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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank accession number for the sequence reported in this paper is AY174185.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
E. coli strain DH5 (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
to B. fragilis as previously described (Guiney et al., 1984
). RVN/F'lac
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.
|
|
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 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
frdC allelic-exchange plasmid pADB247.
pADB247 was transferred by conjugation from DH5 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
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
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 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
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
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 ironsulfur 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 CytophagaFlavobacteriumBacteroides 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
frdC allele (frdC247) lacks coding sequence for transmembrane helices 25, 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
frdB (frdB260) lacks the coding sequence for all three ironsulfur 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 (frdC) and ADB260 (
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
).
|
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. 2
a) 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.
|
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.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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 haptoglobinhaemoglobin 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 |
---|
We thank A. L. Sonenshein, D. W. Lazinski and A. Camilli for their constructive comments on the manuscript.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amann, R., Ludwig, W. & Schleifer, K. H. (1988). -Subunit of ATP-synthase: a useful marker for studying the phylogenetic relationship of eubacteria. J Gen Microbiol 134, 28152821.[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, 647650.[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, 46624667.
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, 149154.[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, 19741983.
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, 332339.[Medline]
Bonner, W. D. (1955). Succinate dehydrogenase. Methods Enzymol 1, 722728.
Boyer, H. W. & Roulland-Dussoix. (1969). A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41, 459472.[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, 16451654.[Medline]
Clark, D. P. (1989). Anaerobic growth defects resulting from gene fusions affecting succinyl-CoA synthetase in Escherichia coli K12. Mol Gen Genet 215, 276280.[Medline]
Claros, M. G. & von Heijne, G. (1994). TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10, 685686.[Medline]
Denger, K. & Schink, B. (1992). Energy conservation by succinate decarboxylation in Veilonella parvula. J Gen Microbiol 138, 967971.[Medline]
Fuller, M. D. & Caldwell, D. R. (1982). Tetrapyrrole utilization by Bacteroides fragilis. Can J Microbiol 28, 13041310.[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, 72037206.[Abstract]
Hanahan, D., Jessee, J. & Bloom, F. R. (1991). Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol 204, 63113.[Medline]
Harris, M. A. & Reddy, C. A. (1977). Hydrogenase activity and the H2-fumarate electron transport system in Bacteroides fragilis. J Bacteriol 131, 922928.[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, 11191128.
Janssen, P. (1992). Growth yield increase and ATP formation linked to succinate decarboxylation in Veillonella parvula. Arch Microbiol 157, 442445.[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, 102109.[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, 377385.[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, 1305113056.
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, 158170.[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, 436442.[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, 58905896.[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, 349355.[Medline]
Ochoa, S. (1955). Malic dehydrogenase from pig heart. Methods Enzymol 1, 735739.
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, 137147.[CrossRef][Medline]
Pan, N. & Imlay, J. A. (2001). How does oxygen inhibit central metabolism in the obligate anaerobe Bacteroides thetaiotaomicron? Mol Microbiol 39, 15621571.[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, 982991.[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, 12481255.[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, 7378.[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, 254264.[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, 30693074.
Sperry, J. F., Appleman, M. D. & Wilkins, T. D. (1977). Requirement of heme for growth of Bacteroides fragilis. Appl Environ Microbiol 34, 386390.[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 -lactamase II. J Bacteriol 172, 25842593.[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, 34693478.[Abstract]
Received 20 January 2003;
revised 13 March 2003;
accepted 14 March 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |