Determination of the size of the Azotobacter vinelandii chromosome

The chromosome of Azotobacter vinelandii UW was digested separately with the rape cutter restriction endonucleases Swal (5′-ATTTAAAT), PmeI (5′GTTTAAAC) and Pacl (5′-TTAATTAA) and the products were separated by pulsed-field gel electrophoresis. The size of the chromosome was determined to be approximately 4.5 megabase pairs (Mb) based on the sum of the sizes of the restriction fragments. This is almost the same as the size of the chromosome of Escherichia coli. The inability of the undigested DNA to enter the gel has led us to infer that the chromosome is circular.     

TheAzotobacter flora of some Czechoslovakian watercourses

The occurrence of Azotobacter in some Szechoslovakian watercourses has been investigated. Several strains belonging to A. chroococcum and A. beijerinckii were isolated from 7 out of 18 samples. A. insignis has been isolated from the flowing water of Lake Machovo at Doksy. This is first report of this strictly aquatic Azotobacter species in Czechoslovakian watercourses. The taxonomy of the genus Azotobacter was discussed against the background of the existing knowledge resulting mainly from other taxonomic techniques than those based on the phenotypically expressed characters.     

Flavodoxin fromAzotobacter vinelandii

Summary A flavoprotein fromAzotobacter vinelandii, named in the literature Shethna flavoprotein or azotoflavin, has recently been shown to catalyze acetylene reduction by a cell-free nitrogenase preparation fromAzotobacter. In this communication this result is confirmed. Moreover, the Shethna flavoprotein is now shown to replace ferredoxin in the catalysis of NADP⁺-reduction by illuminated spinach chloroplasts or by molecular hydrogen and hydrogenase fromClostridium. Evidence is presented that the fully reduced form of the Shethna flavoprotein is involved in the catalysis of NADP⁺-reduction. This indicates that the Shethna flavoprotein functions as a substitute for an one electron carrier shuttling between fully reduced and semiquinoid form. From the data reported here, together with that in the literature, it is concluded that this flavoprotein belongs to the class of flavodoxins. Some preliminary results on ferredoxin fromAzotobacter are reported.A flavoprotein fromAzotobacter commonly referred in the literature as the Shethna flavoprotein or azotoflavin is found to exhibit the characteristics of a flavodoxin.     

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.     

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.     

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.     

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.     

Endogenous Encystment of Azotobacter vinelandii

When young cells of Azotobacter vinelandii are impinged on membrane filters, washed free of carbon substrate, and placed on a mineral salts basal medium, the culture will proceed to encyst although at a slower rate than if n-butanol were supplied as a substrate. The endogenous cysts are depleted in polyβ-hydroxybutyrate and have a narrower intine but show an increased resistance to desiccation and are susceptible to lysis by chelating agents. Membrane-supported cells reveal details of the encystment process such as the formation of a zone within the capsule prior to exine formation and the early deposition of exine structures.     

Inosine nucleosidase from Azotobacter vinelandii

An enzyme catalyzing the hydrolysis of purine nucleosides was found to occur in the extract of Azotobacter vinelandii, strain 0, and was highly purified by ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxylapatite chromatography and gel filtration on Sephadex G-150. A strict substrate specificity of the purified enzyme was shown with respect to the base components. The enzyme specifically attacked the nucleosides without amino groups in the purine moiety: inosine gave the maximum rate of hydrolysis and xanthosine was hydrolyzed to a lesser extent. The pH optimum of inosine hydrolysis was observed from pH 7 to 9, while xanthosine was hydrolyzed maximally at pH 7. The K _m values of the enzyme for inosine were 0.65 and 0.85 mM at pH 7.1 and 9.0, respectively, and the value for xanthosine was 1.2 mM at pH 7.1.Several nucleotides inhibited the enzyme: the phosphate portions of the nucleotides were suggested to be responsible for the inhibition by nucleotides. Although the inhibition of the enzyme by nucleotides was apparently non-competitive type with respect to inosine, allosteric (cooperative) binding of the substrate was suggested in the presence of the inhibitor. The physiological significance of the enzyme was discussed in connection with the degradation and salvage pathways of purine nucleotides.     

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.     

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.     

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.