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Environmental Microbiology (2007) 9(1), 81–92 doi:10.1111/j.1462-2920.2006.01118.x Autotrophic CO2 fixation via the reductive tricarboxylic acid cycle in different lineages within the phylum Aquificae: evidence for two ways of citrate cleavage Michael Hügler,1 Harald Huber,2 Stephen J. Molyneaux,1 Costantino Vetriani3 and Stefan M. Sievert1,4* 1 Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. 2 Lehrstuhl für Mikrobiologie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany. 3 Department of Biochemistry and Microbiology and Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ 08901, USA. 4 NASA Astrobiology Institute, Marine Biological Laboratory, Woods Hole, MA 02543, USA. Summary Autotrophic carbon fixation was characterized in representative members of the three lineages of the bacterial phylum Aquificae. Enzyme activity measurements and the detection of key genes demonstrated that Aquificae use the reductive tricarboxylic acid (TCA) cycle for autotrophic CO2 fixation. This is the first time that strains of the Hydrogenothermaceae and ‘Desulfurobacteriaceae’ have been investigated for enzymes of autotrophic carbon fixation. Unexpectedly, two different mechanisms of citrate cleavage could be identified within the Aquificae. Aquificaceae use citryl-CoA synthetase and citryl-CoA lyase, whereas Hydrogenothermaceae and ‘Desulfurobacteriaceae’ use ATP citrate lyase. The first mechanism is likely to represent the ancestral version of the reductive TCA cycle. Sequence analyses further suggest that ATP citrate lyase formed by a gene fusion of citryl-CoA synthetase and citryl-CoA lyase and subsequently became involved in a modified version of this pathway. However, rather than having evolved within the Aquificae, our phylogenetic analyses indicate that Aquificae obtained their ATP citrate lyase through lateral gene transfer. Aquificae play an important role in biogeochemical processes in a variety of high-temperature habitats. Thus, these findings sub- Received 25 January, 2006; accepted 27 June, 2006. *For correspondence. E-mail [email protected]; Tel. (+1) 508 289 2305; Fax (+1) 508 457 2076. stantiate the hypothesis that autotrophic carbon fixation through the reductive TCA cycle is widespread and contributes significantly to biomass production particularly in hydrothermal habitats. Introduction Autotrophy is a requirement for self-sustaining life and chemolithoautotrophs – i.e. organisms that acquire energy from the oxidation of inorganic compounds and that use inorganic carbon as the source for cell carbon – are likely to have been among the first types of organisms on Earth (e.g. Huber and Wächtershäuser, 1997; Russell and Hall, 1997). Phylogenetic analyses based on 16S rRNA and whole genomes place autotrophic hyperthermophiles at the base of the evolutionary tree of life, lending evidence to the hypothesis that in addition to being autotrophs, the first life forms might have been hyperthermophilic (Pace, 1991; House and Fitz-Gibbon, 2002). Microorganisms carrying out autotrophic carbon fixation at high temperatures, i.e. above 70°C, utilize CO2 fixation pathways other than the Calvin-Benson-Bassham (Calvin) cycle, which is well known from cyanobacteria, plants and a variety of chemolithoautotrophic Proteobacteria (Fuchs, 1989; Madigan et al., 2003). Presently, three of these so-called alternative pathways are known: the 3-hydroxypropionate cycle, the reductive acetyl-CoA pathway and the reductive tricarboxylic acid (TCA) cycle (Madigan et al., 2003). The reductive TCA cycle, which has been proposed as a likely candidate for the earliest autotrophic pathway that evolved on Earth (Wächtershäuser, 1990; Cody et al., 2001; Smith and Morowitz, 2004), was first shown to function in the green sulfur bacterium Chlorobium limicola (Evans et al., 1966; Fuchs et al., 1980; Ivanovski et al., 1980). Subsequently, the operation of the reductive TCA cycle was also confirmed in a variety of anaerobic and microaerobic bacteria, including the sulfate-reducing bacterium Desulfobacter hydrogenophilus (Schauder et al., 1987), and most recently various e-Proteobacteria (Hügler et al., 2005; Takai et al., 2005). Interestingly, two species of the Aquificaceae, Aquifex pyrophilus (Beh et al., 1993) and Hydrogenobacter thermophilus (Shiba et al., 1985) have also been shown to use the reductive TCA cycle for autotrophic carbon fixation. © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd 82 M. Hügler et al. Fig. 1. 16S rRNA based tree showing the phylogenetic position of Aquificaceae, Hydrogenothermaceae and ‘Desulfurobacteriaceae’. The tree was constructed by using the phylogenetic software ARB (Ludwig et al., 2004), using Archaea as an outgroup. Strains used in this study are indicated in bold. Escherichia coli Candidatus Arcobacter sulfidicus Thiomicrospira denitrificans Desulfobacter hydrogenophilus Chlorobium limicola Chlorobium tepidum Chloroflexus aurantiacus Bacillus subtilis Streptomyces griseus Thermus aquaticus Thermoanaerobacter brockii Fervidobacterium islandicum Thermosipho africanus Thermotoga maritima Aquifex aeolicus Aquifex pyrophilus Hydrogenivirga caldilitoris Thermocrinis albus Hydrogenobacter subterraneus Hydrogenobacter hydrogenophilus Hydrogenobacter thermophilus Thermocrinis ruber Hydrogenobaculum acidophilum Aquificaceae Sulfurihydrogenibium subterraneum Sulfurihydrogenibium azorense Hydrogenothermus marinus Hydrogenothermaceae Persephonella hydrogeniphila Persephonella marina Persephonella guaymasensis Desulfurobacterium thermolithotrophum Desulfurobacterium crinifex 'Desulfurobacteriaceae' Thermovibrio ammonificans Balnearium lithotrophicum Thermovibrio ruber Archaeoglobus fulgidus Methanocaldococcus jannaschii Sulfolobus solfataricus 0.1 nucleotide substitutions / site Within the bacteria, the phylum Aquificae is the only group that contains hyperthermophilic autotrophs. Presently it is divided into three major lineages (Eder and Huber, 2002; Takai et al., 2003a), (i) the Aquificaceae comprising the genera Aquifex, Hydrogenobacter, Hydrogenobaculum, Hydrogenivirga and Thermocrinis; (ii) the Hydrogenothermaceae comprising the genera Hydrogenothermus, Persephonella and Sulfurihydrogenibium; and (iii) several genera incertae sedis (‘Desulfurobacteriaceae’) including the genera Balnearium, Desulfurobacterium and Thermovibrio (Fig. 1). Whereas the Aquificaceae and Hydrogenothermaceae contain predominantly microaerophilic chemolithoautotrophs that obtain energy by oxidizing molecular hydrogen or reduced sulfur compounds, all members of the ‘Desulfurobacteriaceae’ are strict anaerobes, obtaining energy by coupling the oxidation of H2 either to the reduction of elemental sulfur or nitrate. In recent years, several studies have shown that members of the Aquificae are likely to play an important role as primary producers in various hightemperature environments (e.g. Reysenbach et al., 1994; 2000a,b; Harmsen et al., 1997; Yamamoto et al., 1998; Eder and Huber, 2002; Blank et al., 2002; Inagaki et al., 2003; Spear et al., 2005), suggesting that carbon fixation through the reductive TCA cycle might be sig- nificant in these habitats. However, at present no information exists on what carbon fixation pathway members of the ‘Desulfurobacteriaceae’ might use and only limited information, in form of carbon isotopic analyses, exists for the Hydrogenothermaceae (Zhang et al., 2002). The reductive TCA cycle is essentially a reversal of the well-known oxidative TCA or Krebs cycle. Three molecules of CO2 are fixed, forming acetyl-CoA and subsequently pyruvate, the precursor of all other central metabolites. While most of the enzymes are shared between the reductive and oxidative TCA cycle, three enzymes are essential to run this cycle in reverse: 2-oxoglutarate : ferredoxin oxidoreductase, fumarate reductase and ATP citrate lyase. The latter enzyme catalyses the ATP- and the CoA-dependent cleavage of citrate into acetyl-CoA and oxaloacetate. ATP citrate lyase is encoded by two separated genes aclA and aclB, arranged in the same way in all organisms studied to date (Fig. 2A). ATP citrate lyase genes have so far only been found in prokaryotes using the reductive TCA cycle for autotrophic carbon fixation, such as the green sulfur bacteria (Antranikian et al., 1982; Wahlund and Tabita, 1997; Kanao et al., 2001), and e-Proteobacteria (Campbell et al., 2003; Hügler et al., 2005; Takai et al., 2005), and thus can at present be considered as indicator genes © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92 Autotrophic CO2 fixation in Aquificae 83 Fig. 2. Reaction schemes of the citrate cleavage reactions within the reductive TCA cycle. A. Reaction catalysed by ATP citrate lyase (ACL) and gene organization of the genes coding for ACL (aclB and aclA). B. Reaction catalysed through the combined action of citryl-CoA synthetase (CCS) and citryl-CoA lyase (CCL) and gene organization of the genes coding for CCS (ccsA and ccsB) and CCL (ccl). for this pathway. In contrast to Chlorobiaceae and e-Proteobacteria, the ATP-dependent citrate cleavage in H. thermophilus is catalysed by two enzymes, citryl-CoA synthetase and citryl-CoA lyase, which catalyse the ATPdependent formation of citryl-CoA from citrate and CoA and the subsequent cleavage of citryl-CoA into acetylCoA and oxaloacetate respectively (Aoshima et al., 2004a,b) (Fig. 2B). The sequence similarity between citryl-CoA synthetase, succinyl-CoA synthetase and ATP citrate lyase on one hand and between citryl-CoA lyase, citrate synthase and ATP citrate lyase on the other hand suggests that these enzymes share an evolutionary history (Aoshima et al., 2004b). Furthermore, it has been argued that the reductive TCA cycle as identified in H. thermophilus constitutes the ancient form, whereas the reductive TCA cycle in Chlorobium involving ATP citrate lyase represents a secondary adaptation that evolved from the oxidative TCA cycle (Aoshima et al., 2004b). In this context, we were interested in investigating which citrate cleavage mechanism might operate in the two other lineages of the Aquificae. In the present study we have investigated the carbon fixation pathways in representative organisms of all major lineages within the phylum Aquificae. We could measure the activities of the enzymes of the reductive TCA cycle and amplify the genes coding for the respective citrate cleaving enzymes, suggesting that all investigated members of the Aquificae utilize the reductive TCA cycle for autotrophic carbon fixation. Unexpectedly, we found that there exist two ways of citrate cleavage within the Aquificae. The evolutionary and ecological implications of these findings are discussed. Results and discussion Activities of enzymes of the reductive TCA cycle in Aquificae We studied carbon fixation in representatives of the three different lineages of the phylum Aquificae. Table 1 provides an overview of the strains that were used in this investigation, including their isolation site and growth requirements. Cell extracts of Aquifex aeolicus, Thermocrinis ruber (both Aquificaceae), Thermovibrio ammonificans, Thermovibrio ruber (both ‘Desulfurobacteriaceae’) and Sulfurihydrogenibium subterraneum (Hydrogenothermaceae) were tested for the activity of enzymes of the reductive and oxidative TCA cycle. In addition, cell extract of Desulfurobacterium thermolithotrophum (‘Desulfurobacteriaceae’) was tested for citrate cleavage, 2-oxoglutarate and pyruvate : benzyl viologen (BV) oxidoreductase activities. The results of these enzyme measurements are presented in Table 2. In general, the activities of all enzymes of a reductive TCA cycle could be detected in all tested organisms, including the key enzymatic activities of ATP-dependent citrate cleavage, fuma- © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92 84 M. Hügler et al. Table 1. Strains used in this study and some characteristics. Strain Family Isolation site Aquifex aeolicus Aquificaceae Balnearium lithotrophicum Desulfurobacterium crinifex Desulfurobacterium thermolithotrophum Hydrogenobacter hydrogenophilus Hydrogenobacter thermophilus Persephonella marina ‘Desulfurobacteriaceae’ Hydrothermal system (Vulcano, Italy) Deep-sea hydrothermal vent chimney (Suiyo Seamount, Japan) Deep-sea hydrothermal vent chimney (Juan de Fuca Ridge) Deep-sea hydrothermal vent chimney (Mid-Atlantic Ridge) Hot spring (Geyser Valley, Kamchatka, Russia) Hot spring (Mine, Izu, Japan) Deep-sea hydrothermal vent chimney (East Pacific Rise) Octopus Spring (Yellowstone National Park) Deep-sea hydrothermal vent chimney (East Pacific Rise) Submarine hydrothermal vent system (Lihir Island, Papua New Guinea) Hot spring (Furnas, Azores, Portugal) Terrestrial, subsurface hot aquifer water (Hishikari gold mine, Japan) ‘Desulfurobacteriaceae’ ‘Desulfurobacteriaceae’ Aquificaceae Aquificaceae Hydrogenothermaceae Thermocrinis ruber Aquificaceae Thermovibrio ammonificans Thermovibrio ruber ‘Desulfurobacteriaceae’ Sulfurihydrogenibium azorense Sulfurihydrogenibium subterraneum Hydrogenothermaceae ‘Desulfurobacteriaceae’ Hydrogenothermaceae Growth temperature (°C) (optimum) Electron donor/ carbon source Electron acceptor 85 H2/CO2 O2 H2/CO2 S H2/CO2 S H2/CO2 O2 H2/CO2 O2 Formate O2 H2/CO2 NO3– H2/CO2 NO3– H2/CO2 NO3– 45–80 (70–75) 50–70 (60–65) 40–75 (70) 50–82 (74–76) 50–79 (70) 55–80 (73) 44–89 (80) 60–80 (75) 50–80 (75) 50–73 (68) 40–70 (65) only very low rates. These enzymes are 2-oxoglutarate, succinate and pyruvate dehydrogenase. In the following sections, the results for the three families are described and discussed in more detail. rate reductase, 2-oxoglutarate : BV oxidoreductase, and pyruvate : BV oxidoreductase. Enzymes of the oxidative TCA cycle, which are not shared with the reductive version of the pathway, could not be detected or exhibited Table 2. Specific activities [nmol min-1 (mg cell protein)-1] of enzymes of the reductive and oxidative TCA cycle.a Enzyme activity tested Assay temperature (°C) Citrate cleavage 2-Oxoglutarate : BV oxidoreductase Pyruvate : BV oxidoreductase Fumarate reductase Isocitrate dehydrogenase NAD+ NADP+ Malate dehydrogenase NADH NADPH Fumarate hydratase Fumarate Malate Succinyl-CoA synthase 2-Oxoglutarate dehydrogenase NAD+ NADP+ Pyruvate dehydrogenase NAD+ NADP+ Succinate dehydrogenase Formate dehydrogenase Aquifex aeolicus Thermocrinis ruber Sulfurihydrogenibium subterraneum 85 71 420 160 1300 80 82 345 140 1010 65 535 1600 160 2690 385 11 145 8 400 52 100 9500 1310 3350 1710 n.d. 100 600 Desulfurobacterium thermolithotrophum 75 485 330 360 – Thermovibrio ammonificans Thermovibrio ruber 75 275 565 175 635 75 340 400 160 1200 – – 115 11 750 185 45 500 6040 2920 – – 9120 4500 13 780 8560 n.d. 81 975 455 235 380 – – – 190 77 580 4500 350 1460 10 11 11 7 n.d. n.d. – – n.d. n.d. 67 52 n.d. 7 n.d. n.d. 1 2 n.d. 140 n.d. n.d. n.d. n.d. – – – – n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. a. Mean values were obtained from at least five measurements. Standard errors were less than ⫾ 20%. n.d., no activity detected, detection limit < 1 nmol min-1 (mg cell protein)-1; –, not determined. © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92 Autotrophic CO2 fixation in Aquificae Aquificaceae. The operation of the reductive TCA cycle for carbon fixation had previously been demonstrated in A. pyrophilus and H. thermophilus, two members of the Aquificaceae (Shiba et al., 1985; Beh et al., 1993). We could measure all the enzymes of the reductive TCA cycle in A. aeolicus and Tc. ruber. Based either on the whole genome sequence (A. aeolicus) or carbon isotopic measurements (Tc. ruber) both organisms were previously suspected to use this pathway, but direct evidence had been lacking (Deckert et al., 1998; Jahnke et al., 2001). Isocitrate dehydrogenase, as well as malate dehydrogenase, was found to be NAD(H)-dependent. ATPdependent citrate lyase activity in cell extracts was found to be optimal around pH 8.3 (data not shown) exhibiting values of 70 nmol min-1 (mg protein-1) for A. aeolicus and 80 nmol min-1 (mg protein-1) for Tc. ruber. These activities are low compared with the values obtained for S. subterraneum, D. thermolithotrophum or the Thermovibrio species. This might reflect difficulties in measuring the coupled reactions of three different enzymes in vitro, namely citryl-CoA synthetase, citryl-CoA lyase and malate dehydrogenase rather than two as in the case of ATP citrate lyase and malate dehydrogenase. Although Tc. ruber was grown with formate as carbon source, the organism still uses the enzymes of the reductive TCA cycle to fix the CO2 derived from formate. In addition, formate dehydrogenase activity could be measured, probably catalysing the formation of CO2 from formate. Hydrogenothermaceae. Sulfurihydrogenibium subterraneum, a representative member of the Hydrogenothermaceae, showed activities of all enzymes of the reductive TCA cycle, including high activities of ATP-dependent citrate lyase and 2-oxoglutarate : BV oxidoreductase. The pH optimum of ATP citrate lyase was determined to be pH 8.3 (data not shown). In contrast to members of the Aquificaceae, isocitrate dehydrogenase was NADPdependent. Activities of the enzymes of the oxidative TCA cycle were not detected. The demonstration of the reductive TCA cycle in a member of the Hydrogenothermaceae is in agreement with stable carbon isotopic analyses and the amplification of a small fragment of aclB from Persephonella marina (Zhang et al., 2002; Campbell et al., 2003). ‘Desulfurobacteriaceae’. All tested members of this family showed high activities of ATP-dependent citrate lyase and 2-oxoglutarate/pyruvate : BV oxidoreductase. The pH optimum of ATP citrate lyase was determined to be pH 8.3 in both Tv. ammonificans and Tv. ruber (data not shown). In both strains isocitrate dehydrogenase was NADP-dependent. This is the first report describing the autotrophic carbon fixation mode in this lineage, which has been proposed to represent an additional order within the 85 phylum Aquificae (L’Haridon et al., 1998; Huber et al., 2002). Based on our data, the use of the reductive TCA cycle for autotrophic carbon fixation emerges as a common feature among the different lineages within the Aquificae. Amplification of citryl-CoA lyase and ATP citrate lyase genes Citrate cleavage is a key reaction of the reductive TCA cycle. It can be catalysed either through ATP citrate lyase (e.g. Chlorobium, Thiomicrospira denitrificans) (Kanao et al., 2001; Hügler et al., 2005), or by the combined action of citryl-CoA synthetase and citryl-CoA lyase (H. thermophilus) (Aoshima et al., 2004a,b) (Fig. 2). To determine which mechanism is present in the different lineages of the Aquificae, we isolated DNA and amplified, cloned and sequenced both subunits of ATP citrate lyase as well as large parts of citryl-CoA lyase using previously published and newly designed primer pairs (see Experimental procedures). Genomic DNA of A. aeolicus, Hydrogenobacter hydrogenophilus, H. thermophilus, Tc. ruber (all Aquificaceae); S. subterraneum (Hydrogenothermaceae); Balnearium lithotrophicum, Desulfurobacterium crinifex, D. thermolithotrophum, Tv. ammonificans and Tv. ruber (all ‘Desulfurobacteriaceae’) was used. In addition, the draft genomes of P. marina and Sulfurihydrogenibium azorense were searched for ATP citrate lyase, citryl-CoA lyase and citryl-CoA synthetase genes (preliminary sequence data were obtained from The Institute for Genomic Research through the website at http://www.tigr. org). Surprisingly, both mechanisms for citrate cleavage seem to be present within the Aquificae. Aquificaceae. As expected, we were not able to amplify fragments of the ATP citrate lyase genes (acl) from A. aeolicus, H. hydrogenophilus, H. thermophilus and Tc. ruber. Instead, we could amplify a fragment of the gene coding for citryl-CoA lyase (ccl) from all four strains using the newly designed primer pairs cclf1/cclr1 or cclf2/ cclr2. This resulted in ~620 bp of sequence for ccl (representing ~80% of the expected ~780 bp). A phylogenetic tree based on the alignment of the protein sequence of citryl-CoA lyase is presented in Fig. 3. The ccl gene has previously been sequenced from H. thermophilus (Aoshima et al., 2004b). Based on this sequence, gene aq_150 (AAC06486) from A. aeolicus (annotated as coding for citrate synthase) could be identified as a ccl gene (Deckert et al., 1998; Aoshima et al., 2004b). The sequenced gene fragments from H. hydrogenophilus and Tc. ruber are closely related to these genes. As no acl gene could be amplified from H. hydrogenophilus and Tc. ruber and no acl genes are found in the genome of A. aeolicus, we assume that these three organisms use the combined action of citryl-CoA synthetase and citryl- © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92 86 M. Hügler et al. Fig. 3. Phylogenetic tree based on ~205 amino acids of the citryl-CoA lyase protein. The tree depicts the phylogenetic relationships between the newly sequenced and translated ccl fragments and sequences obtained from the data base representing citryl-CoA lyase, ATP citrate lyase and citrate synthase sequences. ATP citrate lyase (aclA) P. marina S. azorense T. denitrificans C. limicola S. azorense S. subterraneum A.aeolicus C. tepidum citryl-CoA lyase H. hydrogenophilus H. thermophilus Tc. ruber T. denitrificans P. marina E. coli P. marina C. tepidum S. azorense citrate synthase 0.1 amino acid substitutions / site CoA lyase for the cleavage of citrate into oxaloacetate and acetyl-CoA. Genes coding for citryl-CoA synthetase can be found within the genome sequence of A. aeolicus. They have been assigned to encode succinyl-CoA synthetase [sucC1 (AAC07285) and sucD1 (AAC07686)]. Consequently, A. aeolicus, H. hydrogenophilus and Tc. ruber seem to fix CO2 via the same set of enzymes as H. thermophilus. These include citryl-CoA synthetase, citryl-CoA lyase as well as a NAD-dependent isocitrate dehydrogenase (Aoshima et al., 2004a,b,c). Hydrogenothermaceae. Like in Aquificaceae, we could successfully amplify and sequence a gene fragment of citryl-CoA lyase (~620 bp, representing ~80% of the expected ~780 bp). In addition, we also amplified, cloned and sequenced the genes coding for the a- and b-subunit of ATP citrate lyase (aclA and aclB) from S. subterraneum using the primer pairs F2/R5, 892F/R5 and F1/R1. This way we obtained the complete gene sequence for aclB and a ~1.600 bp fragment of aclA (~90% of the expected ~1.800 bp). The gene arrangement is identical to all known ATP citrate lyase genes (aclBA). BLAST searches of the draft genomes of P. marina and S. azorense revealed that both organisms also possess genes coding for both ATP citrate lyase subunits, strongly suggesting that they also use the reductive TCA cycle for carbon fixation. The ATP citrate lyase genes of all three investigated members of Hydrogenothermaceae show high sequence identities, clustering together in the phylogenetic tree. Interestingly, BLAST searches also revealed a putative gene in the draft genomes of P. marina and S. azorense that could code for citryl-CoA lyase. However, a homologue of the citryl-CoA synthetase could not be identified, indicating that these organisms use ATP citrate lyase to catalyse citrate cleavage. The physiological role of citryl-CoA lyase is presently unclear and warrants further investigations. ‘Desulfurobacteriaceae’. We could amplify the ATP citrate lyase genes from all isolated strains of the ‘Desulfurobacteriaceae’. The entire sequence of aclB and ~90% of aclA was obtained from B. lithotrophicum, D. crinifex and D. thermolithotrophum. In addition, we amplified and sequenced a ~1000 bp fragment of aclA (primer pair F2/R5) from Tv. ammonificans, as well as ~450 bp of the 5′ end of aclB and ~90% of aclA from Tv. ruber (primer pair 892F/R5). The translated sequences obtained from these strains show high identities between each other, as well as high identities and similarities to the sequences from Persephonella and Sulfurihydrogenibium. Using our newly designed primers we failed to amplify a citryl-CoA lyase gene from members of the ‘Desulfurobacteriaceae’. However, at this point we cannot exclude the presence of a citryl-CoA gene within this lineage, as the primers were designed only on a limited number of ccl sequences and thus might not be appropriate for this group. Regardless, the presence of aclBA and the high citrate cleavage activities strongly suggest that ‘Desulfurobacteriaceae’ use ATP citrate lyase to accomplish citrate cleavage. ATP citrate lyase (aclBA) and citryl-CoA lyase (ccl) as phylogenetic and functional gene markers Phylogenetic analyses of available aclA and aclB sequences reveal that the sequences obtained from © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92 Autotrophic CO2 fixation in Aquificae B. lithotrophicum (86/100) Tv. ruber (100/100) D. crinifex (100/100) (100/100) D. thermolithotrophum S. subterraneum (100/100) (100/100) 'Desulfurobacteriaceae' S. azorense (63/-) Hydrogenothermaceae P. marina T. denitrificans (100/100) (100/100) (92/83) 6C6 ε-Proteobacteria 7G3 Neurospora (100/100) 87 Fig. 4. Phylogenetic tree based on ~970 amino acids of both subunits of ATP citrate lyase. The tree depicts the phylogenetic relationships of the newly obtained ATP citrate lyase sequences from members of the Aquificae with sequences from e-Proteobacteria, Chlorobiaceae and eukaryotes. A hypothetical ancestral ATP citrate lyase, constructed by concatenating citryl-CoA synthetase and citryl-CoA lyase from H. thermophilus and A. aeolicus, respectively, to one protein, was used as outgroup. Bootstrap values from both distance analysis (first number) and parsimony analysis (second number) are shown as percentages of 1000 bootstrap replications. Schizosaccharomyces (100/100) (100/100) (100/100) Rattus Eukaryotes Danio (100/100) Arabidopsis Oryza (100/100) C. limicola Chlorobiaceae C. tepidum (100/100) H. thermophilus A. aeolicus 0.05 amino acid substitutions / site members of the phylum Aquificae form a monophyletic group clearly separated from sequences of e-Proteobacteria, Chlorobiaceae and eukaryotes (fungi, plants, animals) (Fig. 4). The ATP citrate lyase sequences from Aquificae are most closely related to sequences from e-Proteobacteria, with which they form a clade of their own. Phylogenetic trees calculated for the individual subunits of ATP citrate lyase show the same overall pattern (data not shown). The ATP citrate lyase based tree is largely in congruence with 16S rRNA phylogeny, with e-Proteobacteria and Aquificae clearly separated from each other, and the sequences from ‘Desulfurobacteriaceae’ (Balnearium, Desulfurobacterium, Thermovibrio) and Hydrogenothermaceae (Persephonella, Sulfurihydrogenibium) forming two branches within the Aquificae. Our expanded data set of ATP citrate lyase genes from well-characterized strains of the Aquificae builds a framework that allows us to assign acl sequences obtained from the environment to particular lineages. Along these lines, we compared the acl sequences obtained in this study with environmental sequences obtained from different hydrothermal vent sites (Campbell and Cary, 2004) (Fig. 5). This way we can clearly classify some sequences as Persephonella-like, while Sulfurihydrogenibium species apparently seem to be absent. This is not too surprising, considering that all Sulfurihydrogenibium species to date have been identified in terrestrial environments. However, no sequences were found that could be assigned to the ‘Desulfurobacteriaceae’, although these organisms have been isolated from hydrothermal vents, and were found to account for 40% of the microorganisms present in chimney walls (e.g. Harmsen et al., 1997). An explanation for this unexpected finding could be the specificity of the primers used by Campbell and Cary (2004), which in our hands failed to amplify aclB from ‘Desulfurobacteriaceae’. In addition, our sequence analyses revealed a cluster of acl sequences that seem to be affiliated with Hydrogenothermaceae, but cannot be directly linked to sequences obtained from either Persephonella or Sulfurihydrogenibium. This strongly suggests that these sequences represent a new group of so far uncultivated Aquificae. Thus, our data provide further evidence for the usefulness of the ATP citrate lyase genes as both phylogenetic and functional gene markers. Based on our limited number of sequences, it appears that citryl-CoA lyase may have the potential to be used in the same way (Fig. 3). Evolutionary implications Surprisingly, two different pathways of citrate cleavage exist within the Aquificae. Members of the genera Aquifex, Hydrogenobacter and Thermocrinis use two enzymes, namely citryl-CoA synthetase and citryl-CoA lyase to accomplish the ATP-dependent cleavage of citrate into acetyl-CoA and oxaloacetate. This has been proposed to represent the ancestral version of the reductive TCA cycle (Aoshima et al., 2004a,b). Furthermore, succinyl-CoA synthetase and citryl-CoA synthetase are believed to have evolved via gene duplication and diversification of an enzyme that possibly catalysed both reactions © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92 88 M. Hügler et al. Tv. ruber B. lithotrophicum 'Desulfurobacteriaceae' D. crinifex D. thermolithotrophum 2E08 Guay 694-12 Fig. 5. Phylogenetic tree based on a ~330 bp fragment of the gene coding for the small subunit of ATP citrate lyase (aclB). The tree depicts the phylogenetic relationships of the newly obtained sequences from Aquificae with other bacterial sequences derived either from the environment (Campbell and Cary, 2004) or from other cultures. 694-4 690-42 Persephonella 729-4 A1 P. marina 729-4 C2 729-4 A5 S. azorense S. subterraneum Sulfurihydrogenibium 820-A8 729-4 C1 820-C7 820-D8 uncultured Aquificae ? 820-C1 820-A12 729-4 H5 820-D5 T. denitrificans Candidatus A. sulfidicus Nautilia sp. Am-H 899-1 ε-Proteobacteria 899-3 6C6 7G3 C. tepidum C. limicola 0.05 nucleotide substitutions / site (Aoshima et al., 2004a). On the other hand, members of the genera Persephonella, Sulfurihydrogenibium (both Hydrogenothermaceae), Balnearium, Desulfurobacterium and Thermovibrio (all ‘Desulfurobacteriaceae’) are using ATP citrate lyase for citrate cleavage. Based on sequence similarity, the origin of the ATP citrate lyase might be the result of a gene fusion of citryl-CoA synthetase and citrylCoA lyase, which themselves are homologues of succinylCoA synthetase and citrate synthase (Fatland et al., 2002; Aoshima et al., 2004a,b). The small subunit of ATP citrate lyase is a homologue of the large subunit of citryl-CoA synthetase, whereas the large subunit of ATP citrate lyase seems to have formed by a fusion of the small subunit of citryl-CoA synthetase and citryl-CoA lyase (Fig. 2). These findings raise the question on the origin of ATP citrate lyase in Hydrogenothermaceae and ‘Desulfurobacteriaceae’. In one possible scenario, ATP citrate lyase might have evolved within the Aquificae from an ancient form of the reductive TCA cycle involving a two-step citrate cleavage, as exemplified presently by H. thermophilus, to a reductive TCA cycle utilizing only one enzyme, like it is operating in the ‘Desulfurobacteriaceae’. In another scenario, ATP citrate lyase might have been transferred into Hydrogenothermaceae and ‘Desulfurobacteriaceae’ via lateral gene transfer. Based on our data, the second scenario seems more likely, because ATP citrate lyase from Hydrogenothermaceae and ‘Desulfurobacteriaceae’ appears to be a derived form of the ATP citrate lyase since it is more similar to the one from e-Proteobacteria than to the evolutionary ancestral enzyme (Figs 3 and 4). Our phylogenetic analyses confirm earlier results of Fatland and colleagues (2002), who concluded that the sequence from C. limicola appears to be closest to the evolutionary ancestral form. Thus, ATP citrate lyase appears to have been acquired by Aquificae via horizontal gene transfer, most likely from e-Proteobacteria. Along these lines it is interesting to note that both groups occur in similar environments, providing an ecological basis for such an event. Ecological implications The results obtained in this study lend further support to the hypothesis that autotrophic carbon fixation through © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92 Autotrophic CO2 fixation in Aquificae the reductive TCA cycle is widespread and contributes significantly to biomass production in certain environments, particularly in hydrothermal habitats. Members of the Aquificae are important in many hot environments, e.g. shallow and deep-sea marine vents, terrestrial hot springs, as well as the terrestrial subsurface (e.g. Harmsen et al., 1997; Reysenbach et al., 2000a,b; Blank et al., 2002; Takai et al., 2002; Huber et al., 2003; Inagaki et al., 2003), and have been isolated from all around the world (Table 1). Extensive research has been particularly carried out at Yellowstone National Park, USA, where Tc. ruber has been identified as an ubiquitous member of microbial communities in silicadepositing hot springs (Blank et al., 2002) and Aquificae in general may dominate the microbial communities at temperatures higher than 70°C (Spear et al., 2005). Our finding that Tc. ruber and other Aquificae do fix CO2 via the reductive TCA cycle suggests that primary production at temperatures higher than 70°C in hot springs in Yellowstone and probably elsewhere occurs mainly via the reductive TCA cycle. Members of the Aquificae have also been shown to constitute an important component of the microbial communities at deep-sea hydrothermal vents, where these organisms seem to be mainly associated with sulfide structures and most likely also colonize the subsurface portion of vents (Harmsen et al., 1997; L’Haridon et al., 1998; Reysenbach et al., 2000a; Götz et al., 2002; Alain et al., 2003; Huber et al., 2003; Takai et al., 2003a; Vetriani et al., 2004). Besides members of the Aquificae, e-Proteobacteria have been identified as a dominant bacterial group at deep-sea vents (e.g. Reysenbach et al., 2000b; Huber et al., 2003; Nakagawa et al., 2005). Interestingly, autotrophic members of this group also use the reductive TCA cycle for carbon fixation (Campbell et al., 2003; Hügler et al., 2005; Takai et al., 2005). As a whole, both groups exhibit similar metabolisms – i.e. the oxidation of reduced sulfur compounds and hydrogen with both oxygen and nitrate or the oxidation of hydrogen with elemental sulfur coupled to the fixation of inorganic carbon – and thus occupy a similar ecological niche. However, they seem to be partitioned by their temperature preference, with e-Proteobacteria dominating the microbial communities at temperatures from 20°C to 60°C, whereas Aquificae seem to be the predominant autotrophs at temperatures higher than 60°C. Taken together, this suggests that a significant fraction of the biomass at deep-sea hydrothermal vents might be produced via carbon fixation through the reductive TCA cycle. It remains to be determined whether this production rivals the fixation occurring through the Calvin cycle, which in the current paradigm forms the base of deep-sea hydrothermal ecosystems. Ongoing studies in our laboratory that aim to couple the identity 89 of the microorganisms carrying out CO2 fixation in situ with concurrent rate measurements are addressing this question. Experimental procedures Bacteria, growth conditions and preparation of cell extracts Table 1 gives an overview about the strains used in this investigation, including their isolation site and growth requirements. Aquifex aeolicus VF5 (Eder and Huber, 2002), B. lithotrophicum 17S (DSMZ 16304) (Takai et al., 2003a), D. crinifex NE1206 (DSMZ 15218) (Alain et al., 2003), D. thermolithotrophum BSA (DSMZ 11699) (L’Haridon et al., 1998), H. hydrogenophilus Z-829 (DSMZ 2913) (Kryukov et al., 1983), H. thermophilus TK-6 (DSMZ 6543) (Kawasumi et al., 1984), Tv. ammonificans HB-1 (DSMZ 15698) (Vetriani et al., 2004), Tv. ruber ED11/3LLK (DSMZ 14644) (Huber et al., 2002) and S. subterraneum HGMK1 (DSMZ 15120) (Takai et al., 2003b) were grown autotrophically as described in references. Thermocrinis ruber OC1/4 (DSMZ 12173) (Huber et al., 1998) was grown with formate as sole carbon source. Biomass was harvested under the exclusion of oxygen. Cell extracts were prepared using a mixer mill (type MM2; Retsch, Haare, Germany) according to Hügler and colleagues (2005). Protein concentration in cell extracts was determined by the method of Bradford (1976) using bovine serum albumin as standard. Enzyme assays Enzyme assays (0.5 ml assay mixture) were performed in stoppered 0.5 ml glass cuvettes. The assay temperature differed according to the organisms and is given in Table 2. Reactions involving pyridine nucleotides were followed spectrophotometrically at 365 nm [e365 nm NAD(P)H = 3.4 ¥ 103 M-1 cm-1]. Reactions involving BV were followed spectrophotometrically at 578 nm (e578 nm BV = 8.6 ¥ 103 M-1 cm-1). The citrate cleavage activity of ATP citrate lyase or the combined activities of citryl-CoA synthetase and citryl-CoA lyase were determined by coupling the reactions to malate dehydrogenase, which reduces the produced oxaloacetate with NADH. The citrate-, CoA- and MgATP-dependent oxidation of NADH was monitored. The assay mixture contained 100 mM HEPES (N-2-Hydroxyethylpiperazine-N⬘-2ethanesulfonic acid)/NaOH, pH 8.3, 5 mM DTE, 5 mM MgCl2, 3 mM ATP, 0.5 mM CoA, 0.4 mM NADH, and 3 mM D-citrate. The reaction was started by the addition of citrate. Buffers used to determine the pH optimum were HEPES/ NaOH (pH 6.5–8.5), and MOPS [3-(N-Morpholino)-2hydroxypropanesulfonic acid]/NaOH (pH 6.5–8.5). 2Oxoglutarate : BV oxidoreductase, pyruvate : BV oxidoreductase, fumarate reductase, malate dehydrogenase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, and pyruvate dehydrogenase were measured according to references (Hügler et al., 2003; 2005). Formate dehydrogenase and succinyl-CoA synthetase were measured as described in Beh and colleagues (1993). Succinate dehydrogenase was determined spectrophotometrically as the reduction of © 2006 The Authors Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 81–92 90 M. Hügler et al. 2,6-dichloroindophenol with succinate at 578 nm according to Schauder and colleagues (1987). Fumarate hydratase was measured in a direct spectrophotometrical assay by following the consumption or the production of fumarate at 240 nm using 0.5 mM fumarate or 5 mM malate as substrate (Schauder et al., 1987; Hügler et al., 2003). DNA extraction, PCR amplification, cloning and sequencing of aclBA and ccl and phylogenetic analyses DNA was extracted with the UltraClean microbial DNA Isolation kit (Mo Bio Laboratories, Solana Beach, CA) according to the manufacturer’s protocol. For amplification of the ATP citrate lyase genes, different primer sets were used in a 35-cycle PCR at an annealing temperature of 42–50°C. The used primers were F2/R5 (annealing at 42°C) (Hügler et al., 2005), 892f/R5 (annealing at 50°C) (Campbell et al., 2003; Hügler et al., 2005) and F1 (5′-ATG GCT CAA AAG GCA ATT AGA GAA T-3′)/R1 (5′-GCA CCA GCA TGA CCA AAC TGA -3′) (annealing at 46°C). For the amplification of the citryl-CoA lyase gene we used newly designed primers cclf1 (5′-CGG CTT CTT GAT CTT GTA GG-3′)/cclr1(5′-TTC TCT GTG GTT AGT TCT TC-3′) or cclf2(5′-CCT TTA ACC CAG ATG ATA GG-3′)/cclr2(5′-GGT TCT TGA GTT AGT TCT TC-3′) in a 30-cycle PCR at an annealing temperature of 50°C. PCR products were purified from agarose gels using a QIAquick gel extraction kit (QIAGEN, Chatswood, CA) and cloned using the TOPO TA cloning kit for sequencing (Invitrogen, Carlsberg, CA) as described by the manufacturer. Colonies were picked, and the plasmid DNA was purified with the QIAprep spin miniprep kit (QIAGEN) as described by the user manual. Plasmids were sent to the BioResource Center at Cornell University (Ithaca, NY) for sequencing. DNA sequencing was performed using the Applied Biosystems Automated 3730 DNA analyser. The big dye terminator chemistry and AmliTaq-FS DNA polymerase were used. Preliminary sequence data of P. marina and S. azorense were obtained from The Institute for Genomic Research through the website at http://www.tigr.org. The sequences were analysed using MacVector (Accelrys, San Diego, CA) and PAUP, version 4.0b10, as described previously (Hügler et al., 2005). Nucleotide sequence accession numbers The nucleotide sequences have been deposited in GenBank. The accession numbers for the ATP citrate lyase genes are: DQ853417 (B. lithotrophicum), DQ853418 (D. crinifex), DQ853419 (D. thermolithotrophum), DQ853420 (Tv. ammonificans), DQ853421 (Tv. ruber), and DQ853422 (S. subterraneum). The accession numbers for the citryl-CoA lyase gene fragments are: DQ853423 (H. hydrogenophilus), DQ853424 (S. subterraneum) and DQ853425 (Tc. ruber). Acknowledgements This study was supported by the NASA Astrobiology Institute (‘From Early Biospheric Metabolism to the Evolution of Complex Systems’; Grant NNA04CC04A), as well as NSF Grants MCB 04-56676 (C.V.), MCB 04-56689 (S.M.S.) and OCE 04-52333 (S.M.S.). M.H. was supported through a post- doctoral scholarship from the Woods Hole Oceanographic Institution. Preliminary sequence data were obtained from The Institute for Genomic Research through the website at http://www.tigr.org. Sequencing of P. marina and S. azorense was accomplished with support from the National Science Foundation. 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