Oligotropha Carboxidovorans Research Articles

Autotrophic nitrite removal in the cathode of microbial fuel cells

Nitrification to nitrite (nitritation process) followed by reduction to dinitrogen gas decreases the energy demand and the carbon requirements of the overall process of nitrogen removal. This work studies autotrophic nitrite removal in the cathode of microbial fuel cells (MFCs). Special attention was paid to determining whether nitrite is used as the electron acceptor by exoelectrogenic bacteria (biologic reaction) or by graphite electrodes (abiotic reaction). The results demonstrated that, after a nitrate pulse at the cathode, nitrite was initially accumulated; subsequently, nitrite was removed. Nitrite and nitrate can be used interchangeably as an electron acceptor by exoelectrogenic bacteria for nitrogen reduction from wastewater while producing bioelectricity. However, if oxygen is present in the cathode chamber, nitrite is oxidised via biological or electrochemical processes. The identification of a dominant bacterial member similar to Oligotropha carboxidovorans confirms that autotrophic denitrification is the main metabolism mechanism in the cathode of an MFC.

Interaction of CO Dehydrogenase with the Cytoplasmic Membrane Monitored by Fluorescence Correlation Spectroscopy

We have investigated the interaction of carbon monoxide dehydrogenase (CODH), an enzyme that catalyses the oxidation of CO in the aerobic eubacterium Oligotropha carboxidovorans, with the cytoplasmic membrane by using fluorescence correlation spectroscopy (FCS). Our results reveal that in vitro this interaction of CODH is specific for cytoplasmic membranes from CO-grown bacteria.

Complete genome and comparative analysis of the chemolithoautotrophic bacterium Oligotropha carboxidovorans OM5

BackgroundOligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium capable of utilizing CO (carbon monoxide) and fixing CO2 (carbon dioxide). We previously published the draft genome of this organism and recently submitted the complete genome sequence to GenBank.ResultsThe genome sequence of the chemolithoautotrophic bacterium Oligotropha carboxidovorans OM5 consists of a 3.74-Mb chromosome and a 133-kb megaplasmid that contains the genes responsible for utilization of carbon monoxide, carbon dioxide, and hydrogen. To our knowledge, this strain is the first one to be sequenced in the genus Oligotropha, the closest fully sequenced relatives being Bradyrhizobium sp. BTAi and USDA110 and Nitrobacter hamburgiensis X14. Analysis of the O. carboxidovorans genome reveals potential links between plasmid-encoded chemolithoautotrophy and chromosomally-encoded lipid metabolism. Comparative analysis of O. carboxidovorans with closely related species revealed differences in metabolic pathways, particularly in carbohydrate and lipid metabolism, as well as transport pathways.ConclusionOligotropha, Bradyrhizobium sp and Nitrobacter hamburgiensis X14 are phylogenetically proximal. Although there is significant conservation of genome organization between the species, there are major differences in many metabolic pathways that reflect the adaptive strategies unique to each species.

Kinetic and Spectroscopic Studies of the Molybdenum-Copper CO Dehydrogenase from Oligotropha carboxidovorans

Carbon monoxide dehydrogenase from the aerobic bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO(2), yielding two electrons and two H(+). The steady-state kinetics of the enzyme exhibit a pH optimum of 7.2 with a k(cat) of 93.3 s(-1) and K(m) of 10.7 microM at 25 degrees C. k(red) for the reductive half-reaction agrees well with k(cat) and exhibits a similar pH optimum, indicating that the rate-limiting step of overall turnover is likely in the reductive half-reaction. No dependence on CO concentration was observed in the rapid reaction kinetics, however, suggesting that CO initially binds rapidly to the enzyme, possibly at the Cu(I) of the active site, prior to undergoing oxidation. A Mo(V) species that exhibits strong coupling to the copper of the active center (I = 3/2) has been characterized by EPR. The signal is further split when [(13)C]CO is used to generate it, demonstrating that substrate (or product) is a component of the signal-giving species. Finally, resonance Raman spectra of CODH reveal the presence of FAD, Fe/S clusters, and a [CuSMoO(2)] coordination in the active site, consistent with earlier x-ray absorption and crystallographic results.

The CoxD Protein of Oligotropha carboxidovorans Is a Predicted AAA+ ATPase Chaperone Involved in the Biogenesis of the CO Dehydrogenase [CuSMoO2] Cluster

CO dehydrogenase from the Gram-negative chemolithoautotrophic eubacterium Oligotropha carboxidovorans OM5 is a structurally characterized molybdenum-containing iron-sulfur flavoenzyme, which catalyzes the oxidation of CO (CO + H(2)O --> CO(2) + 2e(-) + 2H(+)). It accommodates in its active site a unique bimetallic [CuSMoO(2)] cluster, which is subject to post-translational maturation. Insertional mutagenesis of coxD has established its requirement for the assembly of the [CuSMoO(2)] cluster. Disruption of coxD led to a phenotype of the corresponding mutant OM5 D::km with the following characteristics: (i) It was impaired in the utilization of CO, whereas the utilization of H(2) plus CO(2) was not affected; (ii) Under appropriate induction conditions bacteria synthesized a fully assembled apo-CO dehydrogenase, which could not oxidize CO; (iii) Apo-CO dehydrogenase contained a [MoO(3)] site in place of the [CuSMoO(2)] cluster; and (iv) Employing sodium sulfide first and then the Cu(I)-(thiourea)(3) complex, the non-catalytic [MoO(3)] site could be reconstituted in vitro to a [CuSMoO(2)] cluster capable of oxidizing CO. Sequence information suggests that CoxD is a MoxR-like AAA+ ATPase chaperone related to the hexameric, ring-shaped BchI component of Mg(2+)-chelatases. Recombinant CoxD, which appeared in Escherichia coli in inclusion bodies, occurs exclusively in cytoplasmic membranes of O. carboxidovorans grown in the presence of CO, and its occurrence coincided with GTPase activity upon sucrose density gradient centrifugation of cell extracts. The presumed function of CoxD is the partial unfolding of apo-CO dehydrogenase to assist in the stepwise introduction of sulfur and copper in the [MoO(3)] center of the enzyme.

Novel coaggregating microbial consortium: Testing strength for field applications

Acinetobacter johnsonii S35 has an ability to coaggregate with Oligotropha carboxidovorans S23 when grown with favorable carbon substrate and nutrient-rich growth media. However, nutrient-deficient situations or poorly accessible carbon substrates in waste streams could trigger changes in the cell surface properties and thus modify the coaggregation behavior. Thus, in the present study, we test the stability of these novel interactions under various stresses that might transpire in the waste streams. The effect of mineral-component deficiency and shortage of substrate on the growth, cell surface hydrophobicity and coaggregation ability of S35 and O. carboxidovorans S23 has been investigated. As compared to the control, growth of S35 was adversely affected in mineral-deficient medium; the cells had lower hydrophobicity but displayed only slightly lower aggregation index (AI, 75–84%) as compared to control (92%). S35 grown on marginally supplemented Sterile Filtrate of Activated Sludge (SFAS) could also grow well and form larger stable coaggregates with S23. S35 grown with mineral-deficient medium and with the supplemented SFAS still posses the ability to coaggregate with S23. Hence the coaggregation ability of S35 strain is constitutive and is not significantly affected by environmental factors. It would be desirable to work out suitable formulations of such robust and environmentally relevant microbes.

Extracción y purificación de la enzima monóxido de carbono deshidrogenasa a partir de la bacteria carboxidótrofa Oligotropha carboxidovorans

This paper reports the extraction and purification of the carbon monoxide dehydrogenase enzyme from the Oligotropha carboxidovorans bacteria, OM 5 strain. The bacteria grew autotrophically in a mineral medium, in carbon monoxide saturated atmosphere, at 30°C with constant agitation. In order to obtain the crude enzymatic extracts, the crops underwent a breakdown by sonication, solvolysis with non-ionic detergent and centrifugation to eliminate the cellular debris. During the purification process, the crude enzymatic extracts were passed through a molecular exclusion column as well as through an ion-exchange one.

Structural and functional reconstruction in situ of the [CuSMoO2] active site of carbon monoxide dehydrogenase from the carbon monoxide oxidizing eubacterium Oligotropha carboxidovorans

Carbon monoxide dehydrogenase from the bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO(2) at a unique [CuSMoO(2)] cluster. In the bacteria the cluster is assembled post-translational. The integration of S, and particularly of Cu, is rate limiting in vivo, which leads to CO dehydrogenase preparations containing the mature and fully functional enzyme along with forms of the enzyme deficient in one or both of these elements. The active sites of mature and immature forms of CO dehydrogenase were converted into a [MoO(3)] centre by treatment with potassium cyanide. We have established a method, which rescues 50% of the CO dehydrogenase activity by in vitro reconstitution of the active site through the supply of sulphide first and subsequently of Cu(I) under reducing conditions. Immature forms of CO dehydrogenase isolated from the bacterium, which were deficient in S and/or Cu at the active site, were similarly activated. X-ray crystallography and electron paramagnetic resonance spectroscopy indicated that the [CuSMoO(2)] cluster was properly reconstructed. However, reconstituted CO dehydrogenase contains mature along with immature forms. The chemical reactions of the reconstitution of CO dehydrogenase are summarized in a model, which assumes resulphuration of the Mo-ion at both equatorial positions at a 1:1 molar ratio. One equatorial Mo-S group reacts with Cu(I) in a productive fashion yielding a mature, functional [CuSMoO(2)] cluster. The other Mo-S group reacts with Cu(I), then Cu(2)S is released and an oxo group is introduced from water, yielding an inactive [MoO(3)] centre.

Stability of Bacterial Coaggregates in Extreme Environments

The stability of coaggregates formed by the interaction of Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23, both of which are nonflocculating sludge bacteria, was evaluated by a spectrophotometric assay of aggregation index and phase contrast microscopy. Exposure of coaggregates to conditions of various pH (3–11) or temperature (10–50°C) did not cause significant deflocculation and high aggregation index (85–93%) was still maintained as compared to 93.3% under normal conditions. Further, heat pretreatment of A. johnsonii S35 and O. carboxidovorans S23 cells at 60 or 80°C did not prevent their subsequent coaggregation indicating that the interacting cell surface structures are heat-resistant. However, dual exposure of coaggregates to low temperature (10°C) and extreme pH caused disruption of coaggregates coupled with the reduction in aggregation index below 28% (pH 3) and 49% (pH 11). Use of sterile filtrate of settled sewage (SFSS) in place of electrolyte solution as coaggregating medium produced usual large coaggregates (above 100 μm) indicating reproducibility of this coaggregation in sewage treatment plants. SFSS coaggregates incurred lesser deflocculation (50 and 85%, respectively) at extreme pH (3 and 11, respectively) and low temperature. This study demonstrated that although bacterial coaggregates are strong and stable against individual pH or temperature stress, a dual stress might deteriorate the performance and quality of treated waters.

Effect of cultivation time and growth medium on the coaggregation capability of Acinetobacter johnsonii S35

The effects of different cultivation periods and growth media on the cell surface hydrophobicity and coaggregation ability of Acinetobacter johnsonii S35 with Oligotropha carboxidovorans S23 (both sewage sludge isolates) were examined. It was observed that when cultivated in polypeptone medium, A. johnsonii S35 shows high hydrophobicity and strong coaggregation (98% aggregation index) with O. carboxidovorans S23, which remain unaltered after a 1-day, 3-day or 7-day cultivation period. When A. johnsonii S35 was cultivated with different carbon substrates, large coaggregates and a high aggregation index were observed using acetate-grown cells (96%), followed by ethanol-(83%) and vegetable oil-grown cells (72%). Coaggregates were small and narrow in ethanol-grown cells and still smaller in case of the vegetable oil-grown cells. Although no direct correlation could be observed, more hydrophobic cells showed a higher aggregation index. None of the culture supernatants of A. johnsonii S35 showed emulsification of hexadecane or a significant ability to aggregate O. carboxidovorans S23 cells, indicating that the coaggregation factor of A. johnsonii S35 is cell-bound. These results demonstrate that the coaggregation ability of A. johnsonii S35 is constitutive and is not lost under diverse cultivation conditions.

Effect of surfactants on stability of Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 coaggregates

The effect of anionic (sodium dodecyl sulphate or SDS) and cationic (cetyltrimethylammonium bromide or CTAB) surfactants on the stability of binary bacterial coaggregates comprising Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 (both sewage sludge isolates) was studied and compared with that on the complex sewage sludge flocs. Both SDS and CTAB enhanced the bacterial coaggregation at their lower concentrations of 0.2 and 0.07 mg ml −1, respectively. However, complete deflocculation of coaggregates was observed at 1 mg ml −1 SDS and 0.3 mg l −1 CTAB concentrations. Further, sewage sludge flocs did not deflocculate in the presence of CTAB, although a concentration-dependent deflocculation was observed in the presence of SDS. A. johnsonii S35 and O. carboxidovorans S23 cells were separately pretreated (prior to coaggregation) with the surfactants. In spite of the partial (complete) loss of viability during SDS (CTAB) pretreatment, washed cells still retained hydrophobic character and displayed significant coaggregation (aggregation index ranging from 84% to 97% in comparison to 96% in the case of non-treated cells), demonstrating reversibility of the surfactant induced deflocculation. Further, when exposed to lower concentration of surfactants (0.2 mg ml −1 SDS), coaggregates were more resistant (76% viability) as compared to the individual partner (S35: 52%; S23: 39% viability). Since the coaggregates are stable and provide protection from surfactants at lower concentrations (those normally expected in the sewage treatment plants), their presence as well as a sustained role in the sewage sludge bioflocculation is evident.

Complete nucleotide sequence of the circular megaplasmid pHCG3 of Oligotropha carboxidovorans: function in the chemolithoautotrophic utilization of CO, H 2 and CO 2

Oligotropha carboxidovorans harbors the low-copy-number, circular, 133,058-bp DNA megaplasmid pHCG3, which is essential in the chemolithoautotrophic utilization of CO (carboxidotrophy), H 2 (hydrogenotrophy) and CO 2 under aerobic conditions. The complete nucleotide sequence of pHCG3 revealed 125 open reading frames. Of these, 95 were identified as putative structural genes. The plasmid carries the four gene clusters cox (14.54 kb, 12 genes), cbb (13.33 kb, 13 genes), hox (23.35 kb, 19 genes plus one ORF) and tra/trb (25.01 kb, 22 genes plus 2 ORFs), which assemble the functions required for the utilization of CO, CO 2 or H 2, and the conjugal transfer of the plasmid, respectively. The gene clusters cox, cbb and hox form a 51.2-kb chemolithoautotrophy module. The tra/trb cluster on the plasmid pHCG3 of O. carboxidovorans has a similar architecture as the Ti-plasmid of Agrobacterium tumefaciens. The tra/trb cluster is separated from the chemolithoautotrophy module by two regions (25.2 and 29.6 kb) with miscellaneous or mostly unknown functions. These regions carry a number of single genes coding for replication and stabilization of pHCG3 as well as the components of a putative system of global regulation of plasmid replication in O. carboxidovorans. An oriV encodes the replication proteins RepABC. Sequence comparisons of pHCG3-encoded genes suggest that major genetic exchange between O. carboxidovorans and the proteobacteria has occurred.

Intergeneric coaggregations among Oligotropha carboxidovorans and Acinetobacter species present in activated sludge.

The coaggregation traits of two pairs of sewage sludge bacteria were tested and characterized. Oligotropha carboxidovorans S23 coaggregated with two strains of the genus Acinetobacter viz. Acinetobacter junii S33 (56%) and Acinetobacter johnsonii S35 (99%). Coaggregates of O. carboxidovorans S23 and A. junii S33 were small (20-40 microm), weak and susceptible to EDTA and a commercial protease (Actinase E). Actinase/periodate pretreatment of the partners prior to coaggregation revealed that interaction in this case was mediated by protein surface components. Coaggregates of O. carboxidovorans S23 and A. johnsonii S35 were large (above 100 microm), strong and not deflocculated by EDTA or Actinase E. Only periodate pretreatment of A. johnsonii S35 prevented this coaggregation indicating a role for a carbohydrate-containing moiety without the involvement of protein components. The potential mechanisms and strength of bacterial coaggregations seem to be pair dependent.

A novel binuclear [CuSMo] cluster at the active site of carbon monoxide dehydrogenase: characterization by X-ray absorption spectroscopy.

The structurally characterized molybdoenzyme carbon monoxide dehydrogenase (CODH) catalyzes the oxidation of CO to CO2 in the aerobic bacterium Oligotropha carboxidovorans. The active site of the enzyme was studied by Mo- and Cu-K-edge X-ray absorption spectroscopy. This revealed a bimetallic [Cu(I)SMo(VI)(double bond O)2] cluster in oxidized CODH which was converted into a [Cu(I)SMo(IV)(double bond O)OH2] cluster upon reduction. The Cu...Mo distance is 3.70 A in the oxidized form and is increased to 4.23 A upon reduction. The bacteria contain CODH species with the complete and functional bimetallic cluster along with enzyme species deficient in Cu and/or bridging S. The latter are precursors in the posttranslational biosynthesis of the metal cluster. Cu-deficient CODH is the most prominent precursor and contains a [HSMo(double bond O)OH2] cluster. Se-K-edge X-ray absorption spectroscopy demonstrates that Se is coordinated by two C atoms at 1.94-1.95 A distance. This is interpreted as a replacement of the S in methionine residues. In contrast to a previous report [Dobbek, H., Gremer, L., Meyer, O., and Huber, R. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8884-8889] Se was not identified in the active site of CODH.

Catalysis at a dinuclear [CuSMo(==O)OH] cluster in a CO dehydrogenase resolved at 1.1-A resolution.

The CO dehydrogenase of the eubacterium Oligotropha carboxidovorans is a 277-kDa Mo- and Cu-containing iron-sulfur flavoprotein. Here, the enzyme's active site in the oxidized or reduced state, after inactivation with potassium cyanide or with n-butylisocyanide bound to the active site, has been reinvestigated by multiple wavelength anomalous dispersion measurements at atomic resolution, electron spin resonance spectroscopy, and chemical analyses. We present evidence for a dinuclear heterometal [CuSMoO)OH] cluster in the active site of the oxidized or reduced enzyme, which is prone to cyanolysis. The cluster is coordinated through interactions of the Mo with the dithiolate pyran ring of molybdopterin cytosine dinucleotide and of the Cu with the Sgamma of Cys-388, which is part of the active-site loop VAYRC(388)SFR. The previously reported active-site structure [Dobbek, H., Gremer, L., Meyer, O. & Huber, R. (1999) Proc. Natl. Acad. Sci. USA 96, 8884-8889] of an Mo with three oxygen ligands and an SeH-group bound to the Sgamma atom of Cys-388 could not be confirmed. The structure of CO dehydrogenase with the inhibitor n-butylisocyanide bound has led to a model for the catalytic mechanism of CO oxidation which involves a thiocarbonate-like intermediate state. The dinuclear [CuSMo(O)OH] cluster of CO dehydrogenase establishes a previously uncharacterized class of dinuclear molybdoenzymes containing the pterin cofactor.

The effect of intracellular molybdenum in Hydrogenophaga pseudoflava on the crystallographic structure of the seleno-molybdo-iron-sulfur flavoenzyme carbon monoxide dehydrogenase

Crystal structures of carbon monoxide dehydrogenase (CODH), a seleno-molybdo-iron-sulfur flavoprotein from the aerobic carbon monoxide utilizing carboxidotrophic eubacterium Hydrogenophaga pseudoflava, have been determined from the enzyme synthesized at high (Mo plus CODH) and low intracellular molybdenum content (Mo minus CODH) at 2.25 Å and 2.35Å resolution, respectively. The structures were solved by Patterson search methods utilizing the enzyme from Oligotropha carboxidovorans as the initial model. The CODHs from both sources are structurally very much conserved and show the same overall fold, architecture and arrangements of the molybdopterin-cytosine dinucleotide-type of molybdenum cofactor, the type I and type II [2Fe-2S] clusters and the flavin-adenine dinucleotide. Unlike the CODH from O. carboxidovorans, the enzyme from H. pseudoflava reveals a unique post-translationally modified C γ-hydroxy-Arg384 residue which precedes the catalytically essential S-selanyl-Cys385 in the active-site loop. In addition, the Trp193 which shields the isoalloxazine ring of the flavin-adenine dinucleotide in the M subunit of the H. pseudoflava CODH is a Tyr193 in the O. carboxidovorans CODH. The hydrogen bonding interaction pattern of the molybdenum cofactor involves 27 hydrogen bonds with the surrounding protein. Of these, eight are with the cytosine moiety, eight with the pyrophosphate, six with the pyranopterin, and five with the ligands of the Mo ion. The structure of the catalytically inactive Mo minus CODH indicates that an intracellular Mo-deficiency affects exclusively the active site of the enzyme as an incomplete non-functional molybdenum cofactor was synthesized. The 5′-CDP residue was present in Mo minus CODH, whereas the Mo-pyranopterin moiety was absent. In Mo plus CODH the selenium faces the Mo ion and flips away from the Mo site in Mo minus CODH. The different side-chain conformations of the active-site residues S-selanyl-Cys385 and Glu757 in Mo plus and Mo minus CODH indicate a side-chain flexibility and a function of the Mo ion in the proper orientation of both residues.

The role of Se, Mo and Fe in the structure and function of carbon monoxide dehydrogenase.

CO dehydrogenase (EC 1.2.99.2) catalyzes the oxidation of CO according to the following equation: CO + H2O-->CO2 + 2 e- + 2 H+. It is a selenium-containing molybdo-iron-sulfur-flavoenzyme, which has been crystallized and structurally characterized in its oxidized state from the aerobic CO utilizing bacteria Oligotropha carboxidovorans and Hydrogenophaga pseudoflava. Both CO dehydrogenase structures show only minor differences, and the enzymes are dimers of two heterotrimers. Each heterotrimer is composed of a molybdoprotein, a flavoprotein, and an iron-sulfur protein. CO oxidation takes place at the molybdoprotein which contains a 1:1 mononuclear complex of molybdopterin-cytosine dinucleotide and a Mo-ion, along with a catalytically essential S-selanylcysteine. The latter is appropriately positioned in the SeMo-active site by a unique VAYRCSFR active site loop. In H. pseudoflava the arginine preceeding the cysteine in the active site loop is modified to a Cgamma-hydroxy arginine residue which has no obvious function. The substituents in the first coordination sphere of the Mo-ion are the enedithiolate sulfur atoms of the molybdopterin-cytosine dinucleotide, two oxo- and a sulfido-group. Extended X-ray absorption fine structure spectroscopy (EXAFS), along with the crystal structure of CO dehydrogenase (23.2 U mg(-1)) at 1.85 A resolution, have identified a sulfur atom at 2.3 A from the Mo-ion. The sulfur reacts with cyanide yielding thiocyanate. The corresponding inactive desulfo-CO dehydrogenase shows a typical desulfo inhibited-type of Mo-electron paramagnetic resonance (EPR) spectrum. Structural changes at the SeMo-site during catalysis are suggested by the Mo to Se distance of 3.7 A and the Mo-S-Se angle of 113 degrees in the oxidized enzyme which increase to 4.1 A, and 121 degrees, respectively, in the reduced enzyme. The intramolecular electron transport chain in CO dehydrogenase involves the following prosthetic groups and minimal distances: CO-->[Mo of the molybdenum cofactor] - 14.6 A - [2Fe-2S]I - 12.4 A - [2Fe-2S]II - 8.7 A - [FAD].

Binding of Flavin Adenine Dinucleotide to Molybdenum-containing Carbon Monoxide Dehydrogenase from Oligotropha carboxidovorans: STRUCTURAL AND FUNCTIONAL ANALYSIS OF A CARBON MONOXIDE DEHYDROGENASE SPECIES IN WHICH THE NATIVE FLAVOPROTEIN HAS BEEN REPLACED BY ITS RECOMBINANT COUNTERPART PRODUCED IN ESCHERICHIA COLI

The carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is composed of an S-selanylcysteine-containing 88. 7-kDa molybdoprotein (L), a 17.8-kDa iron-sulfur protein (S), and a 30.2-kDa flavoprotein (M) in a (LMS)(2) subunit structure. The flavoprotein could be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate. The resulting M(LS)(2)- or (LS)(2)-structured CO dehydrogenase species could be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli. The formation of the heterotrimeric complex composed of the apoflavoprotein, the molybdoprotein, and the iron-sulfur protein involves structural changes that translate into the conversion of the apoflavoprotein from non-FAD binding to FAD binding. Binding of FAD to the reconstituted deflavo (LMS)(2) species occurred with second-order kinetics (k(+1) = 1350 M(-1) s(-1)) and high affinity (K(d) = 1.0 x 10(-9) M). The structure of the resulting flavo (LMS)(2) species at a 2.8-A resolution established the same fold and binding of the flavoprotein as in wild-type CO dehydrogenase, whereas the S-selanylcysteine 388 in the active-site loop on the molybdoprotein was disordered. In addition, the structural changes related to heterotrimeric complex formation or FAD binding were transmitted to the iron-sulfur protein and could be monitored by EPR. The type II 2Fe:2S center was identified in the N-terminal domain and the type I center in the C-terminal domain of the iron-sulfur protein.

Sequence analysis, characterization and CO-specific transcription of the cox gene cluster on the megaplasmid pHCG3 of Oligotropha carboxidovorans.

Sequence, transcriptional, mutational and physiological analyses indicate that the carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is an integral and unique part of an elaborate CO oxidizing system. It is encoded by the 14.5kb gene cluster coxBCMSLDEFGHIK residing on the 128kb megaplasmid pHCG3. The CO dehydrogenase structural genes coxMSL are flanked by nine accessory genes arranged as the cox gene cluster. The cox genes are specifically and coordinately transcribed under chemolithoautotrophic conditions in the presence of CO as carbon and energy source. With the exception of CoxB and CoxK, all deduced products of the cox genes of O. carboxidovorans have counterparts in so far uncharacterized gene clusters of Pseudomonas thermocarboxydovorans, Hydrogenophaga pseudoflava, Bradyrhizobium japonicum, and Mycobacterium tuberculosis. Transposon mutagenesis suggests a function of CoxH and CoxI in the interaction of CO dehydrogenase with the cytoplasmic membrane. The specific functions of the other accessory Cox proteins are difficult to envisage right now, as the polypeptides do not show significant homologies with functionally characterized proteins in the databases. In addition to the clustered cox genes, mutational analyses have identified the genes lon, cycH and orfX which reside on the plasmid pHCG3. The Lon protease, the CycH protein and the unknown orfX gene product have essential functions in the utilization of CO.

Purification and molecular characterization of the H2 uptake membrane-bound NiFe-hydrogenase from the carboxidotrophic bacterium Oligotropha carboxidovorans.

The membrane-bound hydrogenase of Oligotropha carboxidovorans was solubilized with n-dodecyl-beta-D-maltoside and purified 28-fold with a yield of 29% and a specific activity of 173 to 178 micromol of H2 x min(-1) x mg(-1). It is the first hydrogenase studied in a carboxidotrophic bacterium. The enzyme acts on artificial electron-accepting dyes, such as methylene blue, but is ineffective with pyridine nucleotides or other soluble physiological electron acceptors. Hydrogenase of O. carboxidovorans belongs to class I of hydrogenases and is a heterodimeric 101,692-Da NiFe-protein composed of the polypeptides HoxL and HoxS. Molecular cloning data revealed, that HoxL comprises 604 amino acid residues and has a molecular mass of 67,163 Da. Pre-HoxS comprises 360 amino acid residues and is synthesized as a precursor protein which is cleaved after alanine at position 45, thus producing a mature HoxS of 33,767 Da. The leader sequence corresponds to the signal peptide of small subunits of hydrogenases. The hydropathy plots of HoxL and HoxS were indicative for the absence of transmembranous helices. HoxZ has four transmembranous helices and is considered the potential membrane anchor of hydrogenase in O. carboxidovorans. Hydrogenase genes show the transcriptional order 5' hoxV --> hoxS --> hoxL --> hoxZ 3'. The hox gene cluster as well as the clustered CO dehydrogenase (cox) and Calvin cycle (cbb) genes are arranged within a 30-kb DNA segment of the 128-kb megaplasmid pHCG3 of O. carboxidovorans.