Degradation of 2,4,6-trichlorophenol by Azotobacter sp. strain GP1.
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A bacterium which utilizes 2,4,6-trichlorophenol (TCP) as a sole source of carbon and energy was isolated from soil. The bacterium, designated strain GP1, was identified as an Azotobacter sp. TCP was the only chlorinated phenol which supported the growth of the bacterium. Resting cells transformed monochlorophenols, 2,6-dichlorophenol, and 2,3,6-trichlorophenol. Phenol and a number of phenolic compounds, including 4-methylphenol, all of the monohydroxybenzoates, and several dihydroxybenzoates, were very good carbon sources for Azotobacter sp. strain GP1. The organism utilized up to 800 mg of TCP per liter; the lag phase and time for degradation, however, were severely prolonged at TCP concentrations above 500 mg/liter. Repeated additions of 200 mg of TCP per liter led to accelerated degradation, with an optimum value of 100 mg of TCP per liter per h. TCP degradation was significantly faster in shaken than in nonshaken cultures. The optimum temperature for degradation was 25 to 30 degrees C. Induction studies, including treatment of the cells with chloramphenicol prior to TCP or phenol addition, revealed that TCP induced TCP degradation but not phenol degradation and that phenol induced only its own utilization. Per mol of TCP, 3 mol of Cl- was released. 2,6-Dichloro-p-benzoquinone was detected in the resting-cell medium of Azotobacter sp. strain GP1. By chemical mutagenesis, mutants blocked in either TCP degradation or phenol degradation were obtained. No mutant defective in the degradation of both phenols was found, indicating separate pathways for the dissimilation of the compounds. In some of the phenol-deficient mutants, pyrocatechol was found to accumulate, and in some of the TCP-deficient mutants, 2,6-dichlorohydroquinone was found to accumulate. (+info)
The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii.
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Under diazotrophic conditions in the absence of molybdenum (Mo) and vanadium (V), Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase 3 (encoded by anfHDGK). However, dinitrogenase reductase 2 (encoded by vnfH) is also expressed under these conditions even though this protein is a component of the V-containing alternative nitrogenase. Mutant strains that lack dinitrogenase reductase 2 (VnfH-) grow slower than the wild-type strain in N-free, Mo-, and V-deficient medium. In this medium, these strains synthesize dinitrogenase reductase 1 (a component of the Mo-containing nitrogenase encoded by nifH), even though this component is not normally synthesized in the absence of Mo. Strains that lack both dinitrogenase reductases 1 and 2 (NifH-VnfH-) are unable to grow diazotrophically in Mo- and V-deficient medium. In this medium, NifH- VnfH- strains containing an anfH-lacZ transcriptional fusion exhibited less than 3% of the beta-galactosidase activity observed in the wild type with the same fusion. Beta-Galactosidase activity expressed by VnfH- mutants containing the anfH-lacZ fusion ranged between 57 and 78% of that expressed by the wild type containing the same fusion. Thus, expression of dinitrogenase reductase 2 seems to be required for transcription of the anfHDGK operon, although, in VnfH-mutants, dinitrogenase reductase 1 appears to serve this function. Active dinitrogenase reductase 1 or 2 is probably required for this function since a nifM deletion mutant containing the anfH-lacZ fusion was unable to synthesize beta-galactosidase above background levels. An anfA deletion strain containing the anfH-lacZ fusion exhibited beta-galactosidase activity at 16% of that of the wild type containing the same fusion. However, in the presence of NH4+, the beta-galactosidase activity expressed by this strain more than doubled. This indicates that AnfA is required not only for normal levels of anfHDGK transcription but also for NH4+ -and, to a lesser extent, Mo-mediated repression of this transcription. (+info)
Interaction of lipoamide dehydrogenase with the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.
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The interaction between lipoamide dehydrogenase (E3) and dihydrolipoyl transacetylase (E2p) from the pyruvate dehydrogenase complex was studied during the reconstitution of monomeric E3 apoenzymes from Azotobacter vinelandii and Pseudomonas fluorescens. The dimeric form of E3 is not only essential for catalysis but also for binding to the E2p core, because the apoenzymes as well as a monomeric holoenzyme from P. fluorescens, which can be stabilized as an intermediate at 0 degree C, do not bind to E2p. Lipoamide dehydrogenase from A. vinelandii contains a C-terminal extension of 15 amino acids with respect to glutathione reductase which is, in contrast to E3, presumably not part of a multienzyme complex. Furthermore, the last 10 amino acid residues of E3 are not visible in the electron density map of the crystal structure and are probably disordered. Therefore, the C-terminal tail of E3 might be an attractive candidate for a binding region. To probe this hypothesis, a set of deletions of this part was prepared by site-directed mutagenesis. Deletion of the last five amino acid residues did not result in significant changes. A further deletion of four amino acid residues resulted in a decrease of lipoamide activity to 5% of wild type, but the binding to E2p was unaffected. Therefore it is concluded that the C-terminus is not directly involved in binding to the E2p core. Deletion of the last 14 amino acids produced an enzyme with a high tendency to dissociate (Kd approximately 2.5 microM). This mutant binds only weakly to E2p. The diaphorase activity was still high. This indicates, together with the decreased Km for NADH, that the structure of the monomer is not appreciably changed by the mutation. Rather the orientation of the monomers with respect to each other is changed. It can be concluded that the binding region of E3 for E2p is constituted from structural parts of both monomers and binding occurs only when dimerization is complete. (+info)
Molecular analysis of the Azotobacter vinelandii glnA gene encoding glutamine synthetase.
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The gene encoding glutamine synthetase (GS), glnA, was cloned from Azotobacter vinelandii on a 6-kb EcoRI fragment that also carries the ntrBC genes. The DNA sequence of 1,952 bp including the GS-coding region was determined. An open reading frame of 467 amino acids indicated a gene product of Mr 51,747. Transcription of glnA occurred from a C residue located 32 bases upstream of an ATG considered to be the initiator codon because (i) it had a nearby potential ribosome-binding site and (ii) an open reading frame translated from this site indicated good N-terminal homology to 10 other procaryotic GSs. Sequences similar to the consensus RNA polymerase recognition sites at -10 and -35 were present at the appropriate distance upstream of the transcription initiation site. As expected from earlier genetic studies indicating that expression of A. vinelandii glnA did not depend on the rpoN (ntrA; sigma 54) gene product, no sigma 54 recognition sequences were present, nor was there significant regulation of glnA expression by fixed nitrogen. Repeated attempts to construct glutamine auxotrophs by recombination of glnA insertion mutations were unsuccessful, Although the mutated DNA could be found by hybridization experiments in drug-resistant A. vinelandii transformants, the wild-type glnA region was always present. These results suggest that glnA mutations are lethal in A. vinelandii. In [14C]glutamine uptake experiments, very little glutamine was incorporated into cells, suggesting that glutamine auxotrophs are nonviable because they cannot be supplied with sufficient glutamine to support growth. (+info)
Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria.
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Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases. (+info)
Identification of sulfurtransferase enzymes in Azotobacter vinelandii.
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Rhodanese and 3-mercaptopyruvate sulphurtransferase have been identified in A. vinelandii. Two distinct active fractions of the two sulphur transferases were obtained after FPLC ion-exchange chromatography of material partially purified from crude extracts. Rhodanese has been purified to homogeneity, and it consists of one polypeptide chain of Mr ca 25,000. A partial purification of 3-mercaptopyruvate sulphurtransferase was obtained. (+info)
Effects of aeration on the synthesis of poly(3-hydroxybutyrate) from glycerol and glucose in recombinant Escherichia coli.
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On the FAD-induced dimerization of apo-lipoamide dehydrogenase from Azotobacter vinelandii and Pseudomonas fluorescens. Kinetics of reconstitution.
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The apoenzymes of lipoamide dehydrogenase from pig heart and from Pseudomonas fluorescens were prepared at pH 2.7 and pH 4.0, respectively, using a hydrophobic interaction chromatography procedure recently developed for lipoamide dehydrogenase from Azotobacter vinelandii and other flavoproteins [Van Berkel et al. (1988) Eur. J. Biochem. 178, 197-207]. The apoenzyme from pig heart, having 5% of residual activity, shows an equilibrium between the monomeric and dimeric species. Both the yield and the degree of reconstitution of dimeric holoenzyme is 75% of starting material under optimal conditions. The kinetics of reconstitution of pig heart apoenzyme differ slightly from that obtained with the apoenzyme prepared by acid ammonium sulfate precipitation at pH 1.5 [Kalse, J. F. and Veeger, C. (1968) Biochim. Biophys. Acta 159, 244-256]. The apoenzyme from P. fluorescens is in the monomeric state and shows negligible residual activity. The yield and degree of reconstitution of the dimeric holoenzyme is more than 90% of starting material. Reconstitution of the apoenzymes from A. vinelandii and P. fluorescens involves minimally a two-step sequential process. Initial flavin-binding results in regaining of full dichloroindophenol activity, quenching of tryptophan fluorescence and strong increase of FAD fluorescence polarization. In the second step, dimerization occurs as reflected by regain of lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of the visible absorption spectrum. The kinetics of FAD-induced dimerization are strongly dependent on the apoenzyme used. At 0 degrees C, the monomeric apoenzyme-FAD complex is either stabilized (P. fluorescens) or only transiently detectable (A. vinelandii). Dimerization of P. fluorescens enzyme is strongly stimulated in the presence of NADH. (+info)