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Keywords:

  • Archaeoglobus lithotrophicus;
  • 4-hydroxybutyryl-CoA dehydratase;
  • autotrophy;
  • dicarboxylate/hydroxybutyrate cycle;
  • hydroxypropionate/hydroxybutyrate cycle;
  • ribulose 1,5-bisphosphate carboxylase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Several representatives of the euryarchaeal class Archaeoglobi are able to grow facultative autotrophically using the reductive acetyl-CoA pathway, with ‘Archaeoglobus lithotrophicus’ being an obligate autotroph. However, genome sequencing revealed that some species harbor genes for key enzymes of other autotrophic pathways, i.e. 4-hydroxybutyryl-CoA dehydratase of the dicarboxylate/hydroxybutyrate cycle and the hydroxypropionate/hydroxybutyrate cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the Calvin–Benson cycle. This raised the question of whether only one or multiple autotrophic pathways are operating in these species. We searched for the presence of enzyme activities specific for the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycles in ‘A. lithotrophicus’, but such enzymes could not be detected. Low Rubisco activity was detected that could not account for the carbon dioxide (CO2) fixation rate; in addition, phosphoribulokinase activity was not found. The generation of ribulose 1,5-bisphosphate from 5-phospho-d-ribose 1-pyrophosphate was observed, but not from AMP; these sources for ribulose 1,5-bisphosphate have been proposed before. Our data indicate that the reductive acetyl-CoA pathway is the only functioning CO2 fixation pathway in ‘A. lithotrophicus’.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

To date, six autotrophic carbon dioxide (CO2) fixation pathways have been found in nature, three of which occur in Archaea (Berg et al., 2010a). Whereas Crenarchaeota use either the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycle (Fig. 1), all autotrophic Euryarchaeota studied so far use the reductive acetyl-CoA pathway (Fig. 1c) (Berg et al., 2010a). The functioning of this pathway in Euryarchaeota conforms to the fact that most autotrophic Euryarchaeota are methanogens. They use much of the enzymes involved in energy generation during methanogenesis also for autotrophic acetyl-CoA synthesis. The only known exceptions to this rule are members of Archaeoglobi (Huber & Stetter, 2001) and perhaps Ferroplasma acidiphilum (Golyshina et al., 2000), whose ability to grow autotrophically was questioned (Dopson et al., 2004).

image

Figure 1.  Pathways of autotrophic CO2 fixation in Archaea (Berg et al., 2010a). (a) The dicarboxylate/hydroxybutyrate cycle functioning in Desulfurococcales and Thermoproteales, (b) the hydroxypropionate/hydroxybutyrate cycle functioning in Sulfolobales and (c) the reductive acetyl-CoA pathway functioning in Euryarchaeota. Note that succinyl-CoA reductase in Thermoproteales and Sulfolobales uses NADPH (Kockelkorn & Fuchs, 2009; Ramos-Vera et al., 2009, 2011) and reduced methyl viologen (possibly as a substitute for reduced ferredoxin) in Desulfurococcales (Huber et al., 2008; Ramos-Vera et al., 2009). Enzymes (a, b): 1, pyruvate synthase; 2, pyruvate : water dikinase; 3, PEP carboxylase; 4, malate dehydrogenase (NADH); 5, fumarate hydratase; 6, fumarate reductase (natural electron acceptor is not known); 7, succinyl-CoA synthetase (ADP forming); 8, acetyl-CoA/propionyl-CoA carboxylase; 9, malonyl-CoA reductase (NADPH); 10, malonic semialdehyde reductase (NADPH); 11, 3-hydroxypropionate-CoA ligase (AMP-forming); 12, 3-hydroxypropionyl-CoA dehydratase; 13, acryloyl-CoA reductase (NADPH); 14, methylmalonyl-CoA epimerase; 15, methylmalonyl-CoA mutase; 16, succinyl-CoA reductase (for electron donor, see above); 17, succinic semialdehyde reductase (NADPH); 18, 4-hydroxybutyrate-CoA ligase (AMP forming); 19, 4-hydroxybutyryl-CoA dehydratase; 20, crotonyl-CoA hydratase; 21, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD+); 22, acetoacetyl-CoA β-ketothiolase; (c) 1, formylmethanofuran dehydrogenase; 2, formylmethanofuran:tetrahydromethanopterin formyltransferase; 3, methenyl-tetrahydromethanopterin cyclohydrolase; 4, methylene-tetrahydromethanopterin dehydrogenase; 5, methylene-tetrahydromethanopterin reductase; 6, CO dehydrogenase/acetyl-CoA synthase. Fd, ferredoxin; MF, methanofuran; F420, deazaflavin factor 420.

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Representatives of the class Archaeoglobi (with only one order and one family, Archaeoglobales and Archaeoglobaceae) are hyperthermophiles that grow by means of anaerobic respiration by oxidizing organic substrates or molecular hydrogen (in some cases, also Fe2+ or S2−) (Huber & Stetter, 2001). The Archaeoglobaceae comprise three genera: Archaeoglobus, Ferroglobus and Geoglobus. Besides Archaeoglobus profundus and Archaeoglobus infectus, all species can grow autotrophically, with ‘Archaeoglobus lithotrophicus’ being an obligate autotroph (Stetter et al., 1993). Biochemical studies revealed the presence of the enzymes of the reductive acetyl-CoA pathway in ‘A. lithotrophicus’ and Ferroglobus placidus (Vorholt et al., 1995, 1997). The corresponding genes also exist in the Archaeoglobus fulgidus genome (Klenk et al., 1997), whereas the genome of the heterotrophic A. profundus lacks the gene for the key enzyme of this pathway, CO-dehydrogenase/acetyl-CoA synthase (von Jan et al., 2010). Therefore, these data suggest that autotrophic Archaeoglobaceae use the reductive acetyl-CoA pathway for CO2 fixation.

Interestingly, the genome of A. fulgidus also harbors, besides the genes of the reductive acetyl-CoA pathway, three copies of genes encoding homologues of the 4-hydroxybutyryl-CoA dehydratase. In contrast, this key enzyme of the dicarboxylate/hydroxybutyrate and hydroxypropionate/hydroxybutyrate cycles is absent in the heterotrophic A. profundus. Moreover, genomes of Archaeoglobaceae harbor the genes for B12-dependent mutases and biotin-binding proteins as well, which may encode methylmalonyl-CoA mutase and subunits of acetyl-CoA/propionyl-CoA carboxylase, respectively, of the hydroxypropionate/hydroxybutyrate cycle. In addition, all sequenced strains have the gene encoding archaeal form III ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), leaving the question as to whether only one or multiple pathways are functioning in these species (Berg et al., 2007). The possibility of the functioning of two different pathways of autotrophic CO2 fixation has been shown recently for an uncultured endosymbiont of a deep-sea tube worm (Markert et al., 2007).

The goal of our work was to study the presence of the enzymes of the dicarboxylate/hydroxybutyrate and hydroxypropionate/hydroxybutyrate cycles in A. lithotrophicus. This species was chosen for the study as it is the only strictly autotrophic representative of this group known so far. Also, the possible function of Rubisco in this species was addressed.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Materials and syntheses

Materials were as described previously (Berg et al., 2010b). Acetyl-CoA, propionyl-CoA, succinyl-CoA and crotonyl-CoA were synthesized from the respective anhydrides, and acetoacetyl-CoA from diketene using the method of Simon & Shemin (1953). The dry powders of the CoA-esters were stored at −20 °C. (R)- and (S)-3-hydroxybutyryl-CoA were synthesized using the mixed anhydride method (Stadtman, 1957).

Cell material and growth conditions

‘Archaeoglobus lithotrophicus’ strain TF2 was obtained from the culture collection of the Lehrstuhl für Mikrobiologie, University of Regensburg. Cells were grown autotrophically under anoxic conditions in MGG medium (Huber et al., 1982) at 80 °C and pH 6.0 using sulfate (2 g L−1) as an electron acceptor. In the 300-L fermentor, a gassing rate of 1 L min−1 was applied (using a gas mixture of 80% H2 and 20% CO2, v/v). The cells were harvested by centrifugation in the late exponential growth phase and stored at −70 °C until use. Metallosphaera sedula TH2T (DSMZ 5348) was grown autotrophically as reported before (Alber et al., 2006). Archaeoglobus fulgidus VC16T (DSMZ 4304) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) and grown according to the recommendations of DSMZ.

Preparation of cell extracts and enzyme measurements

Cell extracts were prepared under anoxic conditions using a French pressure cell as described previously (Berg et al., 2010b). Spectrophotometric enzyme assays (0.5 mL assay mixture) were performed in 0.5-mL cuvettes at 65 °C. Radiochemical enzyme assays were performed at 80 °C. Anoxic assays were performed with N2 as the headspace. For the wavelengths and extinction coefficients used in spectrophotometric assays, see Berg et al. (2010b).

Pyruvate and 2-oxoglutarate:acceptor oxidoreductase were measured anoxically as a pyruvate- or 2-oxoglutarate-dependent reduction of methyl viologen and as a 14CO2 exchange reaction with the carboxyl group of pyruvate or 2-oxoglutarate (Ramos-Vera et al., 2009).

Phosphoenolpyruvate (PEP) carboxylase was measured radiochemically as PEP-dependent fixation of 14CO2 (Ramos-Vera et al., 2009). ATP-, GTP- and diphosphate-dependent PEP carboxykinases were measured in a similar manner, but the reaction mixture was supplemented with ADP, GDP or potassium phosphate for ATP-, GTP- or pyrophosphate-dependent PEP carboxykinase, respectively (Ramos-Vera et al., 2009).

Phosphoribulokinase, Rubisco, acetyl-CoA and propionyl-CoA carboxylase were measured under anoxic conditions radiochemically, as described in Berg et al. (2010b).

Pyruvate carboxylase was measured radiochemically by determining pyruvate-dependent fixation of 14CO2 using a modified method of Mukhopadhyay et al. (2001). The reaction mixture (0.35 mL) contained 100 mM Tris/HCl (pH 7.8), 5 mM dithiothreitol, 200 mM KCl, 1 mM MgCl2, 1 mM ATP, 15 mM NaH14CO3 (3.3 kBq μmol−1), 1 mM NADH and cell extract. The reaction was started by the addition of pyruvate (20 mM). Acid-stable 14C was determined as described previously (Hügler et al., 2003).

Succinyl-CoA reductase was measured as succinyl-CoA-dependent oxidation of NAD(P)H (Kockelkorn & Fuchs, 2009) and of reduced methyl viologen, respectively (Huber et al., 2008).

Succinic semialdehyde reductase was measured as succinic semialdehyde-dependent oxidation of NAD(P)H (Kockelkorn & Fuchs, 2009) or of reduced methyl viologen, similar to methyl viologen-dependent succinyl-CoA reductase (Huber et al., 2008), in an assay mixture containing 100 mM MOPS/KOH (pH 7.2), 5 mM MgCl2, 5 mM methyl viologen, 5 mM dithiothreitol and cell extract. The reaction was started by the addition of succinic semialdehyde (2 mM).

4-Hydroxybutyryl-CoA dehydratase activity was measured anoxically using a spectrophotometric assay with 4-hydroxybutyryl-CoA synthetase from Thermoproteus neutrophilus (Tneu_0420, Ramos-Vera et al., 2011) and crotonyl-CoA hydratase/3-hydroxybutyryl-CoA dehydrogenase from M. sedula (Msed_0399, Ramos-Vera et al., 2011) as coupling enzymes. The assay mixture contained 100 mM Tris/HCl (pH 9.0), 5 mM NAD+, 2.5 mM ATP, 1 mM CoA, 1 mM MgCl2, 5 mM dithiothreitol, 2 mM 4-hydroxybutyrate, 0.5 U mL−1 4-hydroxybutyryl-CoA synthetase, 0.5 U mL−1 crotonyl-CoA hydratase/3-hydroxybutyryl-CoA dehydrogenase and cell extract.

3-Hydroxybutyryl-CoA dehydrogenase was measured spectrophotometrically as (S)- or (R)-3-hydroxybutyryl-CoA-dependent reduction of NAD+ (Ramos-Vera et al., 2009) or as acetoacetyl-CoA-dependent oxidation of NADH in the following reaction mixture: 100 mM MOPS/KOH (pH 7.8), 5 mM dithiothreitol, 10 mM MgCl2, 0.5 mM NADH, 0.2 mM acetoacetyl-CoA and cell extract.

5-phospho-d-ribose 1-pyrophosphate (PRPP) conversion to ribulose 1,5-bisphosphate was determined as PRPP-dependent fixation of NaH14CO3 into acid-stable products under anoxic conditions. The reaction mixture (0.35 mL) contained 100 mM Tris/HCl (pH 7.8), 5 mM dithiothreitol, 5 mM MgCl2, 15 mM NaH14CO3 (18 kBq μmol−1) and cell extract. After preincubation for 5 min, the reaction was started by the addition of PRPP (1 mM) and the acid-stable 14C was determined after appropriate time intervals (Hügler et al., 2003). In some experiments, the reaction mixture was supplemented with NAD+ (0.5 mM) and/or recombinant Rubisco from A. fulgidus (0.5 U mL−1).

AMP conversion to ribulose 1,5-bisphosphate was determined as AMP-dependent fixation of NaH14CO3 into acid-stable products under anoxic conditions as described for PRPP, but including 1 mM phosphate and recombinant Rubisco from A. fulgidus (0.5 U mL−1). After preincubation for 5 min, the reaction was started by the addition of AMP (1 mM).

4-Hydroxybutyrate conversion in cell extracts of ‘A. lithotrophicus

The conversion of 4-hydroxybutyrate with ATP and CoA by cell extracts of ‘A. lithotrophicus’ was performed and analyzed by HPLC, as described previously (Berg et al., 2010b). In some experiments, 4-hydroxybutyryl-CoA synthetase from T. neutrophilus was added as a coupling enzyme (0.5 U mL−1).

Heterologous expression of the A. fulgidus Rubisco gene in Escherichia coli

The A. fulgidus Rubisco gene was heterologously expressed in E. coli, as described by Kreel & Tabita (2007). DNA extraction, PCR amplification and control sequencing of the gene were performed as described in Berg et al. (2010b). The enzyme was partly purified by heat precipitation of the extract (15 min, 75 °C), followed by centrifugation (20 000 g) at 4 °C for 15 min. The supernatant was dialyzed and used for enzyme measurements.

Protein analytics

Protein was measured according to the Bradford method, using bovine serum albumin as a standard. Biotinylated proteins in cell extracts were detected with peroxidase-conjugated avidin (Menendez et al., 1999) after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Enzymes of the hydroxypropionate/hydroxybutyrate cycle or the dicarboxylate/hydroxybutyrate cycle

The activity of acetyl-CoA/propionyl-CoA carboxylase, the characteristic carboxylase of the hydroxypropionate/hydroxybutyrate cycle, was not detected in ‘A. lithotrophicus’. In contrast, the key carboxylases of the dicarboxylate/hydroxybutyrate cycle, pyruvate synthase and PEP carboxylase, were detected. Pyruvate synthase activity was 170 or 140 mU mg−1 protein in the 14CO2 exchange or methyl viologen reduction reaction, respectively, and the rate of PEP carboxylase reaction was 4 mU mg−1 protein. However, these enzymes are also involved in the assimilation of acetyl-CoA synthesized by the reductive acetyl-CoA pathway (Vorholt et al., 1995) and therefore cannot be regarded as indicators for the dicarboxylate/hydroxybutyrate cycle. Interestingly, 2-oxoglutarate synthase, pyruvate carboxylase and ADP-, GDP- or phosphate-dependent PEP carboxykinase activities were not detected in ‘A. lithotrophicus’ cell extracts.

The hydroxypropionate/hydroxybutyrate and dicarboxylate/hydroxybutyrate cycles have in common the conversion of succinyl-CoA via 4-hydroxybutyrate to two molecules of acetyl-CoA. Enzyme activities required for this process were not detected: Succinyl-CoA reductase and succinic semialdehyde reductase assays with NADH, NADPH or reduced methyl viologen failed. Furthermore, cell extracts did not convert 4-hydroxybutyrate in the presence of CoA and ATP to 4-hydroxybutyryl-CoA and derived products. As a positive control, we used M. sedula cell extracts (data not shown). The addition of recombinant 4-hydroxybutyryl-CoA synthetase from T. neutrophilus resulted in the formation of 4-hydroxybutyryl-CoA, but neither dehydration to crotonyl-CoA catalyzed by 4-hydroxybutyryl-CoA dehydratase nor any (S)- nor (R)-3-hydroxybutyryl-CoA dehydrogenase activity were observed (data not shown). These findings exclude the functioning of the hydroxypropionate/hydroxybutyrate or the dicarboxylate/hydroxybutyrate cycle in ‘A. lithotrophicus’.

The presence of the 4-hydroxybutyryl-CoA dehydratase gene in Crenarchaeota is always accompanied by autotrophy via either the hydroxypropionate/hydroxybutyrate or the dicarboxylate/hydroxybutyrate cycle. The homologues of this gene in Archaeoglobus (three in A. fulgidus) must play another role. These genes probably encode functional proteins, because putative 4-hydroxybutyryl-CoA dehydratases from A. fulgidus contain conserved amino acid residues that are covalently linked to the Fe atoms of the [4Fe–4S]2+ cluster and are important for catalysis: Cys-99, Cys-103, Cys-299 and His-292 (numbering according to the enzyme from Clostridium aminobutyricum) (Martins et al., 2004; for alignment, see Berg et al., 2007). Interestingly, five genes encoding homologues of 4-hydroxybutyryl-CoA dehydratase were found in the deltaproteobacterium Desulfatibacillum alkenivorans, which degrades alkenes coupled to sulfate reduction (Cravo-Laureau et al., 2004). Similarly, A. fulgidus is able to grow on a wide range of alkenes (Khelifi et al., 2010), and many Archaeoglobaceae were found in or isolated from the environments enriched in aliphatic compounds (Stetter et al., 1993; Kashefi et al., 2002; Slobodkina et al., 2009; Steinsbu et al., 2010). In contrast, A. profundus probably does not metabolize these compounds, because its genome lacks two of four key enzymes for β-oxidation and the 4-hydroxybutyryl-CoA dehydratase gene homologue as well (Von Jan et al., 2010). These circumstances point to a possible role of the Archaeoglobus 4-hydroxybutyryl-CoA dehydratase homologues in the oxidation of aliphatic compounds by adding or eliminating water. Note that 4-hydroxybutyryl-CoA dehydratase also has vinylacetyl-CoA δ-isomerase activity (Scherf et al., 1994). Such an isomerase may play a role in alkene degradation.

Detection of biotin-containing proteins in cell extracts

Proteins from cell extracts of ‘A. lithotrophicus’ and A. fulgidus were separated by SDS-PAGE and blotted to detect biotin-containing proteins using the avidin technique. The cell extract of autotrophically grown M. sedula was used as a positive control for the presence of the biotin carrier protein of acetyl-CoA/propionyl-CoA carboxylase. A single band of biotin-containing protein was detected in ‘A. lithotrophicus’ as well as in A. fulgidus (Fig. 2). Interestingly, the apparent molecular mass of the ‘A. lithotrophicus’ protein (25 kDa) was significantly higher than that of A. fulgidus and M. sedula (20 kDa, respectively). This may indicate a possible difference in the functions of the corresponding proteins in autotrophically grown ‘A. lithotrophicus’ and heterotrophically grown A. fulgidus.

image

Figure 2.  Detection of biotin-containing proteins in cell extracts of ‘Archaeoglobus lithotrophicus’ (1), Archaeoglobus fulgidus (2) and Metallosphaera sedula (3). The proteins (40 μg) were separated by SDS-PAGE, and biotin-containing proteins were stained with peroxidase-conjugated avidin. Note that the theoretical molecular masses of the biotin-containing proteins in A. fulgidus (15.6 kDa) deviate from what is observed here (∼20 kDa).

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The genome of A. fulgidus harbors two biotin-binding proteins (AF2085 and AF2216) with the same calculated molecular mass (15.6 kDa). AF2085 was shown to be a part of the oxaloacetate decarboxylase complex, whereas AF2216 is probably a subunit of methylmalonyl-CoA decarboxylase (Dahinden et al., 2004). Furthermore, AF2085, together with the biotin carboxylase domain protein AF0220 and the carboxytransferase subunit of oxaloacetate decarboxylase, might catalyze the pyruvate carboxylase reaction. Although we detected a biotin-containing protein in ‘A. lithotrophicus’ cell extracts (Fig. 2), neither acetyl-CoA/propionyl-CoA carboxylase nor pyruvate carboxylase activity was found. Because no sequence information is available for ‘A. lithotrophicus’, the function of the biotin-containing protein detected in cell extracts of this species (Fig. 2) remains unknown and requires further investigations.

Enzymes of the Calvin cycle

Rubisco activity was detected at a very low level (5 nmol min−1 mg−1 protein, 80 °C); the results obtained were similar to those for A. fulgidus (Finn & Tabita, 2003). The ‘A. lithotrophicus’ cells studied here grew with a generation time of 2 h, which requires CO2 fixation at 0.4 μmol min−1 mg−1 protein (calculated as described in Ramos-Vera et al., 2009). Hence, the observed Rubisco activity is almost 100 times lower and cannot account for the in vivo CO2 fixation rate, even if optimization of the assay may yield a somewhat higher value. Furthermore, attempts to demonstrate phosphoribulokinase activity failed (Table 1). Archaea containing Rubisco may have other options to form ribulose 1,5-bisphosphate. One option is to transform AMP. In Thermococcus kodakarensis, AMP is cleaved phosphorolytically to ribose 1,5-bisphosphate and adenine, followed by isomerization of ribose 1,5-bisphosphate to ribulose 1,5-bisphosphate (Sato et al., 2007). Archaeoglobus species produce vast amounts of AMP during sulfate reduction via adenosinephosphosulfate (Speich & Trüper, 1988; Dahl et al., 1990), and the genome of A. fulgidus harbors putative genes for enzymes of this pathway (Klenk et al., 1997; Sato et al., 2007). Yet, cell extracts did not catalyze CO2 fixation in the presence of AMP (Table 1). The addition of recombinant A. fulgidus Rubisco to ‘A. lithotrophicus’ cell extracts did not lead to any noticeable AMP-dependent CO2 fixation, thus questioning the participation of Rubisco in AMP metabolism in this species.

Table 1.   Ribulose 1,5-bisphosphate carboxylase and related enzyme activities in ‘Archaeoglobus lithotrophicus’ (in nmol min−1 mg−1 protein) at 80°C (growth temperature 83°C).
Enzyme or activitySpecific activity
Phosphoribulokinase (with recombinant Rubisco)<0.2
Ribulose 1,5-bisphosphate carboxylase5 ± 1
PRPP-dependent 14CO2 fixation<1
PRPP-dependent 14CO2 fixation (with recombinant Rubisco)20 ± 3
PRPP-dependent 14CO2 fixation in the presence of NAD+<1
PRPP-dependent 14CO2 fixation in the presence of NAD+ (with recombinant Rubisco)20 ± 2
AMP-dependent 14CO2 fixation (with recombinant Rubisco)<0.2

The other method of obtaining ribulose 1,5-bisphosphate is through dephosphorylation of PRPP and subsequent isomerization of the resulting ribose 1,5-bisphosphate to ribulose 1,5-bisphosphate (Finn & Tabita, 2004). The first reaction may proceed nonenzymatically at an elevated temperature; the second is catalyzed by Mj0601, whose homologue is present in A. fulgidus (AF0702, 46% identity). The addition of PRPP to ‘A. lithotrophicus’ cell extracts resulted in 14CO2 fixation into acid-stabile products in the presence of recombinant Rubisco from A. fulgidus (Table 1), as in Methanocaldococcus jannaschii (Finn & Tabita, 2004). This PRPP-dependent CO2 fixation was not further stimulated by the addition of NAD+, in contrast to the results obtained in experiments with M. jannaschii (Finn & Tabita, 2004).

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Our data suggest that ‘A. lithotrophicus’ uses only the reductive acetyl-CoA pathway for autotrophic CO2 fixation, at least under the conditions of these experiments, namely anaerobic growth in mineral medium pH 6 at 80 °C with CO2 as a carbon source, hydrogen gas as an energy and electron source, and sulfate as an electron acceptor. The findings corroborate the rule that Euryarchaeota use the reductive acetyl-CoA pathway, whereas Crenarchaeota use the dicarboxylate/hydroxybutyrate cycle (anaerobic Thermoproteales and Desulfurococcales) or the hydroxypropionate/hydroxybutyrate cycle [aerobic Sulfolobales and possibly marine Crenarchaeota (Thaumarchaeota)]. Rubisco in Archaeoglobi may participate in scavenging ribose 1,5-bisphosphate, which spontaneously forms from PRPP at a high temperature and otherwise would be a dead-end product.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References

Thanks are due to Christa Ebenau-Jehle, Freiburg, for keeping the lab running. The DOE Joint Genome Institute is acknowledged for the early release of archaeal genomic sequence data. This work was supported by grants from the Deutsche Forschungsgemeinschaft to G.F. and H.H.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
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