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Interaction of Azotobacter chroococcum 20 cells with clay minerals increased their viability at supraoptimal temperatures. Therefore, clay minerals were used to develop granular bacterial preparations with high viable cell counts and stable compositions during long-term storage. The titers of viable bacteria in the preparations remained 60-70% of the initial level after 12-month storage.
Spontaneous mutants resistant to methionine sulfoximine (Msx), methyl alanine (Mal) and methyl ammonium chloride (Mac) were derived from A. chroococcum strain A103. Msx and Mal-resistant mutants expressed 1.73 to 10.98% of the fully derepressed nitrogenase activity when grown in Burk's medium containing ammonium acetate. Mac-resistant mutants did not express nitrogenase activity in ammonium acetate supplemented medium. The mutants excreted ammonia even after 2 days of growth and some mutants excreted more ammonia as compared to the parent. Selected mutants were inoculated on wheat (Triticum aestivum) and barley (Hordeum vulgare) under field conditions. Majority of the derepressed mutants increased grain yield of wheat and barley varying from 1.2 to 33.3%. However, host-dependent effects on grain yield were observed with different mutants. Two mutants, Mal 27 and Mac 19 showed significant increase in grain yields of both the crops. The results suggest that metabolic analogue-resistant mutants of Azotobacter have potential for use as a biofertilizer for cereal crops.
Cell-free, dialyzed extracts from Azotobacter vinelandii rapidly dephosphorylate [U-14C]ATP to labeled ADP and AMP, which is then degraded to hypoxanthine, the end product of AMP catabolism under the experimental conditions which were used. The intermediates of the pathway from ATP to hypoxanthine have been identified by thin layer chromatography and quantitated by the 14-C content. The concentrations of intermediates present during the production of hypoxanthine are consistent with AMP nucleosidase being responsible for AMP degradation in these extracts. This result was confirmed in experiments which utilized rabbit antibody prepared against purified AMP nucleosidase. The antibody inhibited AMP nucleosidase activity in cell-free extracts but did not inhibit adenine demanase or adenosine deaminase from the same extracts. In the presence of antibody prepared against purified AMP nucleosidase, the dialyzed extracts showed a marked reduction in the production of hypoxanthine from ATP. Other enzymes which could be responsible theoretically for the conversion of AMP to hypoxanthine were not detected by standard assay procedures. These results are consistent with AMP degradation proceeding by way of AMP nucleosidase to yield adenine and ribose 5-phosphate. The adenine is then converted to hypoxanthine by adenine deaminase. Both of these enzymes were present in sufficient quantities to account for the observed rates of hypoxanthine formation. The rate of hypoxanthine formation decreases during the time course of the [U-14-C]ATP degradation experiments, even though the concentration of AMP remains high. This decrease in the rate of hypoxanthine formation as a function of time is attributed to the decreasing ATP and increasing P0-4 concentrations, since ATP is an activator of AMP nucleosidase and P0-4 is an inhibitor. These observations suggest that the in vivo activity of AMP nucleosidase could also be regulated by changes in the relative ratios of ATP:AMP:P0-4.
The enzymatic reduction of molecular nitrogen to ammonia requires high amounts of energy, and the presence of oxygen causes the catalyzing nitrogenase complex to be irreversible inactivated. Thus nitrogen-fixing microorganisms tightly control both the synthesis and activity of nitrogenase to avoid the unnecessary consumption of energy. In the free-living diazotrophs Klebsiella pneumoniae and Azotobacter vinelandii, products of the nitrogen fixation nifLA operon regulate transcription of the other nifoperons. NifA activates transcription of nif genes by the alternative form of RNA-polymerase, sigma54-holoenzyme; NifL modulates the activity of the transcriptional activator NifA in response to the presence of combined nitrogen and molecular oxygen. The translationally-coupled synthesis of the two regulatory proteins, in addition to evidence from studies of NifL/NifA complex formation, imply that the inhibition of NifA activity by NifL occurs via direct protein-protein interaction in vivo. The inhibitory function of the negative regulator NifL appears to lie in the C-terminal domain, whereas the N-terminal domain binds FAD as a redox-sensitive cofactor, which is required for signal transduction of the internal oxygen status. Recently it was shown, that NifL acts as a redox-sensitive regulatory protein, which modulates NifA activity in response to the redox-state of its FAD cofactor, and allows NifA activity only in the absence of oxygen. In K. pneumoniae, the primary oxygen sensor appears to be Fnr (fumarate nitrate reduction regulator), which is presumed to transduce the signal of anaerobiosis towards NifL by activating the transcription of gene(s) whose product(s) function to relieve NifL inhibition through reduction of the FAD cofactor. In contrast, the reduction of A. vinelandii-NifL appears to occur unspecifically in response to the availability of reducing equivalents in the cell. Nitrogen status of the cells is transduced towards the NifL/NifA regulatory system by the GlnK protein, a paralogue PII-protein, which appears to interact with the NifL/NifA regulatory system via direct protein-protein interaction. It is not currently known whether GlnK interacts with NifL alone or affects the NifL/NifA-complex; moreover the effects appear to be the opposite in K. pneumoniae and A. vinelandii. In addition to these environmental signals, adenine nucleotides also affect the inhibitory function of NifL; in the presence of ATP or ADP the inhibitory effect on NifA activity in vitro is increased. The NifL proteins from the two organisms differ, however, in that stimulation of K. pneumoniae-NifL occurs only when synthesized under nitrogen excess, and is correlated with the ability to hydrolyze ATP. In general, transduction of environmental signals to the nif regulatory system appears to involve a conformational change of NifL or the NifL/NifA complex. However, experimental data suggest that K. pneumoniae and A. vinelandii employ significantly different species-specific mechanisms of signal transduction.
The effects of bacteria belonging to the genera Azotobacter and Bacillus in a mixed culture with Bradyrhizobium japonicum strains on formation and function of the legume-rhizobium symbiosis of soybean plants were studied. The data showed that the bacterial compositions B. japonicum 634b + B. subtilis 5, B. japonicum 634b + A. chroococcum 20, and B. japonicum 10k + A. vinelandii 56 with a cell ratio of 1:0.1 increased the number and weight of root nodules as well as the height and weight of the aboveground plant parts in almost all the cases by 22-105% compared with the control variants. These binary microbial cultures may be used for development of combined bacterial preparations for soybean.
Aspects of the external morphology of Azotobacter vinelandii cells during encystment and germination processes were observed with scanning electron microscopy. Most of the vegetative cells have a smooth surface but some have warty surfaces. The intact cysts have wrinkled surfaces and occasional small heaves. Mucoid materials are present on the surface of the cysts. During the encystment process, an extensive peeling-off of coat materials was noted, then the excretion and aggregation of new capsular materials was immediately followed. The germination process was initiated by an expansion of the central body (the cell), then the emerging of this cell from the cyst coats was observed.
The influence of various factors on the electron-transfer activity of ferredoxin I from Azotobacter vinelandii was studied. The method of cyclic voltammetry was used to obtain the data.
Nitrogenase binds and hydrolyzes 2MgATP yielding 2MgADP and 2Pi for each electron that is transferred from the iron protein to the MoFe protein. The iron protein alone binds but does not hydrolyze 2MgATP or 2MgADP and the binding of these nucleotides is competitive. Iron protein amino acid sequences all contain a putatitive mononucleotide-binding region similar to a region found in other mononucleotide-binding proteins. To examine the role of this region in MgATP interaction, we have substituted glutamine and proline for conserved lysine 15. The amino acid substitutions, K15Q and K15P, both yielded a non-N2-fixing phenotype when the genes coding for them were substituted into the Azotobacter vinelandii chromosome in place of the wild-type gene. The iron protein from the K15Q mutant was purified to homogeneity, whereas the protein from the K15P mutant could not be purified in its native form. Unlike wild-type iron protein, the purified K15Q iron protein showed no acetylene reduction, H2 evolution, or ATP hydrolysis activities when complemented with wild-type MoFe protein. The K15Q iron protein and the normal iron protein had a similar total iron content and both proteins showed the characteristic rhombic EPR signal resulting from the reduced state of the single 4Fe-4S cluster bridging the two subunits. Unlike the wild-type iron protein, addition of MgATP to the K15Q iron protein did not result in the perturbation necessary to change the EPR signal of its 4Fe-4S center from a rhombic to an axial line shape. Also unlike the wild-type iron protein, addition of MgATP to K15Q iron protein in the presence of the iron chelator, alpha,alpha'-dipyridyl, did not result in a time-dependent transfer of iron to the chelator. Thus, even though the K15Q iron protein contains a normal 4Fe-4S center, it does not respond to MgATP like the wild-type protein. Examination of the ability of the K15Q iron protein to bind MgADP showed no change from the wild-type iron protein, but its ability to bind MgATP decreased to 35% of the wild-type protein. Thus, in A. vinelandii iron protein, lysine 15 is not needed for interaction with MgADP but is involved in the binding of ATP, presumably through charge-charge interaction with the gamma-phosphate. Based on the above data, this lysine appears to be essential for the MgATP induced conformational change of wild-type iron protein that is required for activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Ferredoxins are proteins which contain iron and inorganic sulfide and are capable of electron transport. They are found in a wide range of organisms, from anaerobic bacteria, to plants and mammals. Although NMR spectroscopy has been used to study ferredoxins since the 1970s, little important structural or biochemical information has resulted from these investigations. The major difficulty has been the effect of the paramagnetic iron-sulfur clusters on the peptide resonances, hindering nuclear Overhauser effect (NOE) studies and causing broad line widths. These effects are most pronounced on resonances arising from the nuclei closest to the iron-sulfur center. Unfortunately, these are likely to be the most interesting nuclei, as they report the events and geometry in the vicinity of the active sites. In this paper, the first direct assignment of beta-cysteinyl 13C resonances for any iron-sulfur protein is reported for the spectrum of Pseudomonas putida ferredoxin. These resonances are of special significance, as they arise from the atoms on the protein closest to the iron centers, with the exception of the directly bound cysteinyl sulfur atoms. In addition, cysteinyl and ring system 1H NMR resonance assignments are made for the spectra of P. putida ferredoxin and Azotobacter vinelandii ferredoxin I.
Production of poly (3-hydroxybutyric acid) [P(3HB)] by Azotobacter beijerinckii DAR-102 isolated in this laboratory has been optimized under batch-culture. The accumulatad polymer attained 58% of cell dry mass during mid-stationary phase with an yield of 0.58 g/l when grown in nitrogen-free medium. The optimum concentration of glucose and fructose for P(3HB) production was 3% (w/v) and 2% (w/v) respectively while that of casamino acid and tryptose was 0.1% (w/v). Phosphate at a concentration suboptimal for growth and limitation of oxygen in the medium favoured P(3HB) accumulation. The production of P(3HB) was maximum with an inoculum dose of 4% (v/v). The accumulated polymer was isolated by direct chloroform extraction of the dry cell mass and purified by precipitation with diethyl ether. The purified polymer has been characterized in terms of its solubility properties, melting temperature, and UV-, IR- and NMR-spectroscopic analyses.
A membrane bioreactor which combines a continuous fermentor and a cross-flow microfiltration module enabling continuous cell recycling and removal of the fermentation products freed of microbial cells was tested for the production of alginate by Azotobacter vinelandii. The molecular weight characteristics of the polysaccharide produced were investigated. The macromolecular characteristics of alginate recovered by first filtering batch fermentation broths through different microporous ceramic membranes with pore sizes varying from 0.45 to 1.4 microm seemed to be independent of the MPS. The 0.8-microm membrane pore size (MPS) was selected for continuous alginate production in the membrane bioreactor. A mean yield value of 0.21 g alginate g(-1) saccharose was obtained and an increase in alginate polydispersity as incubation proceeded was observed, probably due to enzymatic degradation of the polysaccharide.
Azotobacter chroococcum MAL-201 (MTCC 3853), a poly-3-hydroxybutyric acid [P(3HB)] producing organism was subjected to mutagenesis by UV-irradiation and N'-methyl-N'-nitro-N'-nitrosoguanidine (MNNG). A sharp decline in survival percentage of treated cells both in UV (1.2% at 20 sec. exposure) and in MNNG ((0.26% at 30 microg/ml) suggest high degree of sensitivity of the isolate to these mutagens. A total of 124 mutant colonies were isolated from viable population based on their morphological features, antibiotic resistance and dependence on exogenous organic nitrogenous substances. Nature of mutants were confirmed by replica plating and growth on antibiotic and organic nitrogen supplemented Norris agar medium. Majority of mutants were devoid of exopolysaccharide, dependent on organic nitrogen and with weak P(3HB) content. The mutant strain, UC-220, a nitrogen-dependent, chloramphenicol-resistant mutant showed an enhancement of more than 10% (w/w) P(3HB) production compared to that of wild type strain when grown under identical conditions.
We have used site-directed mutagenesis to obtain two variants of Azotobacter vinelandii ferredoxin I (AvFdI), whose x-ray structures are now available. In the C20A protein, a ligand to the [4Fe-4S] cluster was removed whereas in the C24A mutant a free cysteine next to that cluster was removed. Like native FdI, both mutants contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structure of C24A is very similar to that of native FdI, while the structure of C20A is rearranged in the region of the [4Fe-4S] cluster to allow it to use the free Cys-24 as a replacement ligand. Here we compare the properties of the native, C20A, and C24A proteins. Although all three proteins are O2 stable in vitro, the C20A protein is much less stable toward proteolysis than the other two in vivo. Spectroscopic results show that all three proteins exhibit the same general redox behavior during O2-oxidation and dithionite reduction. Electrochemical data show that the [3Fe-4S] clusters in all three proteins have the same pH-dependent reduction potentials (-425 mV versus SHE, pH 7.8), whereas the [4Fe-4S] cluster potentials vary over a approximately 150 mV range from -600 mV (C24A) to -647 mV (native) to -746 mV (C20A). Despite this variation in potential both the C20A and C24A proteins appear to be functional in vivo. Native FdI reacts with three equivalents of Fe(CN)3-(6) to form a paramagnetic species previously proposed to be a cysteinyl-disulfide radical. Neither the C20A nor the C24A variant undergoes this reaction, strongly suggesting that it involves the free Cys-24.
Cytochrome d has been postulated to be the "respiratory protection" oxidase of Azotobacter vinelandii, allowing this organism to fix nitrogen under aerobic growth conditions. We have previously cloned and characterized the structural genes for the A. vinelandii cytochrome d (cydA and cydB). The cyd genes are co-transcribed, yielding an mRNA of approximately 3.6 kilobase pairs. The level of the cyd message was 2-3-fold higher in cells that were fixing nitrogen, as compared with non-nitrogen-fixing cells. RNase protection analysis was used to determine the transcriptional start site at 275 bases upstream of the initiator ATG of cydA, and this start site was the same for nitrogen-fixing and non-nitrogen-fixing cells. The cyd promoter has sequence similarities to the canonical Escherichia coli promoters, which are transcribed by the major sigma 70 form of RNA polymerase. Plasmid-borne lacZ transcriptional fusions were constructed, using approximately 650 base pairs of 5'-upstream sequences of the cyd structural genes. This region had a strong promoter activity which was further up-regulated 1.5-2.5-fold upon the induction of nitrogen fixation. The cyd-lacZ fusions were characterized in a nifA- as well as an ntrA- background. Mutations in neither of these nif regulatory genes affected the constitutive expression of cyd under non-nitrogen-fixing conditions. However, the up-regulation of this promoter during the induction of nitrogen fixation was abolished only in the ntrA- background. Based on these results, the cytochrome d promoter of A. vinelandii belongs to a new class of nitrogen-regulated promoters which, unlike the authentic nif genes, does not require the ntrA gene product for its expression. The up-regulation of this promoter during nitrogen fixation, however, requires the ntrA gene product.
Azotobacter vinelandii (4Fe-4S)2 ferredoxin I (Fd I) is an electron transfer protein with Mr equals 14,500 and Eo equals -420 mv. It exhibits and EPR signal of g equals 2.01 in its isolated form. This resonance is almost identical with the signal that originates from a "super-oxidized" state of the 4Fe-4S cluster of potassium ferricyanide-treated Clostridium ferredoxin. A cluster that exhibits this EPR signal at g equals 2.01 is in the same formal oxidation state as the cluster in oxidized Chromatium High-Potential-Iron-Protein (HiPIP). On photoreduction of Fd I with spinach chloroplast fragments, the resonance at g equals 2.01 vanishes and no EPR signal is observed. This EPR behavior is analogous to that of reduced HiPIP, which also fails to exhibit an EPR spectrum. These characteristics suggest that a cluster in A. vinelandii Fd I functions between the same pair of states on reduction as does the cluster in HiPIP, but with a midpoint reduction potential of -420 mv in contrast to the value of +350 mv characteristic of HiPIP. Quantitative EPR and stoichoimetry studies showed that only one 4Fe-4S cluster in this (4Fe-4S)2 ferredoxin is reduced. Oxidation of Fd I with potassium ferricyanide results in the uptake of 1 electron/mol as determined by quantitative EPR spectroscopy. This indicates that a cluster in Fd I shows no electron paramagnetic resonance in the isolated form of the protein accepts an electron on oxidation, as indicated by the EPR spectrum, and becomes paramagnetic. The EPR behavior of this oxidizable cluster indicates that it also functions between the same pair of oxidation states as does the Fe-S cluster in HiPIP. The midpoint reduction potential of this cluster is approximately +340 mv. A. vinelandii Fd I is the first example of an iron-sulfur protein which contains both a high potential cluster (approximately +340 mv) and a low potential cluster (-420 mv). Both Fe-S clusters appear to function between the same pair of oxidation states as the single Fe-S cluster in Chromatium HiPIP, although the midpoint reduction potentials of the two clusters are approximately 760 mv different.
The crystal structure of the C24A mutant of Azotobacter vinelandii 7Fe ferredoxin (FdI) has been solved and refined at 2.0-A resolution. The structure is isomorphous to native FdI except at the site of mutation where A24 moves toward the [4Fe-4S] cluster. In spite of this inefficient packing results: three of five van der Waals contacts from the S gamma of C24 in native FdI are lost and the remaining two become longer. Consequently, the [4Fe-4S] cluster is either disordered or has a higher temperature factor (B factor) compared to the rest of the C24A FdI molecule. In addition, the entire C24A FdI structure has a higher overall B factor than native FdI. Therefore, in comparison to native FdI, the C24A mutant is isomorphous but exhibits large differences in B factor, especially at the [4Fe-4S] cluster. In contrast, the C20A FdI structure (Martin, A. G., Burgess, B. K., Stout, C. D., Cash, V. L., Dean, D. R., Jensen, G. M., and Stephens, P. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 598-602), which contains large structural rearrangements in the vicinity of the [4Fe-4S] cluster, exhibits essentially no change in B factor. The conformational change observed at residue 24 is similar in both C24A and C20A FdI structures. The solvent accessibility of the Fe atoms in the [3Fe-4S] and [4Fe-4S] clusters is similar in C24A, C20A, and native FdI.
Site-directed mutagenesis and gene replacement procedures were used to construct a mutant strain of Azotobacter vinelandii which expresses a hybrid nitrogenase Fe protein. This hybrid Fe protein has its carboxyl-terminal 18 residues replaced with the 5 analogous residues from the Clostridium pasteurianum Fe protein sequence. The hybrid Fe protein is 13 amino acids smaller than the wild-type A. vinelandii Fe protein and has a net loss of 4 negatively charged residues, resulting in a change in size and charge. The strain which produces the hybrid Fe protein remained capable of diazotrophic growth, albeit at a reduced rate. Also, the purified hybrid Fe protein exhibited a maximum activity about one-half that of native Fe protein. These results demonstrate that the tight, inactive complex which is formed when A. vinelandii MoFe protein and C. pasteurianum Fe protein are mixed in heterologous reconstitution experiments cannot be accounted for only by differences in the A. vinelandii and C. pasteurianum Fe protein primary sequences located at their respective carboxyl termini.
Metallocluster extrusion requirements, interspecies MoFe-protein primary sequence comparisons and comparison of the primary sequences of the MoFe-protein subunits with each other have been used to assign potential P-cluster (Fe-S cluster) domains within the MoFe protein. In each alpha-beta unit of the MoFe protein, alpha-subunit domains, which include potential Fe-S cluster ligands Cys-62, His-83, Cys-88 and Cys-154, and beta-subunit domains, which include potential Fe-S cluster ligands Cys-70, His-90, Cys-95 and Cys-153, are proposed to comprise nearly equivalent P-cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the alpha-subunit Cys-62, His-83, Cys-88 and Cys-154 positions by site-directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates, MoFe-protein acetylene-reduction activities, and whole-cell S = 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe-protein alpha-subunit residues, Cys-62 and Cys-154, are probably essential for MoFe-protein activity but that His-83 and Cys-88 residues are not. These results indicate either that His-83 and Cys-88 do not provide essential P-cluster ligands or that a new cluster-ligand arrangement is formed in their absence.