Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.     

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens

Applied Microbiology and Biotechnology - Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes....     

Genetic characterization of a single bifunctional enzyme for fumarate reduction and succinate oxidation in Geobacter sulfurreducens and engineering of fumarate reduction in Geobacter metallireducens

The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.     

Reduction of Fe(III) (Hydr)oxides with Known Thermodynamic Stability by Geobacter metallireducens

Sulfate-reducing and methanogenic microorganisms become inactive when the concentration of the electron donors drops below a threshold set by the minimum Gibbs free energy required for the bacterial metabolism to be maintained. Thus, their activity is thermodynamically controlled. In this paper we study if the activity of dissimilatory Fe(III) reducing bacteria is also limited by the thermodynamics of the reaction. We synthesized five Fe (III) (hydr)oxides (FHOs) of moderate stability and determined the solubility product (log K _SO (?39.1)-(?41.8)), in order to calculate their standard free energy of formation. K _SO values, estimated from the particle size did not correspond with experimentally determined ones. HCO₃ ^? and PIPES-buffered media, containing 45 mM FHO and either 1, 10, or 100 mM acetate were inoculated with Geobacter metallireducens. At the end of bacterial reduction, the Gibbs free energy of the reaction showed significant differences between the different FHOs. The termination of the bacterial activity was consequently not triggered thermodynamically. However, the non-dissolved Fe(II) (HCl-soluble minus soluble Fe(II)) showed an excellent correlation with the surface of the FHOs (15 μmol m^?2). It is therefore likely that the termination of the reaction was caused by blocking of the FHO surface with insoluble Fe(II), as has been previously reported in the literature. The ecological significance of both thermodynamic limitation and surface availability limitation is discussed for FHOs of different K _SO in environments with approximately neutral pH.     

Purification and characterization of active-site components of the putative p-cresol methylhydroxylase membrane complex from Geobacter metallireducens

p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 ± 15 kDa and a unique αα′β₂ subunit composition, with α and α′ representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and β representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr³⁹⁴ of the α subunit. In contrast, the α′ subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low K_m values (1.7 and 2.7 μM, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a K_ic (competitive inhibition) value of 18 ± 9 μM and a K_iu (uncompetitive inhibition) value of 235 ± 20 μM. A putative functional model for an unusual PCMH enzyme is presented.     

Clinical and molecular characterization of five patients with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is an inborn error of ketone body metabolism and causes episodic ketoacidosis. We report clinical and molecular analyses of 5 patients with SCOT deficiency. Patients GS07, GS13, and GS14 are homozygotes of S405P, L327P, and R468C, respectively. GS17 and GS18 are compound heterozygotes for S226N and A215V, and V404F and E273X, respectively. These mutations have not been reported previously. Missense mutations were further characterized by transient expression analysis of mutant cDNAs. Among 6 missense mutations, mutants L327P, R468C, and A215V retained some residual activities and their mutant proteins were detected in immunoblot analysis following expression at 37 °C. They were more stable at 30 °C than 37 °C, indicating their temperature sensitive character. The R468C mutant is a distinct temperature sensitive mutant which retained 12% and 51% of wild-type residual activities at 37 and 30 °C, respectively. The S226N mutant protein was detected but retained no residual activity. Effects of missense mutations were predicted from the tertiary structure of the SCOT molecule. Main effects of these mutations were destabilization of SCOT molecules, and some of them also affected catalytic activity. Among 5 patients, GS07 and GS18 had null mutations in both alleles and the other three patients retained some residual SCOT activities. All 5 developed a first severe ketoacidotic crisis with blood gas pH < 7.1, and experienced multiple ketoacidotic decompensations (two of them had seven such episodes). In general, the outcome was good even following multiple ketoacidotic events. Permanent ketosis or ketonuria is considered a pathognomonic feature of SCOT deficiency. However, this condition depends not only on residual activity but also on environmental factors.     

The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens

Background  

The genome sequence of Geobacter metallireducens is the second to be completed from the metal-respiring genus Geobacter, and is compared in this report to that of Geobacter sulfurreducens in order to understand their metabolic, physiological and regulatory similarities and differences.     

Plutonium(IV) reduction by the metal-reducing bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1

The bacterial reduction of actinides has been suggested as a possible remedial strategy for actinide-contaminated environments, and the bacterial reduction of Pu(VI/V) has the potential to produce highly insoluble Pu(IV) solid phases. However, the behavior of plutonium with regard to bacterial reduction is more complex than for other actinides because it is possible for Pu(IV) to be further reduced to Pu(III), which is relatively more soluble than Pu(IV). This work investigates the ability of the metal-reducing bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1 to enzymatically reduce freshly precipitated amorphous Pu(IV) (OH)(4) [Pu(IV)(OH)(4(am))] and soluble Pu(IV)(EDTA). In cell suspensions without added complexing ligands, minor Pu(III) production was observed in cultures containing S. oneidensis, but little or no Pu(III) production was observed in cultures containing G. metallireducens. In the presence of EDTA, most of the Pu(IV)(OH)(4(am)) present was reduced to Pu(III) and remained soluble in cell suspensions of both S. oneidensis and G. metallireducens. When soluble Pu(IV)(EDTA) was provided as the terminal electron acceptor, cell suspensions of both S. oneidensis and G. metallireducens rapidly reduced Pu(IV)(EDTA) to Pu(III)(EDTA) with nearly complete reduction within 20 to 40 min, depending on the initial concentration. Neither bacterium was able to use Pu(IV) (in any of the forms used) as a terminal electron acceptor to support growth. These results have significant implications for the potential remediation of plutonium and suggest that strongly reducing environments where complexing ligands are present may produce soluble forms of reduced Pu species.     

Plutonium(IV) Reduction by the Metal-Reducing Bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1

The bacterial reduction of actinides has been suggested as a possible remedial strategy for actinide-contaminated environments, and the bacterial reduction of Pu(VI/V) has the potential to produce highly insoluble Pu(IV) solid phases. However, the behavior of plutonium with regard to bacterial reduction is more complex than for other actinides because it is possible for Pu(IV) to be further reduced to Pu(III), which is relatively more soluble than Pu(IV). This work investigates the ability of the metal-reducing bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1 to enzymatically reduce freshly precipitated amorphous Pu(IV) (OH)₄ [Pu(IV)(OH)_4(am)] and soluble Pu(IV)(EDTA). In cell suspensions without added complexing ligands, minor Pu(III) production was observed in cultures containing S. oneidensis, but little or no Pu(III) production was observed in cultures containing G. metallireducens. In the presence of EDTA, most of the Pu(IV)(OH)_4(am) present was reduced to Pu(III) and remained soluble in cell suspensions of both S. oneidensis and G. metallireducens. When soluble Pu(IV)(EDTA) was provided as the terminal electron acceptor, cell suspensions of both S. oneidensis and G. metallireducens rapidly reduced Pu(IV)(EDTA) to Pu(III)(EDTA) with nearly complete reduction within 20 to 40 min, depending on the initial concentration. Neither bacterium was able to use Pu(IV) (in any of the forms used) as a terminal electron acceptor to support growth. These results have significant implications for the potential remediation of plutonium and suggest that strongly reducing environments where complexing ligands are present may produce soluble forms of reduced Pu species.     

How Geobacteraceae may dominate subsurface biodegradation: physiology of Geobacter metallireducens in slow-growth habitat-simulating retentostats

Geobacteraceae dominate many iron-reducing subsurface environments and are associated with biodegradation of organic pollutants. In order to enhance the understanding of the environmental role played by Geobacteraceae , the physiology of Geobacter metallireducens was investigated at the low growth rates found in its subsurface habitat. Cultivation in retentostats (a continuous culturing device with biomass retention) under electron acceptor and electron donor limitation enabled growth rates as low as 0.0008 h⁻¹. The maximum growth yield was between 0.05 and 0.09 C-mol biomass per C-mol acetate and comparable to that observed in batch experiments. Maintenance energy demand is among the lowest reported for heterotrophic bacteria, under both acetate and AQDS limitation. The cells were able to use alternative electron acceptors directly, without requiring de novo protein synthesis. We discuss how the extremely low maintenance energy demand and the ability to readily use alternative electron acceptors may help Geobacter species to become ubiquitous and dominant microorganisms in many iron-reducing subsurface settings.     

Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida.

beta-Ketoadipate:succinyl-coenzyme A transferase (beta-ketoadipate:succinyl-CoA transferase) (EC 2.8.3.6) carries out the penultimate step in the conversion of benzoate and 4-hydroxybenzoate to tricarboxylic acid cycle intermediates in bacteria utilizing the beta-ketoadipate pathway. This report describes the characterization of a DNA fragment from Pseudomonas putida that encodes this enzyme. The fragment complemented mutants defective in the synthesis of the CoA transferase, and two proteins of sizes appropriate to encode the two nonidentical subunits of the enzyme were produced in Escherichia coli when the fragment was placed under the control of a phage T7 promoter. DNA sequence analysis revealed two open reading frames, designated pcaI and pcaJ, that were separated by 8 bp, suggesting that they may comprise an operon. A comparison of the deduced amino acid sequence of the P. putida CoA transferase genes with the sequences of two other bacterial CoA transferases and that of succinyl-CoA:3-ketoacid CoA transferase from pig heart suggests that the homodimeric structure of the mammalian enzyme may have resulted from a gene fusion of the bacterial alpha and beta subunit genes during evolution. Conserved functional groups important to the catalytic activity of CoA transferases were also identified.     

A specialized citric acid cycle requiring succinyl-coenzyme A (CoA):acetate CoA-transferase (AarC) confers acetic acid resistance on the acidophile Acetobacter aceti

Microbes tailor macromolecules and metabolism to overcome specific environmental challenges. Acetic acid bacteria perform the aerobic oxidation of ethanol to acetic acid and are generally resistant to high levels of these two membrane-permeable poisons. The citric acid cycle (CAC) is linked to acetic acid resistance in Acetobacter aceti by several observations, among them the oxidation of acetate to CO2 by highly resistant acetic acid bacteria and the previously unexplained role of A. aceti citrate synthase (AarA) in acetic acid resistance at a low pH. Here we assign specific biochemical roles to the other components of the A. aceti strain 1023 aarABC region. AarC is succinyl-coenzyme A (CoA):acetate CoA-transferase, which replaces succinyl-CoA synthetase in a variant CAC. This new bypass appears to reduce metabolic demand for free CoA, reliance upon nucleotide pools, and the likely effect of variable cytoplasmic pH upon CAC flux. The putative aarB gene is reassigned to SixA, a known activator of CAC flux. Carbon overflow pathways are triggered in many bacteria during metabolic limitation, which typically leads to the production and diffusive loss of acetate. Since acetate overflow is not feasible for A. aceti, a CO(2) loss strategy that allows acetic acid removal without substrate-level (de)phosphorylation may instead be employed. All three aar genes, therefore, support flux through a complete but unorthodox CAC that is needed to lower cytoplasmic acetate levels.     

Flux analysis of central metabolic pathways in Geobacter metallireducens during reduction of soluble Fe(III)-nitrilotriacetic acid

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.     

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using ¹³C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO₂ via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.     

Plutonium(V/VI) Reduction by the Metal-Reducing Bacteria Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1

We examined the ability of the metal-reducing bacteria Geobacter metallireducens GS-15 and Shewanella oneidensis MR-1 to reduce Pu(VI) and Pu(V). Cell suspensions of both bacteria reduced oxidized Pu [a mixture of Pu(VI) and Pu(V)] to Pu(IV). The rate of plutonium reduction was similar to the rate of U(VI) reduction obtained under similar conditions for each bacteria. The rates of Pu(VI) and U(VI) reduction by cell suspensions of S. oneidensis were slightly higher than the rates observed with G. metallireducens. The reduced form of Pu was characterized as aggregates of nanoparticulates of Pu(IV). Transmission electron microscopy images of the solids obtained from the cultures after the reduction of Pu(VI) and Pu(V) by S. oneidensis show that the Pu precipitates have a crystalline structure. The nanoparticulates of Pu(IV) were precipitated on the surface of or within the cell walls of the bacteria. The production of Pu(III) was not observed, which indicates that Pu(IV) was the stable form of reduced Pu under these experimental conditions. Experiments examining the ability of these bacteria to use Pu(VI) as a terminal electron acceptor for growth were inconclusive. A slight increase in cell density was observed for both G. metallireducens and S. oneidensis when Pu(VI) was provided as the sole electron acceptor; however, Pu(VI) concentrations decreased similarly in both the experimental and control cultures.Effective bioremediation and waste management strategies at nuclear sites require an understanding of the fundamental biogeochemical processes that control the mobility of actinides. Microorganisms can influence the chemical speciation, valence state, and distribution of actinides in subsurface environments (2, 8, 12, 14). Dissimilatory metal-reducing bacteria (DMRB), which derive energy by respiring oxidized metals (Fe and Mn in nature), may play a particularly important role in the mobility of actinides, since the oxidized forms of many radionuclides are more mobile than their reduced forms. Remedial strategies have been proposed to biomineralize radionuclides via direct reduction by DMRB or indirectly by DMRB by-products (9-11). Several DMRB have been shown to conserve energy for anaerobic growth via the reduction of U(VI) (9-11, 14).Plutonium redox chemistry is more complex than that of most other actinides. Under environmental conditions, plutonium can exist in the III, IV, V, and VI oxidation states, and multiple oxidation states can coexist simultaneously (4, 5). The oxidized species of plutonium [Pu(V) and Pu(VI)] generally are much more soluble than the reduced species (4). Predicting the influence DMRB have on plutonium biogeochemistry is complicated by the fact that both Pu(III) and Pu(IV) are possible products of biological reduction. Also, the presence of chelating ligands can greatly influence the oxidation state formed during reduction as well as the reduction rate. The reduction of oxidized Pu species to Pu(IV) is desired, because it is highly insoluble and not very mobile. However, in the presence of complexing ligands and under reducing conditions the production of Pu(III) is favored, and Pu(III) complexes can be quite soluble (2). The conditions leading to the reduction of Pu(V) and Pu(VI) need to be understood and controlled so that they do not lead to the production of Pu(III), if the biological reduction of Pu(V) or Pu(VI) is to be used as an effective remediation strategy.There is little information available concerning the influence DMRB have on plutonium biogeochemistry. Few previous studies have reported the biological reduction of Pu(IV) to Pu(III) (2, 7, 16). During the earlier experiments (16), the solubilization of PuO₂ increased approximately ∼40% in solutions with DMRB. In solutions with DMRB and nitrilotriacetic acid (NTA), approximately 90% of the available Pu was solubilized, but the production of Pu(III) was not observed in any of the cultures, either with or without NTA added (16). The enhanced solubility of Pu was attributed to Pu(IV) reduction, the solubilization of resultant Pu(III), and the reoxidation of Pu(III) to Pu(IV) with the NTA complexation of Pu(III). Since Pu(III) was not observed, the biological reduction of Pu(IV) was inferred from the data (16). The biological reduction of Pu(IV) to Pu(III) was first conclusively documented with the production of Pu(III) in monocultures of G. metallireducens GS-15 and S. oneidensis MR-1 both with and without the addition of a chelating agent (EDTA) (2). In experiments without EDTA, the aqueous concentration of Pu(III) in DMRB cultures was very low (<0.05 mM Pu) (2). The aqueous concentration of Pu(III) increased to approximately 60 to 80% (0.3 to 0.4 mM Pu) of the total Pu(IV) when EDTA was added to the cultures (2). To our knowledge, there are no published studies documenting the biological reduction of Pu(V) or Pu(VI) to either Pu(IV) or Pu(III). However, based on thermodynamics calculations, the reduction of Pu(V) and Pu(VI) by DMRB should be possible and yield greater energy for the bacteria than Pu(IV) reduction (2).The study presented here was designed first to assess the ability of G. metallireducens GS-15 and S. oneidensis MR-1 to reduce Pu(V) and Pu(VI) in monocultures under cell resting and growth conditions. Second, the aqueous and solid phases produced during the experiments were analyzed to determine the extent of biological reduction [i.e., to Pu(IV) or Pu(III)].     

Development of a semiquantitative degenerate real-time pcr-based assay for estimation of numbers of butyryl-coenzyme A (CoA) CoA transferase genes in complex bacterial samples

We describe a new degenerate real-time PCR approach to simultaneously quantify phylogenetically different butyrate-producing bacteria based on the detection of butyryl-coenzyme A (CoA) CoA transferase genes. This pathway is present in numerically important groups of butyrate producers within the human colon, and thus this assay estimates the butyrate-producing ability of the microbiota.