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Home , Carbon monoxide

Net synthesis of acetate from CO2 by Eubacterium acidaminophilum through the reductase pathway

Anne Schneeberger, Jochen Frings, Bernhard Schink *

Fakulta«tfu«r Biologie, Mikrobielle Oë kologie, Universita«t Konstanz, Postfach 5560, D-78457 Konstanz, Germany

Abstract

Eubacterium acidaminophilum combines the oxidation of amino acids such as or valine with the reduction of glycine to acetate in a two-substrate fermentation (Stickland reaction). In the absence of glycine, dense cell suspensions oxidized alanine or valine only to a small extent, with limited production of and acetate. Experiments with 14C-labeled 3 14 revealed that acetate was formed under these conditions by net reduction of CO2/HCO3 ; C-labeled formate was formed as an intermediate. E. acidaminophilum did not grow with hydrogen plus CO2 ; dense cell suspensions under H2/CO2 produced only very small amounts ( 6 0.5 mM) of acetate. There was no activity of dehydrogenase, indicating that the glycine pathway was used for acetate synthesis. The results are explained on the basis of biochemical and energetic considerations. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Glycine reduction; Acetogenesis; CO2 ¢xation; Eubacterium acidaminophilum

1. Introduction tron donor [3]. These activities require su¤cient amounts of in the growth medium because Among the catalyzing the so-called Stick- the enzymes involved contain at least one selenopro- land fermentation of amino acids [1,2], Eubacterium tein [4,5]. The reduction of glycine through this path- acidaminophilum is perhaps the most versatile one. It way proceeds via acetyl phosphate and allows ATP can either carry out the complete combined fermen- synthesis by substrate-level phosphorylation which is tation of e.g. alanine plus glycine to acetate plus quite unusual in a reductive fermentation pathway. plus CO2, or convert alanine to acetate E. acidaminophilum can also dismutate glycine as plus CO2 plus hydrogen if a hydrogen-scavenging sole substrate to acetate, ammonia, and CO2, and partner is present. It can also grow by reduction of contains all enzymes involved in glycine oxidation glycine to acetate plus ammonia, or of betaine to through glycine decarboxylation and the tetrahydro- acetate and trimethylamine, with hydrogen as elec- folate pathway [3]. So far, it has never been shown that this organisms could also catalyze a net synthe-

sis of acetate from CO2, using the same pathway in * Corresponding author. Tel.: +49 (7531) 88 2140; Fax: the opposite direction. During studies on the ener- +49 (7531) 88 2966; E-mail: [email protected] getic limitations of syntrophic fermenta-

FEMSLE 8841 15-7-99 2 tion with this organism, we observed that the fer- performed with either 5 mM alanine or 5 mM valine mentation equations did not balance as expected, as substrate in the presence of 20 mM NaHCO3 14 and could explain this only by a net synthesis of which received in addition 10 WlNa2 CO3 per acetate through CO2 reduction. Here we document 1 ml cell suspension ( = 250 kBq), corresponding to this new metabolic capacity of E. acidaminophilum a speci¢c labeling of 12.5 kBq per Wmol total CO2/ 3 which represents to our knowledge the third known HCO3 . Samples were taken and deep-frozen as de- pathway through which acetate can be formed anaer- scribed above. After centrifugation, the supernatant obically from CO2. was separated by high- liquid chromatogra- phy (Beckman System Gold) on an exchange column (Sykam Aminex HPX-87 H) with 5 mM

2. Materials and methods H2SO4 as eluent, 0.4 ml per min. The chromato- graphic conditions were set to allow quantitative sep-

2.1. Strains source and cultivation aration of CO2, formate, acetate, and higher fatty acids. Quanti¢cation of radiolabeled compounds E. acidaminophilum strain al-2 (DSM 3953, ATCC was performed with a RAMONA-5 on-line liquid 49065) was obtained from the Deutsche Sammlung scintillation detector system (Raytest, Straubenhardt, von Mikroorganismen und Zellkulturen GmbH, Germany) equipped with a 400 Wl glass scintillator Braunschweig, Germany. Cells were grown in a fresh £ow cell that had been calibrated before with 14 mineral medium containing 1 WMNa2SeO3 as [ C]acetate standards (1^150 kBq per ml). described [3]. 2.4. Chemical determinations 2.2. Experiments with cell suspensions Acetate and isobutyrate were quanti¢ed either by Cells were harvested in the late exponential growth HPLC (see above) or by gas chromatography [6]. phase by centrifugation in 125 ml infusion bottles for Alanine was determined enzymatically [7]. Cell 30 min at 3000Ug at 4³C. The pellet was washed growth was measured via optical at 578 nm with 50 mM phosphate bu¡er, pH 7.0, wavelength. Protein was quanti¢ed by the Bradford containing 2.5 mM dithioerythritol as reducing method [8]. Hydrogen was quanti¢ed by gas chroma- agent. Cells were resuspended in the same bu¡er to tography with a Carlo Erba GC 6000 gas chromato- a ¢nal optical density of OD578 = 15. Dense suspen- graph equipped with a thermal conductivity detector. sions (4 ml) were incubated in 8 ml serum bottles, The gas sample was run on a Carbosieve SII column with either 10 mM alanine or 10 mM valine as sub- (Supelco) 60^80 mesh. At 120³C gas was strate. Samples were taken through the butyl rubber used as carrier, the thermal conductivity detector stoppers with Unimetrics microliter syringes, and was run at 130³C, ¢lament temperature 350³C. The deep-frozen at 318³C. Possible dependence of sub- system was calibrated with de¢ned H2/N2 mixtures strate turnover on CO2 availability was checked in at 0.1^2%. either CO2-free assays that had been gassed repeat- Carbon monoxide dehydrogenase was assayed edly with CO2-free N2, or in replicates containing 30 after [9]; benzyl viologen-dependent in mM NaHCO3 supplied from a 0.5 M stock solution an analogous manner [10]. that was kept under CO2 gas atmosphere. Acetate formation from H2/CO2 (80/20) was checked with 2.5. Chemicals and gases similar washed cell suspensions that were incubated under this gas mixture at 30³C. Controls were run All chemicals were of the highest purity that is under N2/CO2 (80/20). commercially available, and were obtained from Bio- mol (Ilvesheim, Germany), Boehringer (Mannheim, 2.3. Labeling experiments Germany), Eastman Kodak (Rochester, NY, USA), Fluka (Neu-Ulm, Germany), Merck (Darmstadt, Assays analogous to those described above were Germany), Pharmacia (Freiburg, Germany), Serva

FEMSLE 8841 15-7-99 3

(Heidelberg, Germany), and Sigma (Deisenhofen, Germany). Gases were purchased from Messer- Griesheim (Darmstadt, Germany), and Sauersto¡- werke Friedrichshafen (Friedrichshafen, Germany).

3. Results

3.1. Experiments in dense suspensions

E. acidaminophilum grew with glycine as sole sub- strate with a doubling time of 1.16 h (W = 0.597 h31) if selenite was included in the medium at 1036 M concentration. Other amino acids such as alanine Fig. 2. Degradation of valine by dense cell suspensions of E. or valine had been reported to be degraded by the acidaminophilum. Formation of acetate (F) and isobutyrate (b)in the presence of valine. Open symbols: formation of acetate (E) pure culture only if e.g. glycine was provided as a and isobutyrate (a) in the absence of valine. hydrogen-scavenging cosubstrate. In experiments to elucidate the energetic limits of this kind of fermen- tation in the absence of a hydrogen-scavenging co- synthesized from CO2 which was the only substrate, we observed the following substrate turn- acceptor available in the medium. In order to exam- over balances: Resting cell suspensions produced ine this hypothesis, we followed the same reactions in 3 from 10 mM alanine, 3 mM acetate plus 0.4 mM the presence and absence of HCO3 /CO2 in the me- H2 ( = 1000 Pa in the headspace) within 24 h; from dium. The rate of acetate formation in the presence 3 10 mM valine, 4.5 mM isobutyrate, 3^3.5 mM ace- of CO2/HCO3 was about twice as high as in its tate and 0.8 mM H2 ( = 2.000 Pa H2) were produced absence (results not shown); obviously, the endoge- in the same time (Figs. 1 and 2). Similar fermenta- nous CO2 production from alanine oxidation could tion balances had been reported in the original de- not cover the CO2 need su¤ciently. scription of this bacterium [3]. The comparably high The cultures were checked for possible contamina- background acetate formation in the absence of va- tions with homoacetogenic bacteria. These tests as line may be due to endogenous acetate formation well as checks for carbon monoxide dehydrogenase from storage compounds. Obviously, acetate was activity were always negative.

3.2. Labeling experiments

Suspension experiments in the presence of radio- labeled carbonate documented that there was indeed 3 a signi¢cant incorporation of CO2/HCO3 into ace- 14 tate. As shown in Fig. 3, CO2-carbon was incorpo- rated into acetate at a high rate in the presence of alanine as electron donor; the rate of incorporation was far lower in the absence of alanine. Formate was identi¢ed transiently as a labeled side product which disappeared later during the incubation experiment. Similar results were obtained with valine as electron donor: Labeled acetate was formed at a high rate in the presence of valine, and formate was again Fig. 1. Degradation of alanine (b) by dense cell suspensions of E. acidaminophilum. Formation of acetate in the presence (F) formed as a labeled transient coproduct (Fig. 4). and in the absence (E) of alanine. The speci¢c radioactivity of the formed acetate was

FEMSLE 8841 15-7-99 4

Fig. 3. Formation of [14C]acetate and [14C]formate from Fig. 4. Formation of [14C]acetate and [14C]formate from 14 14 [ C]Na2CO3 by dense suspensions of E. acidaminophilum with [ C]Na2CO3 by dense suspensions of E. acidaminophilum with alanine as electron donor. Filled symbols: (F) acetate, (b) 14C la- valine as electron donor. Filled symbols: (F) acetate, (b) 14C la- bel in acetate, (8) 14C label in formate in the presence of 5 mM bel in acetate, (8) 14C label in formate in the presence of 5 mM alanine; open symbols: (E) acetate, (a) 14C label in acetate, (7) valine; open symbols: (E) acetate, (a) 14C label in acetate, (7) 14C label in formate in the absence of alanine. 14C label in formate in the absence of valine.

in both cases slightly lower than that of the CO2/ 3 HCO3 used, indicating that the total CO2 pool E. acidaminophilum can catalyze a net synthesis of was diluted by endogenous CO2 from amino acid acetate from CO2. Occurrence of this capacity oxidation (Table 1). among non-homoacetogenic bacteria has been docu- mented before for acidiurici and C. cy- 3.3. Lithotrophic acetate formation lindrosporum [11], and for C. purinolyticum [12]. With that, the glycine pathway represents the third way In growth experiments, we did not observe growth through which acetate can be synthesized by anaero- with H2/CO2 in the absence of organic electron do- bic bacteria. So far, acetate synthesis through the nors, as also observed before [3]. Although there was carbon monoxide dehydrogenase pathway (Acetyl- su¤cient hydrogenase activity in glycine-grown cells CoA or Wood-pathway) has been described which

(0.6 Wmol H2 per min and mg protein), dense cell is used by homoacetogenic, methanogenic, and sev- suspensions under H2/CO2 formed acetate only at a eral sulfate-reducing bacteria in their assimilatory low rate (0.62 nmol acetate per min and mg protein) metabolism [13], and the reversed tricarboxylic acid which was about twice as high as the endogenous cycle that is applied for autotrophic cell carbon syn- acetate production under N2/CO2 (0.29 nmol per thesis by green phototrophic bacteria and few sulfate min and mg protein). Also these ¢ndings con¢rm reducers [13]. earlier observations [3]. Only the homoacetogens are able to couple litho-

trophic acetate formation from hydrogen and CO2 with energy conservation and growth [14,15]; the 4. Discussion other organisms mentioned depend on other energy sources, and may excrete acetate only in traces dur- In the present communication, we document that ing cell matter synthesis.

Table 1 14 14 31 Incorporation of C label from [ C]NaHCO3 (speci¢c radioactivity 12.5 kBq Wmol ) into acetate Electron donor Acetate formed (Wmol) Speci¢c radioactivity in acetate (kBq Wmol31) Alanine (5 mM) 3.26 9.85 Valine (5 mM) 2.07 11.0

FEMSLE 8841 15-7-99 5

In the present study, we observed net acetate syn- CO2, but the overall process does not provide for thesis from CO2 by dense cell suspensions of E. acid- net ATP formation. This explains why growth of aminophilum with alanine or valine as electron do- E. acidaminophilum with H2+CO2 was never ob- nor. Alanine fermentation under these conditions served, and acetate formation from H2+CO2 was should follow the equation found only as a cometabolic side activity. With that, this bacterium di¡ers from the homoacetogenic 3 ‡ Alanine ‡ 2H2O ! acetate ‡ NH4 ‡ CO2‡ bacteria which appear to conserve a fraction of an ATP either in the tetrahydrofolate reduc- 0 31 2H2 vG0 ˆ‡2:7 kJ mol 1† tase reaction [14] or through a ion-translo- cating methyl transferase reaction [17]. This reaction was partly combined with acetogenic The observed excretion of radiolabeled formate

CO2 reduction, according to during acetate formation from alanine or valine 14 with CO2 indicates that the ATP level in the cells 3 ‡ 4H2 ‡ 2CO2 ! CH3COO ‡ H ‡ under these conditions is probably insu¤cient to ini- tiate the ATP-dependent formate activation. Obvi- 0 31 2H2O vG0 ˆ 395 kJ mol 2† ously, the electron £ow through the reductive reac- tion chain is impeded, and this exergonic branch of The overall reaction is exergonic the metabolism (reaction 2) can be employed only to a limited extent. 3 ‡ ‡ 2 Alanine ‡ 2H2O ! 3 Acetate ‡ 2NH4 ‡ In our experiments (alanine 10 mM, NH4 5 mM), the reaction stopped when acetate reached 3 mM ‡ 0 31 H vG0 ˆ 392 kJ mol 3† concentration and hydrogen 0.01 atm, corresponding to a remnant energy in the system (according to re- and should allow ATP synthesis and growth since action 1) in the range of 320 kJ mol31. This is the synthesis of one ATP requires a vG0P of at least minimum amount of energy needed to maintain via- 370 kJ per reaction run [16]. However, we found bility with a fully charged cytoplasmic membrane neither complete substrate turnover nor active [18], and is the same where also the growth under these conditions, and there was also syntrophically fermenting bacteria ¢nd their ener- no lithotrophic growth on the basis of reaction 2, getic limits. Thus, although basically possible, the and only very slow (cometabolic?) acetate formation glycine pathway is not an e¤cient alternative to the from H2 and CO2. carbon monoxide dehydrogenase pathway for a net The biochemical pathway used for glycine dismu- acetate synthesis. tation to CO2 and acetate has been worked out in detail in the past [3], and is probably used as well in the reductive direction, as suggested before for some Acknowledgements non-homoacetogenic clostridia [11,12]: CO2 is re- duced via formate, ATP-dependent linkage to tetra- This study was ¢nanced in part by funds of the hydrofolate, and subsequent reduction to methylene University of Constance and of the Fonds der Chem- tetrahydrofolate. The methylene derivative is reduc- ischen Industrie, Frankfurt/M. tively carboxylated and aminated to form glycine which is subsequently reduced to acetate, releasing ammonia. This step also releases one ATP in a sub- References strate-level phosphorylation reaction, thus recovering the ATP invested before. All reactions, except for the [1] Stickland, L.H. (1934) The by which Clos- one catalyzed by glycine reductase, are known to be tridium sporogenes obtains energy. Biochem. J. 28, 1746^1759. [2] Lengeler, J.W., Drews, G. and Schlegel, H.G. (1999) Biology reversible, with NADPH as electron donor. From of the Prokaryotes. Thieme, Stuttgart. this point of view, it is not surprising that the overall [3] Zindel, U., Freudenberg, W., Rieth, M., Andreesen, J.R., reaction chain can operate to form acetate from Schnell, J. and Widdel, F. (1988) Eubacterium acidaminophi-

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lum sp. nov., a versatile amino acid-degrading anaerobe pro- by mass analysis of the di¡erent types of acetate formed from 13 ducing or utilizing H2 or formate. Description and enzymatic CO2 by heterotrophic bacteria. J. Bacteriol. 109, 633^644. studies. Arch. Microbiol. 150, 254^266. [12] Du«rre, P. and Andreesen, J.R. (1982) Pathway of carbon di- [4] Andreesen, J.R. (1994) Acetate via glycine: a di¡erent form of reduction to acetate without a net energy requirement in acetogenesis. In: Acetogenesis (Drake, H.L., Ed.), pp. 568^ Clostridium purinolyticum. FEMS Microbiol. Lett. 15, 51^56.

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