Chemotactic and adhesive properties of Azotobacter vinelandii and Bacillus subtilis

The effects of some factors on the chemotaxis of Azotobacter vinelandii IMV V-7076 and Bacillus subtilis IMV V-7023 and on their adhesion to cucumber roots have been studied. Glucose chemotaxis and adhesion to roots reach peak values in pH ranges characteristic of each strain. These ranges are 7.0–8.0 for A. vinelandii IMV V-7076 and 6.0–7.0 for B. subtilis IMV V-7023. The adhesion values of each species decrease significantly in their mixed suspension. The interaction of each of the strains with the clay mineral montmorillonite improves their adhesion to cucumber roots. The clay mineral palygorskite improves the adhesion of A. vinelandii but reduces that of B. subtilis.     

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.     

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.     

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.     

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.     

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.     

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.     

Chemical composition of Azotobacter vinelandii cysts

Cysts of Azotobacter vinelandii ATCC 12837 were germinated by exposure to 3.0 mm ethylenediaminetetraacetic acid (EDTA)-tris(hydroxymethyl)aminomethane buffer at pH 7.8, and their outer coats (exines) were purified by differential and isopycnic centrifugation. Electron micrographs of exine showed it to consist of multilayers of a three-membered sheet structure whose thickness was 7.0 to 7.5 nm. The inner, less electron-dense layer (intine) was also prepared from cysts by EDTA treatment, centrifugation, concentration, and dialysis. The exine consisted of 32% carbohydrate, 28% protein, 30% lipid, and 3.2% ash, with the ash comprised of 1.62% calcium, 0.02% magnesium, and 0.34% phosphorus. The amino acid composition of exine was similar to that of gram-negative bacterial cell walls. The intine consisted of 44% carbohydrate, 9.1% protein, 37% lipid, and 4.1% ash, with the ash comprised of 2.45% calcium, 0.02% magnesium, and 0.38% phosphorus. The carbohydrates of both exine and intine contained glucose, mannose, xylose, and rhamnose. Glucosamine and galactosamine were found only in the exines. The fatty acids consisted of normal, iso, and anteiso saturated fatty acids with 10 to 18 carbon atoms and mono-unsaturated C(11), C(16), and C(18) fatty acids. The exines contained mostly bound lipid, but intines contained primarily free lipid.     

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.     

Plasmid transformation of Azotobacter vinelandii OP

Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron- and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif+). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42 degrees C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HincII-digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.     

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.     

The Siderophore Metabolome of Azotobacter vinelandii

In this study, we performed a detailed characterization of the siderophore metabolome, or “chelome,” of the agriculturally important and widely studied model organism Azotobacter vinelandii. Using a new high-resolution liquid chromatography-mass spectrometry (LC-MS) approach, we found over 35 metal-binding secondary metabolites, indicative of a vast chelome in A. vinelandii. These include vibrioferrin, a siderophore previously observed only in marine bacteria. Quantitative analyses of siderophore production during diazotrophic growth with different sources and availabilities of Fe showed that, under all tested conditions, vibrioferrin was present at the highest concentration of all siderophores and suggested new roles for vibrioferrin in the soil environment. Bioinformatic searches confirmed the capacity for vibrioferrin production in Azotobacter spp. and other bacteria spanning multiple phyla, habitats, and lifestyles. Moreover, our studies revealed a large number of previously unreported derivatives of all known A. vinelandii siderophores and rationalized their origins based on genomic analyses, with implications for siderophore diversity and evolution. Together, these insights provide clues as to why A. vinelandii harbors multiple siderophore biosynthesis gene clusters. Coupled with the growing evidence for alternative functions of siderophores, the vast chelome in A. vinelandii may be explained by multiple, disparate evolutionary pressures that act on siderophore production.     

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.     

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