The essential role of fumarate reductase in haem-dependent growth stimulation of Bacteroides fragilis

Anthony D. Baughn and Michael H. Malamy

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA

Correspondence
Michael H. Malamy
michael.malamy{at}tufts.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Haem is required for optimal growth of the bacterial anaerobe Bacteroides fragilis. Previous studies have shown that growth in the presence of haem is coincident with increased yields of ATP from glucose, expression of b-type cytochromes and expression of fumarate reductase activity. This paper describes the identification of the genes that encode the cytochrome, iron–sulfur cluster protein and flavoprotein of the B. fragilis fumarate reductase. These genes, frdC, frdA and frdB, respectively, are organized in an operon. Nonpolar, in-frame deletions of frdC and frdB were constructed in the B. fragilis chromosome. These mutant strains had no detectable fumarate reductase or succinate dehydrogenase activity. In addition, the frd mutant strains showed a threefold increase in generation time, relative to the wild-type strain. Growth of these mutant strains was fully restored to the wild-type rate by the introduction of a B. fragilis replicon containing the entire frd operon. Growth of the frd mutant strains was partially restored by supplementing the growth medium with succinate, indicating that the frd gene products function as a fumarate reductase. During growth on glucose, the frd mutant strains showed a threefold decrease in cell mass yield, relative to the wild-type strain. These data indicate that fumarate reductase is important for both energy metabolism and succinate biosynthesis in B. fragilis.


Abbreviations: FRD, fumarate reductase; SDH, succinate dehydrogenase; SQR, succinate : quinone oxidoreductase

The GenBank accession number for the sequence reported in this paper is AY174185.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The obligately anaerobic, opportunistic pathogen Bacteroides fragilis requires exogenous haem for optimal growth on glucose as a source of carbon and energy (Baughn & Malamy, 2002; Macy et al., 1975; Sperry et al., 1977). Molar growth yield studies indicate that B. fragilis grown in the presence of haem can generate more than twofold more ATP per mole of glucose than that which is generated in the absence of haem (Macy et al., 1975). In addition, the doubling time of this bacterium is reduced by three- to fourfold in the presence of haem, suggesting that haem may also affect the rate of ATP biosynthesis during exponential growth (Baughn & Malamy, 2002; Macy et al., 1975). Studies of B. fragilis central metabolism indicate that haem is essential for the reduction of fumarate to succinate, as well as for the synthesis of b-type cytochromes (Fuller & Caldwell, 1982; Macy et al., 1975). In B. fragilis cell extracts, either NADH or H2 can serve as the electron donor for fumarate reduction (Harris & Reddy, 1977; Macy et al., 1975). Taken together, these observations have led to a model in which fumarate reduction couples the oxidation of NADH or H2 to the generation of ATP.

In the colon, Bacteroides spp. outnumber some eubacteria, such as Escherichia coli, by more than 1000-fold. During anaerobiosis, E. coli produces 2·5 moles of ATP per mole of glucose, whereas B. fragilis produces 4·5 moles of ATP per mole of glucose (Macy et al., 1975). It is predicted that this unique feature of anaerobic energy metabolism affords Bacteroides spp. a fitness advantage over other bacteria, such as E. coli, for growth in the colon.

B. fragilis can generate 2-oxoglutarate by the reductive carboxylation of succinyl-CoA (Allison et al., 1979). Since B. fragilis requires haem for the synthesis of succinate, the precursor of succinyl-CoA, biosynthesis of 2-oxoglutarate by this pathway is predicted to be haem-dependent. In the absence of haem, B. fragilis can generate 2-oxoglutarate by the oxidative decarboxylation of isocitrate (Baughn & Malamy, 2002). Though the oxidative pathway is essential for growth in the absence of haem, it is dispensable for growth in the presence of haem or succinate. These observations indicate that haem is also important for the generation of 2-oxoglutarate.

In this report we define an operon containing the three genes for the fumarate reductase (FRD) of B. fragilis, including the haemoprotein FrdC. We confirm a role for this enzyme in high yields of ATP during growth on glucose. Thus the haem-stimulatory effect is a direct result of the requirement for FRD.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, plasmids and growth conditions.
Bacterial strains used in this study are described in Table 1. B. fragilis strains were grown in an anaerobic chamber (Coy Laboratory Products) at 37 °C with an atmosphere of 5 % CO2, 10 % H2 and 85 % N2 (Airgas Northeast). Strains were grown in brain heart infusion broth supplemented with 0·5 % yeast extract and 15 µg haematin ml-1 (BHIS; Thompson & Malamy, 1990) or in anaerobic minimal medium with 0·5 % glucose (AMM gluc; Baughn & Malamy, 2002). Unless otherwise stated, all AMM gluc media were supplemented with 5 µg haematin ml-1 as the sole source of haem. Haematin stock solution was prepared by dissolving 5 g haemin chloride (Fisher Scientific) in 1 litre of 0·1 M NaOH. Thymine (50 µg ml-1; Sigma) was added for growth of Thy- strains. All glassware used for haem-restricted cultures was baked at 260 °C for 3 h to destroy trace haem contamination.


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Table 1. Bacterial strains and plasmids used in this study

 
Growth experiments were performed in the anaerobic chamber at 37 °C. Strains were first grown in BHIS medium to OD600 ~1. Cultures were diluted 1 to 2000 in AMM gluc containing no haematin and grown to OD600 ~0·5. These cultures were diluted 1 to 2000 in the test media. Culture growth was monitored by plating for colonies on BHIS medium.

E. coli strain DH5{alpha} (Woodcock et al., 1989) was used for cloning, and strain DW1030 (Robillard et al., 1985) containing the RP4 derivative RK231 (Guiney et al., 1984) was used for mobilization of plasmids from DH5{alpha} to B. fragilis as previously described (Guiney et al., 1984). RVN/F'lac{Delta}33-43 (Anthony et al., 1974; Kilbane, 1981; Rotman et al., 1983) was used as the donor strain for Tn1000 mutagenesis of plasmids in E. coli strain HB101 (Boyer & Roulland-Dussoix, 1969). E. coli strains were grown in Luria medium or MacConkey lactose medium (Difco) at 37 °C. Competent cells were prepared by the rubidium chloride method and transformed as described by Hanahan et al. (1991).

Chloramphenicol (25 µg ml-1), ampicillin (100 µg ml-1), streptomycin (50 µg ml-1), spectinomycin (50 µg ml-1), tetracycline (2 µg ml-1 for B. fragilis, 10 µg ml-1 for E. coli), erythromycin (8 µg ml-1), rifampicin (50 µg ml-1), gentamicin (50 µg ml-1) and trimethoprim (100 µg ml-1) were used as indicated.

Sequencing and analysis of the frd operon.
Oligonucleotide primers used in this study are described in Table 2. Oligonucleotides frd01 and frd11 were used as primers for Taq DNA polymerase (Invitrogen) mediated amplification of the frd operon from B. fragilis ADB77, a Thy- mutant of B. fragilis TM4000. Oligonucleotide sequences were based on sequence data obtained from the B. fragilis NCTC 9343 preliminary genome sequence produced by the Pathogen Sequencing Group at the Sanger Centre (http://www.sanger.ac.uk/Projects/B_fragilis). The PCR-amplified fragment containing the frd operon was digested with BamHI and cloned into the BglII site of pJST61 (Thompson & Malamy, 1990) to create plasmid pADB261 (see Fig. 1). Plasmids and PCR products were purified using QIAprep spin columns (Qiagen). Sequencing of the insert in pADB261 was performed with an ABI3100 sequencing apparatus (Perkin-Elmer) by the Tufts University Nucleic Acids and Protein Core Facility. The nucleotide sequence of the frd region of B. fragilis TM4000 has been deposited in GenBank under the accession number AY174185.


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Table 2. Oligonucleotide primers used in this study

 


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Fig. 1. Physical map of the B. fragilis frd operon. Genes encoding the cytochrome b subunit (frdC), catalytic flavoprotein subunit (frdA) and iron–sulfur cluster protein subunit (frdB) for fumarate reductase are depicted by large arrows. The frd promoter sequence (above the map) illustrates the predicted B. fragilis -35 and -10 promoter elements. Consensus B. fragilis -35 and -10 promoter elements (Bayley et al., 2000) are aligned above the promoter sequence. The predicted start codon for frdC is marked in bold. The frdC and frdB deletions are marked ADB247 and ADB260, respectively. The frdCAB fragment cloned into pJST61 is marked pADB261. Tn1000 insertion sites in pADB261 are marked m5 (between nucleotides 412 and 413 of frdC) and m7 (between nucleotides 1616 and 1617 of frdA).

 
All other nucleotide and protein sequence data described in this study were obtained from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/).

The software programs DNA STRIDER 1.2, EDITSEQ and MEGALIGN (DNAstar), MACVECTOR 7.0 (Oxford Molecular) and TOPPRED 2 (Claros & von Heijne, 1994) were used for DNA and protein sequence analysis.

Construction of deletions on the B. fragilis chromosome.
An in-frame deletion of frdC in B. fragilis ADB77 was constructed by allelic exchange between the chromosomal frdC locus and the {Delta}frdC locus of pADB247 using the two-step double-crossover technique as previously described (Baughn & Malamy, 2002). pADB247 was constructed as follows. A fragment consisting of 620 bp upstream of frdC and 115 bp of frdC amino-terminal coding sequence was amplified using the oligonucleotides frd01 and frd02 as primers. The product was digested with BamHI and NcoI. A fragment consisting of 14 bp of carboxyl-terminal frdC coding sequence and 732 bp downstream of frdC was amplified by using frd03 and frd04 as primers. The product was digested with NcoI and HindIII. The digested fragments were ligated into BamHI/HindIII-digested pADB242B to generate the {Delta}frdC allelic-exchange plasmid pADB247.

pADB247 was transferred by conjugation from DH5{alpha} to B. fragilis ADB77. Cointegrants were selected on BHIS medium containing gentamicin, rifampicin and tetracycline. Tetracycline-resistant colonies were screened by the colony PCR method with oligonucleotides frd04 and frd05 and separately with oligonucleotides frd01 and frd06. Isolates that tested positive for recombination of the suicide vector at the frdC locus were chosen for further use. Since pADB247 contains the B. fragilis thyA gene, recombination at frdC results in thymine prototrophy and trimethoprim sensitivity. Cointegrants were grown overnight from single colonies in BHIS with thymine. Overnight cultures were plated on AMM gluc with 50 µg ml-1 haematin, thymine and trimethoprim to select for resolvants. Trimethoprim-resistant isolates were screened for tetracycline sensitivity on BHIS plates containing tetracycline. Tetracycline-sensitive isolates were screened by the colony PCR method using oligonucleotides frd05 and frd06 as primers to distinguish between wild-type frdC resolution products and {Delta}frdC resolution products. Using this primer pair, a 2·1 kb product was amplified from the wild-type frdC locus while a 1·6 kb product was amplified from the {Delta}frdC locus.

A chromosomal in-frame deletion of the frdB gene in B. fragilis ADB77 was constructed by allelic exchange using the procedure described above. The {Delta}frdB allelic-exchange plasmid was constructed as follows. A fragment consisting of 699 bp 5' of frdB and 38 bp of amino-terminal coding sequence was amplified using the oligonucleotides frd07 and frd08, and digested with NcoI and HindIII. A fragment consisting of 31 bp of carboxyl-terminal coding sequence and 708 bp of 3' sequence was amplified using the oligonucleotides frd09 and frd10, and digested with NcoI and BamHI. The digested fragments were ligated with BamHI/HindIII-digested pADB242B to generate the plasmid pADB260. B. fragilis isolates bearing the {Delta}frdB allele were identified by using the colony PCR method with the primers frd07 and frd10. Using this primer pair, a 2·2 kb product was amplified from the wild-type frdB locus while a 1·5 kb product was amplified from the {Delta}frdB locus.

Enzyme assays.
Enzyme assays were performed using crude extracts prepared from anaerobically grown cells, as previously described (Baughn & Malamy, 2002). Exponential-phase cultures were harvested in 50 ml screw-cap conical tubes by centrifugation at 2300 g at 4 °C in a Sorvall RC2-B centrifuge. Cells were resuspended in the anaerobic chamber in 5 ml chilled prereduced buffer A (Macy et al., 1975) and harvested as described above. All subsequent steps were performed in the anaerobic chamber. Washed cells were resuspended in 1·5 ml chilled prereduced buffer A and transferred to 2 ml screw-cap microcentrifuge tubes (product nos 6550 and 6582; Perfector Scientific). Cells were disrupted by sonication for 2 min with 50 % output on a 50 % duty cycle by using a Branson Sonifier 250. Sonication was performed in the anaerobic chamber to protect redox-sensitive enzymes from oxidative inactivation (Pan & Imlay, 2001). Samples were maintained at 4 °C by using an ArcticIce block (USA Scientific). Sonicated samples were clarified by centrifugation for 5 min at 2000 g in a microcentrifuge at 4 °C. Aliquots of clarified sonicates were placed in 1·5 ml microcentrifuge tubes (product no. 05-406-16; Fisher Scientific). Use of tubes other than those described above resulted in inactivation of iron–sulfur cluster containing enzymes. Samples were flash frozen in liquid nitrogen and stored at -80 °C until needed.

Enzyme activities were determined in a reaction buffer containing 50 mM potassium phosphate (pH 7·6) and 5 mM MgCl2 (Macy et al., 1975). When necessary, 200 µM NADH or 44 µM methylene blue was added to the reaction mixture. Reactions were started by addition of cell extract to the reaction mixture. All solutions were prepared in the anaerobic chamber with preboiled distilled water to ensure minimal O2 contamination. Assays were performed in cuvettes that were sealed with Parafilm. Fumarate reductase activity was determined by measuring fumarate-dependent oxidation of NADH to NAD+ as indicated by a decrease in absorbance at 340 nm (Macy et al., 1975). One unit was defined as the amount of enzyme required to oxidize 1 µmol NADH min-1 in the presence of 1 mM sodium fumarate. Malate dehydrogenase activity was determined by measuring oxaloacetate-dependent conversion of NADH to NAD+ (Ochoa, 1955). One unit was defined as the amount of enzyme required to oxidize 1 µmol NADH min-1 in the presence of 0·25 mM oxaloacetate. Succinate dehydrogenase activity was determined by measuring succinate-dependent reduction of methylene blue (Bonner, 1955). One unit was defined as the amount of enzyme required to reduce 1 µmol methylene blue min-1 in the presence of 3 mM sodium succinate.

Molar growth yield determinations.
Molar growth yield determinations were performed (Macy et al., 1975) on B. fragilis cultures in which less than 50 % of the glucose had been consumed. Fifty millilitres of exponential-phase culture was centrifuged for 10 min at 2300 g at 4 °C in a Sorvall RC2-B centrifuge. The supernatant was transferred to a screw-cap tube and stored at -20 °C until needed. Supernatant glucose concentration was determined by the glucose oxidase method. Dry cell mass was determined as follows. Exponential-phase cultures were filtered onto preweighed 0·45 µm nitrocellulose filters (Millipore). The filters were dried for 48 h under vacuum at 75 °C and weighed. Dry cell mass was determined by subtracting the mass of filters from the mass of the filters with dry cells.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
B. fragilis encodes a succinate : quinone oxidoreductase homologue
FRD activity was previously measured in cell extracts prepared from B. fragilis grown in the presence of haem (Macy et al., 1975). In order to characterize the role of FRD in the growth of B. fragilis we set out to identify the genes that encode this enzyme. By using the E. coli K-12 FrdA and FrdB sequences as the query sequences in TBLASTN searches of the unfinished B. fragilis NCTC 9343 genome, a gene cluster encoding homologues of FrdA and FrdB was identified (Fig. 1). The corresponding genes, frdA and frdB, were located downstream of an open reading frame that we designated frdC (Fig. 1).

Because B. fragilis NCTC 9343 is not identical to B. fragilis TM4000, our reference strain for genetic studies, primers were designed for PCR amplification and sequencing of the frd region from strain TM4000. A 4·2 kb PCR product containing the frd region was cloned in pJST61, generating the plasmid pADB261. The insert was completely sequenced on both strands. The frd regions of strains NCTC 9343 and TM4000 were found to share 99·7 % nucleotide identity (data not shown). The deduced amino acid sequences of the corresponding Frd polypeptides were 100 % identical.

Putative B. fragilis -35 and -10 promoter elements (Bayley et al., 2000) were identified 101 and 73 bp upstream of the frdC start codon (Fig. 1). The translation stop codon for frdC and the start codon for frdA were separated by 37 bp. The stop codon for frdA and the start codon for frdB were separated by 29 bp. No apparent B. fragilis promoter elements could be identified upstream of frdA or frdB. A 20 bp inverted repeat that may serve as a transcription terminator was identified 28 bp downstream of the frdB stop codon. These observations indicate that frdCAB may constitute an operon.

By comparing the B. fragilis Frd homologues to the non-redundant database at NCBI, we found that these peptides are most similar to type B succinate : quinone oxidoreductases (SQR) of eubacteria (Lemos et al., 2002). For example, the B. fragilis FrdA was 36 % identical and 51 % similar to SdhA of the Bacillus subtilis succinate dehydrogenase (SDH; not shown); the B. fragilis FrdB was 29 % identical and 49 % similar to SdhB of B. subtilis (not shown); and the B. fragilis FrdC was 20 % identical and 37 % similar to SdhC of B. subtilis (not shown). Subunits of the B. fragilis SQR show conservation of most of the residues important for cofactor binding, substrate binding and catalysis described for the Wolinella succinogens FRD, another type B SQR (not shown; Lancaster et al., 1999).

The membrane topology prediction program TOPPRED2 applied to the B. fragilis FrdC sequence suggests that the protein contains five membrane-spanning helices (not shown). This topology is identical to that of the cytochrome b of the W. succinogenes FRD (Lancaster et al., 1999). In addition, the periplasmic loop that joins helices 1 and 2 of B. fragilis FrdC contains a glutamic acid residue (Glu40). This residue potentially corresponds to Glu66 of the W. succinogenes FrdC, which is known to be essential for FRD-dependent energy metabolism (Lancaster et al., 2000).

When used as query sequences against the NCBI microbial genomes, the subunits of the B. fragilis SQR were found to be most similar to predicted homologues from other Cytophaga–Flavobacterium–Bacteroides group members of eubacteria, such as Porphyromonas gingivalis, Prevotella intermedia, Tannerella (Bacteroides) forsythensis and Cytophaga hutchinsonii. For example, the FrdA homologue of P. gingivalis showed 80 % identity and 87 % similarity to the B. fragilis FrdA (not shown), the FrdB homologue of P. gingivalis showed 79 % identity and 89 % similarity to the B. fragilis FrdB (not shown), and the FrdC homologue of P. gingivalis showed 66 % identity and 80 % similarity to the B. fragilis FrdC (not shown).

The frd genes are essential for FRD and SDH activity
To determine if the SQR homologue described above encodes the B. fragilis FRD, derivatives of strain ADB77 bearing deletions of frdC and frdB were constructed (Fig. 1). Since the {Delta}frdC allele (frdC247) lacks coding sequence for transmembrane helices 2–5, the product of frdC247 should not be capable of haem coordination. In addition, the frdC247 product lacks the two predicted cytoplasmic loops and should not be able to serve as a membrane anchor for the FrdB subunit. Since the {Delta}frdB (frdB260) lacks the coding sequence for all three iron–sulfur cluster coordination sites, the frdB260 product should not be able to mediate electron transfer between the FrdC and FrdA subunits.

Crude cell extracts prepared from B. fragilis ADB77, ADB247 ({Delta}frdC) and ADB260 ({Delta}frdB) grown in AMM gluc were assayed for the presence of FRD activity. Both ADB247 and ADB260 extracts showed a greater than 50-fold reduction in FRD activity relative to the ADB77 extract (Table 3), indicating that frdC and frdB are essential for FRD activity in B. fragilis. It is important to note that all extracts contained similar amounts of malate dehydrogenase activity, indicating that none of the extracts were inactivated during preparation (Table 3).


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Table 3. Fumarate reductase (FRD), malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) activities in crude cell extracts of wild-type B. fragilis and frd mutants

Specific activities are shown as units (mg protein)-1. Values are the mean ± SE for triplicate assays of the same extract. The values presented for FRD and MDH are from one representative experiment that was performed three times independently. SDH assays were performed on a single extract.

 
Consistent with the observation that the phylogenetically related anaerobe Bacteroides thetaiotaomicron expresses SDH activity (Kotarski & Salyers, 1984), B. fragilis crude cell extracts were found to contain this activity (Table 3). SDH activity required the presence of the frd genes (Table 3), indicating that the B. fragilis SQR can function both as an FRD and as an SDH.

The frd genes are essential for optimal growth in the presence of haem
Consistent with previous reports (Baughn & Malamy, 2002; Macy et al., 1975), the generation time of wild-type B. fragilis (ADB77) was approximately 8 h in AMM gluc lacking haematin (Fig. 2a) compared to a generation time of 2·4 h in AMM gluc containing 5 µg haematin ml-1 (Fig. 2b). In contrast, growth of the frd mutant strains (ADB247 and ADB260) was not stimulated by the presence of haematin in the growth medium (Fig. 2a, b), indicating that the frd gene products play a critical role in the haem stimulatory effect.



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Fig. 2. Growth of the B. fragilis frd mutant strains in AMM gluc (a) without haematin; (b, c) with 5 µg haematin ml-1; (d) with haematin and 0·5 % succinate. {square}, ADB77 (wild-type); {lozenge}, ADB247 ({Delta}frdC); {circ}, ADB260 ({Delta}frdB); {blacksquare}, ADB77 with pJST61 (empty vector); {blacklozenge}, ADB247 with pJST61; {bullet}, ADB260 with pJST61; {triangleup}, ADB247 with pADB261 (vector with the frd operon); {triangledown}, ADB260 with pADB261.

 
To ensure that the growth defects of the frd mutants were due to the lack of the frd genes, a plasmid containing the entire frd region (pADB261) was introduced into these strains. Both frd mutant strains were fully complemented by this plasmid (Fig. 2c). Thus, the growth defects displayed by these mutants were due to lack of the respective frd genes.

We previously demonstrated that the growth of haem-limited wild-type B. fragilis is stimulated by the addition of succinate to the growth medium (Baughn & Malamy, 2002). Consistent with this observation, the generation time of both of the frd mutants was decreased to 4 h when the culture medium was supplemented with succinate (Fig. 2d). These results indicate that during growth the frd gene products function as an FRD. Furthermore, both FRD-mediated electron transport and succinate biosynthesis are important for the optimal growth of B. fragilis.

A role for FRD in the generation of ATP
Molar growth yield studies indicate that B. fragilis grown in the presence of haem is capable of generating more than twofold more ATP per mole of glucose than that which is generated in the absence of haem (Macy et al., 1975). To determine if FRD is required for maximum yield of ATP from glucose, molar growth yields were determined for the wild-type strain and the frd mutant strains grown in AMM gluc. The FRD mutant strains showed a growth yield on glucose similar to that of the haem-restricted wild-type strain (Table 4). This yield was approximately threefold lower than that observed for the wild-type strain grown in the presence of haem. These results confirm a role for FRD in the generation of metabolic energy.


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Table 4. Molar growth yield (Ym) determinations for wild-type B. fragilis and frd mutants grown in AMM with 0·5 % glucose

Ym was determined by dividing the dry cell mass (g) by the amount of glucose used (mol). Ym(X)/Ym(wt+haem) is the ratio of Ym to the Ym of the wild-type strain grown in the presence of haem.

 
frdCAB form an operon
To determine if frdCAB form an operon, the complementing plasmid pADB261 was mutagenized with Tn1000 in E. coli. Plasmids containing insertions in the frd region were chosen for further use. Insertion sites were determined by sequencing across the frd–Tn1000 junction. Plasmids with insertions in frdC and frdA (pADB261m5 and pADB261m7, respectively; Fig. 1) were transferred to B. fragilis strains ADB247 ({Delta}frdC) and ADB260 ({Delta}frdB). As expected, pADB261m5 failed to complement ADB247, but pADB261m7 fully complemented this strain (Table 5). Neither pADB261m5 nor pADB261m7 was capable of complementing ADB260 (Table 5), indicating that the insertions in frdC and frdA were polar on frdB expression. Thus, the frdCAB gene cluster forms an operon.


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Table 5. Test for complementation of B. fragilis frd mutant strains ADB247 and ADB260 with pADB261m5 and pADB261m7

Plasmids: 1, pJST61 (vector); 2, pADB261 (pJST61 + frdCAB); 3, pADB261m5 (pADB261 frdC : : Tn1000); 4, pADB261m7 (pADB261 frdB : : Tn1000). Scores: +, Colony size comparable to that of the wild-type strain (>2 mm in diameter) after 3 days incubation on BHIS at 37 °C; -, pinpoint colonies (<1 mm) after 3 days incubation on BHIS at 37 °C.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Several species of the Bacteroides group of eubacteria require exogenous haem for optimal growth (Caldwell et al., 1965; Macy et al., 1975; McKee et al., 1986). Previous studies of the haem requirement of B. fragilis suggested that haem is necessary for the synthesis of a cytochrome-b-dependent FRD (Macy et al., 1975). Analysis of the B. fragilis genome revealed a gene cluster encoding a type B SQR. This gene cluster was found to be highly conserved amongst other species of the Bacteroides group. We have now demonstrated that this gene cluster encodes FRD and is essential for the haem-stimulatory effect in B. fragilis.

It is interesting to note that the B. fragilis SQR most closely resembles oxidoreductases that function physiologically as SDHs. SDH is an enzyme of the Krebs cycle that catalyses the oxidation of succinate to fumarate. During aerobiosis this cycle provides the reducing power required for aerobic respiration. In organisms such as E. coli, SDH plays no role in anaerobic energy metabolism. However, due to the high degree of sequence conservation between SDH and FRD, it is not surprising that these oxidoreductases can function reversibly depending upon environmental conditions. For example, SDH of the strict aerobe B. subtilis can operate as an FRD under anaerobic conditions (Schnorpfeil et al., 2001). Thus, it is not surprising that the B. fragilis SQR can catalyse the reduction of fumarate as well as the oxidation of succinate.

In the anaerobic {varepsilon}-proteobacterium W. succinogenes, fumarate reduction is coupled to the generation of ATP (Reddy & Peck, 1978). Recent studies of the W. succinogenes FRD indicate that this enzyme facilitates the formation of a proton gradient across the cytoplasmic membrane (Biel et al., 2002; Lancaster et al., 2000). Formation of this proton gradient is predicted to be coupled to ATP biosynthesis via an F1F0-type ATP synthase (Bokranz et al., 1985).

Similar to that which is observed for W. succinogenes, the B. fragilis FRD is involved in anaerobic energy metabolism. Moreover, B. fragilis encodes a canonical F-type ATP synthase that could participate in the generation of ATP at the expense of an electrochemical potential (Amann et al., 1988). Given these observations, we predict that the FRD of B. fragilis functions in energy metabolism in a manner similar to that of the FRD of W. succinogenes.

Under anaerobic conditions, organisms such as E. coli require succinate for the synthesis of succinyl-CoA (Clark, 1989), an important cofactor for biosynthesis of methionine, diaminopimelic acid and lysine. Studies of the haem requirement of B. fragilis indicate that FRD is the major source of succinate in this bacterium (Baughn & Malamy, 2002; Macy et al., 1975). However, B. fragilis is capable of growth under conditions that do not permit the reduction of fumarate to succinate. This observation indicates that either succinyl-CoA is not required for the growth of B. fragilis, or succinyl-CoA is synthesized via an alternative pathway. It is known that B. fragilis is capable of the reductive carboxylation of succinyl-CoA (Allison et al., 1979) by 2-oxoglutarate : ferredoxin oxidoreductase (KFOR; Baughn & Malamy, unpublished). In Thermococcus litoralis and Helicobacter pylori this enzyme can also catalyse the oxidative decarboxylation of 2-oxoglutarate to form succinyl-CoA (Hughes et al., 1998; Mai & Adams, 1996). B. fragilis can synthesize 2-oxoglutarate by the oxidative branch of the Krebs cycle (Baughn & Malamy, 2002); thus it is likely that this bacterium can generate succinyl-CoA via the oxidative decarboxylation of 2-oxoglutarate. Since exogenous succinate stimulates growth of FRD-deficient B. fragilis, this alternative pathway does not permit the generation of sufficient amounts of succinate for optimal growth.

In addition to serving as a precursor for cellular metabolites, succinate can also be involved in the conservation of metabolic energy. For example, the anaerobe Veillonella parvula can generate ATP in a process that is coupled to the decarboxylation of succinate (Denger & Schink, 1992; Janssen, 1992). Since succinate supplementation partially restores growth of FRD-deficient B. fragilis, it is likely that this bacterium also uses succinate for the conservation of metabolic energy.

Since FRD plays such an important role in growth of B. fragilis, the ability to obtain haem is likely to be critical for the fitness of this bacterium during infection. B. fragilis can use mammalian haemoproteins, such as haemoglobin, as a source of haem in vitro (Otto et al., 1994). Since haemoglobin is either found within red blood cells or complexed with haptoglobin, this haemoprotein is unavailable to most invading pathogens. However, it has been shown that B. fragilis is capable of using the haptoglobin–haemoglobin complex as a source of haem (Otto et al., 1994). In addition, mammals use haemopexin to tightly sequester any extracellular haem. It has recently been shown that B. fragilis can use haemopexin as a source of haem via the action of a secreted serine protease (Rocha et al., 2001). It is likely that these factors for haem acquisition are critical for the virulence of this anaerobic pathogen.


   ACKNOWLEDGEMENTS
 
This study was supported by Public Health Grant AI-19497 from the National Institute of Allergy and Infectious Disease of the National Institutes of Health.

We thank A. L. Sonenshein, D. W. Lazinski and A. Camilli for their constructive comments on the manuscript.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Allison, M. J., Robinson, I. M. & Baetz, A. L. (1979). Synthesis of {alpha}-ketoglutarate by reductive carboxylation of succinate in Veillonella, Selenomonas, and Bacteriodes species. J Bacteriol 140, 980–986.[Medline]

Amann, R., Ludwig, W. & Schleifer, K. H. (1988). {beta}-Subunit of ATP-synthase: a useful marker for studying the phylogenetic relationship of eubacteria. J Gen Microbiol 134, 2815–2821.[Medline]

Anthony, W. M., Donier, R. C., Lee, H., Hu, S., Ohtsubo, E., Davidson, N. & Broda, P. (1974). Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. J Mol Biol 89, 647–650.[Medline]

Baughn, A. D. & Malamy, M. H. (2002). A mitochondrial-like aconitase in the bacterium Bacteroides fragilis: implications for the evolution of the mitochondrial Krebs cycle. Proc Natl Acad Sci U S A 99, 4662–4667.[Abstract/Free Full Text]

Bayley, D. P., Rocha, E. R. & Smith, C. J. (2000). Analysis of cepA and other Bacteroides fragilis genes reveals a unique promoter structure. FEMS Microbiol Lett 193, 149–154.[CrossRef][Medline]

Biel, S., Simon, J., Gross, R., Ruiz, T., Ruitenberg, M. & Kröger, A. (2002). Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes. Eur J Biochem 269, 1974–1983.[Abstract/Free Full Text]

Bokranz, M., Morschel, E. & Kröger, A. (1985). Phosphorylation and phosphate-ATP exchange catalyzed by the ATP synthase isolated from Wolinella succinogenes. Biochim Biophys Acta 810, 332–339.[Medline]

Bonner, W. D. (1955). Succinate dehydrogenase. Methods Enzymol 1, 722–728.

Boyer, H. W. & Roulland-Dussoix. (1969). A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41, 459–472.[Medline]

Caldwell, D. R., White, D. C., Bryant, M. P. & Doetsch, R. N. (1965). Specificity of the heme requirement for growth of Bacteroides ruminicola. J Bacteriol 90, 1645–1654.[Medline]

Clark, D. P. (1989). Anaerobic growth defects resulting from gene fusions affecting succinyl-CoA synthetase in Escherichia coli K12. Mol Gen Genet 215, 276–280.[Medline]

Claros, M. G. & von Heijne, G. (1994). TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10, 685–686.[Medline]

Denger, K. & Schink, B. (1992). Energy conservation by succinate decarboxylation in Veilonella parvula. J Gen Microbiol 138, 967–971.[Medline]

Fuller, M. D. & Caldwell, D. R. (1982). Tetrapyrrole utilization by Bacteroides fragilis. Can J Microbiol 28, 1304–1310.[Medline]

Guiney, D. G., Hasegawa, P. & Davis, C. E. (1984). Plasmid transfer from Escherichia coli to Bacteroides fragilis: differential expression of antibiotic resistance phenotypes. Proc Natl Acad Sci U S A 81, 7203–7206.[Abstract]

Hanahan, D., Jessee, J. & Bloom, F. R. (1991). Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol 204, 63–113.[Medline]

Harris, M. A. & Reddy, C. A. (1977). Hydrogenase activity and the H2-fumarate electron transport system in Bacteroides fragilis. J Bacteriol 131, 922–928.[Medline]

Hughes, N. J., Clayton, C. L., Chalk, P. A. & Kelly, D. J. (1998). Helicobacter pylori porCDAB and oorDABC genes encode distinct pyruvate : flavodoxin and 2-oxoglutarate : acceptor oxidoreductases which mediate electron transport to NADP. J Bacteriol 180, 1119–1128.[Abstract/Free Full Text]

Janssen, P. (1992). Growth yield increase and ATP formation linked to succinate decarboxylation in Veillonella parvula. Arch Microbiol 157, 442–445.[Medline]

Kilbane, J. J. (1981). F factor mobilization of non-conjugative plasmids in Escherichia coli: general mechanisms and a role for site-specific recA-independent recombination at oriV1. PhD thesis, Tufts University, Boston.

Kotarski, S. F. & Salyers, A. A. (1984). Isolation and characterization of outer membranes of Bacteroides thetaiotaomicron grown on different carbohydrates. J Bacteriol 158, 102–109.[Medline]

Lancaster, C. R. D., Kröger, A., Auer, M. & Michel, H. (1999). Structure of fumarate reductase from Wolinella succinogenes at 2·2 Å resolution. Nature 402, 377–385.[CrossRef][Medline]

Lancaster, C. R. D., Groß, R., Haas, A., Ritter, M., Mäntele, W., Simon, J. & Kröger, A. (2000). Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase. Proc Natl Acad Sci U S A 97, 13051–13056.[Abstract/Free Full Text]

Lemos, R. S., Fernandes, A. S., Pereira, M. M., Gomes, C. M. & Teixeira, M. (2002). Quinol : fumarate oxidoreductases and succinate : quinone oxidoreductases: phylogenetic relationships, metal centres and membrane attachment. Biochim Biophys Acta 1553, 158–170.[CrossRef][Medline]

Macy, J., Probst, I. & Gottschalk, G. (1975). Evidence for cytochrome involvement in fumarate reduction and adenosine 5'-triphosphate synthesis by Bacteroides fragilis grown in the presence of hemin. J Bacteriol 123, 436–442.[Medline]

Mai, X. & Adams, M. W. W. (1996). Characterization of a fourth type of 2-keto acid-oxidizing enzyme from a hyperthermophilic archaeon: 2-ketoglutarate ferredoxin oxidoreductase from Thermococcus litoralis. J Bacteriol 178, 5890–5896.[Abstract]

McKee, A. S., McDermid, A. S., Baskerville, A., Dowsett, A. B., Ellwood, D. C. & Marsh, P. D. (1986). Effect of hemin on the physiology and virulence of Bacteroides gingivalis W50. Infect Immun 52, 349–355.[Medline]

Ochoa, S. (1955). Malic dehydrogenase from pig heart. Methods Enzymol 1, 735–739.

Otto, B. R., Sparrius, M., Wors, D. J., de Graaf, F. K. & MacLaren, D. M. (1994). Utilization of haem from the haptoglobin-haemoglobin complex by Bacteroides fragilis. Microb Pathog 17, 137–147.[CrossRef][Medline]

Pan, N. & Imlay, J. A. (2001). How does oxygen inhibit central metabolism in the obligate anaerobe Bacteroides thetaiotaomicron? Mol Microbiol 39, 1562–1571.[CrossRef][Medline]

Reddy, C. A. & Peck, H. D. (1978). Electron transport phosphorylation coupled to fumarate reduction by hydrogen and Mg2+-dependent adenosine triphosphatase activity in extracts of the rumen anaerobe Vibrio succinogenes. J Bacteriol 134, 982–991.[Medline]

Robillard, N. J., Tally, F. P. & Malamy, M. H. (1985). Tn4400, a compound transposon isolated from Bacteroides fragilis, functions in Escherichia coli. J Bacteriol 164, 1248–1255.[Medline]

Rocha, E. R., Smith, A., Smith, C. J. & Brock, J. H. (2001). Growth inhibition of Bacteroides fragilis by hemopexin: proteolytic degradation of hemopexin to overcome heme limitation. FEMS Microbiol Lett 199, 73–78.[CrossRef][Medline]

Rotman, G. S., Cooney, R. & Malamy, M. H. (1983). Cloning of the pif region of the F sex factor and identification of a pif protein product. J Bacteriol 155, 254–264.[Medline]

Schnorpfeil, M., Janausch, I. G., Biel, S., Kröger, A. & Unden, G. (2001). Generation of a proton potential by succinate dehydrogenase of Bacillus subtilis functioning as a fumarate reductase. Eur J Biochem 268, 3069–3074.[Abstract/Free Full Text]

Sperry, J. F., Appleman, M. D. & Wilkins, T. D. (1977). Requirement of heme for growth of Bacteroides fragilis. Appl Environ Microbiol 34, 386–390.[Medline]

Tang, Y. P. (2000). Identification and characterization of genes in Bacteroides fragilis involved in aerotolerance. PhD thesis, Tufts University, Boston.

Thompson, J. S. & Malamy, M. H. (1990). Sequencing the gene for an imipenem-cefoxitin-hydrolyzing enzyme (CfiA) from Bacteroides fragilis TAL2480 reveals strong similarity between CfiA and Bacillus cereus {beta}-lactamase II. J Bacteriol 172, 2584–2593.[Medline]

Woodcock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S. S., Michael, M. Z. & Graham, M. W. (1989). Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17, 3469–3478.[Abstract]

Received 20 January 2003; revised 13 March 2003; accepted 14 March 2003.



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