Functional domains of theEscherichia coli ferric uptake regulator protein (Fur)

The functions of N- and C-terminal domains of the Fur repressor ofEscherichia coli in promoter recognition and dimerization were studied. We investigated the ability of fusion proteins containing the N- or C-terminal domain of Fur to dimerize and to repress a Fur-regulatedlacZ fusion gene. The N-terminal domain, when fused to the C-terminal domain of the repressor C1857, repressed a Fur-regulatedlacZ fusion. However, the Fur-CI857 fusion was unable to complement the growth defect of anE. coli fur mutant on fumarate and succinate. The C-terminal domain of Fur, when fused to the N-terminus of CI857, repressed a λP, -regulatedlacZ fusion, indicating dimerization of the chimeric protein, which is a prerequisite for Cl activity. Both fusion proteins were fully active under both iron-rich and iron-poor growth conditions. We conclude that the N-terminal domain of Fur is involved in recognition of the Fur-responsive promoter and the C-terminus mediates oligomerization of the repressor.     

Identification of a membrane-bound hydrogenase of Desulfovibrio vulgaris (Hildenborough)

Two hydrogenase activities from Desulfovibrio vulgaris (Hildenborough) could be distinguished immunologically and biochemically. The first activity, described as hydrogenase I, corresponded to the soluble enzyme located in the periplasmic space of D. vulgaris. Hydrogenase I had a high specific activity and was sensitive to inhibition by CO. The second activity, hydrogenase II, was located in the membrane fraction, had a lower specific activity and was not affected by CO. The enzymes exhibited different electrophoretic mobilities in polyacrylamide gels, and reacted differently when exposed to proteases. Antibodies raised against purified periplasmic hydrogenase of D. vulgaris reacted with hydrogenase I, but not with hydrogenase II.     

Extraction of periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough)

Abstract Periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) was extracted according to the method of van der Westen [8] and the effect of trace minerals on the extractability of this enzyme was investigated. The final growth yields in the presence or absence of trace minerals were the same; however, the growth was much faster and the amount of periplasmic hydrogenase extracted was significantly lower in the presence of trace minerals. Polyacrylamide gel electrophoresis showed the presence of 2 hydrogenases in D. vulgaris , one soluble and the other possibly membrane-bound.     

Kinetic properties of hydrogenase isolated from Desulfovibrio vulgaris (Hildenborough)

Hydrogenase of Desulfovibrio vulgaris shows nonlinear kinetics in hydrogen production with both the natural electron carrier, cytochrome c3, and the artificial donor, methyl viologen semiquinone. Increasing concentrations of salt progressively inhibit the hydrogen production, as do increasing amounts of dimethylsulfoxide (Me2SO). Hydrogen consumption activity does not change up to 30% (v/v) of Me2SO. Preincubation in Me2SO up to 55% (v/v) does not affect the hydrogen uptake or production. The production activity of the enzyme shows an optimum around pH 6. When plotted as a function of redox potential the activity can be fitted to a Nernst equation with n = 1. Midpoint potentials calculated at various values follow approximately the hydrogen electrode to pH 6. Thereafter, there is a shift of about 40 mV to higher redox potentials.     

Functional domains of theEscherichia coli ferric uptake regulator protein (Fur)

The functions of N- and C-terminal domains of the Fur repressor ofEscherichia coli in promoter recognition and dimerization were studied. We investigated the ability of fusion proteins containing the N- or C-terminal domain of Fur to dimerize and to repress a Fur-regulatedlacZ fusion gene. The N-terminal domain, when fused to the C-terminal domain of the repressor C1857, repressed a Fur-regulatedlacZ fusion. However, the Fur-CI857 fusion was unable to complement the growth defect of anE. coli fur mutant on fumarate and succinate. The C-terminal domain of Fur, when fused to the N-terminus of CI857, repressed a P, -regulatedlacZ fusion, indicating dimerization of the chimeric protein, which is a prerequisite for Cl activity. Both fusion proteins were fully active under both iron-rich and iron-poor growth conditions. We conclude that the N-terminal domain of Fur is involved in recognition of the Fur-responsive promoter and the C-terminus mediates oligomerization of the repressor.     

The adaptive genome of Desulfovibrio vulgaris Hildenborough

Peculiar attributes revealed by sequencing the genome of Desulfovibrio vulgaris Hildenborough are analyzed, particularly in relation to the presence of a phosphotransferase system (PTS). The PTS is a typical bacterial carbohydrate transport system functioning via group translocation. Novel avenues for investigations are proposed emphasizing the metabolic diversity of D. vulgaris Hildenborough, especially the likely utilization of mannose-type sugars. Comparative analysis with PTS from other Gram-negative and Gram-positive bacteria indicates regulatory functions for the PTS of D. vulgaris Hildenborough, including catabolite repression and inducer exclusion. Chemotaxis towards PTS substrates is considered. Evidence suggests that this organism may not be a strict anaerobic sulfate reducer typical of the ocean, but a versatile organism capable of bidirectional transmigration and adaptation to both water and terrestrial environments.     

Recognition of DNA by three ferric uptake regulator (Fur) homologs in Bacillus subtilis

Bacillus subtilis contains three Fur homologs: Fur, PerR, and Zur. Despite significant sequence similarities, they respond to different stimuli and regulate different sets of genes. DNA target site comparisons indicate that all three paralogs recognize operators with a core 7-1-7 inverted repeat. The corresponding consensus sequences are identical at five or more of the seven defined positions. Using site-directed mutagenesis, the Per box at the mrgA promoter was altered to mimic the core 7-1-7 motif of the Fur and Zur boxes. In vitro, the mrgA promoter containing a Zur box was only recognized by Zur, as demonstrated by DNase I footprinting assays. In contrast, both Fur and PerR bound to the mrgA promoter region containing a consensus Fur box. Expression analysis of these promoters is consistent with the in vitro data demonstrating as few as 1 or 2 base changes per half-site are sufficient to alter regulation. Similarly, the Fur box at the feuA promoter can be converted into a Per or a Zur box by appropriate mutations. While both Fur and PerR could recognize some of the same synthetic operator sequences, no naturally occurring sites are known that are subject to dual regulation. However, the PerR-regulated zosA gene is controlled from a regulatory region that contains both Per and Fur boxes. Although purified Fur protein bound to the candidate Fur boxes, Fur has little effect on zosA expression-possibly due to the location of the Fur boxes relative to the zosA promoter. Together, our results identify two nucleotide positions that are important for the ability of PerR, Fur, and Zur to distinguish among the many closely related operator sites present in the B. subtilis genome.     

Carbon monoxide cycling by Desulfovibrio vulgaris Hildenborough

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, use the reduction of sulfate as a sink for electrons liberated in oxidation reactions of organic substrates. The rate of the latter exceeds that of sulfate reduction at the onset of growth, causing a temporary accumulation of hydrogen and other fermentation products (the hydrogen or fermentation burst). In addition to hydrogen, D. vulgaris was found to produce significant amounts of carbon monoxide during the fermentation burst. With excess sulfate, the hyd mutant (lacking periplasmic Fe-only hydrogenase) and hmc mutant (lacking the membrane-bound, electron-transporting Hmc complex) strains produced increased amounts of hydrogen from lactate and formate compared to wild-type D. vulgaris during the fermentation burst. Both hydrogen and CO were produced from pyruvate, with the hyd mutant producing the largest transient amounts of CO. When grown with lactate and excess sulfate, the hyd mutant also exhibited a temporary pause in sulfate reduction at the start of stationary phase, resulting in production of 600 ppm of headspace hydrogen and 6,000 ppm of CO, which disappeared when sulfate reduction resumed. Cultures with an excess of the organic electron donor showed production of large amounts of hydrogen, but no CO, from lactate. Pyruvate fermentation was diverse, with the hmc mutant producing 75,000 ppm of hydrogen, the hyd mutant producing 4,000 ppm of CO, and the wild-type strain producing no significant amount of either as a fermentation end product. The wild type was most active in transient production of an organic acid intermediate, tentatively identified as fumarate, indicating increased formation of organic fermentation end products in the wild-type strain. These results suggest that alternative routes for pyruvate fermentation resulting in production of hydrogen or CO exist in D. vulgaris. The CO produced can be reoxidized through a CO dehydrogenase, the presence of which is indicated in the genome sequence.     

Coordination and redox properties of a novel triheme cytochrome from Desulfovibrio vulgaris (Hildenborough)

A high molecular weight multiheme c-type cytochrome from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) has been spectroscopically characterized and compared with the tetraheme cytochrome c3. The protein contains a pentacoordinate high-spin heme (gz 6.0) and two hexacoordinate low-spin hemes (gz 2.95, gy 2.27, gx 1.48). From analysis of the g values for the low-spin hemes by the procedure of Blumberg and Peisach (Palmer, 1983) and comparison with with the optical spectra from a variety of c-type cytochromes, it is likely that these low-spin hemes are bound by two histidine residues. The NO derivative displayed typical rhombic EPR features (gx 2.07, gz 2.02, gy 1.99). Addition of azide does not lead to coupling between heme chromophores, but the ligand is accessible to the high-spin heme. The use of a glassy-carbon electrode to perform direct (no promoter) electrochemistry on the cytochrome is illustrated. Differential pulse polarography of the native protein gave two waves with reduction potentials of -59 (5) and -400 (8) mV (versus NHE). The cyanide adduct gave two waves with reduction potentials of -263 (8) and -401 (8) mV. The cytochrome was found to catalyze the reduction of nitrite and hydroxylamine.     

Comparative studies of polyhemic cytochromes c isolated from Desulfovibrio vulgaris (Hildenborough) and Desulfovibrio desulfuricans (Norway)

Cytochrome c3 (Mr 26,000) has been characterized in Desulfovibrio vulgaris (Hildenborough) and its properties compared with polyhemic cytochromes c isolated from the same organism and from D. desulfuricans (Norway). It can be described as an octaheme cytochrome c3 constituted of two identical subunits. Absorption spectrum is similar to cytochrome c3 (Mr 13,000) and individual redox potentials have an average value of -180 mV.3 The N terminal sequence is compared with an homologous cytochrome isolated from D. desulfuricans Norway.     

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (K(A)) of 10(+/-7)x10(6), 5.7(+/-3)x10(6), 2.0(+/-2)x10(6) and 2.0(+/-3)x10(4) M(-1) for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(+/-2)x10(6), 3.2(+/-2)x10(4), 1.76(+/-1)x10(5) and 1.5(+/-2)x10(3) M(-1) respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 degrees C). The stability of metal ion binding to the sensory site follows the Irving-Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.     

Hybrid-cluster protein (HCP) from Desulfovibrio vulgaris (Hildenborough) at 1.6 A resolution

The three-dimensional structure of the hybrid cluster protein from Desulfovibrio vulgaris (Hildenborough) has been determined at 1.6 A resolution using synchrotron X-ray radiation. The protein can be divided into three domains: an N-terminal mainly alpha-helical domain and two similar domains comprising a central beta-sheet flanked by alpha-helices. The protein contains two 4Fe clusters with an edge-to-edge distance of 10.9 A. Four cysteine residues at the N-terminus of the protein are ligands to the iron atoms of a conventional [4Fe-4S] cubane cluster. The second cluster has an unusual asymmetric structure and has been named the hybrid cluster to reflect the variety of protein ligands, namely two mu-sulfido bridges, two mu(2)-oxo bridges, and a further disordered bridging ligand. Anomalous differences in data collected at 1.488 A and close to the iron edge at 1.743 A have been used to confirm the identity of the metal and sulfur atoms. The hybrid cluster is buried in the center of the protein, but is accessible through a large hydrophobic cavity that runs the length of domain 3. Hydrophobic channels have previously been identified as access routes to the active centers in redox enzymes with gaseous substrates. The hybrid cluster is also accessible by a hydrophilic channel. The [4Fe-4S] cubane cluster is close to an indentation on the surface of the protein and can also be approached on the opposite side by a long solvent channel. At the present time, neither the significance of these channels nor, indeed, the function of the hybrid cluster protein is known.     

Crystal structure of the Vibrio cholerae ferric uptake regulator (Fur) reveals insights into metal co-ordination

The ferric uptake regulator (Fur) is a metal-dependent DNA-binding protein that acts as both a repressor and an activator of numerous genes involved in maintaining iron homeostasis in bacteria. It has also been demonstrated in Vibrio cholerae that Fur plays an additional role in pathogenesis, opening up the potential of Fur as a drug target for cholera. Here we present the crystal structure of V. cholerae Fur that reveals a very different orientation of the DNA-binding domains compared with that observed in Pseudomonas aeruginosa Fur . Each monomer of the dimeric Fur protein contains two metal binding sites occupied by zinc in the crystal structure. In the P. aeruginosa study these were designated as the regulatory site (Zn1) and structural site (Zn2). This V. cholerae Fur study, together with studies on Fur homologues and paralogues, suggests that in fact the Zn2 site is the regulatory iron binding site and the Zn1 site plays an auxiliary role. There is no evidence of metal binding to the cysteines that are conserved in many Fur homologues, including Escherichia coli Fur. An analysis of the metal binding properties shows that V. cholerae Fur can be activated by a range of divalent metals.     

Nucleotide sequence of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough)

The nucleotide sequence of the 4.7-kb SalI/EcoRI insert of plasmid pHV 15 containing the hydrogenase gene from Desulfovibrio vulgaris (Hildenborough) has been determined with the dideoxy chain-termination method. The structural gene for hydrogenase encodes a protein product of molecular mass 45820 Da. The NH2-terminal sequence of the enzyme deduced from the nucleic acid sequence corresponds exactly to the amino acid sequence determined by Edman degradation. The nucleic acid sequence indicates that a N-formylmethionine residue precedes the NH2-terminal amino acid Ser-1. There is no evidence for a leader sequence. The NH2-terminal part of the hydrogenase shows homology to the bacterial [8Fe-8S] ferredoxins. The sequence Cys-Ile-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro-Xaa-Xaa-Ala-(Ile) occurs twice both in the hydrogenase and in [8Fe-8S] ferredoxins, where the Cys residues have been shown to coordinate two [4Fe-4S] clusters [Adman, E. T., Sieker, L. C. and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996]. These results, therefore, suggest that two electron-transferring ferredoxin-like [4Fe-4S] clusters are located in the NH2-terminal segment of the hydrogenase molecule. There are ten more Cys residues but it is not clear which four of these could participate in the formation of the third cluster, which is thought to be the hydrogen binding centre. Another gene, encoding a protein of molecular mass 13493 Da, was found immediately downstream from the gene for the 46-kDa hydrogenase. The nucleic acid sequence suggests that the hydrogenase and the 13.5-kDa protein belong to a single operon and are coordinately expressed. Since dodecylsulfate gel electrophoresis of purified hydrogenase indicates the presence of a 13.5-kDa polypeptide in addition to the 46-kDa component, it is proposed that the hydrogenase from D. vulgaris (Hildenborough) is a two-subunit enzyme.     

The presence of multiple intrinsic membrane nickel-containing hydrogenases in Desulfovibrio vulgaris (Hildenborough)

Three intrinsic membrane proteins exhibiting oxygen stable hydrogenase activity have been isolated from D. vulgaris. In contrast to the periplasmic exclusively non-heme iron hydrogenase, all three hydrogenases contain Ni in addition to non-heme iron, have low specific activities and are insensitive to inhibition by CO. None of the three hydrogenases cross react with IgA against the periplasmic hydrogenase of D. vulgaris but two of the new hydrogenases cross react with IgA against the periplasmic nickel containing hydrogenase of D. gigas and the other new hydrogenase cross reacts with IgA against the periplasmic nickel and selenium hydrogenase of D. desulfuricans (Norway -4).     

Induction and partial purification of bacteriophages from Desulfovibrio vulgaris (Hildenborough) and Desulfovibrio desulfuricans ATCC 13541.

Bacteriophages were induced from cultures of Desulfovibrio vulgaris NCIMB 8303 and Desulfovibrio desulfuricans ATCC 13541 by UV light. The optimum time of UV exposure was 1 min and the maximum yield of phage was obtained 9-10 h after UV treatment. The two phage preparations were compared by restriction enzyme analysis and Southern blot hybridization. The nucleic acid from both phages was cut by restriction endonucleases specific for double-stranded DNA. The phage DNAs from D. vulgaris and D. desulfuricans showed different restriction enzyme cleavage patterns. No homology was observed between a 25 kb probe from the D. vulgaris phage DNA and the phage DNA from D. desulfuricans. Protein profiles of the phages from both sources were also studied; the D. vulgaris phage contained two major bands corresponding to Mr values of 37 000 and 56 000 while the D. desulfuricans phage contained only one major band, of Mr 38 000.     

Purification and characterization of Desulfovibrio vulgaris (Hildenborough) hydrogenase expressed in Escherichia coli

Hydrogenase from Desulfovibrio vulgaris (Hildenborough) is a heterologous dimer of molecular mass 46 + 13.5 kDa. Its two structural genes have been cloned on a 4664-base-pair fragment of known sequence in the vector pUC9. Expression of hydrogenase polypeptides in Escherichia coli transformed with this plasmid is poor (approximately 0.1% w/w of total protein). Deletion of up to 1.9 kb of insert DNA brings the gene encoding for the large subunit in close proximity to the lac promotor of pUC9 and results in a 50-fold increased expression of hydrogenase polypeptides in E. coli. The protein formed is inactive and was purified in order to delineate its defect. Complete purification was achieved with a procedure similar to that used for the isolation of active hydrogenase from D. vulgaris H. The derived protein is also an alpha beta dimer and electron-paramagnetic resonance studies indicate the presence of the electron-transferring ferredoxin-type iron-sulfur clusters. In contrast to the native protein from D. vulgaris H, these can only be reduced with dithionite, not with hydrogen, indicating that the hydrogen-binding active centre which also contains an iron-sulfur cluster is missing.