Biological treatment of two-phase olive mill wastewater (TPOMW, alpeorujo): polyhydroxyalkanoates (PHAs) production by Azotobacter strains.
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Azotobacter chroococcum H23 (CECT 4435), Azotobacter vinelandii UWD, and Azotobacter vinelandii (ATCC 12837), members of the family Pseudomonadaceae, were used to evaluate their capacity to grow and accumulate polyhydroxyalkanoates (PHAs) using two-phase olive mill wastewater (TPOMW, alpeorujo) diluted at different concentrations as the sole carbon source. The PHAs amounts (g/l) increased clearly when the TPOMW samples were previously digested under anaerobic conditions. The MNR analysis demonstrated that the bacterial strains formed only homopolymers containing beta-hydroxybutyrate, either when grown in diluted TPOMW medium or diluted anaerobically digested TPOMW medium. COD values of the diluted anaerobically digested waste were measured before and after the aerobic PHA-storing phase, and a clear reduction (72%) was recorded after 72 h of incubation. The results obtained in this study suggest the perspectives for using these bacterial strains to produce PHAs from TPOMW, and in parallel, contribute efficiently to the bioremediation of this waste. This fact seems essential if bioplastics are to become competitive products. (+info)
The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein.
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We have examined three strains of Azotobacter vinelandii, which contain defined deletions within the nifH, nifB, or nifE genes. All three strains accumulate inactive FeMo cofactor-deficient forms of the MoFe protein of nitrogenase. These forms can be activated in vitro by addition of isolated FeMo cofactor in N-methylformamide. Although the phenotypes of these strains are superficially the same, our characterizations demonstrate that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is quite different from that synthesized by either the delta nifB or delta nifE strains. These differences include the following: 1) the activation of the delta nifH protein requires MgATP, whereas the activation of the delta nifB and delta nifE proteins does not; 2) the delta nifH extracts can be activated with FeMo cofactor to wild-type levels of activity, whereas delta nifB and delta nifE extracts cannot; 3) the delta nifH protein is markedly less heat stable than the delta nifB and delta nifE proteins; and 4) the migration of the delta nifH protein on native gels is very different when compared with delta nifB and delta nifE, which look like each other. These data can be explained if the nifB and nifE gene products are only involved in FeMo cofactor biosynthesis, whereas the nifH gene product is involved in both the initial synthesis of FeMo cofactor and in the insertion of preformed FeMo cofactor into the MoFe protein. A model is presented that suggests that the FeMo cofactor-deficient MoFe protein synthesized by the delta nifH strain is the one that normally participates in MoFe protein assembly in wild-type cells. (+info)
Characterization of isolated nitrogenase FeVco.
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A 31P-nuclear-magnetic-resonance study of NADPH-cytochrome-P-450 reductase and of the Azotobacter flavodoxin/ferredoxin-NADP+ reductase complex.
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31P-nuclear-magnetic-resonance spectroscopy has been employed to probe the structure of the detergent-solubilized form of liver microsomal NADPH--cytochrome-P-450 reductase. In addition to the resonances due to the FMN and FAD coenzymes, additional phosphorus resonances are observed and are assigned to the tightly bound adenosine 2'-phosphate (2'-AMP) and to phospholipids. The phospholipid content was found to vary with the preparation; however, the 2'-AMP resonance was observed in all preparations tested. In agreement with published results [Otvos et al. (1986) Biochemistry 25, 7220-7228] for the protease-solubilized enzyme, the addition of Mn(II) to the oxidized enzyme did not result in any observable line-broadening of the FMN and FAD phosphorus resonances. The phospholipid resonances, however, were extensively broadened and the line width of the phosphorus resonance assigned to the bound 2'-AMP was broadened by approximately 70 Hz. The data show that only the phosphorus moieties of the phospholipids and the 2'-AMP, but not the flavin coenzymes are exposed to the bulk solvent. Removal of the FMN moiety from the enzyme substantially alters the 31P-NMR spectrum as compared with the native enzyme. The 2'-AMP is removed from the enzyme during the FMN-depletion procedure and the pyrophosphate resonances of the bound FAD are significantly altered. Reconstitution of the FMN-depleted protein with FMN results in the restoration of the coenzyme spectral properties. Reduction of FMN to its air-stable paramagnetic semiquinone form results in broadening of the FMN and 2'-AMP resonances in the detergent-solubilized enzyme. In agreement with previous results. FMN semiquinone formation had little or no effect on the line width of the FMN phosphorus resonance for the proteolytically solubilized enzyme. 31P-NMR experiments with Azotobacter flavodoxin semiquinone, both in its free form and in a complex with spinach ferredoxin-NADP+ reductase, mimic the differential paramagnetic effects of the flavin semiquinone on the line width of the FMN phosphorus resonance, observed by comparison of the detergent-solubilized and protease-solubilized forms of the reductase. The data demonstrate that assignment of the site of flavin semiquinone formation to a particular flavin coenzyme may not always be possible by 31P-NMR experiments in multi-flavin containing enzymes. (+info)
Analysis of Azotobacter vinelandii strains containing defined deletions in the nifD and nifK genes.
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Strains of Azotobacter vinelandii which contain defined deletions within the nifD and nifK genes which encode, respectively, the alpha and beta subunits of the MoFe protein of nitrogenase were analyzed. When synthesized without its partner, the beta subunit accumulated as a soluble beta 4 tetramer. In contrast, when the alpha subunit was present without its partner, it accumulated primarily as an insoluble aggregate. The solubility of this protein was increased by the presence of a form of the beta subunit which contained a large internal deletion, such that the alpha subunit could participate in the assembly of small amounts of an alpha 2 beta 2 holoprotein. When synthesized alone, the beta subunit was remarkably stable, even when the protein contained a large internal deletion. The alpha subunit, however, was much more rapidly degraded than the beta subunit, both when it was synthesized alone in its native background and when it was synthesized with its beta subunit partner in a foreign background. Antibodies raised against purified alpha 2 beta 2 MoFe protein recognized epitopes only on the nondenatured beta subunit and not on the nondenatured alpha subunit. Our findings that all epitopes for the alpha2beta2 tetramer appeared to be on the beta subunit, that the beta subunit assembled into beta4 tetramers, and that the alpha subunit alone was very insoluble, combined with the previous finding that the Fe protein binds to the beta subunit (A. H. Willing, M. M. Georgiadis, D. C. Rees, and J. B. Howard, J. Biol. Chem. 264:8499-8503, 1989) all suggest that the beta subunit has a more surface location than the alpha subunit in the alpha2beta2 tetramer. (+info)
Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2.
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One of the regions responsible for the stable inheritance of the broad-host-range plasmid RK2 is contained within the PstI C fragment, located from coordinates 30.8 to 37.0 kb (P.N. Saurugger, O. Hrabak, H. Schwab, and R.M. Lafferty, J. Biotechnol. 4:333-343, 1986). Genetic analysis of this 6.2-kb region demonstrated that no function was present that stabilized by selectively killing plasmid-free segregants. The sequence from 36.0 to 37.0 kb mediated a twofold increase in plasmid copy number, but this region was not required for stabilization activity. The PstI C fragment was shown to encode a multimer resolution system from 33.1 to 35.3 kb. The resolution cis-acting site was mapped to 140 bp, sequenced, and observed to contain two directly repeated sequences of 6 and 7 bases and two perfect inverted repeats of 6 and 8 bases. The trans-acting factor(s) was mapped and functionally determined to encode a resolvase capable of catalyzing recombination at high frequency between cis-acting sites in either direct or inverted orientation. Multimer resolution alone did not account for complete plasmid stabilization by the PstI C fragment, since removal of regions adjacent to the 35.3-kb border of the minimal mrs locus dramatically reduced stabilization. The minimal region required for complete stabilization, from 32.8 to 35.9 kb, was capable of fully stabilizing plasmids independently of the replicon or the recA proficiency of the host. Stabilization activity was also fully expressed in several diverse gram-negative bacteria, whereas the F plasmid par locus functioned only in Escherichia coli. On the basis of these observations, we conclude that under the growth conditions used, the minimal stabilization locus encodes both an mrs activity and a stabilization activity that has the properties of a par locus. (+info)
Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: [Fe-S] cluster-driven protein rearrangement.
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Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here we report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure. (+info)