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. 2007 Dec 14;74(5):1447–1452. doi: 10.1128/AEM.01421-07

Degradation of Glyoxylate and Glycolate with ATP Synthesis by a Thermophilic Anaerobic Bacterium, Moorella sp. Strain HUC22-1▿

Shinsuke Sakai 1, Kentaro Inokuma 1, Yutaka Nakashimada 1,†, Naomichi Nishio 1,*
PMCID: PMC2258637  PMID: 18083850

Abstract

The thermophilic homoacetogenic bacterium Moorella sp. strain HUC22-1 ferments glyoxylate to acetate roughly according to the reaction 2 glyoxylate → acetate + 2 CO2. A batch culture with glyoxylate and yeast extract yielded 11.7 g per mol of cells per substrate, which was much higher than that obtained with H2 plus CO2. Crude extracts of glyoxylate-grown cells catalyzed the ADP- and NADP-dependent condensation of glyoxylate and acetyl coenzyme A (acetyl-CoA) to pyruvate and CO2 and converted pyruvate to acetyl-CoA and CO2, which are the key reactions of the malyl-CoA pathway. ATP generation was also detected during the key enzyme reactions of this pathway. Furthermore, this bacterium consumed l-malate, an intermediate in the malyl-CoA pathway, and produced acetate. These findings suggest that Moorella sp. strain HUC22-1 can generate ATP by substrate-level phosphorylation during glyoxylate catabolism through the malyl-CoA pathway.

Glyoxylate, which occurs in green leaves and unripe fruits of many plants and is excreted by certain algal species (37), is often found in forest soils, rhizosphere soils, and leaf molds (14, 34, 39). It is proposed that glyoxylate and other organic acids such as citrate and oxalate (18, 32) increase the solubility of trace metals for plants. Glyoxylate, which is an important intermediate in various metabolic pathways such as the glyoxylate cycle (43), is used by many bacteria as a source of carbon (3, 20, 23-25). In various environments, glyoxylate is finally oxidized, either through formate or not, to CO2 and H2O. This method of oxidation has also been reported as an intermediate step in the oxidation of glyceric acid and allantoin by Pseudomonas spp. and other bacteria isolated from soils (3, 5, 6).

Anaerobic acetogenic bacteria (acetogens) generate acetate as a reduced fermentation product through ATP synthesis by using CO2 as an electron acceptor (13). Such bacteria can metabolize a broad range of organic and inorganic substrates in energy metabolism (10). A thermophilic acetogen, Moorella thermoacetica, utilizes glyoxylate and related two-carbon substrates, such as glycolate and oxalate (9, 34, 35). Some acetogens, including M. thermoacetica, have been isolated from anoxic habitats such as those found in soils, sediments, and the gastrointestinal tracts of humans and animals (13, 19). On the other hand, glycolate, a glyoxylate-related two-carbon substrate, has been observed in thermophilic environments such as the surface of the 55°C cyanobacterial mat in Mushroom Spring in Yellowstone National Park (4). These findings suggest that acetogens participate in the turnover of two-carbon substrates, including glyoxylate in soils and spring sediment environments (34). In addition, acetate produced by acetogens from organic substrates is considered to play an important role in the turnover of carbon in anaerobic soil aggregates (26). However, the mechanics of catabolism of glyoxylate, glycolate, and oxalate by Moorella species is yet to be resolved.

Several pathways for glyoxylate metabolism have been reported. In Escherichia coli, the dicarboxylic acid and the tricarboxylic acid cycles contribute to the oxidative catabolism of glyoxylate under aerobic conditions (25, 31). Glyoxylate is also metabolized by the glycerate pathway and the tricarboxylic acid cycle in E. coli (20). An aerobic pathway similar to the glycerate pathway has been reported in a Pseudomonas sp. (23). In Micrococcus denitrificans, the β-hydroxyaspartate pathway is aerobic (24), where 1 mol of oxaloacetate, an intermediate of the tricarboxylic acid cycle, is directly synthesized from 2 mol of glyoxylate. The anaerobic glycerate and serine pathways are found in anaerobic oxalate-degrading bacterium Oxalobacter formigenes (7). The malyl coenzyme A (malyl-CoA) pathway is also an anaerobic glyoxylate oxidation pathway, found in the unidentified bacterium strain PerGlyox1 (16).

This study, through the measurement of enzyme activities, demonstrates that a thermophilic anaerobic acetogen, Moorella sp. strain HUC22-1 isolated in our laboratory (33), possesses the malyl-CoA pathway for the degradation of both glyoxylate and glycolate. In addition, we report for the first time that thermophilic acetogens can utilize l-malate as well as can previously reported mesophilic acetogens such as Acetobacterium sp. strain AmMan1 and Acetobacterium malicum (38).

MATERIALS AND METHODS

Microorganism and culture medium.

The microorganism used in this study was Moorella sp. strain HUC22-1, which was isolated in our laboratory from a mud sample from Japan (33).

ATCC 1754 PETC medium (http://www.atcc.org) constituted the basal medium to which yeast extract and cysteine·HCl·H2O were added at ratios of 1 and 0.3 g per liter, respectively. Fructose and Na2S·9H2O were eliminated from this medium. The initial pH of the medium was adjusted to 6.3.

Batch culture using serum bottles.

A modified Hungate technique (22) in combination with serum bottles (29) was used for both the subcultures and the batch cultures. These cultures were incubated at 55°C in 125-ml serum bottles containing 25 ml basal medium supplemented with filter-sterilized substrates (glycolate, glyoxylate, l-malate, and oxalate). Free acids were neutralized with 5 N NaOH whenever necessary. When the microorganism was grown on H2 plus CO2, the bottles were flushed and brought to a final pressure of 0.2 MPa with a filter-sterilized gas mixture (80:20, vol/vol) after inoculation. The bottles were then incubated at 55°C with shaking at the rate of 135 strokes per minute.

Preparation of cell extracts and enzyme assays.

Cells were harvested during the mid-exponential growth phase by centrifugation (3,000 × g, 10 min, 4°C). The cells were washed twice and resuspended in 2 ml of anoxic 125 mM triethanolamine buffer of pH 7.4 to avoid precipitation of Mg- or Mn-phosphates. The suspended cells were lysed anoxically according to a method described previously (33).

Malyl-CoA lyase (EC 4.1.3.24) activity was measured by the rate of phenylhydrazone reduction at 324 nm (ɛ324 = 15,000 M−1 cm−1 [28]), as described by Herter et al. (21).

The activity of the malyl-CoA lyase/malyl-CoA synthetase (EC 6.2.1.9)/malic enzyme (EC 1.1.1.38 and EC 1.1.1.40) system was determined as a reaction chain. The reaction involved the conversion of acetyl-CoA and glyoxylate to l-malate via malyl-CoA with the reaction between malyl-CoA lyase, malyl-CoA synthetase, and malic enzyme. The cell extracts contained the activities of all the three enzymes. The activity of this enzyme system was measured by the rate of NAD+ or NADP+ reduction (ɛ340 = 6.3 mM−1 cm−1), as described by Friedrich and Schink (16). The reverse reaction involved the conversion of l-malate to acetyl-CoA and glyoxylate via malyl-CoA with the malyl-CoA synthetase and malyl-CoA lyase reactions. The activities of both malyl-CoA lyase and malyl-CoA synthetase were measured by the rate of phenylhydrazone formation at 324 nm [ɛ324 = 15,000 M−1 cm−1 [28]), as described by Tuboi and Kikuchi (42).

Malic enzyme activity was measured by the rate of NAD+ or NADP+ reduction, as described by Stams et al. (36).

Pyruvate synthase (EC 1.2.7.1) activity was measured by the rate of benzyl viologen reduction with pyruvate (ɛ578 = 8.65 mM−1 cm−1), as described by Odom and Peck (30).

Oxaloacetate decarboxylase (EC 4.1.1.2) activity was measured by the rate of NADH or NADPH oxidation (ɛ340 = 6.3 mM−1 cm−1), as described by Dimroth (11).

Glyoxylate reductase (EC 1.1.1.26) activity was measured by the rate of NADH or NADPH oxidation, as described by Zeltich (44).

Glycolate dehydrogenase (EC 1.1.99.14) activity was measured with various electron acceptors (NAD+, NADP+, methylene blue, methyl viologen, and benzyl viologen), as described by Friedrich and Schink (17).

The protein concentrations in the cell extracts were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). All assays were carried out in triplicate under anaerobic conditions with the appropriate controls at 45°C.

ATP determination.

The reaction of the malyl-CoA lyase/malyl-CoA synthetase system was carried out at 45°C. The reaction mixture of 1 ml contained 100 mM of potassium phosphate buffer (pH 7.5), 10 mM of MgCl2, 2 mM of glyoxylate, 4 mM of acetyl-CoA, 5 mM of ADP, and cell extracts. Samples of 25 μl were withdrawn and cooled on ice to stop the reaction. The amount of ATP generated was measured by luciferase-driven bioluminescence using the CLS II ATP bioluminescence assay kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. The intensity of luminescence was monitored using the Wallac ARVO SX 1420 multilabel counter (Perkin-Elmer, Waltham, MA), and the data were normalized to the protein content.

Additional analytical methods.

Cell growth was monitored turbidimetrically by optical density at 660 nm (1 unit = 0.46 g dry cells per liter). The maximum specific growth rate was calculated from the exponential growth phase.

Acetate, glycolate, glyoxylate, l-malate, and oxalate concentrations were measured using high-performance liquid chromatography (27). The concentrations of H2 and CO2 in the gas phase were measured as described previously (33).

RESULTS

Energetics of growth on glyoxylate and related substrates.

In glyoxylate with yeast extract, the growth yield per substrate (YX/S) was 11.7 g per mol, while YX/S was 0.68 g per mol of hydrogen in H2 plus CO2 with yeast extract (Table 1). If strain HUC22-1 mainly uses the Wood-Ljungdahl pathway during glyoxylate degradation, then YX/S in glyoxylate cultures should be at the most twice that in H2-plus-CO2 cultures, because 1 mol of glyoxylate is equal to 2 mol of H2 and both substrates are used only for the catabolism in the complex medium containing yeast extract. This suggests that the microorganism possessed an unidentified pathway for ATP generation in addition to the Wood-Ljungdahl pathway in glyoxylate cultures. This hypothesis is supported by the calculation based on the free energy change of ATP hydrolysis and the degradation of glyoxylate to H2 plus CO2 by the following reaction: C2HO3− + 3H2O → 2HCO3− + 2H2 + H+ (ΔG°′ = −34 kJ reaction−1). Since the ΔG°′ of ATP hydrolysis is −31.8 kJ mol−1 (40), theoretically 1.1 mol of additional ATP can be obtained through the degradation of glyoxylate to H2 plus CO2. In a previous study of hexose fermentation by M. thermoacetica (former name, Clostridium thermoaceticum), Andreesen et al. used a YATP of 10.5 for calculation of cell yield per substrate and speculated that there was an unknown source of ATP (2). The YATP in glyoxylate might be lower than that in hexose, since glyoxylate is a simpler substrate than hexose and the YATP of Acetobacterium woodii in H2 plus CO2 with yeast extract is calculated to be 4.3 (41). If the YATP of strain HUC22-1 in glyoxylate with yeast extract is between 4.3 and 10.5, then the YATP/S in glyoxylate with yeast extract for HUC22-1 is calculated to be between 2.7 and 1.1. The value calculated using a YATP of 10.5 is in good agreement with the theoretical ATP yield per glyoxylate as presented above.

TABLE 1.

Fermentation profiles and various yields in batch culture of Moorella sp. strain HUC22-1 grown at the expense of glyoxylate, l-malate, and other substrates with yeast extracta

Substrate Culture time (h) Dry cells (g liter−1) Substrate degraded (mM) Product formed (mM)
Acetate yield per substrate (YP/S) (mol mol−1) Cell yield per substrate (YX/S) (g mol−1) Electron recoveryb (%) Carbon recoveryc (%)
Acetate Ethanol
Glyoxylate 72 0.15 ± 0.01 12.8 ± 1.0 5.8 ± 0.7 ND 0.46 ± 0.02 11.7 ± 0.06 91 ± 3 96 ± 2
Glycolate 72 0.12 ± 0.01 17.5 ± 0.7 11.1 ± 1.2 ND 0.64 ± 0.05 7.02 ± 0.31 85 ± 6 89 ± 5
Oxalate 72 0.11 ± 0.02 19.3 ± 1.7 4.2 ± 0.5 ND 0.22 ± 0.01 5.64 ± 0.54 86 ± 2 97 ± 1
l-Malate 60 0.16 ± 0.01 20.2 ± 0.3 27.6 ± 0.3 ND 1.36 ± 0.02 7.80 ± 0.36 91 ± 2 93 ± 1
H2-CO2 108 0.17 ± 0.01 260 ± 20 (H2) 56.7 ± 2.1 1.5 ± 0.3 0.22 ± 0.01 (H2) 0.68 ± 0.04 (H2) 92 ± 5 95 ± 9
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a

Cultures were incubated in 125-ml serum bottles (medium, 25 ml; initial pH, 6.3) containing H2 and CO2 (80:20, vol/vol; 0.2 MPa) or 20 to 40 mM other substrate and kept at 55°C. Values represent the averages and standard deviations from at least three replicate experiments. ND, no compound was detected.

b

Electron recovery was calculated from electron equivalents of substrate and products.

c

Carbon recovery was calculated from substrate and products, including an amount of CO2 estimated according to the stoichiometry (34, 38).

Among several pathways previously reported for glyoxylate degradation, the malyl-CoA pathway is a candidate for glyoxylate catabolism in Moorella sp. strain HUC22-1. When 1 mol of glyoxylate is oxidized to 2 mol of CO2 by the malyl-CoA pathway, 2 mol of electron acceptors is reduced. Since 2 mol of reducing equivalents is used for CO2 reduction to acetate stoichiometrically, 0.5 mol of acetate should be produced through 1 mol of glyoxylate oxidation. In a batch culture of strain HUC22-1 with glyoxylate and yeast extract, the acetate yield was 0.46 mol per mol of glyoxylate (Table 1). This is in good agreement with the theoretical value. Furthermore, the malyl-CoA pathway can generate 1 mol of ATP by substrate-level phosphorylation coupled to the reaction of malyl-CoA lyase/malyl-CoA synthetase during the degradation of 1 mol of glyoxylate.

If glyoxylate is oxidized by the malyl-CoA pathway in strain HUC22-1, then this bacterium should use l-malate, which is a key intermediate in this pathway, although the utilization of l-malate by Moorella species has not been reported so far (12). When the batch culture was performed using serum bottles under basal medium with yeast extract and including l-malate, this bacterium consumed 20.2 mM of l-malate and produced 27.6 mM of acetate after 60 h of cultivation (Table 1). A maximum of 0.16 g per liter of cells (dry weight) was obtained after 60 h of cultivation. The maximum specific growth rate was 0.072 h−1. The acetate yield per l-malate degraded was 1.36 mol per mol, which was similar to that obtained for A. malicum grown on l-malate (38). M. thermoacetica DSM 521 and M. thermoautotrophica DSM 1974, which are closely related to strain HUC22-1, also utilized l-malate (data not shown).

Moorella sp. strain HUC22-1 also catabolizes glycolate and oxalate. In batch culture experiments with yeast extract, the YX/S in glycolate and oxalate cultures was 7.02 and 5.64 g per mol, respectively, which was much larger than that in H2-plus-CO2 cultures (Table 1). These findings also suggest that strain HUC22-1 possesses an unidentified pathway for ATP generation in addition to the Wood-Ljungdahl pathway, as for glyoxylate-grown cells.

Enzymatic analysis of the pathway for glyoxylate metabolism with ATP generation.

The activities of the enzymes involved in the malyl-CoA pathway were measured using cell extracts of strain HUC22-1 grown on glyoxylate and various other substrates with yeast extract, in order to ascertain whether HUC22-1 possesses the malyl-CoA pathway. The activity of the malyl-CoA lyase/malyl-CoA synthetase/malic enzyme system was significant in cells grown on glyoxylate and glycolate (Table 2). In l-malate- and oxalate-grown cells, this enzyme system had very low levels of activity. In H2-plus-CO2-grown cells, no activity of this enzyme system was observed. This enzyme system was ADP and NADP+ dependent and did not reduce NAD+. In addition, the CoA-SH- and ATP-dependent reverse reaction, forming acetyl-CoA and glyoxylate from l-malate, was also detected in cell extracts grown on glyoxylate and glycolate by monitoring phenylhydrazone formation as a glyoxylate trap (Table 2). Furthermore, the individual activity of malyl-CoA lyase was also detected in cell extracts grown on glyoxylate and glycolate and was an acetyl-CoA-dependent reaction (Table 2). NADP+-dependent malic enzyme activity was detected in cells grown on all substrates tested (Table 2). Moreover, the activity levels of this enzyme in glyoxylate- and glycolate-grown cells were higher than those with other substrates tested. No NAD+-dependent malic enzyme activity was detected in cells grown on any of the substrates tested. Similar levels of pyruvate synthase activity, which ranged from 0.54 to 0.42 μmol per min per mg protein, were observed in cells grown on all substrates tested (Table 2). NADH- and NADPH-dependent glyoxylate reductases, which catalyze the reduction of glyoxylate to glycolate, were detected in glyoxylate- and glycolate-grown cells, whereas no activities of these enzymes were observed with other substrates tested. No oxaloacetate-decarboxylating enzyme activity was detected in cells grown on any of the substrates tested (data not shown). In the dicarboxylic acid cycle found in E. coli (25), glyoxylate condenses with acetyl-CoA to form malate, which is oxidized via oxaloacetate and pyruvate to regenerate the acetyl-CoA required for the initial condensation. Since l-malate is not converted to oxaloacetate, it is assumed that strain HUC22-1 does not possess the dicarboxylic acid cycle with malate.

TABLE 2.

Enzyme activities detected in cell extracts of Moorella sp. strain HUC22-1 grown at the expense of glyoxylate, l-malate, and other substratesa

Enzyme and reaction Sp act (μmol min−1 mg protein−1) after growth on the following substrate:
Glyoxylate Glycolate Oxalate l-Malate H2-CO2
Malyl-CoA lyase (EC 4.1.3.24), glyoxylate + acetyl- CoA → l-malyl-CoA 10.0 (1.4) 20.9 (1.1) 0.54 (0.04) 5.6 (0.18) 1.6 (0.09)
Malyl-CoA lyase/malyl-CoA synthetase/malic enzyme (NAD+, forward direction), glyoxylate + acetyl- CoA + NAD+ + ADP + Pi → pyruvate + CO2 + NADH + CoA + ATP ND ND ND ND ND
Malyl-CoA lyase/malyl-CoA synthetase/malic enzyme (NADP+, forward direction), glyoxylate + acetyl-CoA + NADP+ + ADP + Pi → pyruvate + CO2 + NADPH + CoA + ATP 0.15 (0.005) 0.26 (0.013) 0.02 (0.002) 0.05 (0.003) ND
Malyl-CoA lyase/malyl-CoA synthetase (reverse direction), l-malate + CoA +ATP → glyoxylate + acetyl-CoA + ADP + Pi 0.23 (0.018) 0.38 (0.02) 0.04 (0.003) 0.10 (0.006) 0.08 (0.004)
Malic enzyme (EC 1.1.1.38 or EC 1.1.1.40)
    l-Malate + NAD+ → pyruvate + CO2 + NADH (EC 1.1.1.38) ND ND ND ND ND
    l-Malate + NADP+ → pyruvate + CO2 + NADPH (EC 1.1.1.40) 0.16 (0.031) 0.12 (0.01) 0.03 (0.001) 0.05 (0.003) 0.03 (0.004)
Pyruvate synthase (EC 1.2.7.1), pyruvate + CoA + BVox → acetyl-CoA + CO2 + BVred 0.44 (0.012) 0.54 (0.012) 0.42 (0.015) 0.48 (0.077) 0.48 (0.047)
Glyoxylate reductase (EC 1.1.1.26 or EC 1.1.1.79)
    Glyoxylate + NADH → glycolate + NAD+ (EC 1.1.1.26 or 79) 0.25 (0.011) 0.04 (0.006) ND ND ND
    Glyoxylate + NADPH → glycolate + NADP+ (EC 1.1.1.79) 0.31 (0.027) 0.10 (0.009) ND ND ND
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a

Cells were grown in serum bottles containing a basal medium with yeast extract at 55°C until the mid-exponential growth phase. Values represent the averages (standard deviations) from at least three replicate experiments. BVox, oxidized benzyl viologen; BVred, reduced benzyl viologen; ND, no activity was detected.

ATP generation during the malyl-CoA lyase/malyl-CoA synthetase reaction was measured by a luciferase-driven bioluminescence assay. In glyoxylate- and glycolate-grown cells, glyoxylate- and acetyl-CoA-dependent ATP generations of 0.50 and 0.98 μmol per min per mg protein, respectively, were detected (Table 3), which were significantly higher than those in oxalate- and l-malate-grown cells. However, a high level of glyoxylate- and acetyl-CoA-dependent ATP generation was also detected in H2-plus-CO2-grown cells. This is in disagreement with the results for the enzyme activities. The reason for this discrepancy is yet to be resolved.

TABLE 3.

ATP generation during malyl-CoA lyase/malyl-CoA synthetase reaction (forward direction) with cell extracts of Moorella sp. strain HUC22-1 grown at the expense of glyoxylate, l-malate, and other substratesa

Growth substrate ATP generation (μmol min−1 mg protein−1)
Glyoxylate 0.50 ± 0.07
Glycolate 0.98 ± 0.11
Oxalate ND
l-Malate 0.16 ± 0.01
H2-CO2 0.49 ± 0.05
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a

Cells were grown in serum bottles containing a basal medium with yeast extract at 55°C until the mid-exponential growth phase. Values represent the averages and standard deviations from at least three replicate experiments. ND, no compound was detected.

DISCUSSION

This study demonstrates that a thermophilic anaerobic acetogen, Moorella sp. strain HUC22-1, possesses the malyl-CoA pathway for glyoxylate degradation. The experimental results from the enzyme assay suggest that strain HUC22-1 catabolizes glyoxylate via the malyl-CoA pathway (Fig. 1). First, glyoxylate is condensed with acetyl-CoA to form malyl-CoA by malyl-CoA lyase. The formation of l-malate from malyl-CoA by malyl-CoA synthetase is ADP dependent. l-Malate is oxidized to pyruvate by NADP+-dependent malic enzyme. Finally, pyruvate synthase converts pyruvate to acetyl-CoA and CO2. Acetyl-CoA is used again for a new condensation with glyoxylate. Conversion of l-malate from glyoxylate yields 1 mol of ATP through substrate-level phosphorylation in the malyl-CoA pathway. Portions of the reducing equivalents and CO2 released in this pathway are assumed to be used for the Wood-Ljungdahl pathway. ATP synthesis during the reaction of malyl-CoA lyase/malyl-CoA synthetase was actually observed (Table 3) with the luciferase-driven bioluminescence assay.

FIG. 1.

FIG. 1.

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Proposed pathway for metabolism of glycolate and glyoxylate by Moorella sp. strain HUC22-1, showing a scheme proposed by Friedrich and Schink slightly modified from reference 17 with permission from Blackwell Publishing. 1, malyl-CoA lyase (EC 4.1.3.24); 2, malyl-CoA synthetase (EC 6.2.1.9); 3, malic enzyme (EC 1.1.1.40); 4, pyruvate synthase (EC 1.2.7.1). Fdox, oxidized ferredoxin; Fdred, reduced ferredoxin; X, unidentified electron acceptor.

Recently, the preliminary genome sequence of M. thermoacetica ATCC 39073 was published by the DOE Joint Genome Institute (http://www.jgi.doe.gov/index.html). In this database, the putative gene encoding malic enzyme was reported. Furthermore, in our search of this database with BLAST (Basic Local Alignment Search Tool) (1), three amino acid sequences were found to have a high identity with the malyl-CoA synthetase α chain (accession number P53595), the malyl-CoA synthetase β chain (P53594), and malyl-CoA lyase (AAB58884) from Methylobacterium extorquens. Interestingly, these three genes and one gene encoding malic enzyme line up on the genome and have the same direction of transcription. Therefore, these four genes might be present in one gene cluster.

In glyoxylate metabolism pathways reported so far, the mesophilic anaerobic bacterium Syntrophobotulus glycolicus FlGlyR (DSM 8271) can obtain ATP not only by the malyl-CoA pathway but also by glyoxylate respiration with hydrogen as the electron donor (15). In glyoxylate respiration, it is suggested that a proton motive force generated by glyoxylate reduction to glycolate drives ATP synthesis, in which H2 serves as the electron donor. As shown in Table 2, both NADH- and NADPH-dependent glyoxylate reductases were detected in glyoxylate- and glycolate-grown cells of HUC22-1, apparently indicating the existence of glyoxylate respiration. However, we assume that there is no or little glyoxylate respiration activity in HUC22-1. NADPH-dependent glyoxylate reductase activity was also detected in strain PerGlyox1 (16), but this bacterium does not synthesize ATP by glyoxylate respiration, since only a small amount of glycolate is formed and the cell yield for degraded substrate was smaller than that in S. glycolicus FlGlyR, which possesses both the malyl-CoA pathway and glyoxylate respiration (15). On the same score, strain HUC22-1 would not reduce glyoxylate to glycolate to generate ATP through glyoxylate respiration. The functional significance of the glyoxylate reductase observed in glyoxylate cultures is not yet understood.

The β-hydroxyaspartate pathway found by Kornberg and Morris (24) does not involve substrate-level phosphorylation with ATP generation during the aerobic conversion of 2 mol of glyoxylate to 1 mol of oxaloacetate. The dicarboxylic acid cycle (25) can generate 1 mol of ATP by substrate-level phosphorylation coupled to the reaction of pyruvate kinase during the aerobic oxidation of 1 mol of glyoxylate, but it requires 1 mol of ATP or GTP for the phosphoenolpyruvate carboxykinase reaction. Since these pathways do not generate ATP, it is considered that Moorella sp. strain HUC22-1 does not possess these pathways.

On the other hand, the glycerate and serine pathways found in Oxalobacter formigenes by Cornick and Allison (7) could cause glyoxylate catabolism in Moorella sp. strain HUC22-1. A maximum of 0.5 mol of ATP should be generated per 1 mol of glyoxylate via these pathways, because 1 mol of ATP and 1 mol of reducing equivalent are used out of 2 mol of ATP and 1 mol of reducing equivalent generated from 2 mol of glyoxylate when all 3-phosphoglycerates convert to acetate and CO2. In this experiment, however, the YATP/S (1.1) calculated using a YATP of 10.5 is twice that (maximum, 0.5) to be generated via these pathways. In addition, Seifritz et al. (35) reported that glyoxylate carboligase and serine aminotranferase, the enzymes involved in the glycerate and the serine pathways, were not detected in cell extracts of M. thermoacetica grown on glyoxylate and oxalate. Therefore, it is likely that even though Moorella sp. strain HUC22-1 might possess the glycerate and serine pathways, their contribution during glyoxylate catabolism is low.

The key enzyme activities and ATP generation through the malyl-CoA pathway were also detected in glycolate cultures of strain HUC22-1 (Tables 2 and 3), suggesting that this bacterium oxidizes glycolate to glyoxylate and then glyoxylate is degraded by the malyl-CoA pathway (Fig. 1). However, the activity of glycolate dehydrogenase, which catalyzes the oxidation of glycolate to glyoxylate, was not detected in cell extracts of glycolate cultures with NAD+, NADP+, methylene blue, methyl viologen, and benzyl viologen as electron acceptors (data not shown). NADH- and NADPH-dependent glyoxylate reductases, the reverse enzyme reactions, were detected, however (Table 2). The oxidation of glycolate to glyoxylate with NAD(P)H is described by the following reaction (45): C2H3O3− + NAD(P)+ → C2HO3− + NAD(P)H + H+ (ΔG°′ = +85 kJ reaction−1). This highly endergonic reaction in standard conditions proceeds when the intercellular concentration of NAD(P)H is low; otherwise, since oxidation of glycolate to glyoxylate with NAD(P)H (as shown in the equation) requires energy, this process must be coupled to an exergonic reaction to drive this endergonic reaction. Indeed, YX/S in glycolate cultures was approximately half of that in glyoxylate cultures (Table 1). This difference also lends support to the observation that glycolate-grown cells require greater consumption of ATP for substrate catabolism than glyoxylate-grown cells.

In oxalate-grown cells, the activity of the malyl-CoA lyase/malyl-CoA synthetase/malic enzyme system was not detected (Table 2). In addition, no ATP generation through the malyl-CoA pathway was observed (Table 3). These observations indicate that the malyl-CoA pathway is not involved in oxalate degradation. In whole-cell and cell extract experiments with M. thermoacetica ATCC 39073, Daniel et al. (8) reported that CoA-level intermediates were not involved in oxalate metabolism. Therefore, the oxalate catabolism pathway might be different from that of glyoxylate for strain HUC22-1.

Seifritz et al. (35) reported that glyoxylate and oxalate metabolisms in M. thermoacetica were compared with or without NO3−. In glyoxylate-grown cells, 14CO2 incorporated into biomass decreased, and no acetate synthesis was observed in the presence of NO3−. They claimed that the Wood-Ljungdahl pathway is blocked by NO3− through the repression of a membranous b-type cytochrome, an electron carrier. On the other hand, in oxalate-grown cells cultivated with or without NO3−, cells incorporated similar amounts of 14CO2 into biomass and acetate, and these amounts were greater than those observed for glyoxylate-grown cells. These observations also suggest that oxalate is catabolized by a pathway distinct from that for glyoxylate. Although l-malate and pyruvate are intermediates of the malyl-CoA pathway, they are important building blocks for biomass synthesis, since both are generated without CO2 fixation through the malyl-CoA pathway by coupling of glyoxylate with acetyl-CoA supplied from the Wood-Ljungdahl pathway (Fig. 1). This suggests that the malyl-CoA pathway not only might play a role in cleavage of glyoxylate to generate reducing power and ATP but also might participate in cell anabolism in the presence of NO3−.

Footnotes

▿

Published ahead of print on 14 December 2007.

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