Molecular mechanisms of regulation by a ß-alanine-responsive Lrp-type transcription factor from Acidianus hospitalis.

The leucine-responsive regulatory protein (Lrp) family of transcriptional regulators is widespread among prokaryotes and especially well-represented in archaea. It harbors members with diverse functional mechanisms and physiological roles, often linked to the regulation of amino acid metabolism. BarR is an Lrp-type regulator that is conserved in thermoacidophilic Thermoprotei belonging to the order Sulfolobales and is responsive to the non-proteinogenic amino acid ß-alanine. In this work, we unravel molecular mechanisms of the Acidianus hospitalis BarR homolog, Ah-BarR. Using a heterologous reporter gene system in Escherichia coli, we demonstrate that Ah-BarR is a dual-function transcription regulator that is capable of repressing transcription of its own gene and activating transcription of an aminotransferase gene, which is divergently transcribed from a common intergenic region. Atomic force microscopy (AFM) visualization reveals a conformation in which the intergenic region appears wrapped around an octameric Ah-BarR protein. ß-alanine causes small conformational changes without affecting the oligomeric state of the protein, resulting in a relief of regulation while the regulator remains bound to the DNA. This regulatory and ligand response is different from the orthologous regulators in Sulfolobus acidocaldarius and Sulfurisphaera tokodaii, which is possibly explained by a distinct binding site organization and/or by the presence of an additional C-terminal tail in Ah-BarR. By performing site-directed mutagenesis, this tail is shown to be involved in ligand-binding response.

Affitins: Ribosome Display for Selection of Aho7c-Based Affinity Proteins.

Engineered protein scaffolds have made a tremendous contribution to the panel of affinity tools owing to their favorable biophysical properties that make them useful for many applications. In 2007, our group paved the way for using archaeal Sul7d proteins for the design of artificial affinity ligands, so-called Affitins. For many years, Sac7d and Sso7d have been used as molecular basis to obtain binders for various targets. Recently, we characterized their old gifted protein family and identified Aho7c, originating from Acidianus hospitalis, as the shortest member (60 amino-acids) with impressive stability (96.5 °C, pH 0-12). Here, we describe the construction of Aho7c combinatorial libraries and their use for selection of binders by ribosome display.

Genome analysis of the thermoacidophilic archaeon Acidianus copahuensis focusing on the metabolisms associated to biomining activities.

BACKGROUND: Several archaeal species from the order Sulfolobales are interesting from the biotechnological point of view due to their biomining capacities. Within this group, the genus Acidianus contains four biomining species (from ten known Acidianus species), but none of these have their genome sequenced. To get insights into the genetic potential and metabolic pathways involved in the biomining activity of this group, we sequenced the genome of Acidianus copahuensis ALE1 strain, a novel thermoacidophilic crenarchaeon (optimum growth: 75 °C, pH 3) isolated from the volcanic geothermal area of Copahue at Neuquén province in Argentina. Previous experimental characterization of A. copahuensis revealed a high biomining potential, exhibited as high oxidation activity of sulfur and sulfur compounds, ferrous iron and sulfide minerals (e.g.: pyrite). This strain is also autotrophic and tolerant to heavy metals, thus, it can grow under adverse conditions for most forms of life with a low nutrient demand, conditions that are commonly found in mining environments. RESULTS: In this work we analyzed the genome of Acidianus copahuensis and describe the genetic pathways involved in biomining processes. We identified the enzymes that are most likely involved in growth on sulfur and ferrous iron oxidation as well as those involved in autotrophic carbon fixation. We also found that A. copahuensis genome gathers different features that are only present in particular lineages or species from the order Sulfolobales, some of which are involved in biomining. We found that although most of its genes (81%) were found in at least one other Sulfolobales species, it is not specifically closer to any particular species (60-70% of proteins shared with each of them). Although almost one fifth of A. copahuensis proteins are not found in any other Sulfolobales species, most of them corresponded to hypothetical proteins from uncharacterized metabolisms. CONCLUSION: In this work we identified the genes responsible for the biomining metabolisms that we have previously observed experimentally. We provide a landscape of the metabolic potentials of this strain in the context of Sulfolobales and propose various pathways and cellular processes not yet fully understood that can use A. copahuensis as an experimental model to further understand the fascinating biology of thermoacidophilic biomining archaea.

[Adaptation of Acidianus hospitalis W1 to oligotrophic and acidic hot spring environments].

OBJECTIVE: To study the adaptation of A. hospitalis W1 to oligotrophic and acidic hot spring environments at the whole genome level. METHODS: We annotated the gene functions and constructed metabolic pathways of strain W1 by using different databases, such as NCBI non-redundant database (NRDB), UniProt, Sulfolobus protein database and Kyoto Encyclopedia of Genes and Genomes (KEGG). The metabolic pathways were polished according to the results of comparative genomics. RESULTS: Strain W1 grew autotrophically by fixing CO2 as carbon source through 3-hydroxypropionate/4-hydroxybutyrate or dicarboxylate-4-hydroxybutyrate cycle, and gained energy for growth by oxidation of reduced inorganic sulfur compounds (RISCs). Strain W1 differenced from A. ambivalens because its genome did not possess sulfur-metabolizing genes encoding sulfite: acceptor oxidoreductase, adenosine phosphosulfate reductase, sulfate adenylyl transferase and phosphoadenosine phosphosulfate reductase. Glucose was metabolized by strain W1 through non- phosphorylated Entner-Doudoroff pathway and tricarboxylic acid cycle. In addition, the sugar and amino acids transporters, as well as related hydrolysis enzymes were identified in the genome. These results suggest that strain W1 could also grow facultative autotrophically. Strain W1 cannot use H2 as electron donor due to lack of hydrogenase encoding genes. CONCLUSION: The versatile metabolic patterns afforded A. hospitalis W1 the ability to adapt to oligotrophic and acidic hot spring environments. Furthermore, the unique metabolic features of strain W1 will help to better understand the metabolic diversities of Acidianus.

Bacterial CS2 hydrolases from Acidithiobacillus thiooxidans strains are homologous to the archaeal catenane CS2 hydrolase.

Carbon disulfide (CS(2)) and carbonyl sulfide (COS) are important in the global sulfur cycle, and CS(2) is used as a solvent in the viscose industry. These compounds can be converted by sulfur-oxidizing bacteria, such as Acidithiobacillus thiooxidans species, to carbon dioxide (CO(2)) and hydrogen sulfide (H2S), a property used in industrial biofiltration of CS(2)-polluted airstreams. We report on the mechanism of bacterial CS(2) conversion in the extremely acidophilic A. thiooxidans strains S1p and G8. The bacterial CS(2) hydrolases were highly abundant. They were purified and found to be homologous to the only other described (archaeal) CS(2) hydrolase from Acidianus strain A1-3, which forms a catenane of two interlocked rings. The enzymes cluster in a group of ß-carbonic anhydrase (ß-CA) homologues that may comprise a subclass of CS(2) hydrolases within the ß-CA family. Unlike CAs, the CS(2) hydrolases did not hydrate CO(2) but converted CS(2) and COS with H(2)O to H(2)S and CO(2). The CS(2) hydrolases of A. thiooxidans strains G8, 2Bp, Sts 4-3, and BBW1, like the CS(2) hydrolase of Acidianus strain A1-3, exist as both octamers and hexadecamers in solution. The CS(2) hydrolase of A. thiooxidans strain S1p forms only octamers. Structure models of the A. thiooxidans CS(2) hydrolases based on the structure of Acidianus strain A1-3 CS(2) hydrolase suggest that the A. thiooxidans strain G8 CS(2) hydrolase may also form a catenane. In the A. thiooxidans strain S1p enzyme, two insertions (positions 26 and 27 [PD] and positions 56 to 61 [TPAGGG]) and a nine-amino-acid-longer C-terminal tail may prevent catenane formation.

Solution structure of an archaeal DNA binding protein with an eukaryotic zinc finger fold.

While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

Structure and function of AvtR, a novel transcriptional regulator from a hyperthermophilic archaeal lipothrixvirus.

The structural and functional analysis of the protein AvtR encoded by Acidianus filamentous virus 6 (AFV6), which infects the archaeal genus Acidianus, revealed its unusual structure and involvement in transcriptional regulation of several viral genes. The crystal structure of AvtR (100 amino acids) at 2.6-Å resolution shows that it is constituted of a repeated ribbon-helix-helix (RHH) motif, which is found in a large family of bacterial transcriptional regulators. The known RHH proteins form dimers that interact with DNA using their ribbon to create a central ß-sheet. The repeated RHH motifs of AvtR superpose well on such dimers, but its central sheet contains an extra strand, suggesting either conformational changes or a different mode of DNA binding. Systematic evolution of ligands by exponential enrichment (SELEX) experiments combined with systematic mutational and computational analysis of the predicted site revealed 8 potential AvtR targets in the AFV6 genome. Two of these targets were studied in detail, and the complex role of AvtR in the transcriptional regulation of viral genes was established. Repressing transcription from its own gene, gp29, AvtR can also act as an activator of another gene, gp30. Its binding sites are distant from both genes' TATA boxes, and the mechanism of AvtR-dependent regulation appears to include protein oligomerization starting from the protein's initial binding sites. Many RHH transcriptional regulators of archaeal viruses could share this regulatory mechanism.

Physiologic versatility and growth flexibility as the main characteristics of a novel thermoacidophilic Acidianus strain isolated from Copahue geothermal area in Argentina.

A novel thermoacidophilic archaeal strain has been isolated from three geothermal acidic hot springs in Copahue, Argentina. One of the most striking characteristic of ALE1 isolate is its metabolic versatility. It grows on sulphur, tetrathionate, iron (II) and sucrose under aerobic conditions, but it can also develop under anaerobic conditions using iron (III) or sulphur as electron acceptors and sulphur or hydrogen as electron donors autotrophically. A temperature of 75 °C and a pH between 2.5 and 3.0 are strain ALE1 optimal growth conditions, but it is able to oxidise iron (II) even at pH 1.0. Cells are irregular cocci surrounded by a regularly arrayed glycoprotein layer (S-layer). Phylogenetic analysis shows that strain ALE1 belongs to the family Sulfolobaceae in the class Thermoprotei, within the phylum Crenarchaeota. Based on 16S rRNA gene sequence similarity on NCBI database, ALE1 does not have closely related relatives, neither in culture nor uncultured, which is more surprising. Its closest related species are strains of Acidianus hospitalis (91 % of sequence similarity), Acidianus infernus (90 %), Acidianus ambivalens (90 %) and Acidianus manzanensis (90 %). Its DNA base composition of 34.5 % mol C + G is higher than that reported for other Acidianus species. Considering physiological and phylogenetic characteristics of strain ALE1, we considered it to represent a novel species of the genus Acidianus (candidatus "Acidianus copahuensis"). The aim of this study is to physiologically characterise this novel archaea in order to understand its role in iron and sulphur geochemical cycles in the Copahue geothermal area and to evaluate its potential applications in bioleaching and biooxidation.

Evolution of a new enzyme for carbon disulphide conversion by an acidothermophilic archaeon.

Extremophilic organisms require specialized enzymes for their exotic metabolisms. Acid-loving thermophilic Archaea that live in the mudpots of volcanic solfataras obtain their energy from reduced sulphur compounds such as hydrogen sulphide (H(2)S) and carbon disulphide (CS(2)). The oxidation of these compounds into sulphuric acid creates the extremely acidic environment that characterizes solfataras. The hyperthermophilic Acidianus strain A1-3, which was isolated from the fumarolic, ancient sauna building at the Solfatara volcano (Naples, Italy), was shown to rapidly convert CS(2) into H(2)S and carbon dioxide (CO(2)), but nothing has been known about the modes of action and the evolution of the enzyme(s) involved. Here we describe the structure, the proposed mechanism and evolution of a CS(2) hydrolase from Acidianus A1-3. The enzyme monomer displays a typical ß-carbonic anhydrase fold and active site, yet CO(2) is not one of its substrates. Owing to large carboxy- and amino-terminal arms, an unusual hexadecameric catenane oligomer has evolved. This structure results in the blocking of the entrance to the active site that is found in canonical ß-carbonic anhydrases and the formation of a single 15-Å-long, highly hydrophobic tunnel that functions as a specificity filter. The tunnel determines the enzyme's substrate specificity for CS(2), which is hydrophobic. The transposon sequences that surround the gene encoding this CS(2) hydrolase point to horizontal gene transfer as a mechanism for its acquisition during evolution. Our results show how the ancient ß-carbonic anhydrase, which is central to global carbon metabolism, was transformed by divergent evolution into a crucial enzyme in CS(2) metabolism.

Genomic analysis of Acidianus hospitalis W1 a host for studying crenarchaeal virus and plasmid life cycles.

The Acidianus hospitalis W1 genome consists of a minimally sized chromosome of about 2.13 Mb and a conjugative plasmid pAH1 and it is a host for the model filamentous lipothrixvirus AFV1. The chromosome carries three putative replication origins in conserved genomic regions and two large regions where non-essential genes are clustered. Within these variable regions, a few orphan orfB and other elements of the IS200/607/605 family are concentrated with a novel class of MITE-like repeat elements. There are also 26 highly diverse vapBC antitoxin-toxin gene pairs proposed to facilitate maintenance of local chromosomal regions and to minimise the impact of environmental stress. Complex and partially defective CRISPR/Cas/Cmr immune systems are present and interspersed with five vapBC gene pairs. Remnants of integrated viral genomes and plasmids are located at five intron-less tRNA genes and several non-coding RNA genes are predicted that are conserved in other Sulfolobus genomes. The putative metabolic pathways for sulphur metabolism show some significant differences from those proposed for other Acidianus and Sulfolobus species. The small and relatively stable genome of A. hospitalis W1 renders it a promising candidate for developing the first Acidianus genetic systems.

Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1.

At present very little is known about interactions between extrachromosomal genetic elements in Archaea. Here we describe an Acidianus strain which carries naturally a novel 28 kb conjugative plasmid-like element, pAH1, and also serves as a laboratory host for lipothrixvirus AFV1. In an attempt to establish a system for studying plasmid-virus interactions we characterized the genome of pAH1 which closely resembles those of the Sulfolobus conjugative plasmids pARN3 and pARN4. pAH1 integrates site specifically into, and excises from, the host chromosome indicating a dynamic interaction with the latter. Although nucleotide sequence comparisons revealed extensive intergenomic exchange during the evolution of archaeal conjugative plasmids, pAH1 was shown to be stably maintained suggesting that the host system is suitable for studying plasmid-virus interactions. AFV1 infection and propagation leads to a loss of the circular form of pAH1 and this effect correlates positively with the increase in the intracellular quantity of AFV1 DNA. We infer that the virus inhibits plasmid replication since no pAH1 degradation was observed. This mechanism of archaeal viral inhibition of plasmid propagation is not observed in bacteria where relevant bacteriophages either are dependent on a conjugative plasmid for successful infection or are excluded by a resident plasmid.

Viruses in acidic geothermal environments of the Kamchatka Peninsula.

Screening for viruses in samples taken from acidic hot springs of Kamchatka (Russia) revealed a collection of morphotypes, including linear, spherical and complex fusiform shapes, which show partial similarity to those found in acidic geothermal environments in other geographical locations. One of the viruses, Acidianus filamentous virus 9, AFV9, was isolated and its structure and genome were studied in detail.

The dihydrolipoamide dehydrogenase from the crenarchaeon Acidianus ambivalens.

A dihydrolipoamide dehydrogenase (DLDH) was purified and characterized for the first time from a crenarchaeon, Acidianus ambivalens. The holoenzyme consists of two identical subunits with a molecular mass of 45.4 kDa per monomer. It contains FAD as a prosthetic group and uses NAD+ as the preferential substrate, but can also reduce NADP+. The Michaelis-Menten constants of the forward (NAD+ reduction) and reverse (NADH oxidation) reactions were KM (dihydrolipoamide)=0.70 mM, KM (NAD+)=0.71 mM, KM (lipoamide)=1.26 mM and KM (NADH)=3.15 microM. A comparative study of NADH:lipoamide oxidoreductase and NADH:K3[Fe(CN)6] oxidoreductase activities was performed, the optimal temperature and pH being different for each: 55 degrees C, pH 7 and 89 degrees C, pH 5.5, respectively. Although DLDH is generally part of the alpha-ketoacid dehydrogenase complexes in Bacteria and Eukarya, none of these complexes has yet been isolated from Sulfolobales. The metabolic role of DLDH in these organisms is discussed.

Acidianus sulfidivorans sp. nov., an extremely acidophilic, thermophilic archaeon isolated from a solfatara on Lihir Island, Papua New Guinea, and emendation of the genus description.

A novel, extremely thermoacidophilic, obligately chemolithotrophic archaeon (strain JP7(T)) was isolated from a solfatara on Lihir Island, Papua New Guinea. Cells of this organism were non-motile, Gram-negative staining, irregular-shaped cocci, 0.5-1.5 microm in size, that grew aerobically by oxidation of sulfur, Fe(2+) or mineral sulfides. Cells grew anaerobically using Fe(3+) as a terminal electron acceptor and H(2)S as an electron donor but did not oxidize hydrogen with elemental sulfur as electron acceptor. Strain JP7(T) grew optimally at 74 degrees C (temperature range 45-83 degrees C) and pH 0.8-1.4 (pH range 0.35-3.0). On the basis of 16S rRNA gene sequence similarity, strain JP7(T) was shown to belong to the Sulfolobaceae, being most closely related to the type strains of Acidianus ambivalens (93.7 %) and Acidianus infernus (93.6 %). Cell-membrane lipid structure, DNA base composition and 16S rRNA gene sequence similarity data support the placement of this strain in the genus Acidianus. Differences in aerobic and anaerobic metabolism, temperature and pH range for growth, and 16S rRNA gene sequence differentiate strain JP7(T) from recognized species of the genus Acidianus, and an emendation of the description of the genus is proposed. Strain JP7(T) is considered to represent a novel species of the genus Acidianus, for which the name Acidianus sulfidivorans sp. nov. is proposed. The type strain is JP7(T) (=DSM 18786(T)=JCM 13667(T)).

Identification of core active site residues of the sulfur oxygenase reductase from Acidianus ambivalens by site-directed mutagenesis.

The sulfur oxygenase reductase (SOR) is the initial enzyme in the sulfur oxidation pathway of Acidianus ambivalens. The SOR is composed of 308 aa residues, three of which are cysteines, and contains a mononuclear non-heme iron site. Mutations of the suspected iron-binding residues H86, H90 and E114 to alanine resulted in inactive enzyme with no iron incorporated, whereas an E114D mutant showed 1% of wild type activity. The mutation of C31 to alanine and serine caused inactivity of the enzyme, however, the iron content was the same as in the wild type. C101A, C104S/A, and C101/104S/A double mutants caused a decrease in specific activity to 10-43% of the wild type while the C101S mutant showed only 1% activity of the wild type. The drop in activity of the C101S and E114D mutants was accompanied with a proportional decrease in iron content. In all cases the oxygenase and reductase partial reactions were equally affected. It was concluded that the Fe site with H86, H90 and E114 as ligands and C31 constitute the core active site whereas C101 and C104 optimize reaction conditions.

Prokaryotic sulfur oxidation.

Recent biochemical and genomic data differentiate the sulfur oxidation pathway of Archaea from those of Bacteria. From these data it is evident that members of the Alphaproteobacteria harbor the complete sulfur-oxidizing Sox enzyme system, whereas members of the beta and gamma subclass and the Chlorobiaceae contain sox gene clusters that lack the genes encoding sulfur dehydrogenase. This indicates a different pathway for oxidation of sulfur to sulfate. Acidophilic bacteria oxidize sulfur by a system different from the Sox enzyme system, as do chemotrophic endosymbiotic bacteria.

The sulfur oxygenase reductase from Acidianus ambivalens is an icosatetramer as shown by crystallization and Patterson analysis.

The sulfur oxygenase reductase (SOR) is the initial enzyme in the aerobic sulfur metabolism of the thermoacidophilic and chemolithoautotrophic crenarchaeote Acidianus ambivalens. Single colorless polyhedral crystals were obtained under two crystallization conditions from SOR preparations heterologously overproduced in Escherichia coli. They belonged to space-group I4 and diffraction data were collected up to 1.7 A resolution. Their Patterson symmetry shows additional 4-, 3- and 2-fold non-crystallographic symmetry rotation axes, characteristic of the point group 432. Taking into account the molecular mass of SOR, the crystal unit cell volume, the non-crystallographic symmetry operators and previous electron microscopy studies of the SOR, it was deduced that the quaternary structure of the functionally active enzyme is an icosatetramer with 871 kDa molecular mass.

Key role of cysteine residues in catalysis and subcellular localization of sulfur oxygenase-reductase of Acidianus tengchongensis.

Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C(31) and C(101)-X-X-C(104); numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn(2+) strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C(31) and C(101)-X-X-C(104), in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.

Coupling of the pathway of sulphur oxidation to dioxygen reduction: characterization of a novel membrane-bound thiosulphate:quinone oxidoreductase.

Thiosulphate is one of the products of the initial step of the elemental sulphur oxidation pathway in the thermoacidophilic archaeon Acidianus ambivalens. A novel thiosulphate:quinone oxidoreductase (TQO) activity was found in the membrane extracts of aerobically grown cells of this organism. The enzyme was purified 21-fold from the solubilized membrane fraction. The TQO oxidized thiosulphate with tetrathionate as product and ferricyanide or decyl ubiquinone (DQ) as electron acceptors. The maximum specific activity with ferricyanide was 73.4 U (mg protein)(-1) at 92 degrees C and pH 6, with DQ it was 397 mU (mg protein)(-1) at 80 degrees C. The Km values were 2.6 mM for thiosulphate (k(cat) = 167 s(-1)), 3.4 mM for ferricyanide and 5.87 micro M for DQ. The enzymic activity was inhibited by sulphite (Ki = 5 micro M), metabisulphite, dithionite and TritonX-100, but not by sulphate or tetrathionate. A mixture of caldariella quinone, sulfolobus quinone and menaquinone was non-covalently bound to the protein. No other cofactors were detected. Oxygen consumption was measured in membrane fractions upon thiosulphate addition, thus linking thiosulphate oxidation to dioxygen reduction, in what constitutes a novel activity among Archaea. The holoenzyme was composed of two subunits of apparent molecular masses of 28 and 16 kDa. The larger subunit appeared to be glycosylated and was identical to DoxA, and the smaller was identical to DoxD. Both subunits had been described previously as a part of the terminal quinol:oxygen oxidoreductase complex (cytochrome aa3).

Occurrence, biochemistry and possible biotechnological application of the 3-hydroxypropionate cycle.

The 3-hydroxypropionate cycle, a pathway for autotrophic carbon dioxide fixation, is reviewed with special emphasis on the biochemistry of CO2 fixing enzymes in Acidianus brierleyi, a thermophilic and acidophilic archeon. In the 3-hydroxypropionate cycle, two enzymes, acetyl-CoA carboxylase and propionyl-CoA carboxylase, catalyze CO2 fixation. It has been shown in A. brierleyi, and subsequently in Metallosphaera sedula, that acetyl-CoA carboxylase is promiscuous, acting equally well on acetyl-CoA and propionyl-CoA. The subunit structure of the acyl-CoA carboxylase was shown to be alpha4beta4gamma4. Gene cloning revealed that the genes encoding the three subunits are adjacent to each other. accC encodes the beta-subunit (59 kDa subunit, biotin carboxylase subunit), accB encodes the gamma-subunit (20 kDa subunit, biotin carboxyl carrier protein), and pccB encodes the alpha-subunit (62 kDa subunit, carboxyltransferase subunit). Sequence analyses showed that accC and accB are co-transcribed and that pccB is transcribed separately. Potential biotechnological applications for the 3-hydroxypropionate cycle are also presented.