The major features of the C. sticklandii genome are listed in Table 1 (comparison with other clostridial species - Additional file 1). Its genome consists of a single circular chromosome of 2,715,461 bp, which carries 2573 coding sequences (CDS). The number of CDS in such a small genome and the low level of repeated regions (2.1%) explain the high protein coding density of 89.2%. Of the 2573 CDS, 33% were annotated as conserved hypothetical or hypothetical proteins. Two members of the genus Alkaliphilus (A. metalliredigens QIMF and A. oremlandii OhILas) and Clostridium difficile 630 share the highest number of orthologous genes with C. sticklandii (Additional file 2).

Annotation of the C. sticklandii genome reveals that most biosynthetic pathways considered as essential for viability seem to be present (e.g., biosynthesis of amino acids, purines, pyrimidines and cofactors). However, it should be noted that genes involved in synthesis of biotin and pantothenate are absent, confirming the absolute requirement of these two vitamins for growth observed by Golovchenko et al. 23 . These authors also report a requirement for cyanocobalamin and thiamin. The absence of some genes involved in the biosynthesis of these cofactors is in agreement with these results. Pyridoxal phosphate synthesis proceeds through the proteins encoded by pdxS and pdxT 24 and not by the pathway existing in E. coli and many other bacteria. C. sticklandii is unable to assimilate sulphate, since genes involved in this pathway are absent (cysN, cysD, cysC, and cysH ). This inability is also found in some other clostridia (Additional file 1). In C. sticklandii, the sulphur requirement for cysteine biosynthesis can be met by sulphide (data not shown) since genes coding for serine O-acetyltransferase (cysE) and O-acetylsulfhydrylase (cysK) are present. The only genes of the upper portion of the tricarboxylic acid cycle found with certainty in this bacterium are the adjacent genes encoding Si-citrate synthase and isocitrate dehydrogenase. Since glutamate is excreted (see section below), there is no need for glutamate formation via the tricarboxylic acid cycle. Glutamate could be formed by histidine degradation since all the genes coding for the enzymes of histidine biosynthesis and degradation are present. Additionally, 2-ketoglutarate could be formed by reductive carboxylation of succinyl-CoA using reduced ferredoxin by one of the many 2-ketoacid ferredoxin oxidoreductases. All genes involved in the glycolysis pathway were detected. However, we found that C. sticklandii is not able to grow on glucose (data not shown) although a slight fermentation of glucose has been described 21 . This is explained by a defect in some transport components 25 . Indeed, genome analysis shows the absence of genes coding for protein II of the PTS system. The genes of the non-oxidative pentose phosphate pathway are present, accounting for the synthesis of hexose and pentose phosphates respectively. A cluster of genes encoding a ribose transporter and a ribokinase could lead to ribose 5-phosphate, which can be completely converted into glycolytic intermediates.

As a proteolytic species, C. sticklandii contains about 12 proteases and 38 peptidases and is highly dependent on the utilization of amino acids (see section below). Annotation of its genome has confirmed the presence of several biochemically described amino acid degradation pathways (Additional file 3). Similar amino acid degradation pathways are found in the metal-reducing strain Alkaliphilus metalliredigens QIMF, in the arsenic-resistant strain Alkaliphilus oremlandii OhILas 26 27 as well as in the pathogenic strains of C. difficile and C. botulinum (Additional file 4). It should be noticed that arginine deiminase is missing in the genome of C. difficile, although this species contains all the genes involved in the subsequent steps in the arginine breakdown pathway. The genes coding for several transporters for amino acids were found in C. sticklandii (e.g., ABC transporters for oligopeptides, methionine, and branched-chain amino acids, a serine/threonine symporter as well as an arginine/ornithine antiporter). The degradation of the purines (adenine, xanthine and uric acid) by C. sticklandii has been reported 19 20 . However, xanthine dehydrogenase, the key enzyme of purine degradation, was not found in this genome. We cannot exclude that purine compounds are converted by a totally different 2-ketopurine dehydrogenase. This protein has been purified from several species of peptostreptococci 28 , but the gene is not known.

Important features of amino acid metabolism by C. sticklandii are depicted in Figure 1A. It oxidizes ornithine, arginine, threonine, serine and reduces proline and glycine 2 12 15 . C. sticklandii can utilize glycine and ornithine both as an electron donor and an electron acceptor in the Stickland reaction. Ornithine can be reduced to 5-aminovaleric acid through the formation of D-proline as an intermediate, or it can be oxidized to acetate, D-alanine and ammonia 15 29 30 . Glycine can be directly oxidized by NAD⁺ into methylene-THF, CO₂ and ammonia via the glycine cleavage system (gcv-system) or reduced to acetyl phosphate by glycine reductase 31 . Electrons from the gcv-system can be directly utilized by glycine reductase as observed for Eubacterium acidaminophilum 32 .

Since a large variety of amino acids can be utilized by C. sticklandii as nutrient and energy sources, it was interesting to determine which amino acids are preferentially metabolized by this bacterium. For this purpose C. sticklandii was cultivated in a defined medium with the amino acids at the same concentration (see Methods). Using Liquid Chromatography-Mass Spectrometry (LC-MS) analysis (Figure 1B and Additional file 5) we found that arginine, serine, threonine, cysteine, proline and glycine disappear rapidly from the medium in the exponential growth phase. In contrast, lysine, histidine, asparagine and valine disappear at the onset of the stationary growth phase. The concentrations of the aromatic amino acids remained stable: they were not metabolized under our growth conditions. Glutamate, aspartate and L- and D-alanine are excreted. These results show that C. sticklandii seems to prefer certain amino acids and degrades them in a specific order.

Subsequently, we tried to associate amino acid utilization with the analysis of genes coding for proteins involved in the degradation pathways. The rapid utilization of threonine can be explained by the presence of three pathways through which this amino acid is catabolized. Threonine dehydrogenase oxidizes threonine into 2-amino-3-ketobutyrate 33 which is split into glycine and acetyl-CoA. Threonine aldolase converts threonine into glycine and acetaldehyde, whereas threonine dehydratase transforms threonine into ammonia and 2-ketobutyrate. The latter compound is oxidized to propionate and CO₂ or reductively aminated to 2-aminobutyrate (Figure 1A), typically formed by C. sticklandii 1 and detected in extracellular medium by LC-MS (data not shown). Arginine is quickly metabolized through the arginine deiminase pathway into ornithine via citrulline. This is a generally favoured pathway by anaerobes, for they can conserve energy without any redox reaction by splitting the deaminated intermediate citrulline by ornithine carbamoyl-phosphate transferase and conserve ATP by carbamate kinase. As expected, C. sticklandii contains all the genes involved in the arginine deiminase pathway as well as the ornithine oxidative and reductive pathways 29 30 . Like C. botulinum and C. sporogenes 34 , C. sticklandii possesses an ornithine cyclodeaminase that directly produces proline from ornithine. Dehydration of serine leads to pyruvate which is decarboxylated into acetyl-CoA by pyruvate ferredoxin oxidoreductase. The rapid utilization of cysteine may be correlated with its degradation to pyruvate, ammonia and hydrogen sulphide by a putative L-cysteine sulphide lyase. In the degradation of lysine into acetate, butyrate, and ammonia 35 36 37 , the highly exergonic reduction of crotonyl-CoA to butyryl-CoA contributes to energy conservation 38 . However, lysine is not used in the exponential growth phase (Additional file 5). One could hypothesize that during this growth phase crotonyl-CoA is formed from the condensation of two acetyl-CoAs via butyrate fermentation, since all the genes involved in this pathway are present in C. sticklandii. Acetyl-CoA could be generated from serine, arginine and cysteine during the exponential growth phase. Thus, after depletion of these amino acids (at the beginning of the stationary growth phase) crotonyl-CoA is produced from lysine fermentation. It has been reported that C. sticklandii degrades aromatic and branched chain amino acids by oxidative means 14 17 18 . However, under our experimental conditions, aromatic amino acids do not appear to be metabolized (Additional file 5) whereas valine, leucine, and (or) isoleucine (the two latter compounds are not distinguishable by mass spectrometry) disappear from the medium. They are probably transaminated to the corresponding 2-ketoacids, which are further degraded by the branched-chain fatty acid ferredoxin oxidoreductase. Alanine, which was not added to the medium, may be formed by the breakdown of ornithine. This amino acid is excreted in the exponential phase, but disappears slowly in the stationary growth phase. Stadtman has shown that the Stickland reaction amino acid pair, alanine and glycine, is not operative in C. sticklandii 16 . A gene coding for alanine dehydrogenase is actually absent in C. sticklandii. Glutamate might be formed by degradation of histidine but in contrast to other clostridial species, especially C. tetanomorphum and C. cochlearium 39 , glutamate is not used by C. sticklandii in accordance with previous results 1 2 . Genes encoding methylaspartate mutase or 2-hydroxyglutaryl-CoA dehydratase, the key enzymes of the two major glutamate fermentation pathways 40 , have not been detected in the genome.

Based on the results of amino acid utilization, we tried to grow C. sticklandii in three different media. The following amino acid combinations were utilized: the first contained amino acids utilized in the exponential growth phase, the second contained those utilized in the exponential and stationary growth phase and the third was equivalent to the second complemented with amino acids that apparently were not metabolized (Additional file 6). Surprisingly, no growth was observed in the medium with the second amino acid combination. Similar experiments have been performed by Golovchenko et al. using C. sticklandii strain CSG 23 . Thus, it appears that the combination of amino acid influences the growth of C. sticklandii. There are probably regulatory properties implying that the presence or absence of some amino acids could repress the utilization or the biosynthesis of other amino acids. In particular, it has been shown that in some clostridial species that perform the Stickland reaction, the type of amino acid pair is quite important for growth 41 .

Selenoproteins are characterized by the replacement of a cysteine residue by a selenocysteine (Sec) encoded by the UGA stop-codon. A complex machinery is required for translational insertion of Sec in response to the UGA codon. In fact, the first selenoprotein, glycine reductase A (GrdA), was discovered in C. sticklandii 42 43 . Analysis of the C. sticklandii genome revealed that all known components of the Sec insertion machinery (Additional file 3) and the Sec tRNA are present. Using the gene fusion-fission functionality of MaGe 44 , we identified eight selenoproteins in C. sticklandii (Table 2). Interestingly, one of these proteins, PrdC, had never been described as a selenoprotein before, although the proline reductase genes have been studied in detail in both C. difficile and C. sticklandii 22 45 46 . PrdC is located in the D-proline reductase operon and is annotated as an electron transfer protein 45 . This role has also been suggested by Eversmann 47 . Evidence for the exact biochemical role of PrdC is now provided by the discovery of a C-terminal redox center -CxxU-. The amino acid sequence of PrdC exhibits homology to RnfC, the protein involved in the NADH-dependent Rnf electron transfer complex. Recently, Kim et al. have identified RnfC as a selenoprotein in Alkaliphilus oremlandii OhILAs (accession no. YP_001511593). However, the genomic context and the alignment of this sequence with the PrdC of several clostridial species (Figure 2) show that this selenoprotein (RnfC) is actually PrdC. This alignment also shows that the selenocysteine-residue is present in all PrdC proteins in the terminal -CxxU-motif with two exceptions: there is a deviation of the selenocysteine position in PrdC of C. botulinum, and PrdC of C. difficile does not contain a selenocysteine but a cysteine residue, thus emphasizing that the presence of selenocysteine in PrdC is not a necessity, but is probably catalytically advantageous as has been shown e.g. for methionine sulphoxide reductases 48 .

D-Proline acts as the electron acceptor in the Stickland reaction. The D-proline reductase has been biochemically studied only in C. sticklandii 46 49 and in C. difficile 22 . It catalyzes the reductive ring cleavage of D-proline into 5-aminovalerate, which is subsequently excreted. In C. sporogenes, the reduction of D-proline was described to be coupled to transmembrane proton ejection. However, it has not been demonstrated whether this proton gradient is coupled to ATP synthesis 50 . This is possibly due to a very low proton ejection. The D-proline reductase activity of C. sticklandii was detected in the cytoplasmic fraction 46 . In contrast to ATP formation by the glycine reductase reaction 8 , no obvious energy conservation step was identified in the D-proline reductase reaction 45 46 51 . Surprisingly, despite this energetic disadvantage, C. sticklandii preferentially uses proline when glycine and proline are both present in the synthetic medium 31 52 . It seems likely that D-proline reductase is used as an electron sink and plays a role in the redox-balance of the cell 51 . NADH has been suggested as the natural electron donor 51 53 . Genes involved in D-proline reduction are clustered together in an operon and the organization of the genes is conserved in all the organisms studied (Additional file 7). PrdF encodes the proline racemase necessary for D-proline generation in order to discriminate it from the L-proline used for protein synthesis. PrdA undergoes a post-translational modification forming a catalytically active pyruvoyl group 47 54 that binds the D-proline 45 46 (Figure 3). The dissociated, nucleophilic selenocysteine moiety in PrdB could then attack the α-C-atom of proline. This may lead to a reductive cleavage of the C-N-bond of the pyrrolidine ring and the formation of a selenoether. The selenoether would then be cleaved by a cysteine moiety of PrdB resulting in a mixed selenide-sulphide group. PrdC contains NADH- and FMN-binding sites as well as an iron-sulphur cluster as suggested before 46 51 53 and has been hypothesized to function as an electron transport protein 45 47 . With the detection of the potential redox-active center including a selenocysteine in PrdC, we now propose the following extension of the reaction: PrdC will use NADH to reduce its special terminal sulphide-selenide group into the thiol-selenol form (Figure 3). The latter seems to be optimal to reduce the oxidized selenide-sulphide bond of PrdB by an exchange reaction. A similar type of dithiol-disulphide exchange reaction (partly with an involvement of selenocysteine) occurs during glycine reduction by NADPH via thioredoxin reductase, thioredoxin, GrdA and GrdB, where selenocysteine replaces one cysteine in GrdA and GrdB 8 55 . PrdD and PrdE correspond quite significantly in their sequences to the middle and C-terminal part of proprotein PrdA. Both genes are present in an operon-like structure in all genomes of organisms containing a D-proline reductase (Additional file 7). However, both genes are absent in the second set of D-proline reductase genes in C. botulinum, pointing to a more indirect, protein-stabilizing role as shown for the related two proteins derived from proprotein GrdE in glycine reductase 8 55 . Finally, 5-aminovalerate is formed by hydrolysis to regenerate the pyruvoyl moiety 46 . PrdG is a potential membrane-bound protein and might be responsible for the excretion of 5-aminovalerate.

Clostridia are described in textbooks as obligate anaerobes since oxygen is harmful or lethal to these bacteria. For example, pyruvate ferredoxin oxidoreductase, a central enzyme of their metabolism is quickly inactivated in the presence of oxygen. Apparently, this results from damage to its exposed iron-sulphur sites 56 . However, some obligate anaerobes can tolerate small amounts of oxygen thanks to the presence of some proteins involved in oxygen detoxification 10 . In C. acetobutylicum genes coding for some of these proteins are under control of the peroxide repressor PerR, whose deletion results in a prolonged aerotolerance 57 . Genome analysis of C. sticklandii has revealed the presence of a protein similar to PerR as well as several proteins involved in the oxidative stress response. Beside the classical Mn-superoxide dismutase and superoxide reductase, C. sticklandii possesses genes encoding an alkyl hydroperoxide reductase, a regular rubrerythrin, two putative glutathione peroxidases, a seleno-peroxiredoxin as well as a thioredoxin-dependent peroxidase. The methionine sulfoxide reductases A and B involved in the repair of oxidative damaged methionine have also been detected. This well-supplied protection system could enhance the survival of C. sticklandii under microaerophilic conditions. In fact, we observed slight growth when C. sticklandii was cultivated in an aerobic atmosphere (data not shown).

The energy conservation of C. sticklandii via amino acid fermentation and especially by the Stickland reaction, has been the subject of many investigations. In this context, glycine reductase seems to play a central role 8 . The reduction of glycine leads to ATP formation by SLP with acetate as the final product. SLP is also an important step in some fermentation processes (Figure 1A). Energy conservation via electron-transport phosphorylation (ETP) in C. sticklandii is performed by the Rnf complex. This complex, that was recently discovered in various bacteria and archaea, is a membrane-bound system showing strong similarity to the Rhodobacter capsulatus nitrogen fixation and some NADH:quinone oxidoreductase systems. It catalyzes the electron flow from reduced ferredoxin to NAD⁺. This process is coupled to ion-translocation across the membrane, thus allowing chemiosmotic energy generation. Proton-pumping Rnf clusters have been integrated into the energy-conserving systems of Clostridium tetani 58 , Clostridium kluyveri 38 and Acetobacterium woodii 59 . In C. sticklandii, reduced ferredoxin is formed in several metabolic processes such as the highly exergonic NADH-dependent reduction of crotonyl-CoA to butyryl-CoA in the butyrate and lysine fermentation pathways 60 61 and the reactions catalysed by numerous ferredoxin oxidoreductases present in the C. sticklandii genome (Figure 1A). In addition to the possible ion-gradient driven chemiosmotic ATP generation via the Rnf complex, C. sticklandii possesses a Na⁺-dependent F0F1-ATPase and an additional V-ATPase. This is a quite unusual combination for bacteria 62 . Further energy conservation can be provided by a reaction catalyzed by a putative methylmalonyl-CoA decarboxylase. This membrane-bound protein couples the decarboxylation of methylmalonyl-CoA to the efflux of Na⁺ ions, which re-enter the cell by the ATPase systems. A third chemiosmotic energy-conserving process is catalyzed by a membrane-bound pyrophosphatase 63 . An alternative energy-conserving system in anaerobes is provided by the presence of hydrogenases. The C. sticklandii genome contains two cytoplasmic iron-only hydrogenases (HydA and HymABC). The multisubunit hydrogenase HymABC exhibits similarity with the hydrogenase identified in Eubacterium acidaminophilum 64 . The structural gene for the second hydrogenase HydA is clustered together with genes coding for a formate dehydrogenase (FdhA, FdhB), making it possible for these proteins to act as a formate hydrogen lyase. Formate addition to the culture medium increased growth efficiency of C. sticklandii 12 . A formate transporter is present, as in E. acidaminophilum 64 . Electrons from the oxidation of formate to CO₂ are transferred directly to the hydrogenase via ferredoxin to reduce H⁺ ions to hydrogen inside the cell, thus lowering the proton concentration. Genes encoding the hydrogenase maturation proteins HydE, HydF, and HydG are localized downstream from the hydrogenase gene hydA.

A particular metabolic feature of C. sticklandii is the simultaneous presence of genes encoding the complete Wood-Ljungdahl pathway and the glycine synthase (reversed gcv system)/glycine reductase pathway (Figure 1A). The Wood-Ljungdahl pathway is the main characteristic feature of acetogenic bacteria, making them capable of using CO₂ as an electron acceptor. Many of these organisms can also grow autotrophically utilizing CO₂. In certain purine- or amino acid-fermenting "acetogenic" bacteria acetate is synthesized from C1-carbon units via the glycine synthase/glycine reductase pathway 65 66 . It has been reported that the glycine synthase/glycine reductase pathway is not present in acetogenic bacteria containing the Wood-Ljungdahl pathway 67 . Since C. sticklandii possesses both pathways, a possible function of the Wood-Ljungdahl pathway may be the formation of methylene-THF from CO₂, which is then used in the glycine synthase/glycine reductase pathway as described for amino acid-fermenting "acetogenic" bacteria. However, this does not lead to a net energy conservation, since ATP formed via the glycine reductase reaction is consumed for the activation of formate to form formyl-THF. It also seems likely that the whole Wood-Ljungdahl pathway is turned off completely under fermentative conditions 68 , since metabolites involved in fermentation can act as terminal electron acceptors, thus removing the electrons necessary for the reduction of CO₂ to acetate. Therefore, we assume that C. sticklandii cannot grow autotrophically like acetogenic bacteria.

Two main processes of chemiosmotic energy conservation have been described in acetogenic bacteria. The first, observed in Moorella thermoacetica, assumes that the methylene-THF reductase is associated with the cytoplasmic membrane and is coupled to a proton translocating NADH:quinone oxidoreductase with cytochromes and menaquinone. The second energy conservation system, described in A. woodii, suggests that the methyl group transfer from methyl-THF to the carbon monoxide dehydrogenase/acetyl-CoA synthase by a membrane-bound corrinoid protein is accompanied by an efflux of Na⁺ ions. In both systems energy is conserved by the ion-motive force-driven ATPase system. In C. sticklandii the methylene-THF reductase does not seem to be attached to the cytoplasmic membrane, cytochromes are missing, menaquinone is not synthesized and membrane-bound corrinoids are absent. Recently, Schmidt et al. proposed that in A. woodii energy could be conserved in the Wood-Ljungdahl pathway by the membrane-bound Rnf reaction 59 . As mentioned above, the Rnf complex catalyzes an electron flow from reduced ferredoxin to NAD⁺, a reaction coupled to proton or sodium efflux. Reduced NAD⁺ often provides electrons for the reductive steps of the methyl-branch of the Wood-Ljungdahl pathway. The regeneration of NADH via the reduced ferredoxin-dependent Rnf complex leads to chemiosmotic energy conservation. Appropriate experiments are necessary to determine whether either the Wood-Ljungdahl pathway or the glycine synthase/glycine reductase pathway, or both, are active in C. sticklandii.

In any case, it was interesting to see whether other microorganisms contain this particular combination. For this purpose, the characteristic protein sequences of the carbon monoxide dehydrogenase/acetyl-CoA synthase from the Wood-Ljungdahl pathway, the gcv- system and the glycine reductase from C. sticklandii were compared with completely sequenced bacterial genomes and those available as a draft. This analysis reveals that, in addition to C. sticklandii, only four bacterial species contain the genes coding for these proteins (Additional file 8): Alkaliphilus metalliredigens, Carboxydothermus hydrogenoformans, Clostridium carboxidivorans, and several strains of Clostridium difficile. Of these C. hydrogenoformans can grow autotrophically using carbon monoxide as the sole carbon and energy source and presumably synthesizes acetate from CO₂ via the Wood-Ljungdahl pathway 69 70 . Altogether, these results clearly demonstrate that the coexistence of genes encoding the Wood-Ljungdahl pathway and the glycine synthase/glycine reductase pathway, as found in C. sticklandii, is not widespread.

A Sanger/pyrosequencing hybrid approach was used for whole-genome sequencing of C. sticklandii DSM 519. A shotgun library was constructed with a 10 kb size fraction obtained by mechanical shearing of total genomic DNA. The fragments were cloned into vector pCNS (pSU18 derived). Insert ends of the recombinant plasmids (21504 reads, ~6× coverage) were sequenced by dye terminator chemistry using ABI3730 sequencers. Next, total genomic sheared DNA was sequenced (~21× coverage) using the Roche GS20 sequencer. For the assembly, we used the Arachne "HybridAssemble" (Broad Institute, http://www.broad.mit.edu) that combines the 454 contigs with Sanger reads.

Annotation was performed using MaGe 44 71 , which allows graphic visualization of the C. sticklandii DSM 519 annotations enhanced by a simultaneous representation of synteny groups in other genomes chosen for comparisons. Coding sequences (CDS) likely to encode proteins were predicted with AMIGene 72 .

C. sticklandii strain DSM 519 was grown at 37°C under a nitrogen atmosphere in the synthetic medium described by Wagner and Andreesen which contains required trace elements such as selenite and tungstate 33 . Cultures for the analysis of amino acid utilization were prepared in a medium containing all amino acids (except alanine and aspartate) at a concentration of 2 mM, as well as the cofactor and trace element solutions used in the synthetic medium 33 . Samples were collected at different growth phases (exponential, the end of the exponential, stationary, and the end of the stationary phases), centrifuged for 10 min at 20,000 g, and the supernatant was analyzed by LC-MS. The majority of the amino acids were analyzed directly and some were derivatized with o-phthalic aldehyde and N-acetyl L-cysteine as described by Brückner et al. 73 . Additional file 5 shows the amino acid utilization profiles obtained.

Chromatographic separations of free amino acids were performed using an Aquity UPLC BEH C18 column (150 × 2.1 mm, 1.7 μm; Waters). The amino acids were eluted with 10 mM ammonium acetate solution containing 0.1% formic acid (A) and methanol containing 0.1% formic acid (B). Chromatographic separations of the derived amino acids were performed employing an XBridge C18 column (150 × 2.1 mm, 5 μm; Waters). These were eluted with a 30 mM ammonium acetate solution (A) and methanol (B). All LC-MS analyses were performed using a LTQ/Orbitrap high resolution mass spectrometer coupled to an Accella LC system (Thermo-Fisher Scientific).

The genome sequence reported in this article has been deposited in the EMBL database [EMBL:FP565809]. The genome annotation and features are available at http://www.genoscope.cns.fr/agc/microscope/Anaeroscope.