Interaction of Agromyces ramosus with Other Bacteria in Soil

Agromyces ramosus occurs in very high numbers in most soils and, based on studies of laboratory isolates, does not require host cells for growth. Nevertheless, it attacked and destroyed most of the gram-positive and gram-negative bacterial species tested as possible host organisms. A. ramosus also attacked and destroyed Saccharomyces cerevisiae. The possibility of attack on fungi was unclear. Among the bacteria serving as hosts were the important soil species Azotobacter vinelandii, Rhizobium leguminosarum, Rhizobium meliloti, and Agrobacterium tumefaciens. Dead cells were not attacked. A. vinelandii cysts were attacked but left unharmed. To some extent, A. vinelandii seemed to survive this attack by encysting. Attack by A. ramosus occurred in natural soil and over a broad range of nutritional levels in laboratory media. The attack did not seem to be a means for obtaining an increased supply of commonly available nutrients. Instead, it seemed to be a means of obtaining something produced, perhaps in small amounts, by a variety of organisms, but not by all organisms. Several types of culture filtrates were tested for activity. The filtrates neither stimulated nor inhibited the growth of A. ramosus or the host organisms. The availability of catalase activity in host organisms did not seem to be involved. It is not known whether the attack by Agromyces ramosus in soil can be manipulated to cause a decrease in numbers of Agrobacterium tumefaciens or other pathogens without simultaneously depressing the numbers of beneficial organisms in this habitat.     

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.     

Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6

The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6''s genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6''s yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound.     

Growth of Azotobacter vinelandii on Soil Nutrients

Azotobacter vinelandii cells grew well in a medium made from soil and distilled water which contained little or no carbohydrate. They utilized p-hydroxybenzoic acid and other phenolic acids, soil nitrogen, and water-soluble mineral substances. Seventeen soils which supported excellent growth of A. vinelandii contained 11 to 18 different phenolic acids each, including p-hydroxybenzoic, m-hydroxybenzoic, vanillic, p-coumeric, syringic, cis- and trans-ferrulic, and other unidentified aromatic acids. Three white, chalky “caliche” soils which were taken from areas where no plants grew failed to support the growth of A. vinelandii, and these contained no, two, and three phenolic acids, respectively. A. vinelandii did not fix nitrogen when growing in dialysates of soils which contained numerous phenolic acids. Growth was ample and rapid in most of the soils tested, but cell morphology was different from that usually seen in chemically defined, nitrogen-free media which contain glucose.     

Phenotypic and molecular characterisation of efficient nitrogen-fixing Azotobacter strains from rice fields for crop improvement

Biological nitrogen fixation (BNF) is highly effective in the field and potentially useful to reduce adverse effects chemical fertilisers. Here, Azotobacter species were selected via phenotypic, biochemical and molecular characterisations from different rice fields. Acetylene reduction assay of Azotobacter spp. showed that Azotobacter vinelandii (Az3) fixed higher amount of nitrogen (121.09 nmol C₂H₄?mg^-1 bacteria h^-1). Likewise, its plant growth functions, viz. siderophore, hydrogen cyanide, salicylic acid, IAA, GA₃, zeatin, NH₃, phosphorus solubilisation, ACC deaminase and iron tolerance, were also higher. The profile of gDNA, plasmid DNA and cellular protein profile depicted inter-generic and inter-specific diversity among the isolates of A. vinelandii. The PCR-amplified genes nifH, nifD and nifK of 0.87, 1.4 and 1.5 kb , respectively, were ascertained by Southern blot hybridisation in isolates of A. vinelandii. The 16S rRNA sequence from A. vinelandii (Az3) was novel, and its accession number (JQ796077) was received from NCBI data base. Biofertiliser formulation of novel A. vinelandii isolates along with commercial one was evaluated in rice (Oriza sativa L. var. Khandagiri) fields. The present finding revealed that treatment T4 (Az3) (A. vinelandii) are highly efficient to improved growth and yield of rice crop.     

Azotobacter vinelandii gene clusters for two types of peptidic and catechol siderophores produced in response to molybdenum

Aim: To characterize the complementary production of two types of siderophores in Azotobacter vinelandii. Methods and Results: In an iron‐insufficient environment, nitrogen‐fixing A. vinelandii produces peptidic (azotobactin) and catechol siderophores for iron uptake to be used as a nitrogenase cofactor. Molybdenum, another nitrogenase cofactor, was also found to affect the production level of siderophores. Wild‐type cells excreted azotobactin into molybdenum‐supplemented and iron‐insufficient medium, although catechol siderophores predominate in molybdenum‐free environments. Two gene clusters were identified to be involved in the production of azotobactin and catechol siderophores through gene annotation and disruption. Azotobactin‐deficient mutant cells produced catechol siderophores under the molybdenum‐supplemented and iron‐insufficient conditions, whereas catechol siderophore–deficient mutant cells extracellularly secreted excess azotobactin under iron‐deficient condition independent of the concentration of molybdenum. This evidence suggests that a complementary siderophore production system exists in A. vinelandii. Conclusions: Molybdenum was found to regulate the production level of two types of siderophores. Azotobacter vinelandii cells are equipped with a complementary production system for nitrogen fixation in response to a limited quantity of metals. Significance and Impact of the Study: This is the first study identifying A. vinelandii gene clusters for the biosynthesis of two types of siderophores and clarifying the relationship between them.     

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.     

H2-Uptake Activity, Spectra, Reduction Potentials, and Kinetics of Iron Release on the Surface of Iron Core from Azotobacter vinelandii Bacterial Ferritin

Bacterial ferritin from Azotobacter vinelandii (AvBF_o has a function in H₂ uptake. The Fe³⁺ reduction on the surface of the iron core from AvBF_o is accompanied simultaneously by H₂ uptake, with a maximum activity of H₂ uptake of 450 H₂/AvBF_o. A reduction potential of ?402 mV for iron reduction on the surface of the core is found. A shift to the red the protein absorbance peaks ranging from 280 to 290 nm is observed between pH5 and 9 under 100% H₂ reduction. The reduction potential for iron release becomes negative at a rate of 0.025 mV/Fe²⁺ released. The kinetics of iron release on the surface of the core is a first-order reaction.     

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.     

Effect of transformation ofAzotobacter vinelandii with the low copy number plasmid pRK290

We previously observed that whenAzotobacter vinelandii was transformed by different broad-host-range plasmids, normal cellular functions such as growth and siderophore production are impaired. In the present work, whenA. vinelandii was transformed with the low copy number plasmid pRK290, the extent of this metabolic impairment was lessened, as evidenced by increased siderophore production and moderate levels of growth on medium that lacks added iron. It is concluded that the severity of the plasmid-induced metabolic load reflects the relative level of expression of plasmid-encoded proteins.     

Immunological studies with nitrogenase from Azotobacter and bacterial nitrate reductases.

Antibodies raised to purified Component I of nitrogenase (Mo-Fe-S) protein from Azotobacter vinelandii cross-reacted not only with this protein but also with nitrate reductases from a number of bacteria. Antibodies raised for a purified nitrate reductase from Escherichia coli also formed precipitin bands with this Component I of nitrogenase. Antibodies to Component I, however, did not react with nitrate reductases from either a blue-green alga Anabaena cylindrica or with higher plants, or with aldehyde dehydrogenase and xanthine oxidase from animal sources.     

Development of a New Biofertilizer with a High Capacity for N2 Fixation,Phosphate and Potassium Solubilization and Auxin Production

Biofertilizers that possess a high capacity for N₂ fixation (Azotobacter tropicalis), and consist of phosphate solubilizing bacteria (Burkhoderia unamae), and potassium solubilizing bacteria (Bacillus subtilis) and produce auxin (KJB9/2 strain), have a high potential for growth and yield enhancement of corn and vegetables (Chinese kale). For vegetables, the addition of biofertilizer alone enhanced growth 4 times. Moreover, an enhancement of growth by 7 times was observed due to the addition of rock phosphate and K-feldspar, natural mineral fertilizers, in combination with the biofertilizer.     

A large, efficient and inexpensive bacterial growth chamber

The construction of a 200 liter bacterial growth chamber for a total cost of less than $400 is described. The chamber was designed for growth of aerobic organisms and therefore provides high rates of aeration and mixing. The growth of Escherichia coli K₁₂ in the chamber was similar to growth in small flasks on a rotary shaker. Azotobacter vinelandii OP growth is slower in the growth chamber than in small, shaken flasks. Under the growth conditions described, kilogram quantities of bacterial cells are obtained from a single culture.     

Chemotaxis of Azotobacter vinelandii and Bacillus subtilis in a mixed culture

In the mixed culture of Azotobacter vinelandii and Bacillus subtilis, chemotaxis of Azotobacter to glucose remained unchanged, while that of bacilli decreased. Microelectrophoresis demonstrated adhesion of the A. vinelandii polysaccharide on the surface of B. subtilis cells. In the presence of 0.05–1.0 g/L of this biopolymer, the chemotaxis of bacilli to glucose decreased 2.6 to 6.8 times. A. vinelandii polysaccharide molecules adherent on the surface of B. subtilis cells were suggested to block bacillary chemotactic receptors, resulting in a decrease in their directed motility in the mixed culture.     

Polysaccharide production and the possible occurrence of GDP-d-mannose dehydrogenase inAzotobacter vinelandii

During the growth ofAzotobacter vinelandii in batch culture in Burk's 2% glucose medium supplemented with 50mg EDTA per litre, water-insoluble capsular polysaccharide material accumulated in cultures prior to the appearance of water-soluble polysaccharide in the culture medium. On isolation, hydrolysis and chromatography, both these polysaccharides were observed to be composed of carbohydrate monomers having the same chromatographic mobilities as glucose, rhamnose, guluronic acid and mannuronic acid. The activity of GDP-d-mannose dehydrogenase recorded in crude cell-free extracts fromAzotobacter vinelandii, when these polysaccharides were produced, may indicate a close similarity between the biosynthetic pathway of alginate synthesis in marine Phaeophyceae and this soil microorganism.     

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.     

A rapid and sensitive paper electrophoresis assay for the detection of microbial siderophores elicited in solid-plating culture

A rapid and sensitive assay for the detection of microbial siderophores (iron-binding compounds) is described. Nine representative fungal and bacterial cultures including Ustilago sphaerogena, Penicillium sp., Fusarium roseum, Rhodotorula pilimanae, Bacillus subtilis W 23, Bacillus subtilis W 168, Bacillus megaterium, Azotobacter vinelandii OP, and Escherichia coli B, were nutritionally stressed for iron by sequential transfers on iron-deficient solid-plating media. In response to Fe-stress conditions, the microorganisms excreted siderophore compounds into the extracellular solid culture medium. The solid agar matrix effectively concentrated and restricted the migration of the siderophore compounds to the region immediately adjacent to colonial growth. Agar-block samples from this region were removed and placed at the origin of an electrophoresis paper strip. The resultant absorbed material from the agar-block sample was subjected to high-voltage paper electrophoresis which separated the siderophore compounds by size and molecular net charge. Phenolic acid (“catechol”)-type siderophores were detected by fluorescence under uv light. Hydroxamic acid-type siderophores were visualized by spraying the electrophoretogram with ferric iron solution.     

Gene Deletions Resulting in Increased Nitrogen Release by Azotobacter vinelandii: Application of a Novel Nitrogen Biosensor

Azotobacter vinelandii is a widely studied model diazotrophic (nitrogen-fixing) bacterium and also an obligate aerobe, differentiating it from many other diazotrophs that require environments low in oxygen for the function of the nitrogenase. As a free-living bacterium, A. vinelandii has evolved enzymes and transporters to minimize the loss of fixed nitrogen to the surrounding environment. In this study, we pursued efforts to target specific enzymes and further developed screens to identify individual colonies of A. vinelandii producing elevated levels of extracellular nitrogen. Targeted deletions were done to convert urea into a terminal product by disrupting the urease genes that influence the ability of A. vinelandii to recycle the urea nitrogen within the cell. Construction of a nitrogen biosensor strain was done to rapidly screen several thousand colonies disrupted by transposon insertional mutagenesis to identify strains with increased extracellular nitrogen production. Several disruptions were identified in the ammonium transporter gene amtB that resulted in the production of sufficient levels of extracellular nitrogen to support the growth of the biosensor strain. Further studies substituting the biosensor strain with the green alga Chlorella sorokiniana confirmed that levels of nitrogen produced were sufficient to support the growth of this organism when the medium was supplemented with sufficient sucrose to support the growth of the A. vinelandii in coculture. The nature and quantities of nitrogen released by urease and amtB disruptions were further compared to strains reported in previous efforts that altered the nifLA regulatory system to produce elevated levels of ammonium. These results reveal alternative approaches that can be used in various combinations to yield new strains that might have further application in biofertilizer schemes.     

Effects of Mannose on the Growth of N2-Fixing Azotobacter vinelandii

Mannose is not a suitable substrate for N₂-fixing Azotobacter vinelandii. However, when H₂ gas is provided, A. vinelandii can grow mixotrophically with H₂ as the energy source and mannose as the carbon source (T.-Y. Wong and R. J. Maier, J. Bacteriol. 163:528-533, 1985). In this report, seven sugars were used to determine whether A. vinelandii could derive energy from these sugars for mannose utilization. Supplementation of fructose- or galactose-limited medium with mannose did not influence the biomass produced by N₂-fixing A. vinelandii. The presence of mannose in glucose- or maltose-limited cultures increased cell yield slightly. The addition of mannose decreased the total biomass in the melibiose-limited culture slightly. Mannose was a potent inhibitor of growth when sucrose or turanose was used as the primary sugar. The inhibitory effect of mannose on utilization of sucrose and turanose seems to be related to the energy requirement of the N₂-fixing processes.