Improving Arsenic Tolerance of Pyrococcus furiosus by Heterologous Expression of a Respiratory Arsenate Reductase.

Arsenate is a notorious toxicant that is known to disrupt multiple biochemical pathways. Many microorganisms have developed mechanisms to detoxify arsenate using the ArsC-type arsenate reductase, and some even use arsenate as a terminal electron acceptor for respiration involving arsenate respiratory reductase (Arr). ArsC-type reductases have been studied extensively, but the phylogenetically unrelated Arr system is less investigated and has not been characterized from Archaea Here, we heterologously expressed the genes encoding Arr from the crenarchaeon Pyrobaculum aerophilum in the euryarchaeon Pyrococcus furiosus, both of which grow optimally near 100°C. Recombinant P. furiosus was grown on molybdenum (Mo)- or tungsten (W)-containing medium, and two types of recombinant Arr enzymes were purified, one containing Mo (Arr-Mo) and one containing W (Arr-W). Purified Arr-Mo had a 140-fold higher specific activity in arsenate [As(V)] reduction than Arr-W, and Arr-Mo also reduced arsenite [As(III)]. The P. furiosus strain expressing Arr-Mo (the Arr strain) was able to use arsenate as a terminal electron acceptor during growth on peptides. In addition, the Arr strain had increased tolerance compared to that of the parent strain to arsenate and also, surprisingly, to arsenite. Compared to the parent, the Arr strain accumulated intracellularly almost an order of magnitude more arsenic when cells were grown in the presence of arsenite. X-ray absorption spectroscopy (XAS) results suggest that the Arr strain of P. furiosus improves its tolerance to arsenite by increasing production of less-toxic arsenate and nontoxic methylated arsenicals compared to that by the parent.IMPORTANCE Arsenate respiratory reductases (Arr) are much less characterized than the detoxifying arsenate reductase system. The heterologous expression and characterization of an Arr from Pyrobaculum aerophilum in Pyrococcus furiosus provides new insights into the function of this enzyme. From in vivo studies, production of Arr not only enabled P. furiosus to use arsenate [As(V)] as a terminal electron acceptor, it also provided the organism with a higher resistance to arsenate and also, surprisingly, to arsenite [As(III)]. In contrast to the tungsten-containing oxidoreductase enzymes natively produced by P. furiosus, recombinant P. aerophilum Arr was much more active with molybdenum than with tungsten. It is also, to our knowledge, the only characterized Arr to be active with both molybdenum and tungsten in the active site.

Dissimilatory sulfate reduction in the archaeon 'Candidatus Vulcanisaeta moutnovskia' sheds light on the evolution of sulfur metabolism.

Dissimilatory sulfate reduction (DSR)-an important reaction in the biogeochemical sulfur cycle-has been dated to the Palaeoarchaean using geological evidence, but its evolutionary history is poorly understood. Several lineages of bacteria carry out DSR, but in archaea only Archaeoglobus, which acquired DSR genes from bacteria, has been proven to catalyse this reaction. We investigated substantial rates of sulfate reduction in acidic hyperthermal terrestrial springs of the Kamchatka Peninsula and attributed DSR in this environment to Crenarchaeota in the Vulcanisaeta genus. Community profiling, coupled with radioisotope and growth experiments and proteomics, confirmed DSR by 'Candidatus Vulcanisaeta moutnovskia', which has all of the required genes. Other cultivated Thermoproteaceae were briefly reported to use sulfate for respiration but we were unable to detect DSR in these isolates. Phylogenetic studies suggest that DSR is rare in archaea and that it originated in Vulcanisaeta, independent of Archaeoglobus, by separate acquisition of qmoABC genes phylogenetically related to bacterial hdrA genes.

A Novel highly thermostable branched-chain amino acid aminotransferase from the crenarchaeon Vulcanisaeta moutnovskia.

A new fold-type IV branched-chain amino acid aminotransferase VMUT0738 from the hyperthermophilic Crenarchaeon Vulcanisaeta moutnovskia was successfully expressed in Escherichia coli. Purified VMUT0738 showed activity toward numerous aliphatic and aromatic l-amino acids and 2-oxo acids at optimal pH 8.0. Distinguishing features of the VMUT0738 compared with typical BCAT are the absence of activity toward acidic substrates, high activity toward basic ones, and low but detectable activity toward the (R)-enantiomer of α-methylbenzylamine (0.0076U/mg) The activity of VMUT0738 increases with a rise in the temperature from 60°C to 90°C. VMUT0738 showed high thermostability (after 24h incubation at 70°C the enzyme lost only 27% of the initial activity) and the resistance to organic solvents. The sequence alignment revealed two motifs (V/I)xLDxR and PFG(K/H)YL characteristic of BCATs from species of the related genera Vulcanisaeta, Pyrobaculum and Thermoproteus that might be responsible for the unique substrate recognition profile of the enzyme.

Characterization of a phosphotriesterase-like lactonase from the hyperthermoacidophilic crenarchaeon Vulcanisaeta moutnovskia.

The phosphotriesterase-like lactonase (PLL) encoded by Vmut_2255 in the hyperthermoacidophilic crenarchaeon Vulcanisaeta moutnovskia (VmutPLL), represents the only hyperthermophilic PLL homologue identified so far in addition to the previously characterized thermophilic PLLs from Sulfolobus spp. The Vmut_2255 gene was cloned, heterologously expressed in Escherichia coli; the resultant protein purified and characterized as a 82kDa homodimer (36kDa subunits). The VmutPLL converted lactones and acyl-homoserine lactones (AHLs) with comparable activities. Towards organophosphates (OP) VmutPLL showed a promiscuous but significantly lower activity and only minor activity was observed with carboxylesters. The catalytic activity strictly depended on bivalent cations (Cd(2+)>Ni(2+)>Co(2+)>Mn(2+)>Zn(2+)). Furthermore, VmutPLL showed a pH optimum around 8.0, a temperature optimum of 80°C, and thermostability with a half-life of 26min at 90°C. In this work, the stereoselectivity of a PLL enzyme was investigated for the first time using enantiopure lactones. The VmutPLL showed a slight preference but not an exclusive specificity for the (R)-enantiomers of capro- and valerolactone. The high thermal stability as well as the broad substrate spectrum towards lactones, AHLs and OPs underlines the high biotechnological potential of VmutPLL.

Modeling the Thermoproteaceae RNase P RNA.

The RNA component of the RNase P complex is found throughout most branches of the tree of life and is principally responsible for removing the 5' leader sequence from pre-tRNA transcripts during tRNA maturation. RNase P RNA has a number of universal core features, however variations in sequence and structure found in homologs across the tree of life require multiple Rfam covariance search models to detect accurately. We describe a new Rfam search model to enable efficient detection of the diminutive archaeal Type T RNase P RNAs, which are missed by existing Rfam models. Using the new model, we establish effective score detection thresholds, and detect four new RNase P RNA genes in recently completed genomes from the crenarchaeal family Thermoproteaceae.

Enzymology of base excision repair in the hyperthermophilic archaeon Pyrobaculum aerophilum.

DNA of all living organisms is constantly modified by exogenous and endogenous reagents. The mutagenic threat of modifications such as methylation, oxidation, and hydrolytic deamination of DNA bases is counteracted by base excision repair (BER). This process is initiated by the action of one of several DNA glycosylases, which removes the aberrant base and thus initiates a cascade of events that involves scission of the DNA backbone, removal of the baseless sugar-phosphate residue, filling in of the resulting single nucleotide gap, and ligation of the remaining nick. We were interested to find out how the BER process functions in hyperthermophiles, organisms growing at temperatures around 100 degrees C, where the rates of these spontaneous reactions are greatly accelerated. In our previous studies, we could show that the crenarchaeon Pyrobaculum aerophilum has at least three uracil-DNA glycosylases, Pa-UDGa, Pa-UDGb, and Pa-MIG, that can initiate the BER process by catalyzing the removal of uracil residues arising through the spontaneous deamination of cytosines. We now report that the genome of P. aerophilum encodes also the remaining functions necessary for BER and show that a system consisting of four P. aerophilum encoded enzymes, Pa-UDGb, AP endonuclease IV, DNA polymerase B2, and DNA ligase, can efficiently repair a G.U mispair in an oligonucleotide substrate to a G.C pair. Interestingly, the efficiency of the in vitro repair reaction was stimulated by Pa-PCNA1, the processivity clamp of DNA polymerases.

Distribution of 16S rRNA introns among the family Thermoproteaceae and their evolutionary implications.

Novel 16S rRNA introns were detected in four new strains within the family Thermoproteaceae. Pyrobaculum oguniense TE7(T) and Thermoproteus sp. IC-062 housed introns of 32 and 665-668 bp after positions 1205 and 1213 ( Escherichia coli numbering system), respectively. Caldivirga maquilingensis IC-167(T) had two introns of 37 and 140 bp after positions 901 and 908, respectively. Vulcanisaeta distributa IC-065 had a 691-bp intron after position 1391. All the introns larger than 650 bp encoded the LAGLI-DADG type proteins. The intron-encoded proteins of P. oguniense TE7(T) and Thermoproteus sp. IC-062 are cognate with the proteins encoded by introns inserted at the same position in other Pyrobaculum/ Thermoproteus strains and phylotypes. The intron-encoded protein of V. distributa IC-065 is partially related to that of a Pyrobaculum phylotype. A large-scale deletion in the second intron of Caldivirga maquilingensis IC-167(T) is suspected. Based on these newly found introns and hitherto known 16S rRNA introns, the evolutionary movements of the 16S rRNA introns and the encoded LAGLI-DADG type proteins are discussed.

Structure and assembly of an augmented Sm-like archaeal protein 14-mer.

To better understand the roles of Sm proteins in forming the cores of many RNA-processing ribonucleoproteins, we determined the crystal structure of an atypical Sm-like archaeal protein (SmAP3) in which the conserved Sm domain is augmented by a previously uncharacterized, mixed alpha/beta C-terminal domain. The structure reveals an unexpected SmAP3 14-mer that is perforated by a cylindrical pore and is bound to 14 cadmium (Cd(2+)) ions. Individual heptamers adopt either "apical" or "equatorial" conformations that chelate Cd(2+) differently. SmAP3 forms supraheptameric oligomers (SmAP3)(n = 7,14,28) in solution, and assembly of the asymmetric 14-mer is modulated by differential divalent cation-binding in apical and equatorial subunits. Phylogenetic and sequence analyses substantiate SmAP3s as a unique subset of SmAPs. These results distinguish SmAP3s from other Sm proteins and provide a model for the structure and properties of Sm proteins >100 residues in length, e.g., several human Sm proteins.

Regulation of the aerobic respiratory chain in the facultatively aerobic and hyperthermophilic archaeon Pyrobaculum oguniense.

The aerobic respiratory chain of Pyrobaculum oguniense is expressed constitutively even under anaerobic conditions. The membranes of both aerobically and anaerobically grown cells show oxygen consumption activity with NADH as substrate, bovine cytochrome c oxidase activity and TMPD oxidase activity. Spectroscopic analysis and haem analysis of membranes of aerobically grown cells show the presence of cytochrome b(559), cytochrome c(551) and haem Op1 containing cytochrome c oxidase in aerobically and anaerobically grown cells, and haem As containing cytochrome c oxidase in aerobically grown cells. The gene clusters of SoxB-type and SoxM-type haem copper oxidase and cytochrome bc complex have been cloned and sequenced and the regulation of these genes was analysed. The Northern blot analysis indicated that the constitutive transcription of the gene cluster of SoxB-type haem-copper oxidase and cytochrome bc complex is observed under both aerobic and anaerobic conditions, and the transcription of the operon of SoxM-type haem-copper oxidase was stimulated under aerobic conditions. Furthermore, the presence of the binding residues for CuA in subunit II of both SoxB- and SoxM-type haem-copper oxidase suggests that these haem-copper oxidases are cytochrome c oxidases.

The hexokinase of the hyperthermophile Thermoproteus tenax. ATP-dependent hexokinases and ADP-dependent glucokinases, teo alternatives for glucose phosphorylation in Archaea.

The phosphorylation of glucose by different sugar kinases plays an essential role in Archaea because of the absence of a phosphoenolpyruvate-dependent transferase system characteristic for Bacteria. In the genome of the hyperthermophilic Archaeon Thermoproteus tenax a gene was identified with sequence similarity to glucokinases of the so-called ROK family (repressor protein, open reading frame, sugar kinase). The T. tenax enzyme, like the recently described ATP-dependent "glucokinase" from Aeropyrum pernix, shows the typical broad substrate specificity of hexokinases catalyzing not only phosphorylation of glucose but also of other hexoses such as fructose, mannose, or 2-deoxyglucose, and thus both enzymes represent true hexokinases. The T. tenax hexokinase shows strikingly low if at all any regulatory properties and thus fulfills no important control function at the beginning of the variant of the Embden-Meyerhof-Parnas pathway in T. tenax. Transcript analyses reveal that the hxk gene of T. tenax is cotranscribed with an upstream located orfX, which codes for an 11-kDa protein of unknown function. Growth-dependent studies and promoter analyses suggest that post-transcriptional RNA processing might be involved in the generation of the monocistronic hxk message, which is observed only under heterotrophic growth conditions. Data base searches revealed T. tenax hexokinase homologs in some archaeal, few eukaryal, and many bacterial genomes. Phylogenetic analyses confirm that the archaeal hexokinase is a member of the so-called ROK family, which, however, should be referred to as ROK group because it represents a group within the bacterial glucokinase fructokinase subfamily II of the hexokinase family. Thus, archaeal hexokinases represent a second major group of glucose-phosphorylating enzymes in Archaea beside the recently described archaeal ADP-dependent glucokinases, which were recognized as members of the ribokinase family. The distribution of the two types of sugar kinases, differing in their cosubstrate as well as substrate specificity, within Archaea is discussed on the basis of physiological constraints of the respective organisms.

Engineering soluble proteins for structural genomics.

Structural genomics has the ambitious goal of delivering three-dimensional structural information on a genome-wide scale. Yet only a small fraction of natural proteins are suitable for structure determination because of bottlenecks such as poor expression, aggregation, and misfolding of proteins, and difficulties in solubilization and crystallization. We propose to overcome these bottlenecks by producing soluble, highly expressed proteins that are derived from and closely related to their natural homologs. Here we demonstrate the utility of this approach by using a green fluorescent protein (GFP) folding reporter assay to evolve an enzymatically active, soluble variant of a hyperthermophilic protein that is normally insoluble when expressed in Escherichia coli, and determining its structure by X-ray crystallography. Analysis of the structure provides insight into the substrate specificity of the enzyme and the improved solubility of the variant.

Vulcanisaeta distributa gen. nov., sp. nov., and Vulcanisaeta souniana sp. nov., novel hyperthermophilic, rod-shaped crenarchaeotes isolated from hot springs in Japan.

Seventeen strains of rod-shaped, heterotrophic, anaerobic, hyperthermophilic crenarchaeotes were isolated from several hot spring areas in eastern Japan, and eight representative strains were characterized further. Cells of these strains were straight to slightly curved rods, 0.4-0.6 microm in width. Occasionally, cells were branched or bore spherical bodies at the poles. They grew optimally at 85-90 degrees C and at pH 4.0-4.5. They utilized yeast extract, peptone, beef extract, Casamino acids, gelatin, starch, maltose and malate as carbon sources and sulfur and thiosulfate as possible electron acceptors. The DNA G+C contents of the novel isolates were 43.9-46.2 mol%. The lipids were mainly cyclic and acyclic tetraether core lipids. Phylogenetic analysis of the 16S rDNA sequences revealed that they represented an independent lineage in the family Thermoproteaceae. Moreover, comparison of the 16S rDNA sequences and a DNA-DNA hybridization study showed that they comprised two species, which could also be differentiated by the maximal growth temperature and degrees of NaCl tolerance. Therefore, a new genus, Vulcanisaeta gen. nov., in the family Thermoproteaceae is proposed to accommodate two novel species, Vulcanisaeta distributa sp. nov. and Vulcanisaeta souniana sp. nov. The type species is V. distributa and the type strains are V. distributa IC 017T (= JCM 11212T = DSM 14429T) and V. souniana IC-059T (= JCM 11219T = DSM 14430T).

Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon.

We have found that the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene (est(Pc)) from strain VA1. est(Pc) consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As est(Pc) showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of P. calidifontis (Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90 degrees C), but also at ambient temperature (1,050 U/mg at 30 degrees C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of P. calidifontis showed higher hydrolytic activity towards esters with short to medium chains, with p-nitrophenyl caproate (C(6)) the best substrate among the p-nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester tert-butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of P. calidifontis in future applications.

A novel uracil-DNA glycosylase with broad substrate specificity and an unusual active site.

Uracil-DNA glycosylases (UDGs) catalyse the removal of uracil by flipping it out of the double helix into their binding pockets, where the glycosidic bond is hydrolysed by a water molecule activated by a polar amino acid. Interestingly, the four known UDG families differ in their active site make-up. The activating residues in UNG and SMUG enzymes are aspartates, thermostable UDGs resemble UNG-type enzymes, but carry glutamate rather than aspartate residues in their active sites, and the less active MUG/TDG enzymes contain an active site asparagine. We now describe the first member of a fifth UDG family, Pa-UDGb from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum, the active site of which lacks the polar residue that was hitherto thought to be essential for catalysis. Moreover, Pa-UDGb is the first member of the UDG family that efficiently catalyses the removal of an aberrant purine, hypoxanthine, from DNA. We postulate that this enzyme has evolved to counteract the mutagenic threat of cytosine and adenine deamination, which becomes particularly acute in organisms living at elevated temperatures.

Genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum.

We determined and annotated the complete 2.2-megabase genome sequence of Pyrobaculum aerophilum, a facultatively aerobic nitrate-reducing hyperthermophilic (T(opt) = 100 degrees C) crenarchaeon. Clues were found suggesting explanations of the organism's surprising intolerance to sulfur, which may aid in the development of methods for genetic studies of the organism. Many interesting features worthy of further genetic studies were revealed. Whole genome computational analysis confirmed experiments showing that P. aerophilum (and perhaps all crenarchaea) lack 5' untranslated regions in their mRNAs and thus appear not to use a ribosome-binding site (Shine-Dalgarno)-based mechanism for translation initiation at the 5' end of transcripts. Inspection of the lengths and distribution of mononucleotide repeat-tracts revealed some interesting features. For instance, it was seen that mononucleotide repeat-tracts of Gs (or Cs) are highly unstable, a pattern expected for an organism deficient in mismatch repair. This result, together with an independent study on mutation rates, suggests a "mutator" phenotype.

Structure of HisF, a histidine biosynthetic protein from Pyrobaculum aerophilum.

HisF (imidazole glycerol phosphate synthase) is an important branch-point enzyme in the histidine biosynthetic pathway of microorganisms. Because of its potential relevance for structure-based drug design, the crystal structure of HisF from the hyperthermophilic archaeon Pyrobaculum aerophilum has been determined. The structure was determined by molecular replacement and refined at 2.0 A resolution to a crystallographic R factor of 20.6% and a free R of 22.7%. The structure adopts a classic (beta/alpha)(8) barrel fold and has networks of surface salt bridges that may contribute to thermostability. The active site is marked out by the presence of two bound phosphate ions and two glycerol molecules that delineate a long groove at one end of the (beta/alpha)(8) barrel. The two phosphate ions, 17 A apart, are bound to sequence-conserved structural motifs that seem likely to provide much of the specificity for the two phosphate groups of the HisF substrate. The two glycerol molecules bind in the vicinity of other sequence-conserved residues that are likely to be involved in binding and/or catalysis. Comparisons with the homologous HisF from Thermatoga maritima reveal a displaced loop that may serve as a lid over the active site.

Archaeal fructose-1,6-bisphosphate aldolases constitute a new family of archaeal type class I aldolase.

Fructose-1,6-bisphosphate (FBP) aldolase activity has been detected previously in several Archaea. However, no obvious orthologs of the bacterial and eucaryal Class I and II FBP aldolases have yet been identified in sequenced archaeal genomes. Based on a recently described novel type of bacterial aldolase, we report on the identification and molecular characterization of the first archaeal FBP aldolases. We have analyzed the FBP aldolases of two hyperthermophilic Archaea, the facultatively heterotrophic Crenarchaeon Thermoproteus tenax and the obligately heterotrophic Euryarchaeon Pyrococcus furiosus. For enzymatic studies the fba genes of T. tenax and P. furiosus were expressed in Escherichia coli. The recombinant FBP aldolases show preferred substrate specificity for FBP in the catabolic direction and exhibit metal-independent Class I FBP aldolase activity via a Schiff-base mechanism. Transcript analyses reveal that the expression of both archaeal genes is induced during sugar fermentation. Remarkably, the fbp gene of T. tenax is co-transcribed with the pfp gene that codes for the reversible PP(i)-dependent phosphofructokinase. As revealed by phylogenetic analyses, orthologs of the T. tenax and P. furiosus enzyme appear to be present in almost all sequenced archaeal genomes, as well as in some bacterial genomes, strongly suggesting that this new enzyme family represents the typical archaeal FBP aldolase. Because this new family shows no significant sequence similarity to classical Class I and II enzymes, a new name is proposed, archaeal type Class I FBP aldolases (FBP aldolase Class IA).

Leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum.

We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S(1) nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from -50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the -1 position and a BRE element with more frequent A bases at position -29 to -32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.