Project description:Chlorite dismutase from Dechloromonas aromatica RCB, a novel b-type hemoprotein that catalyzes O-O bond formation, has been crystallized. Synchrotron X-ray diffraction data have been collected to 3.0 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 122.7, b = 202.9, c = 247.1 A.
Project description:Salinity and pH have direct and indirect impacts on the growth and metabolic activities of microorganisms. In this study, the effects of salt and alkaline stresses on the kinetic balance between nitrous oxide (N2O) production and consumption in the denitrification pathway of Dechloromonas aromatica strain RCB were examined. N2O accumulated transiently only in insignificant amounts at low salinity (≤0.5% NaCl) and circumneutral pH (7.0 and 7.5). As compared to these control conditions, incubation at 0.7% salinity resulted in substantially longer lag phase and slower growth rate, along with the increase in the amounts of transiently accumulated N2O (15.8 ± 2.8 μmoles N2O-N/vessel). Incubation at pH 8.0 severely inhibited growth and resulted in permanent accumulation of 29.9 ± 1.3 μmoles N2O-N/vessel from reduction of 151 ± 20 μmoles NO3 -/vessel. Monitoring of temporal changes in nirS 1, nirS 2, and nosZ transcription suggested that the nosZ/(nirS 1+nirS 2) ratios were indicative of whether N2O was produced or consumed at the time points where measurements were taken. The salt and alkaline stresses altered the N2O consumption kinetics of the resting D. aromatica cells with expressed nitrous oxide reductases. The N2O consumption rates of the cells subjected to the salt and alkaline stress conditions were significantly reduced from 0.84 ± 0.007 μmoles min-1 mg protein-1 of the control to 0.27 ± 0.02 μmoles min-1 mg protein-1 and 0.31 ± 0.03 μmoles min-1 mg protein-1, respectively, when the initial dissolved N2O concentration was 0.1 mM. As the rates of N2O production from NO2 - reduction was not significantly affected by the stresses (0.45-0.55 μmoles min-1 mg protein-1), the N2O consumption rate was lower than the N2O production rate at the stress conditions, but not at the control condition. These results clearly indicate that the altered kinetics of expressed nitrous oxide reductase and the resultant disruption of kinetic balance between N2O production and consumption was another cause of enhanced N2O emission observed under the salt and alkaline stress conditions. These findings suggest that canonical denitrifiers may become a significant N2O source when faced with abrupt environmental changes.
Project description:BACKGROUND: Initial interest in Dechloromonas aromatica strain RCB arose from its ability to anaerobically degrade benzene. It is also able to reduce perchlorate and oxidize chlorobenzoate, toluene, and xylene, creating interest in using this organism for bioremediation. Little physiological data has been published for this microbe. It is considered to be a free-living organism. RESULTS: The a priori prediction that the D. aromatica genome would contain previously characterized "central" enzymes to support anaerobic aromatic degradation of benzene proved to be false, suggesting the presence of novel anaerobic aromatic degradation pathways in this species. These missing pathways include the benzylsuccinate synthase (bssABC) genes (responsible for fumarate addition to toluene) and the central benzoyl-CoA pathway for monoaromatics. In depth analyses using existing TIGRfam, COG, and InterPro models, and the creation of de novo HMM models, indicate a highly complex lifestyle with a large number of environmental sensors and signaling pathways, including a relatively large number of GGDEF domain signal receptors and multiple quorum sensors. A number of proteins indicate interactions with an as yet unknown host, as indicated by the presence of predicted cell host remodeling enzymes, effector enzymes, hemolysin-like proteins, adhesins, NO reductase, and both type III and type VI secretory complexes. Evidence of biofilm formation including a proposed exopolysaccharide complex and exosortase (epsH) are also present. Annotation described in this paper also reveals evidence for several metabolic pathways that have yet to be observed experimentally, including a sulphur oxidation (soxFCDYZAXB) gene cluster, Calvin cycle enzymes, and proteins involved in nitrogen fixation in other species (including RubisCo, ribulose-phosphate 3-epimerase, and nif gene families, respectively). CONCLUSION: Analysis of the D. aromatica genome indicates there is much to be learned regarding the metabolic capabilities, and life-style, for this microbial species. Examples of recent gene duplication events in signaling as well as dioxygenase clusters are present, indicating selective gene family expansion as a relatively recent event in D. aromatica's evolutionary history. Gene families that constitute metabolic cycles presumed to create D. aromatica's environmental 'foot-print' indicate a high level of diversification between its predicted capabilities and those of its close relatives, A. aromaticum str EbN1 and Azoarcus BH72.
Project description:Chlorite dismutase (Cld) is a heme enzyme capable of rapidly and selectively decomposing chlorite (ClO(2) (-)) to Cl(-) and O(2). The ability of Cld to promote O(2) formation from ClO(2) (-) is unusual. Heme enzymes generally utilize ClO(2) (-) as an oxidant for reactions such as oxygen atom transfer to, or halogenation of, a second substrate. The X-ray crystal structure of Dechloromonas aromatica Cld co-crystallized with the substrate analogue nitrite (NO(2) (-)) was determined to investigate features responsible for this novel reactivity. The enzyme active site contains a single b-type heme coordinated by a proximal histidine residue. Structural analysis identified a glutamate residue hydrogen-bonded to the heme proximal histidine that may stabilize reactive heme species. A solvent-exposed arginine residue likely gates substrate entry to a tightly confined distal pocket. On the basis of the proposed mechanism of Cld, initial reaction of ClO(2) (-) within the distal pocket generates hypochlorite (ClO(-)) and a compound I intermediate. The sterically restrictive distal pocket probably facilitates the rapid rebound of ClO(-) with compound I forming the Cl(-) and O(2) products. Common to other heme enzymes, Cld is inactivated after a finite number of turnovers, potentially via the observed formation of an off-pathway tryptophanyl radical species through electron migration to compound I. Three tryptophan residues of Cld have been identified as candidates for this off-pathway radical. Finally, a juxtaposition of hydrophobic residues between the distal pocket and the enzyme surface suggests O(2) may have a preferential direction for exiting the active site.
Project description:Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance but research into the mechanisms has been stymied by a lack of a genetically tractable pure culture which unequivocally does not use molecular oxygen to activate benzene. Geobacter metallireducens grew in a medium in which benzene was the sole electron donor and Fe(III) was the sole electron acceptor with a stoichiometry of benzene loss and Fe(III) reduction consistent with benzene oxidation to carbon dioxide coupled with Fe(III) reduction. Phenol labeled with 18O was produced when the medium was labeled with H218O, as expected for a true anaerobic conversion of benzene to phenol. Gene expression patterns indicated that benzene was metabolized through a phenol intermediate rather than benzoate or toluene. Deletion of ppcB, which encodes a subunit of the phenylphosphate carboxylase, an enzyme required for phenol metabolism, inhibited metabolism of benzene. Deleting genes specific for benzoate or toluene metabolism did not. Comparison of gene expression patterns in cells grown on benzene versus cells grown on phenol revealed genes specifically expressed in benzene-grown cells. Deletion of one of these, Gmet_3376, inhibited anaerobic benzene oxidation, but not the metabolism of phenol, benzoate, or toluene. The availability of a genetically tractable pure culture that can anaerobically convert benzene to phenol with oxygen derived from water should significantly accelerate elucidation of the mechanisms by which benzene can be activated in the absence of molecular oxygen. Total RNA from three separate cultures of G. metallireducens grown with 250 µM benzene three separate cultures of G. metallireducens grown with 500 µM phenol three separate cultures of G. metallireducens grown with 1 mM benzoate three separate cultures of G. metallireducens grown with 500 µM toluene three separate cultures of G. metallireducens grown with 10 mM acetate were used to study [1] Anaerobic oxidation of benzene by G. metallireducens (Benzene vs. acetate, Benzene vs. benzoate, Benzene vs. phenol, Benzene vs. toluene) [2] Anaerobic oxidation of benzoate by G. metallireducens (Benzoate vs. acetate) [3] Anaerobic oxidation of phenol by G. metallireducens (Phenol vs. acetate) [4] Anaerobic oxidation of toluene by G. metallireducens (Toluene vs. acetate) Each chip measures the expression level of 3,627 genes from G. metallireducens DSM 7210 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:This SuperSeries is composed of the following subset Series: GSE28549: Anaerobic Oxidation of Benzene by the Hyperthermophilic Archaeon Ferroglobus placidus (Phenol vs. Benzoate) GSE30798: Anaerobic Oxidation of Benzene by the Hyperthermophilic Archaeon Ferroglobus placidus (Benzene vs. Acetate) GSE30799: Anaerobic Oxidation of Benzene by the Hyperthermophilic Archaeon Ferroglobus placidus (Benzene vs. Phenol) GSE30801: Anaerobic Oxidation of Benzene by the Hyperthermophilic Archaeon Ferroglobus placidus (Benzene vs. Benzoate) Refer to individual Series
Project description:Anaerobic activation of benzene is expected to represent a novel biochemistry of environmental significance but research into the mechanisms has been stymied by a lack of a genetically tractable pure culture which unequivocally does not use molecular oxygen to activate benzene. Geobacter metallireducens grew in a medium in which benzene was the sole electron donor and Fe(III) was the sole electron acceptor with a stoichiometry of benzene loss and Fe(III) reduction consistent with benzene oxidation to carbon dioxide coupled with Fe(III) reduction. Phenol labeled with 18O was produced when the medium was labeled with H218O, as expected for a true anaerobic conversion of benzene to phenol. Gene expression patterns indicated that benzene was metabolized through a phenol intermediate rather than benzoate or toluene. Deletion of ppcB, which encodes a subunit of the phenylphosphate carboxylase, an enzyme required for phenol metabolism, inhibited metabolism of benzene. Deleting genes specific for benzoate or toluene metabolism did not. Comparison of gene expression patterns in cells grown on benzene versus cells grown on phenol revealed genes specifically expressed in benzene-grown cells. Deletion of one of these, Gmet_3376, inhibited anaerobic benzene oxidation, but not the metabolism of phenol, benzoate, or toluene. The availability of a genetically tractable pure culture that can anaerobically convert benzene to phenol with oxygen derived from water should significantly accelerate elucidation of the mechanisms by which benzene can be activated in the absence of molecular oxygen.
Project description:The heme-based chlorite dismutases catalyze the unimolecular decomposition of chlorite (ClO2−) to yield Cl− and O2. The work presented here shows that chlorite dismutase from Dechloromonas aromatica (DaCld) also catalyzes the decomposition of bromite (BrO2−) with the evolution of O2 (kcat = (2.0±0.2)×102 s−1; kcat/KM = (1.2±0.2)×105 M−1 s−1 at pH 5.2). Stopped-flow studies of this BrO2− decomposition as a function of pH show that 1) the two-electron oxidized heme, compound I (Cpd I), is the primary accumulating heme intermediate during O2 evolution in acidic solution, 2) Cpd I and its one-electron reduction product, compound II (Cpd II) are present in varying ratios at intermediate pHs, and 3) only Cpd II is observed at pH 9.0. The pH dependences of Cpd I and Cpd II populations both yield a pKa of 6.7±0.1 in good agreement with the pKa of DaCld activity with ClO2−. The observation of a protein-based amino acid radical (AA•) whose appearance coincides with that of Cpd II supports the hypothesis that conversion of Cpd I to Cpd II occurs via proton-coupled electron transfer (PCET) from a heme-pocket amino acid to the oxidized porphyrinate of Cpd I to yield a dead-end decoupled state in which the holes decay at different rates. The site of the amino acid radical is tentatively assigned to Y118, which serves as a H-bond donor to propionate 6 (P6). The favoring of Cpd II:AA• accumulation in alkaline solution is consistent with the amino acid oxidation being rate limited by transfer of its proton to P6 having pKa 6.7. Examination of reaction mixtures comprising DaCld and ClO2− by resonance Raman and electron paramagnetic resonance spectroscopy reveal formation of Cpd II and •ClO2, which forms in preference to the analogous to AA• in the BrO2− reaction. Addition of ClO− to Cpd II did not yield O2. Together these results are consistent with heterolytic cleavage of the O–BrO− and O–ClO− bonds yielding Cpd I, which is the catalytically active intermediate. The long-lived Cpd II that forms subsequently, is inactive toward O2 production, and diminishes the amount of enzyme available to cycle through the active Cpd I intermediate.
Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms.