In vitro investigation to explore the toxicity of different groups of pesticides for an agronomically important rhizosphere isolate Azotobacter vinelandii.

In this work, an attempt was made to evaluate the effect of pesticides on growth pattern, surface morphology, cell viability and growth regulators of nitrogen fixing soil bacterium. Pesticide tolerant Azotobacter vinelandii strain AZ6 (Accession no. MG028654) was found to tolerate maximum level of pesticide and displayed multifarious PGP activities. At higher concentrations, pesticides triggered cellular/structural damage and reduced the cell viability as clearly shown under SEM and CLSM. With increase in concentration, pesticides exhibited a significant (p < 0.05) decrease in PGP traits of strain AZ6. Among all three groups of pesticides, herbicides glyphosate and atrazine were most toxic. Kitazin, hexaconazole, metalaxyl, glyphosate, quizalofop, atrazine, fipronil, monocrotophos and imidacloprid at 2400, 1800, 1500, 900, 1200, 900, 1800, 2100 and 2700 µg mL-1, respectively, decreased the production of IAA by 19.5 ±â€¯1.9 (61%), 18.1 ±â€¯1.2 (64%), 36.4 ±â€¯3.4 (28%), 13.1 ±â€¯0.8 (74%), 15.6 ±â€¯1.0 (69%), 7.6 ±â€¯0.5 (83%), 11.9 ±â€¯0.8 (76%), 24.7 ±â€¯1.7 (51%) and 32 ±â€¯2.3 (37%) µg mL-1, respectively, over control (50.7 ±â€¯3.6 µg mL-1). A maximum reduction of 8.4 ±â€¯1.2 (46%), 5.8 ±â€¯0.6 (62%) and 4 ±â€¯0.2 (74%) µg mL-1 in 2, 3-DHBA at 300 (1×), 600 (2×) and 900 (3×) µg mL-1 glyphosate, respectively, While, 32.8 ±â€¯2.7 (19%), 27.2 ±â€¯2 (33%) and 21.5 ±â€¯1.3 (47%) µg mL-1, respectively in the production of SA was observed at 300 (1×), 600 (2×) and 900 (3×) µg mL-1 atrazine, respectively. Likewise, with increase in concentration of pesticides, decrease in P solubilization ability and change in pH of broth was detected. The order of pesticide toxicity to PSE (percent decline over control) at highest concentration was: atrazine (45) > kitazin (44) > metalaxyl (43) > monocrotophos (43) > glyphosate (41) > hexaconazole (39) > quizalofop (33) > imidacloprid (31) > fipronil (25). The present study undoubtedly suggests that even at higher doses of pesticides, A. vinelandii maintained secreting plant growth regulators and this property makes this strain agronomically important microbe for enhancing the growth of plants.

Silver Nanoparticles Induced Cell Apoptosis, Membrane Damage of Azotobacter vinelandii and Nitrosomonas europaea via Generation of Reactive Oxygen Species.

Silver nanoparticles (AgNPs) is widely used as an antibacterial agent, but the specific antibacterial mechanism is still conflicting. This study aimed to investigate the size dependent inhibition of AgNPs and the relationship between inhibition and reactive oxygen species (ROS). Azotobactervinelandii and Nitrosomonaseuropaea were exposed to AgNPs with different particles size (10 nm and 50 nm). The ROS production was measured and the results showed that the generation of ROS related to the particle size and concentrations of AgNPs. At 10 mg/L of 10 nm Ag particles, the apoptosis rate of A. vinelandii and N. europaea were 20.23% and 1.87% respectively. Additionally, the necrosis rate of A. vinelandii and N. europaea reached to 15.20% and 42.20% respectively. Furthermore, transmission electron microscopy images also indicated that AgNPs caused severely bacterial cell membrane damage. Together these data suggested that the toxicity of AgNPs depends on its particle size and overproduction of ROS.

Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion.

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.

Effects of tungsten and titanium oxide nanoparticles on the diazotrophic growth and metals acquisition by Azotobacter vinelandii under molybdenum limiting condition.

The acquisition of essential metals, such as the metal cofactors (molybdenum (Mo) and iron (Fe)) of the nitrogenase, the enzyme responsible for the reduction of dinitrogen (N(2)) to ammonium, is critical to N(2) fixing bacteria in soil. The release of metal nanoparticles (MNPs) to the environment could be detrimental to N(2) fixing bacteria by introducing a new source of toxic metals and by interfering with the acquisition of essential metals such as Mo. Since Mo has been reported to limit nonsymbiotic N(2) fixation in many ecosystems from tropical to cold temperate, this question is particularly acute in the context of Mo limitation. Using a combination of microbiology and analytical chemistry techniques, we have evaluated the effect of titanium (Ti) and tungsten (W) oxide nanoparticles on the diazotrophic growth and metals acquisition in pure culture of the ubiquitous N(2) fixing bacterium Azotobacter vinelandii under Mo replete and Mo limiting conditions. We report that under our conditions (≤10 mg·L(-1)) TiO(2) NPs have no effects on the diazotrophic growth of A. vinelandii while WO(3) NPs are highly detrimental to the growth especially under Mo limiting conditions. Our results show that the toxicity of WO(3) NPs to A. vinelandii is due to an interference with the catechol-metalophores assisted uptake of Mo.

The acetylation degree of alginates in Azotobacter vinelandii ATCC9046 is determined by dissolved oxygen and specific growth rate: studies in glucose-limited chemostat cultivations.

Alginates are polysaccharides that may be used as viscosifiers and gel or film-forming agents with a great diversity of applications. The alginates produced by bacteria such as Azotobacter vinelandii are acetylated. The presence of acetyl groups in this type of alginate increases its solubility, viscosity, and swelling capability. The aim of this study was to evaluate, in glucose-limited chemostat cultivations of A. vinelandii ATCC9046, the influence of dissolved oxygen tension (DO) and specific growth rate (µ) on the degree of acetylation of alginates produced by this bacterium. In glucose-limited chemostat cultivations, the degree of alginate acetylation was evaluated under two conditions of DO (1 and 9 %) and for a range of specific growth rates (0.02-0.15 h⁻¹). In addition, the alginate yields and PHB production were evaluated. High DO in the culture resulted in a high degree of alginate acetylation, reaching a maximum acetylation degree of 6.88 % at 9 % DO. In contrast, the increment of µ had a negative effect on the production and acetylation of the polymer. It was found that at high DO (9 %) and low µ, there was a reduction of the respiration rate, and the PHB accumulation was negligible, suggesting that the flux of acetyl-CoA (the acetyl donor) was diverted to alginate acetylation.

[Influence of natural minerals on growth of Azotobacter vinelandii IMV B-7076].

It was shown that the cultivation of Azotobacter vinelandii IMV B-7076 in the presence of natural minerals significantly increased the growth of bacteria activity. Adding in the medium glauconite and saponite in optimal concentration (10 g/l) conditioned the increase of cell growth six and four times compared with control, accordingly. The maximum stimulating effect was observed when the concentration of phosphates was 5.0 g/l, herewith the number of viable cells increased 5 times. The presence of contact interaction of bacterial cells with investigated particles minerals was shown.

Chelate chemistry governs ion-specific stiffening of Bacillus subtilis B-1 and Azotobacter vinelandii biofilms.

Unwanted formation of bacterial biofilms can cause problems in both the medical sector and industrial settings. However, removing them from surfaces remains an ongoing challenge since biofilm bacteria efficiently protect themselves from external influences such as mechanical shear forces by embedding themselves into a matrix of extracellular polymeric substances. Here, we discuss microscopic principles, which are responsible for alterations in the viscoelastic properties of biofilms upon contact with metal ions. We suggest that it is a combination of mainly two parameters, that decides if biofilm stiffening occurs or not: the ion size and the detailed configuration of polyanionic macromolecules from the biofilm matrix. Our results provide new insights in the molecular mechanisms that govern the mechanical properties of biofilms. Also, they indicate that hydrogels comprising purified biopolymers can serve as suitable model systems to reproduce certain aspects of biofilm mechanics - provided that the correct biopolymer is chosen.

Tungsten effects on microbial community structure and activity in a soil.

Tungsten, once deposited onto a soil as a result of private, industrial, and military activities, may persist as tungstate anion or, via polymerization, as a variety of poly-tungstate species, each with varying solubility and soil sorption characteristics. In this study, the impact of weathered tungsten on a soil microbial community was measured. Fatty acid analyses indicated that weathered tungsten at < or =2500 mg kg(-1) was associated with a significant increase in microbial biomass and that concentrations up to 6500 mg kg(-1) did not result in a significant decrease in measured biomass, relative to the control. Analysis of cellular fatty acids also identified significant microbial community shifts between 0 and 325, 1300 and 2600, and 3900 and 6500 mg W kg(-1) soil. In general, the positive effect of tungsten on microbial biomass coincided with an increase in Gram-negative bacterial fatty acids, whereas fatty acids indicative of actinomycetes and Gram-positive bacteria were more abundant at the highest soil tungsten concentrations. The weathered tungsten also inhibited N2 fixing activity of a free living diazotroph at > or =1300 mg W kg(-1) soil. These results indicate that tungsten in soil can alter both the structure and the function of an indigenous soil microbial community.

[Influence of titanium dioxide on growth of Azotobacter vinelandii IMV V-7076].

Adding highly dispersed titanium dioxide particles of the culture medium has a stimulatory effect on growth of Azotobacter vinelandii. The maximum effect was observed when using 5 g/l and 10 g/l concentrations of this component. Under these conditions the number of grown bacteria is several times more than in the control variants. It was shown that the stimulatory effect could not be the consequence of saccharose sorption on the surface of particles of the dispersed compound. Possibly, the contact interaction of bacterial cell with titanium dioxide causes an increase of cell wall permeability which leads to an increase of substrates transport to the cell.

Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion.

Diazotrophic bacteria are an attractive biological alternative to synthetic nitrogen fertilizers due to their remarkable capacity to fix atmospheric nitrogen gas to ammonium via nitrogenase enzymes. However, how diazotrophic bacteria tailor central carbon catabolism to accommodate the energy requirement for nitrogenase activity is largely unknown. In this study, we used Azotobacter vinelandii DJ and an ammonium excreting mutant, AV3 (ΔNifL), to investigate central carbon metabolism fluxes and central cell bioenergetics in response to ammonium availability and nitrogenase activity. Enabled by the powerful and reliable methodology of 13C-metabolic flux analysis, we show that the respiratory TCA cycle is upregulated in association with increased nitrogenase activity and causes a monotonic decrease in specific growth rate. Whereas the activity of the glycolytic Entner-Doudoroff pathway is positively correlated with the cell growth rate. These new observations are formulated into a 13C-metabolic flux model which further improves the understanding and interpretation of intracellular bioenergetics. This analysis leads to the conclusion that, under aerobic conditions, respiratory TCA metabolism is responsible for the supply of additional ATP and reducing equivalents required for elevated nitrogenase activity. This study provides a quantitative relationship between central carbon and nitrogen metabolism in an aerobic diazotroph for the first time.

Identification of two catalases in Azotobacter vinelandii: a KatG homologue and a novel bacterial cytochrome c catalase, CCCAv.

Azotobacter vinelandii produces two detectable catalases during growth on minimal medium. The heat-labile catalase expressed during exponential growth phase was identified as a KatG homologue by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a mixed protein sample. The second catalase was heat resistant and had substantial residual activity after treatment at 90 degrees C. This enzyme was purified by anion-exchange and size exclusion chromatography and was found to exhibit strong absorption at 407 nm, which is often indicative of associated heme moieties. The purified protein was fragmented by proteinase K and identified by LC-MS/MS. Some identity was shared with the MauG/bacterial cytochrome c peroxidase (BCCP) protein family, but the enzyme exhibited a strong catalase activity never before observed in this family. Because two putative c-type heme sites (CXXCH) were predicted in the peptide sequence and were demonstrated experimentally, the enzyme was designated a cytochrome c catalase (CCC(Av)). However, the local organization of the CCC(Av) heme motifs differed significantly from that of the BCCPs as the sites were confined to the C-terminal half of the catalase. A possible Ca2+ binding motif, previously described in the BCCPs, is also present in the CCC(Av) peptide sequence. Some instability in the presence of EGTA was observed. Expression of the catalase was abolished in cccA mutants, resulting in a nearly 8,700-fold reduction in peroxide resistance in stationary phase.

RpoS expression and the general stress response in Azotobacter vinelandii during carbon and nitrogen diauxic shifts.

The general stress response mediated by the sigma factor RpoS is important for survival of bacteria in adverse environments. A mutant unable to produce RpoS was constructed using the diazotrophic bacterium Azotobacter vinelandii strain UW. Under nondesiccating, solid-medium growth conditions the wild type was culturable for 16.5 years, while the rpoS mutant remained viable for only 10 months. The rpoS mutant exhibited reduced survival compared to the wild type following hydrogen peroxide stress, and stationary phase cells were killed rapidly by 15 mM H2O2. Three catalases (Kat1, Kat2, and Kat3) were expressed in the wild type under the conditions used. Kat2 was expressed in exponential phase during shake flask growth and could be induced under highly aerated conditions in all growth phases, suggesting that there was induction by reactive oxygen intermediates. Kat3 was possibly an isoform of Kat2. In contrast, Kat1 was expressed in an RpoS-dependent manner during the mid-exponential to late stationary phases. RpoS expression did not occur exclusively in stationary phase but was influenced by changes in carbon and nitrogen source availability. There was 26- to 28-fold induction of the RpoS protein during acetate-to-glucose and ammonium-to-N2 diauxic shifts. Following recovery of growth on the alternative carbon or nitrogen source, RpoS protein concentrations declined rapidly to a basal level. However, rpoS mRNA levels did not correlate directly to RpoS levels, suggesting that there was posttranscriptional regulation. Evidence obtained using the RpoS-dependent reporter Kat1 suggested that there is regulation of the RNAP:RpoS holoenzyme at the level of complex formation or activity.

Azotobacter vinelandii metal storage protein: "classical" inorganic chemistry involved in Mo/W uptake and release processes.

The release of Mo (as molybdate) from the Mo storage protein (MoSto), which is unique among all existing metalloproteins, is strongly influenced by temperature and pH value; other factors (incubation time, protein concentration, degree of purity) have minor, though significant effects. A detailed pH titration at 12 degrees C revealed that three different steps can be distinguished for the Mo-release process. A proportion of approximately 15% at pH 6.8-7.0, an additional 25% at pH 7.2-7.5 and ca. 50% (up to 90% in total) at pH 7.6-7.8. This triphasic process supports the assumption of the presence of different types of molybdenum-oxide-based clusters that exhibit different pH lability. The complete release of Mo was achieved by increasing the temperature to 30 degrees C and the pH value to >7.5. The Mo-release process does not require ATP; on the contrary, ATP prevents, or at least reduces the degree of metal release, depending on the concentration of the nucleotide. From this point of view, the intracellular ATP concentration is suggested to play-in addition to the pH value-an indirect but crucial role in controlling the extent of Mo release in the cell. The binding of molybdenum to the apoprotein (reconstitution process) was confirmed to be directly dependent on the presence of a nucleotide (preferably ATP) and MgCl2. Maximal reincorporation of Mo required 1 mM ATP, which could partly be replaced by GTP. When the storage protein was purified in the presence of ATP and MgCl2 (1 mM each), the final preparation contained 80 Mo atoms per protein molecule. Maximal metal loading (110-115 atoms/MoSto molecule) was only achieved, if Mo was first completely released from the native protein and subsequently (re-) bound under optimal reconstitution conditions: 1 h incubation at pH 6.5 and 12 degrees C in the presence of ATP, MgCl2 and excess molybdate. A corresponding tungsten-containing storage protein ("WSto") could not only be synthesized in vivo by growing cells, but could also be constructed in vitro by a metalate-ion exchange procedure by using the isolated MoSto protein. The high W content of the isolated cell-made WSto (approximately 110 atoms/protein molecule) and the relatively low amount of tungstate that was released from the protein under optimal "release conditions", demonstrates that the W-oxide-based clusters are more stable inside the protein cavity than the Mo-oxide analogues, as expected from the corresponding findings in polyoxometalate chemistry. The optimized isolation of the W-loaded protein form allowed us to get single crystals, and to determine the crystal X-ray structure. This proved that the protein contains remarkably different types of polyoxotungstates, the formation of which is templated in an unprecedented process by the different protein pockets. (Angew. Chem. Int. Ed. 2007, 46, 2408-2413).

Size-dependent cytotoxicity of silver nanoparticles to Azotobacter vinelandii: Growth inhibition, cell injury, oxidative stress and internalization.

The influence of nanomaterials on the ecological environment is becoming an increasingly hot research field, and many researchers are exploring the mechanisms of nanomaterial toxicity on microorganisms. Herein, we studied the effect of two different sizes of nanosilver (10 nm and 50 nm) on the soil nitrogen fixation by the model bacteria Azotobacter vinelandii. Smaller size AgNPs correlated with higher toxicity, which was evident from reduced cell numbers. Flow cytometry analysis further confirmed this finding, which was carried out with the same concentration of 10 mg/L for 12 h, the apoptotic rates were20.23% and 3.14% for 10 nm and 50 nm AgNPs, respectively. Structural damage to cells were obvious under scanning electron microscopy. Nitrogenase activity and gene expression assays revealed that AgNPs could inhibit the nitrogen fixation of A. vinelandii. The presence of AgNPs caused intracellular reactive oxygen species (ROS) production and electron spin resonance further demonstrated that AgNPs generated hydroxyl radicals, and that AgNPs could cause oxidative damage to bacteria. A combination of Ag content distribution assays and transmission electron microscopy indicated that AgNPs were internalized in A. vinelandii cells. Overall, this study suggested that the toxicity of AgNPs was size and concentration dependent, and the mechanism of antibacterial effects was determined to involve damage to cell membranes and production of reactive oxygen species leading to enzyme inactivation, gene down-regulation and death by apoptosis.

Effect of organic matter on nitrogenase metal cofactors homeostasis in Azotobacter vinelandii under diazotrophic conditions.

Biological nitrogen fixation can be catalysed by three isozymes of nitrogenase: molybdenum (Mo)-nitrogenase, vanadium (V)-nitrogenase and iron-only (Fe)-nitrogenase. The activity of these isozymes strongly depends on their metal cofactors, molybdenum, vanadium and iron, and their bioavailability in ecosystems. Here, we show how metal bioavailability can be affected by the presence of tannic acid (organic matter), and the subsequent consequences on diazotrophic growth of the soil bacterium Azotobacter vinelandii. In the presence of tannic acids, A. vinelandii produces a higher amount of metallophores, which coincides with an active, regulated and concomitant acquisition of molybdenum and vanadium under cellular conditions that are usually considered not molybdenum limiting. The associated nitrogenase genes exhibit decreased nifD expression and increased vnfD expression. Thus, in limiting bioavailable metal conditions, A. vinelandii takes advantage of its nitrogenase diversity to ensure optimal diazotrophic growth.

Planktonic and biofilm-grown nitrogen-cycling bacteria exhibit different susceptibilities to copper nanoparticles.

Proper characterization of nanoparticle (NP) interactions with environmentally relevant bacteria under representative conditions is necessary to enable their sustainable manufacture, use, and disposal. Previous nanotoxicology research based on planktonic growth has not adequately explored biofilms, which serve as the predominant mode of bacterial growth in natural and engineered environments. Copper nanoparticle (Cu-NP) impacts on biofilms were compared with respective planktonic cultures of the ammonium-oxidizing Nitrosomonas europaea, nitrogen-fixing Azotobacter vinelandii, and denitrifying Paracoccus denitrificans using a suite of independent toxicity diagnostics. Median inhibitory concentration (IC50) values derived from adenosine triphosphate (ATP) for Cu-NPs were lower in N. europaea biofilms (19.6 ± 15.3 mg/L) than in planktonic cells (49.0 ± 8.0 mg/L). However, in absorbance-based growth assays, compared with unexposed controls, N. europaea growth rates in biofilms were twice as resilient to inhibition than those in planktonic cultures. Similarly, relative to unexposed controls, growth rates and yields of P. denitrificans in biofilms exposed to Cu-NPs were 40-fold to 50-fold less inhibited than those in planktonic cells. Physiological evaluation of ammonium oxidation and nitrate reduction suggested that biofilms were also less inhibited by Cu-NPs than planktonic cells. Furthermore, functional gene expression for ammonium oxidation (amoA) and nitrite reduction (nirK) showed lower inhibition by NPs in biofilms relative to planktonic-grown cells. These results suggest that biofilms mitigate NP impacts, and that nitrogen-cycling bacteria in wastewater, wetlands, and soils might be more resilient to NPs than planktonic-based assessments suggest.

Effect of Oxygen Tension and Medium Components on Monomer Distribution of Alginate.

Alginate is a natural biopolymer composed of mannuronic and guluronic acid monomers. It is produced by algae and some species of Azotobacter and Pseudomonas. This study aims to investigate the effect of dissolved oxygen tension (DOT) and growth medium substrate and calcium concentrations on the monomeric composition of alginate produced by Azotobacter vinelandii ATCC® 9046 in a fermenter. Results showed that alginate production increased with increasing DOT from 1 to 5 %. The highest alginate production was obtained as 4.51 g/L under 20 g/L of sucrose and 50 mg/L of calcium at 5 % DOT. At these conditions, alginate was rich in mannuronic acid (up to 61 %) and it was particularly high at low calcium concentration. On the other hand, at extreme conditions such as high DOT level (10 % DOT) and low sucrose concentration (10 g/L), guluronic acid was dominant (ranging between 65 and 100 %).

Mutagenesis of a gene encoding a cytochrome o-like terminal oxidase of Azotobacter vinelandii: a cytochrome o mutant is aero-tolerant during nitrogen fixation.

The amino acid sequence obtained by translating the nucleotide sequence of a 0.55 kb fragment, amplified from Azotobacter vinelandii chromosomal DNA by PCR, was 57% identical to part of the Escherichia coli cyoB gene, encoding subunit I of the cytochrome bo-type quinol oxidase. This fragment was mutated in vitro by insertion of a kanamycin-resistance cassette and introduced into the chromosome of A. vinelandii by homologous recombination. The mutant contained no spectrally detectable cytochrome o. However, in the stationary phase of growth, the level of the alternative oxidase (cytochrome bd) was 11-fold higher than in the wild-type strain. Respiration of the mutant was insensitive to chlorpromazine, an inhibitor thought to act specifically on cytochrome o. Cytochrome o-deficient mutants fixed nitrogen in air, clearly distinguishing the role of this oxidase from that of cytochrome bd, which is required for respiratory protection of oxygen-labile nitrogenase.

Regulation of the transcriptional activators AnfA and VnfA by metals and ammonium in Azotobacter vinelandii.

Transcription of the genes encoding molybdenum (Mo)-independent nitrogenases 2 and 3 of Azotobacter vinelandii requires the activators VnfA and AnfA, respectively. The effect of NH4+, Mo, or V (vanadium) was tested on the expression of vnfA-lacZ and anfA-lacZ transcriptional fusions. Mo repressed expression of both fusions whereas NH4+ and V repressed the anfA-lacZ fusion, but not the vnfA-lacZ fusion. Thus the repressive effect on transcription of the anfHDGKOR operon by NH4+, Mo, or V is mediated through their effect on transcription of anfA and the repressive effect of Mo on the vnfHFd and vnfDGK operons is mediated through Mo repression of vnfA transcription. Mo-dependent repression of anfA transcription is influenced but not entirely mediated by the Mo-responsive regulator ModE.

A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels.

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygen-sensitive nitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cells were cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen. The bacteria did not grow at oxygen concentrations >75% but grew well in the presence of 5% nitrogen/25% oxygen. Growth of wild-type and alginate-deficient strains when cultured with 50% oxygen did not differ substantially, indicating that alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogen levels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. The encystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine. Immunoelectron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. By contrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel bleb-dependent polysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels.