Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii

A part of the gene encoding cbb ₃-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c ₄ and c ₅. Thus, the A. vinelandii respiratory chain is shown to contain cbb ₃-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.     

Use of 3,3′-diaminobenzidine as a biochemical electron donor for studies on terminal cytochrome oxidase activity inAzotobacter vinelandii

Azotobacter vinelandii cells readily oxidize the dye 3,3′-diaminobenzidine (DAB), which has been previously used as an electron donor for studies on the mitochondrial cytochromec oxidase reaction. The DAB oxidase activity inA. vinelandii cells was 10-fold lower than that noted for theN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase reaction, which is commonly used to measure terminal oxidase activity both in bacteria and mitochondria. Analyses of cell-free extracts show that DAB oxidase activity is concentrated almost exclusively in theA. vinelandii membrane fractions, most notably in the “R₃” electron transport particle (ETP). Oxidation studies, which employed both whole cells and the ETP fraction, show DAB oxidase activity to be markedly sensitive to KCN, NaN₃, and NH₂OH. A manometric assay system was developed which readily measured DAB oxidase activity in bacteria. Preliminary studies indicate that ascorbate-DAB oxidation inAzotobacter vinelandii measures terminal cytochrome oxidase activity in a manner similar to the TMPD oxidase reaction.     

Purification and characterization of cytochrome o from Azotobacter vinelandii

The membrane-bound cytochrome o has been solubilized from the Azotobacter vinelandii electron transport particle and further purified by use of conventional chromatographic procedures. The spectral characteristics as well as the other properties noted for purified cytochrome o are reported herein.     

Cloning of ferredoxin I gene fromAzotobacter vinelandii using synthetic oligonucleotide probes

Two synthetic oligonucleotide probe mixtures, whose sequences were inferred from two separate stretches of amino acids, one closer to the carboxy terminal and the other closer to the amino terminal, of ferredoxin I protein ofAzotobacter vinelandii, were used to select ferredoxin I gene clones from a cosmid gene library ofAzotobacter vinelandii. Restriction analysis revealed that 7 out of 10 selected clones were of the same type. All these clones were found to hybridize withfixABCX genes ofRhizobium meliloti.     

Respiratory Protection of Nitrogenase Activity in Azotobacter vinelandii—Roles of the Terminal Oxidases

Nitrogen fixation by aerobic prokaryotes appears paradoxical: the nitrogen-fixing enzymes—nitrogenases—are notoriously oxygen-labile, yet many bacteria fix nitrogen aerobically. This review summarises the evidence that cytochrome bd, a terminal oxidase unrelated to the mitochondrial and many other bacterial oxidases, plays a crucial role in aerotolerant nitrogen fixation in Azotobacter vinelandii and other bacteria by rapidly consuming oxygen during uncoupled respiration. We review the pertinent properties of this oxidase, particularly its complement of redox centres, the catalytic cycle of oxygen reduction, the affinity of the oxidase for oxygen, and the regulation of cytochrome bd gene expression. The roles of other oxidases and other mechanisms for limiting damage to nitrogenase are assessed.     

Azotobacter vinelandii strains of disparate origin produce azotobactin siderophores with identical structures

Summary The yellow green fluorescent siderophore, azotobactin, was purified from cultures of twoAzotobacter vinelandii strains. Structural analysis of azotobactin from the North AmericanA. vinelandii strains O and its capsule negative variant UW (also called OP) revealed that both strains produced azotobactins with identical structures. Moreover, azotobactin produced by these two strains was structurally identical to azotobactin D, the fluorescent siderophore previously isolated from the EuropeanA. vinelandii strain D (CCM 289). Unlike strains of fluorescentPseudomonas which produce structurally diverse pyoverdins, strains ofA. vinelandii of disparate origin produced azotobactins of identical structure. Lactonization of azotobactin did not interfere with the ability of this compound to function as a siderophore.     

Comparative study of the response ofAzotobacter vinelandii andAcetobacter diazotrophicus to changes in pH

Summary Curves were established for the pH response of respiration on eleven substrates byAzotobacter vinelandii andAcetobacter diazotrophicus. With every substrate the optimal pH forA. diazotrophicus was lower than forA. vinelandii. The optimal hydrogen ion concentration forA. diazotrophicus was 5 fold to 365 fold greater than forA. vinelandii depending upon the substrate. In general,A. diazotrophicus supports respiration over a wider pH range than doesA. vinelandii.Dedicated to the memory of Professor John G. Torrey     

Growth and nitrogenase activity of Azotobacter vinelandii in the presence of several phenolic acids

Growth and nitrogenase activity (acetylene reduction) of Azotobacter vinelandii in chemically defined N-free media were studied in the presence of p-hydroxybenzoic, vanillic, p-coumaric, and ferulic acids at concentrations from 0.01 to 1% (w/v). Growth and nitrogenase activity were only detected when the microorganism was cultured on p-hydroxybenzoic acid either as sole carbon source or mixed with other phenolic acids, suggesting that p-hydroxybenzoic acid could be utilized as a carbon source by A. vinelandii for growing under certain environmental conditions.     

Whole-cell fluorescence for observing reductive metabolism from carbon compounds and hydrogen inAzotobacter vinelandii

The change in fluorescence of intactAzotobacter vinelandii was observed to study the oxidation and reduction of flavin and pyridine nucleotides resulting from carbon and hydrogen metabolism. Metronidazole, acetaldehyde, and oxygen each oxidized flavin. Flavin oxidized by metronidazole or acetaldehyde was reduced by addition of mannitol or ethanol, but not by acetate or hydrogen. The fluorescence induced by oxygen was transient. Mannitol, ethanol, acetate, acetaldehyde, and hydrogen shortened the duration of the oxygen-dependent fluorescence and supported respiration. The changes in redox state of pyridine nucleotides corresponded to the changes in flavin redox state. This indicates that the use of reducing equivalents from uptake hydrogenase is limited to the respiratory electron transport system inAzotobacter vinelandii.     

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.     

Nitrate reductase in cell-free extracts ofAzotobacter

Summary 1. Nitrate reductase activity in cell-free extracts ofAzotobacter vinelandii was obtained. The enzyme is TPNH-linked and shows some stimulation by molybdenum and FMN.2. The cell-free preparations showed a strong DPNH-oxidase activity and also a slight TPNH oxidation following the addition of distilled water. The latter activity could, however, be removed by ultra-centrifugation of the enzyme extracts. However, nitrate reductase seems to be only to a small extent soluble as its main activity was associated with particles. The particles spun down were red in colour suggesting the presence of cytochromes.3. Thick cell suspensions ofA. vinelandii, A. agile, andA. chroococcum showed similar cytochrome spectra. The _max. observed suggest the presence of cytochromes of thec type (_max. at 524 and 552 mµ) anda type (_max. at 590 and 632 mµ).4. No apparent differences were observed between the cytochrome spectra ofA. vinelandii cells grown on molecular and nitrate nitrogen.     

OriVRK2 replicon function in the absence oftrfA inAzotobacter vinelandii

The oriV_RK2 does not need the function of either trfA⁺ ortrfA Operon for replication and maintenance of an oriV_RK2-containing plasmid inAzotobacter vinelandii.     

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.     

Field response of tomato (Lycopersicon esculentum Mill ‘Pusa Ruby’) to inoculation with a VA mycorrhizal fungusGlomus fasiculatum and withAzotobacter vinelandii

Summary The response of tomato (Lycopersicon esculentum Mill) to inoculation with the vasicular arbuscular mycorrhizal (VAM) fungusGlomus fasiculatum andAzotobacter vinelandii singly and in combination was tested in the field. It was found thatG. fasiculatum as well asA. vinelandii significantly increased leaf area, shoot dry weight, nitrogen content phosphorus content and yield in respect to uninoculated control. While, VAM fungal treatment alone could bring about substantial increase in growth, nitrogen content, phosphorus content and yield, its combination withA. vinelandii produced additional effects on leaf area, shoot dry weight, phosphorus content and yield. Contribution No. 304/83 of Indian Institute of Horticultural Research, Bangalore-89.     

Toxicity of dicamba (2-methoxy-3,6-dichlorobenzoic acid) toAzotobacter vinelandii

The effect of dicamba was studied in N-free medium inoculated withAzotobacter vinelandii ATCC 12837. Nitrogen fixation was determined by acetylene reduction. Dicamba at a concentration of 500 μg/mL had a strong inhibitory effect on nitrogenase activity. However, no inhibitory effect on microbial respiration was detected.     

Expression from symbiotic promoters ofRhizobium meliloti inAzotobacter vinelandii andAzospirillum brasilense

InRhizobium meliloti, the promoter P1 of thenif HDK operon, and also the promoter P2, have earlier been shown to be active in the bacteria present in alfalfa root nodules, but not in the bacteria grown aerobically in culture. Here we have looked at the expression from P1 and P2 in two non-symbiotic nitrogen-fixing bacteria,Azotobacter vinelandii andAzospirillum brasilense, using constructions in which the promoters are fused upstream of theβ-galactosidase gene. The promoter P1, but not P2, is active inA. vinelandii, while neither P1 nor P2 is active inAzospirillum brasilense.     

Accumulation of ppGpp in symbiotic and free-living nitrogen-fixing bacteria following amino acid starvation

Following amino acid or ammonium starvation, ppGpp is accumulated by Rhizobium meliloti strain 1021 but not by R. meliloti strain 41 or Rhizobium tropici. Azorhizobium caulinodans ORS571 produced ppGpp following amino acid deprivation; however, the free-living nitrogen-fixing bacteria Azotobacter vinelandii and Azomonas agilis did not produce ppGpp. Western blot analysis using anti-RelA antibody demonstrated that R. meliloti strain 1021, Azotobacter vinelandii and Azorhizobium caulinodans cross-reacted under conditions that detected RelA in Escherichia coli CF1648. Cross-reaction was not observed in R. meliloti strain 41, R. tropici, or Azomonas agilis. All strains that accumulated ppGpp also produced high intracellular levels of ATP. Received: 28 August 1998 / Accepted: 11 November 1998     

Mobilization of sulfane sulfur from cysteine desulfurases to the <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> sulfurtransferase RhdA

Mobilization of the l-cysteine sulfur for the persulfuration of the rhodanese of Azotobacter vinelandii, RhdA, can be mediated by the A. vinelandii cysteine desulfurases, IscS and NifS. The amount of cysteine was higher in mutant strains lacking rhdA (MV474) than in wild type. The diazotrophic growth of MV474 was impaired. Taking into account the functional results about rhodanese-like proteins and RhdA itself, it is suggested that RhdA-dependent modulation of l-cysteine levels must deal with a redox-related process.     

Use of the plasmid pRK 2013 as a vehicle for transposition inAzotobacter vinelandii

Plasmid stability inAzotobacter vinelandii has been determined and a way to introduce transposon into these cells using the plasmid pRK 2013 has been devised. Transposition of both Tn3 and Tn10 has been attained.     

Influence of soil water potential on respiration and nitrogen fixation ofAzotobacter vinelandii

Summary Respiration and N₂-fixation (acetylene reduction) ofAzotobacter vinelandii have been studied at a variety of soil water potentials. Both processes were strictly linked and strongly reduced at water potentials between –0.6 and –1.3 MPa. Complete inhibition occurred below –2.1MPa. Osmotic potentials in soil compared to matric potentials of the same value were less inhibitory to respiration and acetylene reduction by Azotobacter. The N₂-fixing efficiency (mg N/g glucose) was not influenced by water potentials ranging from –0.1 to –2.1 MPa.