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1 Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg Universität Mainz, 55 099 Mainz, Germany
2 Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
3 MPI für Marine Mikrobiologie, Celsiusstr. 1, 28359 Bremen, Germany
Correspondence
Gottfried Unden
unden{at}uni-mainz.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, reduced benzyl viologen; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCPIP, dichlorophenol indophenol; DMN, 2,3-dimethyl-1,4-naphthoquinone; TRAP, tripartite ATP-independent periplasmicThe GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is DQ643793.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kröger et al., 2002; Cecchini et al., 2002). Bacillus subtilis, which contains menaquinone as the only respiratory quinone, has been shown to contain a specific type of succinate dehydrogenase (Schirawski & Unden, 1998; Schnorpfeil et al., 2001). In contrast to the classical succinate dehydrogenase consisting of four subunits (SdhABCD), the enzyme from B. subtilis is composed of three subunits (SdhABC). Catalytic subunit A and the Fe–S cluster subunit B are exposed to the cytoplasm. Subunit C serves as the membrane anchor and contains the active site for menaquinone reduction. SdhC of the B. subtilis enzyme carries two haem B molecules in a transmembrane arrangement: one of the haem B molecules is close to the outer, the other to the inner aspect of the membrane (Hägerhäll & Hederstedt 1996; Hägerhäll et al., 1995; Körtner et al., 1990; Lancaster et al., 1999; Simon et al., 1998). The haem B molecules are bound by four conserved His residues.
Succinate oxidation by menaquinone is endergonic, since succinate (E0=+30 mV) is more electropositive than the acceptor menaquinone (E0=–80 mV). The reaction requires a transmembrane H+-potential (
p) for function (Schirawski & Unden, 1998). Succinate oxidation (succinate
fumarate+2H++2e–) takes place in the cytoplasm, whereas menaquinone reduction (MK+2H++2e–MKH2) is believed to occur close to the outer aspect of the membrane (Schirawski & Unden, 1998). An active site for menaquinone close to the outer aspect is supported by the presence of a conserved Glu residue which is homologous to Glu66 in subunit FrdC of Wolinella succinogenes fumarate reductase. This residue is at the active site for menaquinol oxidation (Lancaster et al., 2000). Location of the oxidative and reductive half-reactions on opposite sides of the membrane requires a transmembrane electron transfer, which is effected by the two haem B molecules in subunit C. The overall reaction according to this scheme consumes and releases protons on opposite sides of the membrane, and the enzyme functions by a reverse (p-driven) redox loop mechanism (Schirawski & Unden, 1998). Sdh of this type is active only in intact (energized) cells, and is inactivated by addition of an uncoupler, or in membrane preparations (Lemma et al., 1990; Schirawski & Unden 1998; Schnorpfeil et al., 2001). The reaction of ubiquinone (E0=+110 mV)-dependent succinate dehydrogenase, on the other hand, is exergonic. Bacterial and mitochondrial enzymes of this type retain activity with the quinones in the presence of uncouplers and in membrane preparations, and contain only one haem (with two conserved His residues) close to the inner aspect of the membrane. In addition, the membrane anchor consists of two subunits (subunits CD) which are smaller in size (for an overview see Hägerhäll, 1997; Lancaster, 2002b).
Sulphate-reducing bacteria contain only menaquinone as the respiratory quinone (Collins & Widdel, 1986). The anaerobic Gram-negative bacteria are physiologically and phylogenetically largely distant from the (facultatively) aerobic Gram-positive genus Bacillus, raising the question whether succinate dehydrogenase is of the same type as in the genus Bacillus. Genomic sequences of several sulphate- and sulphur-reducing bacteria are available, and the genomes contain gene clusters similar to the sdhCAB genes of B. subtilis. The gene cluster is similar to the frdCAB genes encoding fumarate reductase in W. succinogenes (see Kröger et al., 2002, Lancaster & Simon, 2002 for a review). Fumarate reductase from W. succinogenes is a three-subunit enzyme (FrdABC) and catalyses fumarate reduction by menaquinol (Unden et al., 1980; Kröger et al., 2002; Biel et al., 2002). FrdC of W. succinogenes carries two haem B molecules with a transmembrane arrangement similar to SdhC of B. subtilis (Körtner et al., 1990; Simon et al., 1998). The SdhABC and FrdABC enzymes are similar in sequence and identical in the prosthetic groups.
To identify the role of the enzymes encoded by the sdh/frd genes, their function was determined for selected strains. Desulfovibrio desulfuricans (strain Essex 6) used the enzyme for fumarate respiration and a new form of fumarate disproportionation. In Desulfovibrio vulgaris on the other hand, the enzyme functions as succinate dehydrogenase. Succinate dehydrogenase of D. vulgaris had characteristic properties of the B. subtilis succinate dehydrogenase and required the proton potential for function, which appears to be a general property of menaquinone-dependent succinate dehydrogenases.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) in Sovirell tubes under an atmosphere of N2 at 37 °C. Modified medium 63 consists of 0.5 g K2HPO4 l–1, 1.0 g NH4Cl l–1, 0.1 g CaCl2.2H2O l–1, 1.0 g yeast extract l–1 (Serva no. 24540), 1.0 mg resazurin l–1, 0.6 g trisodium citrate l–1, 50 mg EDTA l–1, 2.5 mg FeSO4.7H2O l–1, 0.1 g sodium thioglycolate l–1 and 0.1 g ascorbic acid l–1 at pH 7.8. Sterile medium (400 ml) in infusion bottles (500 ml) was made anoxic by repeated degassing under vacuum and gassing with N2 before inoculation. For growth on sulphate, the medium contained in addition sulphate (25 mM disodium sulphate, 25 mM magnesium sulphate) plus lactate (50 mM sodium DL-lactate) or succinate (50 mM disodium succinate) as indicated in the individual experiments. For growth on fumarate or formate+fumarate (50 mM disodium fumarate without or with 50 mM sodium formate), the disodium/magnesium sulphate was replaced by 17 mM NaCl and 21 mM MgCl2. The media (400 ml) were inoculated with 7 ml of a subculture grown with lactate plus sulphate for 24 h, and incubated for about 24 h.
Cell suspensions and cell-free extract for measurement of enzyme activities.
The bacteria were sedimented from the growth media by centrifugation for 30 min at 10 000 g, and washed three times with anoxic potassium phosphate (50 mM at pH 7.8). To prepare the cell suspension, the bacteria were suspended in the same buffer at 400 mg wet weight of bacteria per l. For preparing the cell-free extract, the sedimented and washed bacteria (100 mg wet weight) were mixed with 180 µl of the same buffer and 0.5 g glass beads (0.13 mm in diameter, Zirkonia-silica, Roth). The suspension was swirled rapidly on a mixer for 30 s followed by incubation on ice for 5 min. The procedure was repeated six times; the debris and glass beads were sedimented by centrifugation. The glass beads were extracted once with the anoxic buffer, centrifuged, and the supernatants were combined.
Enzyme activities.
Fumarate reductase was measured with reduced benzylviologen (BV) as the electron donor (Lemma et al., 1990; Schirawski & Unden, 1998; Schnorpfeil et al., 2001). Measurement of succinate dehydrogenase activity with the dye dichlorophenol indophenol (DCPIP) or the menaquinone analogue 2,3-dimethyl-1,4-naphthoquinone (DMN) was as described previously (Schirawski & Unden, 1998; Schnorpfeil et al., 2001). All enzyme tests were performed at 37 °C under anoxic conditions in cuvettes sealed with rubber stoppers, and 1 unit corresponds to the consumption of 1 µmol fumarate or succinate min–1 (g protein)–1. Protein concentrations of cell suspensions were determined by the biuret method with KCN (Bode et al., 1968), and those of cell-free extracts by the Bradford assay (Bradford, 1976). The uncoupler CCCP (carbonyl cyanide m-chlorophenylhydrazone, 10 µM final concentration, stock solution 1 mM in ethanol) was added as indicated.
Fumarate disproportionation in cell suspensions and growing bacteria.
D. vulgaris and D. desulfuricans were grown in modified medium 63 with lactate+sulphate and harvested in the late exponential growth phase. Cells were sedimented by centrifugation, washed twice and resuspended at OD578 8–10 in anoxic MOPS buffer (100 mM, pH 7.8). Cells were transferred into rubber-stoppered bottles and degassed and gassed by N2 for three cycles. Growth was started by the addition of anoxic fumarate solution (5 mM final concn). Samples were withdrawn by syringes as required and centrifuged at 10 000 g for 5 min. The supernatants were used for HPLC analysis of the substrates and of the fermentation products (Richter et al., 2001).
For measurement of the products of fumarate disproportionation by growing bacteria, samples were withdrawn from the anaerobic cultures in the serum bottles. After removal of bacteria and particles by centrifugation, the medium or buffer was used for quantitative measurement of the substrates and products by HPLC using a Aminex HPX 87H column (Richter et al., 2001).
Amplification of the sdhC fragment of D. desulfuricans (strain Essex 6).
For amplification of sdhC, genomic DNA of D. desulfuricans (Essex 6) was amplified with primers CGCAAGATGCCCTTCCGCA and TCG GAN CCT TTG ACS GTR TC (N=A/C/G/T, S=G/C; R=A/G). The primers are derived from the sequences of SdhC of D. desulfuricans G20 and D. vulgaris encoding the predicted loop between transmembrane helices 2 and 3, and from a conserved sequence in SdhA of D. desulfuricans G20, D. vulgaris and Desulfobacterium autotrophicum. PCR products of the expected size (645 bp) were amplified in a second step using the same primers and sequenced after purification.
Phylogenetic analysis of protein sequences.
Sequences of TRAP genes were obtained from the ERGO (Overbeek et al., 2003), NCBI and UniProt databases. The contigs from the Rhodobacter capsulatus genome sequence were analysed using the Artemis package (Berriman & Rutherford, 2003). For phylogenetic analyses, sequences were aligned in CLUSTAL X (Thompson et al., 1997) and the output file used in PHYLIP (Felsenstein, 1989) to produce a bootstrapped distance matrix tree, which was viewed in TREEVIEW (Page, 1996).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
The proteins encoded by the sdhA/frdA and sdhB/frdB homologues showed 27 to 66 % sequence identity for subunit A, and 22 to 61 % identity for subunit B to the corresponding subunits of the B. subtilis and W. succinogenes enzymes. The FAD-binding sites in subunit A and the cysteine clusters in subunit B which bind the Fe–S clusters were conserved. The sequences allowed no prediction whether the enzyme from the sulphate-reducing bacteria represents Frd or Sdh. The SdhC/FrdC-subunits show a lower degree of sequence identity to FrdC of W. succinogenes (15.4 to 44.9 % identical residues), and to SdhC of B. subtilis (18.3 to 24.8 % identical residues) (Fig. 1). By the TMHMM program (www.cbs.dtu.dk/services/TMHMM/) five transmembrane helices are predicted for the C subunits, similar to the corresponding proteins from B. subtilis and W. succinogenes (Hägerhäll et al., 1995; Gross et al., 1998, Lancaster, 2002b; Lancaster & Simon, 2002; Hägerhäll, 1997). The four His ligands for the haem B molecules in Sdh/FrdC of B. subtilis and W. succinogenes are conserved in subunits C of the sulphate-reducing bacteria, suggesting a similar structure and topology. Some of the subunits contain also a Glu residue which is homologous to Glu180 of the E-pathway in the FrdC subunit of W. succinogenes (Lancaster, 2002a). D. desulfuricans strain G20, for which the genomic sequence is available, is not closely related to strain Essex 6, which was used for the physiological and biochemical experiments (Pires et al., 2003). Therefore the sequence of part of FrdC from D. desulfuricans (Essex 6) was determined (Fig. 1). The sequence comprised the fragment with the third and fourth of the His residues which represent one of the ligands to each of the two haem B groups (Hägerhäll & Hederstedt, 1996; Simon et al., 1998). It can be concluded that both haem groups are present in FrdC of D. desulfuricans (strain Essex). The Glu residue homologous to Glu180 from the E-pathway, however, was not conserved. Glu66 of W. succinogenes, which is at the menaquinone/menaquinol site close to the outer aspect of the membrane (Lancaster et al., 2000), is found in all SdhC/FrdC sequences of the sulphate-reducing bacteria. The gene and sequence properties therefore suggest that the sulphate and sulphur reducers contain Sdh or Frd enzymes of the SdhABC or FrdABC type.
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, Table 1). D. desulfuricans (Essex 6) was able to grow on fumarate when H2 or formate were supplied as electron donors at rates comparable to growth on sulphate. Succinate on the other hand stimulated growth on sulphate only marginally. D. vulgaris showed the best growth on lactate plus sulphate, whereas growth on fumarate was very low. The growth experiments therefore suggest that the enzyme of D. desulfuricans (Essex 6) functions as fumarate reductase, and that of D. vulgaris as succinate dehydrogenase.
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), corresponding to approx. 0.5 ATP/fumarate, which is comparable to the growth yield by fumarate respiration of Escherichia coli and W. succinogenes (Kröger et al., 2002; Mell et al., 1982; Bernhard & Gottschalk, 1978).
The major product of fumarate respiration was succinate, but in addition acetate (and CO2) were produced (Table 2). The (idealized) growth reactions (a, b) suggest that D. desulfuricans (Essex 6) uses about 70 to 80 % of the fumarate for fumarate reduction when formate or H2 is present. Acetate production indicates oxidation of some fumarate.
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). Growth was slower than by fumarate respiration (Table 1), but for D. desulfuricans the final cell densities were comparable. The type of products changed depending on the growth phase (Fig. 3, Table 2). In the early growth phase of D. vulgaris most of the fumarate was converted to malate and small amounts of acetate. In the second phase, the production of acetate and succinate increased at the expense of malate. Overall, about two-thirds of the fumarate was excreted as succinate, the residue as acetate when malate is not considered (Table 2). Product formation from fumarate by D. desulfuricans was also biphasic, but the differences between the first phase with and the second phase without malate production were less pronounced. Malate production was much lower than for D. vulgaris (Fig. 3a). In the stationary phase about two-thirds of the fumarate was converted to succinate and one third to acetate (Table 2).
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). In the early phase, D. vulgaris excreted mainly malate. In the second phase succinate was the major product and acetate the only further product. D. desulfuricans converted fumarate at much higher rates than D. vulgaris. Succinate and acetate were the main products, with small amounts of malate. In the late phase only succinate and acetate were produced, in an approximate ratio of 2 : 1. Malate seems to be an intermediate of fumarate fermentation and accumulates in the early phase due to limitation in subsequent metabolic steps. The limitation was significant in D. vulgaris and in cell suspensions.
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p for function). The activities of D. vulgaris were low and showed only a small variation (factor <3) after growth by fumarate or sulphate respiration, or in the presence of succinate. Fumarate reductase activities of D. desulfuricans were two orders of magnitude higher than those of D. vulgaris, and also showed no strong variation in response to the growth substrates. The ratio of fumarate reductase vs succinate dehydrogenase activities was significantly higher for D. desulfuricans than for D. vulgaris (mean value 25 versus 4), suggesting that the enzyme of D. desulfuricans resembles a fumarate reductase, and that of D. vulgaris a succinate dehydrogenase.
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, Table 5) after growth on sulphate or fumarate. DMN is very similar to menaquinone in structure and midpoint potential (
) and can be used as a water-soluble analogue of menaquinone. The activity with DMN was lost after addition of the uncoupler CCCP to the bacterial cells (Fig. 4, Table 5), or breaking the cells and preparing cell homogenates (Table 5), presumably due to dissipation of the proton potential, which was shown earlier as the driving force for DMN (or menaquinone) : succinate oxidoreductase in B. subtilis and other Gram-positive bacteria (Schirawski & Unden, 1998; Schnorpfeil et al., 2001). In contrast, high activity of succinate dehydrogenase with the artificial electron acceptor DCPIP was retained in the cell homogenate [75 U (g protein) –1]. It is concluded that this activity is, in contrast to succinate : DMN reductase, not sensitive to breaking the cell membrane.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; He et al., 1986; Lemos et al., 2002), for which, however, no genome, gene or protein sequences are available. Fumarate reductase of the sulphate- and sulphur-reducing bacteria therefore is of the same type (FrdABC with two haem B groups in subunit C) as in other 
-proteobacteria (Lancaster & Simon, 2002).
Fumarate disproportionation by the bacteria allows balanced growth by oxidation of 1 mol fumarate to acetate and reduction of 2 mol fumarate to succinate (reaction (c)) (Fig. 5). The [H] for succinate production is derived from pyruvate oxidation (Fdred, H2 or formate).
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) (www.jgi.doe.gov; www.tigr.org/), and presumably also in D. desulfuricans (strain Essex 6) due to the similar fermentation pattern. Malic enzyme, fumarase and acetate kinase were demonstrated also by their enzymic activities (Lewis & Miller, 1977; Brown & Akagi, 1966). Malate, which was suggested earlier as a product of fumarate disproportionation (Miller & Wakerley, 1966), apparently is only an intermediate. A similar form of fumarate disproportionation with succinate, acetate and CO2 as the products was described for Clostridium formicoaceticum (Dorn et al., 1978), whereas fumarate disproportionation by Proteus rettgeri produced only succinate but no acetate (Kröger, 1974).
Succinate : menaquinone reductase of D. vulgaris: a reversed redox loop mechanism
The succinate : quinone oxidoreductase of D. vulgaris is a succinate dehydrogenase due to the lack of fumarate respiration and the high ratio of Sdh/Frd activity. D. vulgaris is not capable of complete oxidation of organic matter (Postgate, 1984) and does not encode all enzymes of the citric acid cycle and of related pathways (www.jgi.doe.gov; http://www.genome.jp/kegg/; www.tigr.org/; http://www.microbesonline.org/). Therefore succinate dehydrogenase activity might be important for specific metabolic reactions, but not for degradation of citric acid cycle intermediates, which would explain their poor growth on succinate. The sensitivity of succinate : DMN (but not succinate : DCPIP) reductase activity of D. vulgaris to dissipation of the proton potential is reminiscent of succinate dehydrogenase of B. subtilis and other aerobic Gram-positive bacteria (Schirawski & Unden, 1998). Therefore the need for a proton potential is common to menaquinone-dependent succinate dehydrogenases from aerobic Gram-positive and anaerobic Gram-negative bacteria. The same type of succinate dehydrogenase (SdhABC), the His ligands for transmembrane arrangement of two haem B groups and the conserved Glu residue of the external menaquinone site suggest that these phylogentically and physiologically different bacteria use the same principle of a reverse redox loop mechanism to drive the reduction of menaquinone by succinate. Enzymes with an established structure constituting a redox loop are represented by formate dehydrogenase and nitrate reductase from E. coli (Jormakka et al., 2002, 2003). Both enzymes generate p by the release of H+ at the periplasmic side of the membrane during oxidation of one substrate and consumption of H+ in the cytoplasm during reduction of the second substrate.
General presence of the ‘E-pathway’ in fumarate reductases of the FrdABC type?
Fumarate reductase from W. succinogenes shows a transmembrane arrangement of the two haem B groups in FrdC (Körtner et al., 1990; Gross et al., 1998; Lancaster et al., 1999). The enzyme, however, does not generate a proton potential by menaquinol : fumarate reduction (Biel et al., 2002; Kröger et al., 2002). Glu180 in transmembrane helix IV of W. succinogenes FrdC is part of a H+ shortcut (‘E-pathway’) which dissipates the proton potential generated by fumarate reductase (Lancaster, 2002a; Lancaster et al., 2005; Haas et al., 2005). Glu180 is conserved in the enzyme of D. desulfuricans G20 (and of D. autotrophicum), which is in agreement with the function of the enzyme as fumarate reductases. The enzyme from D. vulgaris (and similarly those from B. subtilis, G. sulfurreducens and Desulfotalea psychrophila) lacks the ‘uncoupling’ Glu180 residue, which is in accordance with their role as a ‘coupling’ succinate dehydrogenase.
D. desulfuricans (strain Essex 6), which was used for the biochemical and physiological experiments, is not closely related to strain G20 (Pires, et al. 2003), which is reflected by the different sequences of the FrdC subunits. FrdC of strain Essex 6 lacks the Glu180 homologue, which is in agreement with the p-dependence of succinate : DMN oxidoreductase activity as determined experimentally. Further experiments will have to show how these properties are compatible with the physiological function of the enzyme of strain Essex 6 in fumarate respiration.
Lack of DcuB-type fumarate/succinate antiporter in D. desulfuricans
The genome of D. desulfuricans G20 contains no genes for anaerobic fumarate/succinate antiporters DcuB, DcuA or DcuC (Engel et al., 1994; Six et al., 1994; Zientz et al., 1999; Janausch et al., 2002; Golby et al., 1998; Unden & Kleefeld, 2004). DcuB catalyses electroneutral fumarate/succinate antiport. DcuB is typical for bacteria growing by fumarate respiration and can be replaced by the homologues DcuA and DcuC.
D. desulfuricans contains genes for five putative periplasmic solute-binding proteins, four of which are clustered with homologues of the dctQ and/or dctM genes encoding membrane-bound components of TRAP carriers (tripartite ATP-independent periplasmic carriers). In D. vulgaris, genes for three solute-binding proteins and homologues of dctQ and dctM genes are found. TRAP transporters represent secondary carriers which are driven by a H+ gradient and use a periplasmic binding protein to recruit the solute (Kelly & Thomas, 2001; Forward et al., 1997). Using CLUSTAL X-aligned sequences and PHYLIP analysis the solute-binding proteins of TRAP transporters have been used for constructing a phylogenetic tree, which showed clustering of C4-dicarboxylate-binding proteins (Thomas et al., 2006). By the same method the DctP homologues of D. desulfuricans and D. vulgaris were used to construct an unrooted bootstrapped tree with sequences from those TRAP binding proteins, where biochemical evidence for the identity of the ligand bound is available (Fig. 6). The proteins of D. vulgaris and D. desulfuricans cluster into six distinct groups. The clustering is significant since the known C4-dicarboxylate-binding proteins from phylogenetically distinct bacteria cluster together. The D. vulgaris and D. desulfuricans proteins DVU2822 and Dde0127, respectively, are closely related to known C4-dicarboxylate-binding proteins, and are good candidates for C4-dicarboxylate-binding proteins. The other TRAP solute-binding proteins of D. desulfuricans and D. vulgaris cluster with binding proteins for 2,3-diketogulonate, 2-oxoacids, ectoine or sialate (Fig. 6) (see Kelly & Thomas, 2001; Severi et al., 2005; Thomas et al., 2006). W. succinogenes encodes a DctPQM TRAP carrier in addition to DcuB and DcuA. DctPQM catalysed fumarate uptake, but did not support growth by fumarate respiration (Ullmann et al., 2000). The TRAP carriers could be involved in C4-dicarboxylate uptake during aerobic growth, but in fumarate respiration the function of alternative secondary carriers is more likely. The genes Dde0326, 0693, 0808, 1204 and 1252 of D. desulfuricans encode secondary carriers similar to the di-/tricarboxylate carrier CitT or the Na+/H+ dicarboxylate symporter GltP (Pos et al., 1998; http://www.jgi.doe.gov/). Carriers of the CitT family can be used for C4-dicarboxylate antiport (O. B. Kim & G. Unden, unpublished). The glutamate carrier GltP (Tolner et al., 1995) is homologous to C4-dicarboxylate carriers and the homologues in the sulphate-reducing bacteria represent candidates for C4-dicarboxylate transport. The genome of D. vulgaris encodes carriers with similarity to malonate carriers (predicted protein DVU1401) or an oxalate/formate antiporter (DVU1163) (http://www.membranetransport.org). Thus both Desulfovibrio strains contain candidate genes different from the dcu genes which could function in C4-dicarboxylate transport.
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ACKNOWLEDGEMENTS |
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Received 16 January 2006;
revised 13 April 2006;
accepted 25 April 2006.
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), succinate+sulphate (
), lactate+sulphate (
) and sulphate (
) as the substrates for growth.
) and the end products [succinate (
) and acetate (
