli. On the basis of this observation, it was hypothesized that A. vinelandii has at least 40 to 80 identical chromosomes per cell. However, the gene dosage analysis in A. vinelandii cells suggested that many genetic operations can be performed in these cells without the constraints expected in a polyploid bacterium. In an attempt to explain this apparent discrepancy, we have done systematic analysis of the relationship between the DNA content and the cell volume of this bacterium. Since a linear correlation is observed between the DNA content and the cell size in many other cell types, we hypothesized that if A. vinelandii is polyploid in nature, it should have a much larger cell volume to accommodate such a large amount of DNA. Our scanning electron microscopic analysis revealed that the cell volume of the vegetative cells of A. vinelandii is about 16 times larger than the cell volume of E. coli. This result is apparently consistent with the concept that the A. vinelandii is a polyploid bacterium. It was also reported that the encysted cells of A. vinelandii contain about 25% of the DNA content of the vegetative cells. This would mean that an encysted cell of A. vinelandii could contain about 10 copies of its chromosome. Since the estimated molecular weight of A. vinelandii chromosome is very similar to that of E. coli chromosome, the DNA content of the encysted cells also should be about 10 times higher than that of E. coli cells. If we assume that the relationship between the DNA content and the cell size is linear, then the encysted cells should have a cell volume larger than that of E. coli and smaller than that of the vegetative cells of A. vinelandii. However our scanning electron microscopic analysis showed that the cell volume of the encysted cells of A. vinelandii is in fact very similar to the cell volume of E. coli.