Diverse Energy-Conserving Pathways in Clostridium difficile: Growth in the Absence of Amino Acid Stickland Acceptors and the Role of the Wood-Ljungdahl Pathway

Abstract

Clostridium difficile is the leading cause of hospital-acquired antibiotic-associated diarrhea and is the only widespread human pathogen that contains a complete set of genes encoding the Wood-Ljungdahl pathway (WLP). In acetogenic bacteria, synthesis of acetate from 2 CO₂ molecules by the WLP functions as a terminal electron accepting pathway; however, C. difficile contains various other reductive pathways, including a heavy reliance on Stickland reactions, which questions the role of the WLP in this bacterium. In rich medium containing high levels of electron acceptor substrates, only trace levels of key WLP enzymes were found; therefore, conditions were developed to adapt C. difficile to grow in the absence of amino acid Stickland acceptors. Growth conditions were identified that produce the highest levels of WLP activity, determined by Western blot analyses of the central component acetyl coenzyme A synthase (AcsB) and assays of other WLP enzymes. Fermentation substrate and product analyses, enzyme assays of cell extracts, and characterization of a ΔacsB mutant demonstrated that the WLP functions to dispose of metabolically generated reducing equivalents. While WLP activity in C. difficile does not reach the levels seen in classical acetogens, coupling of the WLP to butyrate formation provides a highly efficient system for regeneration of NAD⁺ "acetobutyrogenesis," requiring only low flux through the pathways to support efficient ATP production from glucose oxidation. Additional insights redefine the amino acid requirements in C. difficile, explore the relationship of the WLP to toxin production, and provide a rationale for colocalization of genes involved in glycine synthesis and cleavage within the WLP operon.IMPORTANCEClostridium difficile is an anaerobic, multidrug-resistant, toxin-producing pathogen with major health impacts worldwide. It is the only widespread pathogen harboring a complete set of Wood-Ljungdahl pathway (WLP) genes; however, the role of the WLP in C. difficile is poorly understood. In other anaerobic bacteria and archaea, the WLP can operate in one direction to convert CO₂ to acetic acid for biosynthesis or in either direction for energy conservation. Here, conditions are defined in which WLP levels in C. difficile increase markedly, functioning to support metabolism of carbohydrates. Amino acid nutritional requirements were better defined, with new insight into how the WLP and butyrate pathways act in concert, contributing significantly to energy metabolism by a mechanism that may have broad physiological significance within the group of nonclassical acetogens.

Keywords: CO dehydrogenase; CODH; Clostridium difficile; Stickland reaction; Wood-Ljungdahl pathway; acetate; acetyl-CoA synthase; acsB; butyrate; carbon monoxide dehydrogenase.

FIG 1

(A) The Wood-Ljungdahl pathway and the conversion of its product acetyl-CoA to ethanol, acetate, and butyrate. Enzyme abbreviations are given in blue and gene names are in black with locus tags (19) in red. H₄F stands for tetrahydrofolate. Separate methyl and carbonyl branches converge in the formation of acetyl-CoA. In the methyl branch, a reversible hydrogen-dependent CO₂ reductase complex (HDCR), which contains formate dehydrogenase (fdh) and [Fe-Fe] hydrogenase components, reduces CO₂ to formate using H₂ as its direct electron donor. Formate is activated to produce N¹⁰-formyl-H₄F (HCO-H₄F) in an ATP-requiring reaction by N¹⁰-formyl-H₄F synthetase (FTS). N⁵,N¹⁰-methenyl-H₄F (CH≡H₄F) is then generated via cyclization and dehydration by N⁵,N¹⁰-methenyl-tetrahydrofolate 5-hydrolase (formyl-H₄F cyclohydrolase; fchA) (MTC). Bifunctional FolD protein, which includes N⁵,N¹⁰-methylene-H₄F dehydrogenase (MTD) and additional formyl-H₄F cyclohydrolase activity, converts CH≡H₄F to N⁵,N¹⁰-methylene-H₄F (CH₂=H₄F), which is then further reduced to N⁵-methyl-H₄F (CH₃-H₄F) by N⁵,N¹⁰-methylene-H₄F reductase (MTR) with catalytic subunit MetF and an accessory Fe/S- and Zn-containing subunit MetV. CH₃-H₄F then transfers its methyl group to a corrinoid iron-sulfur protein to form Co³⁺-methyl corrinoid iron-sulfur protein (CH₃-CFeSP) catalyzed by CH₃-H₄F:corrinoid methyltransferase (MeTr). In the carbonyl branch, carbon monoxide dehydrogenase (CODH) produces CO contained within a protein channel, depicted as [CO], by reduction of CO₂ using low-potential reduced ferredoxin (${\text{Fd}}_{\text{red}}^{-2}$). Acetyl-CoA, shown at the center in the shaded oval, is then produced by condensation of the corrinoid methyl and channel-bound CO groups by acetyl-CoA synthase (ACS) acsB. In the pathway leading to ethanol, acetyl-CoA is reduced in two steps by bifunctional acetaldehyde-CoA/alcohol dehydrogenase (AdhE). An alternative pathway would be to convert acetate to ethanol by way of aldehyde dehydrogenase (Aldh). Acetate formation from acetyl-CoA is via phosphotransacetylase (Pta/Ptb) that produces acetyl phosphate (CD2683 is annotated in the KEGG database as a putative phosphotransacetylase ortholog, and other candidates potentially active with acetate could include phosphotransbutyrylase CD0112 and the two other annotated ptb homologs CD0715 and CD2425). Acetyl phosphate is then used to generate ATP by acetate kinase or butyrate kinase active with acetate (Ack/Buk). Formation of butyrate follows the typical pathway in which thiolase (acetyl-CoA acetyltransferase) (THL) generates acetoacetyl-CoA that is acted on by 3-hydroxybutyryl-CoA dehydrogenase (HBD) followed by 3-hydroxybutyryl-CoA dehydratase (crotonase) (CRT) to form crotonyl-CoA. The electron-bifurcating butyryl-CoA dehydrogenase (Bcd) CD1054 in complex with electron-transferring flavoprotein subunits EtfB and EtfA (CD1055 and CD1056), complex (Bcd-EtfAB)₄, then generates butyryl-CoA (75–77). Thereafter, butyrate is produced by butyryl-CoA:acetate CoA-transferase activity (CoA transfer) using free acetate to reform one molecule of acetyl-CoA. (B) The Wood-Ljungdahl pathway operon in C. difficile. A 15-gene, 18.4-kb region (CD0716 to CD0730) is shown according to data in references and . Gene designations and corresponding products are indicated.

FIG 2

Trace levels of acetyl-CoA synthase (AcsB) detected during growth of C. difficile in BHIS. Growth was measured on four separate BHIS cultures prepared as described in Materials and Methods. (Inset) Western blot of a 12% acrylamide SDS-PAGE gel with samples taken from cultures at the times indicated (hours). Ten nanograms of purified recombinant AcsB was included as a reference on the outside lanes.

FIG 3

Adaptation of C. difficile to growth on glucose in the absence of proline and leucine. To adapt cells to grow without proline and leucine, cells harvested from a BHIS day culture were used to inoculate adaptation medium (ADM) which contained 5 mM glucose, 10 mM glycine, and low concentrations of leucine, isoleucine, valine, proline, methionine, and tryptophan in a basal salt mixture supplemented with trace elements and vitamins, as described in Materials and Methods. Growths were monitored in triplicate by OD₆₀₀ (*, □, and ×), aliquots were removed over time and centrifuged, and the supernatants were assayed for amino acids and glucose. (A) Concentrations are given for glucose (Glc), glycine (Gly), alanine (Ala), and ammonia (NH₃). (B) The same cultures as in panel A, assayed for leucine (Leu), isoleucine (Ile), valine (Val), proline (Pro), and 5-aminovalerate (5-AV).

FIG 4

Glycine-alanine fermentation of C. difficile growing in the absence of proline and leucine. Cells obtained from ADM cultures grown to an OD₆₀₀ of ca. 0.6 were used to inoculate glycine-alanine (Gly/Ala) medium lacking proline and leucine and in which glycine was the only remaining Stickland acceptor (see Materials and Methods). The growth substrates available in Gly/Ala medium were 5 mM glucose, 10 mM glycine, 10 mM alanine, and low concentrations of isoleucine, valine, methionine, and tryptophan. In addition, 2 mM sodium acetate and 3.9 mM NH₄Cl were present. Triplicate growths were monitored, and amino acids and glucose were assayed as indicated for Fig. 3. OD₆₀₀ (*, □, and ×) and concentrations of glucose (Glc), glycine (Gly), alanine (Ala), and ammonia (NH₃) are shown.

FIG 5

Growth of C. difficile on glucose in the absence of Stickland acceptors. A glucose-containing medium in which both glycine and alanine were omitted (Glc only) was used to grow C. difficile in the absence of all amino acid Stickland acceptors. Glc-only medium was similar to Gly/Ala medium without glycine and alanine. OD₆₀₀ (* and □) and concentrations (mM) of glucose (Glc), glycine (Gly), alanine (Ala), and ammonia (NH₃) are shown. Gly/Ala cultures were used to inoculate the Glc-only tubes, accounting for the small amounts of glycine and alanine present initially.

FIG 6

Western blot analysis of AcsB levels in different growth media. Cell extracts prepared from the large-scale growths indicated were analyzed by SDS-PAGE. Gels containing 12.5 μg total protein per lane were stained with Coomassie blue for protein (bottom, lanes 1 to 6), and 37.5 μg per lane of the same samples was applied for Western blot assays of AcsB (top). Gly/Ala 2.2 and Gly/Ala 0.5 indicate Gly/Ala growths with 2.2 and 0.5 mM glucose remaining at the time of harvest. Purified AcsB, 10 ng, was applied on the outer lanes of the Western blot, and molecular weight markers (M) were included on the stained gel. The estimation of equivalent amounts of protein loaded was verified by densitometric analysis of the stained gel (center).

FIG 7

Enzyme activities in C. difficile that reflect metabolic pathways active under different growth conditions. Enzymes involved in a number of key pathways were assayed in anaerobic cell extracts prepared from large-scale growths of C. difficile in BHIS and defined media indicated at the top. Gly/Ala 2.2 and Gly/Ala 0.5 indicate Gly/Ala growths with 2.2 and 0.5 mM glucose remaining at the time of harvest. The substrates and products in bold font were measured directly in the assays described in Materials and Methods. Enzyme specific activities are given in units per milligram extract protein. MV, methyl viologen; BV, benzyl viologen; H₄F, tetrahydrofolate.

FIG 8

C. difficile wild-type and ΔacsB mutant growth, glucose consumption, and acetate production in glucose-only (Glc-only) medium under acetate-limiting conditions. Glc-only medium containing decreasing amounts of sodium acetate was used to compare growth characteristics of the wild type (closed symbols) and the ΔacsB mutant (open symbols). Graphs on the left show growth (OD₆₀₀, black symbols) and glucose consumption (red symbols), whereas those on the right show acetate production (blue symbols) from the same growths and the corresponding glucose consumption curves for comparison.

FIG 9

Fermentation product comparisons of C. difficile wild type and ΔacsB mutant. Samples removed from cultures of C. difficile wild type and ΔacsB mutant grown in Glc-only medium at different levels of added acetate (0 to 2 mM), presented in Fig. 8, were taken over time and analyzed for glucose and volatile organic compounds by GC, HPLC, and enzymatic assays, as described in Materials and Methods. The maximum rates of glucose consumption during the growths are compared, along with the final concentrations of products acetate, butyrate, lactate, ethanol, and formate present at the end of the fermentations. The averages from duplicate cultures are plotted, error bars indicate the differences between them.

FIG 10

Relative TcdA toxin levels in cells grown in BHIS and in defined media. Cell extracts prepared from the large-scale growths indicated were analyzed by SDS-PAGE on an 8% acrylamide gel with equivalent amounts of protein, 37.5 μg, applied per lane. The loading pattern was the same as for Fig. 6. Western blot analysis employed mouse monoclonal anti-TcdA as the primary antibody and was developed and imaged as indicated in Materials and Methods. Relative TcdA band intensities were 10.8, 13.0, 1.00, 1.66, 2.11, and 1.55 for the BHIS 12 h, BHIS 17 h, Glc+Gly, Glc-only, Gly/Ala 2.2, and Gly/Ala 0.5 extracts, respectively.

FIG 11

Approximate ATP yields from glucose in various anaerobic fermentation pathways and using the Wood-Ljungdahl pathway coupled to butyrate formation. The efficiencies of ATP production from glucose are compared. For simplicity, contributions from chemiosmotic mechanisms and membrane complexes, such as Rnf, are excluded. In general, the efficiency of ATP production is highest when organic products of the pathway are not required to serve as electron acceptors, which increases the amount of acetyl-CoA available to produce ATP via SLP. A maximal level of 4.0 ATPs/glucose is expected, provided that another pathway is used to fully regenerate NAD⁺ and assuming that pyruvate is converted to acetyl-CoA without producing additional redox equivalents either as NADH or reduced ferredoxin. This assumption is reasonable for C. difficile given that formate is produced even in the absence of added H₂ and that glycine and alanine are consumed in a 1:1 stoichiometry in the Gly/Ala fermentation (see Results and Fig. 4). By comparison, the lactic acid fermentation yields the lowest ATP/glucose ratio of 2.0, because no SLP from acetyl-CoA is possible. Diversion of one-half of the acetyl-CoA to ethanol regenerates the necessary amount of NAD⁺ and improves the ratio to 3.0. Connection of the WLP pathway to the pathway for butyrate formation from acetyl-CoA results in a further increase to 3.6, closer to the theoretical maximum. A total of 5 NADHs are consumed per mol butyrate produced by this arrangement: 2 NADHs are used by the WLP to produce each acetate, which enters into the butyryl-CoA:acetate CoA transferase reaction to form butyrate and regenerate acetyl-CoA, and 3 NADHs are taken up for each acetoacetyl-CoA converted to butyryl-CoA (1 NADH from the 3-hydroxybutyryl-CoA dehydrogenase reaction, and 2 NADHs from the reduction of crotonyl-CoA involving electron bifurcation by the Bcd-EtfAB complex that also conserves energy by producing 1 ${\text{Fd}}_{\text{red}}^{-2}$, which is then used to reduce CO₂ to CO in the WLP). However, only 2 NAD⁺s instead of 5 are needed per glucose oxidized so that formation of 0.4 mol butyrate is sufficient to balance the fermentation using butyrate/WLP coupling.