Siderophores Produced by Nitrogen-Fixing Azotobacter vinelandii OP in Iron-Limited Continuous Culture

Azotobacter vinelandii requires a high complement of iron and an efficient iron acquisition system to support nitrogen fixation. To circumvent problems inherent in batch culture trace metal studies, continuous cultures were used to measure the response of A. vinelandii to iron stress. Iron was found to be growth limiting for nitrogen-fixing A. vinelandii at a concentration as high as 12.5 muM; iron was growth sufficient at 25 muM. Iron-stressed A. vinelandii in continuous culture formed 2,3-hydroxybenzoic acid (DHB), 2-N,6-N-di-(2,3-dihydroxybenzoyl)-l-lysine (DHBL), and a chromophoric yellow-green fluorescent peptide (YGFP). At a fixed dilution rate of 0.1 h, steady-state growth occurred at growth-limiting iron concentrations. DHB and DHBL were quantitatively measured during iron-limited steady states and iron-sufficient states by Arnow colorimetric assays. YGFP was determined by absorbance measurements taken at 380 nm, and the concentration was calculated from the reported specific absorption coefficient. Biomass increased and DHBL, DHB, and YGFP concentrations decreased as the concentration of growth-limiting iron was increased in the culture vessel and medium reservoirs. DHBL was the major siderophore and YGFP was the minor siderophore species produced during iron-limited equilibrium growth. A low level of DHB and YGFP, but no DHBL, was formed under iron-sufficient conditions. These results provide further physiological evidence that DHB, YGFP, and especially DHBL may function as siderophores in nitrogen-fixing A. vinelandii.     

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.     

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.     

Cross-linking site in Azotobacter vinelandii complex

The Fe-protein and the MoFe-protein of the Azotobacter vinelandii nitrogenase complex can be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Willing, A., Georgiadis, M.M., Rees, D. C., and Howard, J. B. (1989) J. Biol. Chem. 264, 8499-8503). In this reaction, one of the identical subunits of the Fe-protein dimer is linked by an isopeptide bond to each beta-subunit of the MoFe-protein tetramer. The reaction has been found to be highly specific with greater than 85% of amino acid residues Glu-112 (Fe-protein) and Lys-399 (MoFe-protein) cross-linked to each other. Although Glu-112 is located in a highly conserved amino acid sequence, it is found in only half of the known Fe-protein sequences. Likewise, Lys-399 is not a conserved residue in the MoFe-protein. Glu-112 appears to be part of an anionic cluster of nine carboxylic acids which is located between the proposed thiol ligands for the Fe:S center. In contrast, the basic residue cluster which includes Lys-399 has been found in only in the Azotobacter MoFe-protein. Thus, this crosslinking reaction either is unique to Azotobacter nitrogenase or must involve other residues in the MoFe-protein of other species. Because Lys-399 and Glu-112 form a specific cross-link, it is probable that they are part of the interaction site leading to productive complex formation. This information should be useful for the model building of the complex from the crystallographic structures of the individual components.     

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.     

Tn10 mutagenesis in Azotobacter vinelandii

Summary The tetracycline-resistant transposon Tn10 and its high-hopper derivative Tn10HH104 were introduced into the Azotobacter vinelandii genome using suicide conjugative plasmids derived from pRK2013. Several types of mutants induced by either of these elements are described. Nif^- mutants (deficient in nitrogen fixation) were easily isolated, whereas the isolation of other mutant types (auxotrophs, sugar non-users) required special selection conditions. The characterization of the mutations as transposon insertions was often complicated and sometimes required a combination of genetic and physical tests. A common source of complication, the existence of double inserts, was found among the mutants induced by Tn10HH104 but not among those induced by Tn10. Both the high-hopper and the wild-type element proved to undergo secondary transpositions, albeit at different frequencies. Another type of complication, the existence of heterozygotes, occurred because of the high level of redundancy of the A. vinelandii genome.     

Formation of protoplasts in Azotobacter vinelandii

Protoplasts of Azotobacter vinelandii were formed by incubating whole cells in lysozyme and EDTA in Tris-HCl buffer (0.05 M, pH 8.0) supplemented with sucrose (15% w/v). This appeared to be related to the special chelating ability of EDTA and Tris-HCl since substitution of the former by nitrilotriacetic acid or by trisodium citrate and the latter by veronal-acetate buffer or tris-maleate buffer over a pH range of 5.2 to 8.6 yielded only spheroplasts. Of nine strains of Azotobacter studied, only A. vinelandii strain 12837 and strain 0 formed protoplasts.     

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.     

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.     

Growth of Azotobacter vinelandii UWD in Fish Peptone Medium and Simplified Extraction of Poly-beta-Hydroxybutyrate

Azotobacter vinelandii UWD was grown in a fermentor with glucose medium with and without 0.1% fish peptone (FP) in batch and fed-batch cultures for the production of the natural bioplastic poly-beta-hydroxybutyrate (PHB). Strain UWD formed PHB five times faster than cell protein during growth in glucose and NH(4), but PHB synthesis stopped when NH(4) was depleted and nitrogen fixation started. When FP was added to the same medium, PHB accumulated 16 times faster than cell protein, which in turn was inhibited by 40%, and PHB synthesis was unaffected by NH(4) depletion. Thus, FP appeared to be used as a nitrogen source by these nitrogen-fixing cells, which permitted enhanced PHB synthesis, but it was not a general growth stimulator. The addition of FP to the medium led to the production of large, pleomorphic, osmotically sensitive cells that demonstrated impaired growth and partial lysis, with the leakage of DNA into the culture fluid, but these cells were still able to synthesize PHB at elevated rates and efficiency. When FP was continuously present in fed-batch culture, the yield in grams of polymer per gram of glucose consumed was calculated to range from 0.43 g/g, characteristic of nongrowing cells, to an unprecedented 0.65 g/g. Separation of an FP-free growth phase from an FP-containing growth phase in fed-batch culture resulted in better growth of these pleomorphic cells and good production of PHB (yield, 0.32 g/g). The fragility of these cells was exploited in a simple procedure for the extraction of high-molecular-weight PHB. The cells were treated with 1 N aqueous NH(3) (pH 11.4) at 45 degrees C for 10 min. This treatment removed about 10% of the non-PHB mass from the pellet, of which 60 to 77% was protein. The final product consisted of 94% PHB, 2% protein, and 4% nonprotein residual mass. The polymer molecular weight (1.7 x 10 to 2.0 x 10) and dispersity (1.0 to 1.9) were not significantly affected (P = 0.05) by this treatment. In addition, the NH(3) extraction waste could be recycled in the fermentation as a nitrogen source, but it did not promote PHB production like FP. A scheme for improved downstream extraction of PHB as well as the merits of using pleomorphic cells in the production of bioplastics is discussed.     

Chemotactic Behavior of Azotobacter vinelandii

Chemotaxis was exhibited by Azotobacter vinelandii motile cells. Exposure of cells to sudden increases in attractant concentration suppressed the frequency of tumbling and resulted in smooth swimming. Cells responded chemotactically to a chemical gradient produced during metabolism. Motility occurred over a temperature range of 25 to 37°C with an optimum pH range of between pH 7.0 and 8.0. The average speed of motile cells was determined to be 74 μm/s or 37 body lengths per s. The speed of cells appeared to increase as a function of attractant concentration. Chemotactic systems for fructose, glucose, xylitol, and mannitol were inducible. A. vinelandii exhibited chemotaxis for a number of compounds, including hexoses, hexitols, pentitols, pentoses, disaccharides, and amino sugars. We conclude from these studies that A. vinelandii exhibits a temporal chemotactic sensing system.     

Protection of Nitrogenase in Azotobacter vinelandii

The site or sites that protect nitrogenase from O(2) inactivation in vivo are sensitive to sodium azide or 2,4-dinitrophenol. Both components of nitrogenase can be synthesized when oxidative phosphorylation is disrupted.     

Morphogenesis of Cysts in Azotobacter vinelandii

Cultures of Azotobacter vinelandii were induced to encystment with β-hydroxybutyrate. The morphological events in the transition from cell to cyst were observed by electron microscopy of thin sections. Upon induction of encystment, cells became rounded and nonmotile. The exine coat developed by the continuous excretion of membranous components into the capsule surrounding the cell.