Azotobacter Vinelandii Strain Research Articles

Firefly luciferase as a reporter enzyme for measuring gene expression in vegetative and symbiotic Rhizobium meliloti and other Gram-negative bacteria

A DNA segment carrying a cDNA copy of the luciferase gene ( luc) of the North American firefly Photinus pyralis, fused to the λ p R promoter and expressed in Escherichia coli [de Wet et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7870–7873], was inserted into a broad-host-range plasmid vector and established in a variety of Gram-negative bacteria. Luciferase activity, expressed from the λ p R promoter, was detected in both intact cells and extracts prepared from cells of strains of Rhizobium meliloti, R. phaseoli, R. fredii, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Acinetobacter calcoaceticus and Azotobacter vinelandii. The highest levels of activity, determined by measurements of both intact cells and extracts, were observed for P. aeruginosa and the three species of Rhizobium examined. Expression of luciferase activity also was relatively high in R. meliloti bacteroids of mature alfalfa nodules. This activity was readily detectable in intact nodules using x-ray film or in extracts prepared from purified bacteroids.

Construction and characterization of an Azotobacter vinelandii strain with mutations in the genes encoding flavodoxin and ferredoxin I.

Flavodoxin and ferredoxin I have both been implicated as components of the electron transport chain to nitrogenase in the aerobic bacterium Azotobacter vinelandii. Recently, the genes encoding flavodoxin (nifF) and ferredoxin I (fdxA) were cloned and sequenced and mutants were constructed which are unable to synthesize either flavodoxin (DJ130) or ferredoxin I (LM100). Both single mutants grow at wild-type rates under N2-fixing conditions. Here we report the construction of a double mutant (DJ138) which does not synthesize either flavodoxin or ferredoxin I. When plated on ammonium-containing medium, this mutant had a very small colony size when compared with the wild type, and in liquid culture with ammonium, this double mutant grew three times slower than the wild type or single mutant strains. This demonstrated that there is an important metabolic function unrelated to nitrogen fixation that is normally carried out by either flavodoxin or ferredoxin. If either one of these proteins is missing, the other can substitute for it. The double mutant phenotype can now be used to screen site-directed mutant versions of ferredoxin I for functionality in vivo even though the specific function of ferredoxin I is still unknown. The double mutant grew at the same slow rate under N2-fixing conditions. Thus, A. vinelandii continues to fix N2 even when both flavodoxin and ferredoxin I are missing, which suggests that a third as yet unidentified protein also serves as an electron donor to nitrogenase.

Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium.

We have constructed a strain of Azotobacter vinelandii which has deletions in the genes for both the molybdenum (Mo) and vanadium (V) nitrogenases. This strain fixed nitrogen in medium that did not contain Mo or V. Growth and nitrogenase activity were inhibited by Mo and V. In highly purified medium, growth was limited by iron. Addition of other metals (Co, Cr, Cu, Mn, Ni, Re, Ti, W, and Zn) did not stimulate growth. Like the V-nitrogenase, the nitrogenase synthesized by the double deletion strain reduced acetylene to both ethylene and ethane (C2H6/C2H4 ratio, 0.046). There was an approximately 10-fold increase in ethane production when Mo was added to the deletion strain grown in medium lacking Mo and V. This change in reactivity may be due to the incorporation of an Mo-containing cofactor into the nitrogenase synthesized by the double-deletion strain. A strain synthesizing the V-nitrogenase did not show a similar increase in ethane production. The growth characteristics of the double-deletion strain, together with the metal composition reported for a nitrogenase isolated from a tungstate-tolerant strain lacking genes for the molydenum enzyme grown in the absence of Mo and V (J. R. Chisnell, R. Premakumar, and P. E. Bishop, J. Bacteriol. 170:27-33, 1988) show that A. vinelandii can synthesize a nitrogenase which lacks both Mo and V. Reduction of dinitrogen by nitrogenase can therefore occur at a center lacking both these metals.

Production of poly-?-hydroxybutyrate by Azotobacter vinelandii strain UWD during growth on molasses and other complex carbon sources

Azotobacter vinelandii strain UWD formed >2 mg/ml poly-β-hydroxybutyrate (pHB) during exponential growth in media containing ammonium acetate and 1% w/v glucose, fructose, sucrose, or maltose, and >1.5 mg/ml with 1% w/v sodium gluconate or glycerol. After acetate exhaustion, pHB formation accompanied carbohydrate utilization and pHB rapidly accounted for 53%–70% of the cell mass. Strain UWD also formed >2 mg/ml pHB when it was grown with 2% w/v corn syrup, cane molasses, beet molasses, or malt extract. Beet molasses had a growth stimulatory effect which promoted higher yields of pHB/ml and a high ratio of pHB/protein. Malt extract also promoted higher yields of pHB/ml. In this case, pHB formation was no longer subject to acetate repression and the cells contained a higher ratio of pHB/protein. This study shows that unrefined carbon sources support pHB formation in strain UWD and that the yields of pHB were comparable to or better than those obtained with refined carbon sources.

Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii

A 3.8-kilobase-pair EcoRI fragment which corrects the mutations carried by the NifB- Azotobacter vinelandii strains CA30 and UW45 was cloned, and its nucleotide sequence was determined. Four complete open reading frames (ORFs) and two partial ORFs were found. The translation product of the first partial ORF is the carboxy-terminal end of a protein homologous to the nifA gene product from Klebsiella pneumoniae. A 285-base-pair sequence containing a potential nif promoter and nif regulatory sites separates this nifA gene from the first complete ORF which encodes a protein homologous to nifB gene products from K. pneumoniae and Rhizobium species. The Tn5 insertion in strain CA30 and the nif-45 mutation of strain UW45 are located within this nifB gene. The ORF downstream from nifB predicts an amino acid sequence with a cysteine residue pattern that is characteristic of ferredoxins. No similarities were found between the translation product of the third complete ORF and those of nif genes from other organisms. At the carboxy-terminal end of the predicted translation product of the fourth complete ORF, 30 of 60 amino acid residues were identical with the sequence of the nifQ gene product from K. pneumoniae. The partial ORF located at the end of the fragment encodes the N-terminal part of a potential protein with an unknown function. Northern (RNA) blot analysis indicated that transcripts from the region containing the four complete ORFs were NH4+ repressible and that the transcription products were identical in cells derepressed under conditions of Mo sufficiency or Mo deficiency or in the presence of vanadium. In contrast to the NifB- strain CA30, which is Nif- under all conditions, mutants that carry mutations affecting the C-terminal end of nifB or genes located immediately downstream from nifB, grew under all N2-fixing conditions. However, in the presence of Mo, most of the strains required 1,000 times the amount of molybdate that is sufficient for maximal growth of the wild-type strain CA under N2-fixing conditions. Growth data from strain CA37, which carries a Kanr insertion in nifQ, indicate that nifQ in A. vinelandii is not required for N2 fixation in the presence of V2O5 or under Mo-deficient conditions. Growth studies and acetylene reduction assays performed on two nifEN deletion strains showed that nifE and nifN are required for N2 fixation under Mo sufficiency, as previously observed (K. E. Brigle, M. C. Weiss, W. E. Newton, and D. R. Dean, J. Bacteriol. 169:1547-1553, 1987), but not under conditions of Mo deficiency or in the presence of 50 nM V2O5.

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii.

A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (alpha, Mr 58,000, and beta, Mr 50,000) which assemble into at least two active configurations: alpha 2 beta 2 (dinitrogenase 3s) and alpha 1 beta 2 (dinitrogenase 3F). Dinitrogenase 3s contains 24 Fe and 18 acid-labile S2-ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2-ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 complex lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ greater than N2 greater than C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing.

Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium.

Nitrogenase was isolated and purified from wild-type and a tungsten-resistant mutant (LM2) of Azotobacter vinelandii strain OP derepressed on medium containing 1-10 mM W. While the enzyme from the wild-type strain contained the polypeptides of the conventional enzyme, metal analysis of component 1 demonstrated the existence of one atom each of molybdenum and tungsten. Furthermore, the ESR spectrum of this protein contained three signals, two of which originated from S = 3/2 spin states. One of these signals is nearly identical to that of the conventional MoFe-protein while the other is hypothesized to originate from a W-containing cofactor. In spite of the presence of W, the substrate reduction pattern of this enzyme is the same as that of the conventional enzyme.

Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii.

Wild-type Azotobacter vinelandii strain UW was transformed with plasmid pDB12 to produce a species (LS10) unable to synthesize the structural proteins of component 1 and component 2 of native nitrogenase. A spontaneous mutant of this strain was isolated (LS15) which can grow by nitrogen fixation in the presence or absence of either Mo or W. It is proposed that LS15 fixes nitrogen solely by an alternative nitrogen-fixing system which previously has been hypothesized to exist in A. vinelandii. Under nitrogen-fixing conditions, LS15 synthesizes a protein similar to component 2 (Av2) of native nitrogenase in that it can complement native component 1 (Av1) for enzymatic activity. Isolation and characterization of this second component 2 shows it to be a 4Fe-4S protein of molecular mass about 62 kDa and is antigenically similar to Av2. This protein is also similar to Av2 in that in the reduced state it possesses a rhombic ESR spectrum in the g = 2 region, which changes to an axial spectrum upon addition of MgATP. It is suggested that this second Fe-protein is associated with the alternative nitrogen-fixing system in A. vinelandii.

Effect of limiting substrate concentration, growth rate and aeration on alginate composition and production by Azotobacter vinelandii in continuous culture

The extracellular production of the acetylated alginate of Azotobacter vinelandii strain E was studied in PO 4 2− limited continuous cultures. The uronate composition and fine structure of the isolated alginates was determined using 1H-n.m.r. Cultures maintained at low phosphate concentrations (0.24 mM) produced less biomass and polysaccharide than those maintained at higher PO 4 2− concentrations (2.0 mM). The ratio of polysaccharide produced:dry cell wt was not influenced by the degree of PO 4 2− limitation. Alginate production rates were not influenced greatly by changes in culture dilution rates but at very low (<0.08/h) and very high (>0.25/h) dilution rates, reduced production was observed. At low (100 ml/min) and high (800 ml/min) aeration rates alginate production was less than at intermediate aeration rates (300–500 ml/min). Alginates with a great diversity of uronate composition and fine structure were isolated from the cultures although no relationship between composition and growth rate or aeration rate was observed. From cultures with 2.0 mM PO 4 2−, alginates with high mannuronate levels (60–70%, with an essentially polymannuronate and heteropolymeric structure) were isolated whilst those from cultures with 0.24 mM PO 4 2− had high guluronate levels (70%, with essentially polyguluronate and heteropolymeric structure). Increasing the dilution rate of cultures (0.05–0.3/h) resulted in the isolation of alginates with increasing degrees of acetylation.

Monomer sequence and acetylation pattern in some bacterial alginates

The sequential structures and acetylation patterns of alginates from several strains of Azotobacter vinelandii and Pseudomonas species, including P. aeruginosa, P. putida, P. fluorescens, and P. mendocina, have been studied by 1H-n.m.r. spectroscopy. O-Acetyl groups were exclusively associated with the d-mannuronic acid residues and the degree of acetylation varied in the range 4–5%, depending upon the proportion of this acid in the polymer. 1H-N.m.r. spectroscopy of a naturally occurring and an artifically acetylated d-mannuronan made it possible to determine the degrees of acetylation at O-2, O-3, and O-2,3. The most conspicuous difference between alginates from A. vinelandii and the four Pseudomonas species was the complete absence of consecutive l-guluronic acid residues in the latter.

Influence of calcium on alginate production and composition in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii strain E was cultivated in PO4--limited continuous cultures. The influence of growth medium Ca++ levels on dry cell weight and alginate production and composition was examined. Low Ca++ concentrations ( 1.0 g l-1), the production of alginate with a predominantly heteropolymeric structure was favoured by increasing Ca++ levels. In cultures containing less polysaccharide (<1.0 g l-1) increasing Ca++ levels (0.068–0.34 mM Ca++) resulted in the production of alginates high in polyguluronate. With further increases in Ca++ levels (0.34–2.72 mM Ca++) synthesis of alginates with a more heteropolymeric structure occurred. It is proposed that extracellular epimerisation of alginate is influenced by intermolecular associations, the formation of which is mediated by both Ca++ concentration and the concentration of the polymer itself.

Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK).

Steady-state chemostat cultures of Azotobacter vinelandii strain CA11, carrying a deletion of genes encoding the structural polypeptides of nitrogenase nifHDK, were established in a simple defined medium chemically purified to minimize contamination by Mo. The medium contained no utilizable N source. Growth was dependent on N2 (1.1 X 10(8) viable cells X ml-1 at D = 0.176 h-1), and was inhibited by Mo (20 nM). DNA hybridization showed the deletion to be stable during prolonged (55 days) growth in the chemostat (132 doublings). Since batch cultures, using unsupplemented 'spent' chemostat medium, showed good growth (1.9 X 10(8) cells X ml-1), no requirement for subnanomolar concentrations of Mo was found. The biomass yield, as the dilution rate (D) was varied, showed that the N content of the culture, protein and dry wt. increased as D was decreased, indicating that neither N2 nor O2 were limiting growth. The limiting nutrient was not identified. Substantial amounts of H2 were evolved by the chemostat cultures, probably as the result of inhibition of O2-dependent hydrogenase activity by nitrilotriacetic acid present in the medium. Over a range of D values approx. 50% of the electron flux through the alternative system was allocated to H+ reduction. C2H2 was a poor substrate, being reduced at 0.14-0.1 times the rate of N2 fixation, calculated from the N content of the cells. SO4(2-)-limited steady-state continuous cultures of strain UW136 (wild-type for nifHDK) had a 2-fold greater biomass in the presence of MoO4(2-) (1 microM). The significance of this finding for 'Mo-limited' continuous cultures [Eady & Robson (1984) Biochem. J. 224, 853-862] is discussed.

A second nitrogen fixation system in Azotobacter vinelandii

Nif − strains of Azotobacter vinelandii lacking active nitrogenase undergo phenotypic reversal to Nif + under molybdenum-deficient conditions. This phenotypic reversal phenomenon has been cited as evidence that A. vinelandii possesses two nitrogen fixation systems; the conventional molybdo-enzyme system and an alternative N 2 fixation system. Recent experiments with nifHDK deletion strains support this conclusion.

The amino acid sequence of the dihaem cytochrome c4 from the bacterium Azotobacter vinelandii.

An amino acid sequence is proposed for the cytochrome c4 from the bacterium Azotobacter vinelandii strain OP. It is a single polypeptide chain of 190 residues, with two sets of haem-attachment cysteine residues at positions 14/17 and 119/122. Proteins with similar sequences are also present in denitrifying pseudomonads. There is similarity in sequence between the two halves of the cytochrome c4 molecule, and each half also shows similarity to the sequences of certain monohaem cytochromes c isolated from organisms that are not obviously closely related to A. vinelandii. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50125 (17 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment.

Consequences of the association of calcium with alginate during batch culture of Azotobacter vinelandii

Azotobacter vinelandii strain E was grown in batch culture in the presence of radioactive Ca45. The partitioning of Ca45 in solution and associated with extracellular polysaccharide (alginate) during the growth was studied. The amount of alginate produced was estimated and its composition was determined by 1H-n.m.r. Alginate produced early in the growth cycle was characterised by high guluronic acid block mole fraction (0.45) whilst later, alginate with a preponderance of mannuronic acid blocks was observed. Mixed block synthesis occurred throughout the incubation. The level of free Ca2+ fell rapidly (from 97% to 22%) during the first 33 h of incubation.

Formation of cystlike structures by iron-limited Azotobacter vinelandii strain UW during prolonged storage

Slant or liquid cultures of nonencapsulated Azotobacter vinelandii strain UW survived at least 2 years at 20 °C, whereas parallel cultures died after 6 months storage at 4 °C. Survival was decreased at 20 °C when the growth medium was acidic and it was the least at 4 °C when the growth medium was iron limited. Although strain UW was capable of forming cyclopropyl fatty acids and alkyl resorcinols, these indicators of encystment were neither abundant nor present, respectively, in 1.7-year-old cells. Only lauric acid was more abundant in lipid extracts from old cells than from vegetative cells or mature cysts. The old cells were very fragile and rapidly lost viability upon dilution or dehydration. Electron microscopy revealed the production of fragile cystlike structures in iron-limited medium after 1.7 years at 20 °C. These structures had a distinct exine layer composed of membranelike plates, a poorly defined intine, and an extremely dense central body lacking storage granules or internal membranes. Iron-sufficient cultures produced similar but free central bodies, although cystlike structures were rarely seen.

Isolation and characterization of Azotobacter vinelandii mutant strains with potential as bacterial fertilizer

Mutant strains of Azotobacter vinelandii which might have potential for use as bacterial fertilizer have been isolated and fall into two categories: constitutive mutants that synthesize nitrogenase in the presence of ammonium and mutants that overproduce nitrogenase when grown in nitrogen-free medium. The constitutive mutants described in this paper were isolated from the wild type as methylalanine-resistant strains and express up to 23% of the fully derepressed nitrogenase level when grown in medium containing excess ammonium. By contrast, ammonium-grown cultures of wild type have less than 0.003% of the fully derepressed level. Strains which fix more N2 than the wild type in nitrogen-free medium were isolated as mefhylammonium-resistant mutants. Although the methylammonium-resistant mutant strains fix more N2 than the wild type, they grow no faster. The excess nitrogen produced by these mutants is excreted into the medium, resulting in up to 60% more nitrogen than in the medium of the wild type. Higher nitrogenase activity in the methylammonium-resistant mutant strains was found to be a result of increased levels of nitrogenase protein, suggesting that regulation of nitrogenase synthesis may be altered.

Taxonomy of the Azotobacteraceae Determined by Using Immunoelectrophoresis

The similarities of various strains of Azotobacter spp. and Azomonas spp. to reference strains of Azotobacter paspali, Azotobacter vinelandii, Azotobacter chroococcum, Azomonas agilis, Azomonas insignis, and Azomonas monocytogenes were determined by rocket line immunoelectrophoresis. The strains of Azotobacter paspali and Azotobacter vinelandii used were immunologically more homogeneous than the strains of Azotobacter chroococcum studied, possibly due to the more diverse geographical origins of the Azotobacter chroococcum strains. Low values were obtained for the mean immunological distances (1 – proportion of immunoprecipitation bands shared between strains) between Azotobacter paspali and Azotobacter vinelandii strains, suggesting that these two species are immunologically closely related. Immunological distances from the Azotobacter chroococcum reference strain were similar for Azotobacter paspali and for other undisputed members of the genus Azotobacter, which makes it reasonable to retain Azotobacter paspali in this genus. When the three Azotobacter antisera were used, all Azotobacter species had mean immunological distances of less than 0.5, whereas the Azomonas species were immunologically more distant, showing that the six species of Azotobacter form an immunologically related group which is distinct from the Azomonas species. Our results with the three Azomonas antisera show that each species of Azomonas is immunologically distant from the other species, as well as from the Azotobacter species. We compare our immunoelectrophoretic results with the molecular biological results of De Smedt et al. (Int. J. Syst. Bacteriol. 30:106–122, 1980) and the numerical taxonomic analysis of Thompson and Skerman (Azotobacteraceae: the Taxonomy and Ecology of the Aerobic Nitrogen-Fixing Bacteria, Academic Press, Inc., London, 1979).

Effect of O2 limitation on growth and respiration of the wild type and an ascorbate-tetramethyl-p-phenylenediamine-oxidase-negative mutant strain of Azotobacter vinelandii.

Azotobacter vinelandii strain AVOP (wild type) and an ascorbate-N,N,N',N'-tetramethylene-p-phenylenediamine oxidase-negative mutant (AV11) were each grown in O2-limited chemostat cultures. The results showed that the mutant strain grew and used O2 less efficiently than the wild-type strain. Respiration rates of membrane particles with NADH or malate as the substrate were similar for each strain. Succinate oxidase activity was about fourfold lower in membrane particles prepared from mutant than from wild-type strain. Cyanide at a concentration that completely inhibited ascorbate-TMPD oxidase activity resulted in a 50% inhibition of NADH oxidase activity in membrane particles of AVOP. These data suggest that the cytochrome o, a1, oxidase branch of the respiratory chain may be important in the physiology of A. vinelandii under O2-limiting growth conditions.

Molybdenum enhancement of nitrogen fixation in a Mo-starved Azotobacter vinelandii Nif− mutant

Molybdenum (Mo)-starved wild-type and Nif− strains of Azotobacter vinelandii reduced acetylene (fixed nitrogen) in Mo-limited nitrogen-free medium. Vanadate enhanced this activity in all of the strains. Molybdate caused repression of nitrogenase activity in the Nif− mutants and enhanced the nitrogenase activity in the wild type. The nitrogenase activity in the Nif− mutant UW3, however, was enhanced by Mo, became maximal after 3 h, and then declined to zero after 10 h of incubation. The activation of nitrogenase by Mo followed a 5- to 10-min lag and was inhibited when streptomycin or rifampin was added with Mo. Examination of Mo-starved nitrogen-fixing UW3 cell extracts by two-dimensional polyacrylamide gel electrophoresis revealed molecular weight 57 000, 50 000, and 30 000 proteins that were Mo and NH4+ repressive. The molecular weight 30 000 protein appeared in the same position on the gel as the wild-type dinitrogenase reductase, although UW3 did not produce this protein under Mo-sufficient nitrogen-fixing conditions. Cell extracts prepared 3 h after Mo addition lacked the molecular weight 57 000 and 50 000 proteins but contained a new protein corresponding to the β subunit of dinitrogenase. When UW3 nitrogenase activity was lost, the dinitrogenase reductase-like protein also was absent. The results suggest that a complex active in nitrogen fixation may form between components of the traditional Mo-sufficient and alternative Mo-starved cell nitrogen fixation systems.