Archaeon Sulfolobus Solfataricus P2 Research Articles

Hyperthermophilic flavin reductase from Sulfolobus solfataricus P2: Production and biochemical characterization.

Nicotinamide adenine dinucleotide phosphate (NAD(P)H)-flavin oxidoreductases (flavin reductases) catalyze the reduction of flavin by NAD(P)H and provide the reduced form of flavin mononucleotide (FMN) to flavin-dependent monooxygenases. Based on bioinformatics analysis, we identified a putative flavin reductase gene, sso2055, in the genome of hyperthermophilic archaeon Sulfolobus solfataricus P2, and further cloned this target sequence into an expression vector. The cloned flavin reductase (EC. 1.5.1.30) was purified to homogeneity and characterized further. The purified enzyme exists as a monomer of 17.8kDa, free of chromogenic cofactors. Homology modeling revealed this enzyme as a TIM barrel, which is also supported by circular dichroism measurements revealing a beta-sheet rich content. The optimal pH for SSO2055 activity was pH 6.5 in phosphate buffer and the highest activity observed was at 120°C within the measurable temperature. We showed that this enzyme can use FMN and flavin adenine dinucleotide (FAD) as a substrate to generate their reduced forms. The purified enzyme is predicted to be a potential flavin reductase of flavin-dependent monooxygenases that could be involved in the biodesulfurization process of S. solfataricus P2.

Carboxylic acid reductase enzymes

A new thermostable extracellular lipolytic enzyme, induced from the thermoacidophilic archaeon Sulfolobus solfataricus P1 using corn oil as an inducer, was purified to apparent homogeneity by butanol extraction and two column chromatographies using DEAE-Sepharose followed by Butyl-Sepharose. The purified enzyme assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and gel filtration was approximately 45 kDa and monomeric. The maximal activity examined using p-nitrophenyl palmitate as a substrate was observed at 98 °C and pH 6.0. The enzyme showed remarkable thermostability: It retained 51% of its activity after 120 h at 80 °C. In addition, the enzyme displayed extremely high stability against water-miscible alcohols, SDS, and urea, even at high concentrations. This high stability of the enzyme against protein-denaturing agents indicates a high potential in industrial applications. The enzyme has broad substrate specificity, exhibiting not only carboxylesterase activity toward short-chain acyl esters but also lipase activity toward long-chain acyl esters including triacylglycerols regardless of saturated and unsaturated fatty acids. The kcat/Km ratios of the enzyme for p-nitrophenyl palmitate (C16), the most preferable substrate among all tested, was 93.4 s−1 μM−1. Together, it was identified by thin-layer chromatography (TLC) that the enzyme can hydrolyze all positions of the three ester bonds in triolein. The enzyme is a serine esterase belonging to the α/β hydrolase family containing a typical catalytic triad composed of serine, histidine, and aspartic acid in the active site of the enzyme. The enzyme is the first purified inducible extracellular lipolytic enzyme from archaea.

Discovery and characterization of a second extremely thermostable (+)-γ-lactamase from Sulfolobus solfataricus P2

A thermostable formamidase from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was revealed to be a novel, thermostable (+)-γ-lactamase. This (+)-γ-lactamase (Sso2810) is composed of only 318 amino acid residues, in contrast to a previously reported (+)-γ-lactamase (Sso2122) with 504 amino acid residues from the same strain. Herein, we demonstrate that a single strain may contain diverse (+)-γ-lactamases. The gene of this thermostable (+)-γ-lactamase was cloned, functionally expressed in Escherichia coli BL21 and purified by a simple yet effective heat treatment method. Sso2810 was biochemically characterized and compared to Sso2122, with phylogenetic analysis indicating different evolutionary histories for the two encoding genes. This newly found thermostable enzyme shows promising properties for industrial applications; specifically, it could be used for the production of chirally pure (-)-γ-lactam for the synthesis of well-known carbocyclic nucleoside antiretroviral agents like Abacavir and Peramivir. The chiral product of the enzyme was purified to >99% enantiomeric excess.

Purification and characterization of a new inducible thermostable extracellular lipolytic enzyme from the thermoacidophilic archaeon Sulfolobus solfataricus P1

A new thermostable extracellular lipolytic enzyme, induced from the thermoacidophilic archaeon Sulfolobus solfataricus P1 using corn oil as an inducer, was purified to apparent homogeneity by butanol extraction and two column chromatographies using DEAE-Sepharose followed by Butyl-Sepharose. The purified enzyme assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and gel filtration was approximately 45kDa and monomeric. The maximal activity examined using p-nitrophenyl palmitate as a substrate was observed at 98°C and pH 6.0. The enzyme showed remarkable thermostability: It retained 51% of its activity after 120h at 80°C. In addition, the enzyme displayed extremely high stability against water-miscible alcohols, SDS, and urea, even at high concentrations. This high stability of the enzyme against protein-denaturing agents indicates a high potential in industrial applications. The enzyme has broad substrate specificity, exhibiting not only carboxylesterase activity toward short-chain acyl esters but also lipase activity toward long-chain acyl esters including triacylglycerols regardless of saturated and unsaturated fatty acids. The kcat/Km ratios of the enzyme for p-nitrophenyl palmitate (C16), the most preferable substrate among all tested, was 93.4s−1μM−1. Together, it was identified by thin-layer chromatography (TLC) that the enzyme can hydrolyze all positions of the three ester bonds in triolein. The enzyme is a serine esterase belonging to the α/β hydrolase family containing a typical catalytic triad composed of serine, histidine, and aspartic acid in the active site of the enzyme. The enzyme is the first purified inducible extracellular lipolytic enzyme from archaea.

Activation of SsoPK4, an Archaeal eIF2α Kinase Homolog, by Oxidized CoA.

The eukaryotic protein kinase (ePK) paradigm provides integral components for signal transduction cascades throughout nature. However, while so-called typical ePKs permeate the Eucarya and Bacteria, atypical ePKs dominate the kinomes of the Archaea. Intriguingly, the catalytic domains of the handful of deduced typical ePKs from the archaeon Sulfolobus solfataricus P2 exhibit significant resemblance to the protein kinases that phosphorylate translation initiation factor 2α (eIF2α) in response to cellular stresses. We cloned and expressed one of these archaeal eIF2α protein kinases, SsoPK4. SsoPK4 exhibited protein-serine/threonine kinase activity toward several proteins, including the S. solfataricus homolog of eIF2α, aIF2α. The activity of SsoPK4 was inhibited in vitro by 3ʹ,5ʹ-cyclic AMP (Ki of ~23 µM) and was activated by oxidized Coenzyme A, an indicator of oxidative stress in the Archaea. Activation enhanced the apparent affinity for protein substrates, Km, but had little effect on Vmax. Autophosphorylation activated SsoPK4 and rendered it insensitive to oxidized Coenzyme A.

Revisiting the biodesulfurization capability of hyperthermophilic archaeon Sulfolobus solfataricus P2 revealed DBT consumption by the organism in an oil/water two-phase liquid system at high temperatures

The ability of the hyperthermophilic archaeon Sulfolobus solfataricus P2 to grow on organic and inorganic sulfur sources was investigated. A sulfur-free mineral medium was employed with different sources of carbon. The results showed that inorganic sulfur sources display growth curve patterns significantly different from the curves obtained with organic sulfur sources. Solfataricus has the ability to utilize DBT and its derivatives, but it lacks BT utilization. Solfataricus} utilizes DBT at a rate of 1.23 \mu mol 2-HBP h^{-1} g DCW^{-1} even at 78 °C, at which DBT is known to be unstable. After enabling DBT stabilization using a two-phase culture system, stable microbial growth was achieved showing a desulfurization rate of 0.34 \mu M DBT g DCW^{-1} h^{-1}. Solfataricus offers beneficial properties compared to the other desulfurizing mesophilic/moderate thermophilic bacteria due to its capacity to utilize DBT and its derivatives under hyperthermophilic conditions.

Autodisplay of an archaeal γ-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (-) vince lactam.

At present, autotransporter protein mediated surface display has opened a new dimension in the development of whole-cell biocatalysts. Here, we report the identification of a novel autotransporter Xcc_Est from Xanthomonas campestris pv campestris 8004 by bioinformatic analysis and application of Xcc_Est as an anchoring motif for surface display of γ-lactamase (Gla) from thermophilic archaeon Sulfolobus solfataricus P2 in Escherichia coli. The localization of γ-lactamase in the cell envelope was monitored by Western blot, activity assay and flow cytometry analysis. Either the full-length or truncated Xcc_Est could efficiently transport γ-lactamase to the cell surface. Compared with the free enzyme, the displayed γ-lactamase exhibited optimum temperature of 30°C other than 90°C, with a substantial decrease of 60°C. Under the preparation system, the engineered E. coli with autodisplayed γ-lactamase converted 100g racemic vince lactam to produce 49.2g (-) vince lactam at 30°C within 4h. By using chiral HPLC, the ee value of the produced (-) vince lactam was determined to be 99.5%. The whole-cell biocatalyst exhibited excellent stability under the operational conditions. Our results indicate that the E. coli with surface displayed γ-lactamase is an efficient and economical whole cell biocatalyst for preparing the antiviral drug intermediate (-) vince lactam at mild temperature, eliminating expensive energy cost performed at high temperature.

A PilT N-terminus domain protein SSO1118 from hyperthemophilic archaeon Sulfolobus solfataricus P2

The PilT N terminus (PIN) domains with about 130 amino acids in length comprise a very large protein family present in all three kingdoms of life (Arcus et al. 2011). In the Pfam database, the PIN-domain family (PF01850) currently contains 8,807 members from bacteria, archaea, and eukaryotes. The biological functions of PIN-domains are diverse in various species. Most PIN domains have ribonuclease activity involved in different biological process. In eukaryotes, PIN domains are involved in nonsense mediated mRNA decay (NMD), RNA interference (RNAi), ribosomal RNA processing, and RNA degradation in immune response regulation (Bleichert et al. 2006; Xu et al. 2012). In prokaryotes, the majority of PIN domain proteins are the toxic components of VapBC-type toxinantitoxin systems for stress response, while the toxic activity comes from their nuclease activity (Arcus et al. 2011; Blower et al. 2011). Recently, PIN domains were found in Chp1 of RITS (the RNA-induced initiation of transcriptional gene silencing) complex (Schalch et al. 2011) and Rrp44 in the yeast exosome (Makino et al. 2013). PIN-domains have poor sequence conservation but a conserved three-dimensional structure (Arcus et al. 2011). As is shown by the determined structures of many PINdomain proteins using X-ray crystallography, PIN-domains from various organisms have a 3-layer a/b/a sandwich structure which contains a 5-stranded parallel b-sheet with the order 32145 (Arcus et al. 2011). The revealed structural fold of PIN-domains has significant similarity with the Rossmann fold, a nucleotide-binding module existing in many dehydrogenases, kinases, and flavodoxins (Rossmann et al. 1974). Besides the conserved core structure, PINdomain proteins often contain structural decorations and variations of loop and secondary structure elements, such as different a-helix orientation, different length of b-stand, and additional a-helix or b-stand (Takeshita et al. 2007; Bunker et al. 2008). Despite the poor sequence conservation, PIN domains contain a highly conserved active site constituted by several acidic residues for metal binding and ribonuclease activity (Arcus et al. 2011). Protein SSO1118 with the full length of 111 residues from hyperthermophilic archaeon Sulfolobus solfataricus P2 was annotated as a hypothetical protein conserved in Sulfolobale (Fig. 1a). Our previous sequence analysis and NMR chemical shift assignment studies suggested that SSO1118 is a novel putative PIN domain protein (Xuan et al. 2011). Most archaeal PIN domains are from VapC gene of VapBC toxinantitoxin pair whose genes are in an operon in genome, but the gene of SSO1118 is alone in the genome of S. solfataricus P2. Blast search in PDB does not give significant hit. This indicates that SSO1118 has no significant homology with structure-known proteins. In the present study, the solution structure of SSO1118 was determined by NMR J. Xuan (&) X. Song Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Beijing 100083, China e-mail: jsxuan@sas.ustb.edu.cn

Cloning, expression, purification, and characterization of a thermostable esterase from the archaeon Sulfolobus solfataricus P1

A genomic library of the thermoacidophilic archaeon Sulfolobus solfataricus P1 was constructed using 3–5 kb BamHI-fragments into pUC118 vector and the recombinant plasmids were hosted in Escherichia coli. One positive clone showing thermostable esterase activity was directly selected on tributyrin-emulsified agar plates. The open reading frame of the esterase gene isolated from this clone was composed of 942 nucleotides encoding 314 amino acids. The recombinant enzyme stably expressed in E. coli was purified to apparent homogeneity by two column chromatographies using butyl Sepharose followed by Q-Sepharose. The molecular mass of the enzyme, estimated to be approximately 35 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration, agreed with that deduced by sequence analysis (34,260 Da). Maximal activity was observed at 80 °C and pH 8.0. The enzyme was extremely stable without significant change in its activity up to 120 h at 50 °C, and even at 80 °C almost 30% of its activity remained after 120 h. Among the p-nitrophenyl esters (C4–C16) tested, the best substrate was p-nitrophenyl caprate (C10) with Km and kcat values of 24.0 mM and 2337 s−1, respectively. At 30 °C, the enzyme displayed remarkable stability against up to 90% methanol, ethanol, and 2-propanol, and also withstood, to a certain extent, 5% SDS and 8 M urea. Site-directed mutagenesis revealed that the enzyme contains a catalytic triad composed of Ser144, Asp266, and His295 in the active site. The S. solfataricus P1 esterase is an archaeal esterase grouped into family V as well as B-type esterases.

Identification and Characterization of Small RNAs in the Hyperthermophilic Archaeon Sulfolobus solfataricus

The term RNA silencing (RNA interference, RNAi) describes a set of mechanisms that regulate gene expression in eukaryotes. Small interfering RNAs (siRNA) and microRNAs (miRNAs) are two major types of RNAi-associated small RNAs (smRNAs) found in most eukaryotic organisms. Despite the presence of a plethora of non-coding RNAs longer than 50-nucleotide (nt) in length in various species of Archaea, little is known about smRNAs in archaea that resemble the 20–24-nt long smRNAs found in eukaryotes, which have been implicated in the post-transcriptional control of gene expression. Here, we report the finding of a large number of smRNAs approximatelly 20-nt in length, including phased smRNAs and potential miRNAs, from the hyperthermophilic archaeon Sulfolobus solfataricus p2 (Ssp2) based on deep sequencing. The expression of some of the miRNA candidates in Ssp2 was confirmed. Consistent with the Ssp2 hyperthermophilic properties, we found that higher temperatures more efficiently induced the production of the miRNA candidates in an in vitro system using the putative foldback precursor transcripts incubated with Ssp2 extract. Although we initially predicted putative target genes of some miRNA candidates, further analysis mapped the cleavage sites downstream of the miRNA candidate complementary regions, similar to those involved in plant miRNA-mediated TAS transcript cleavage. We also identified smRNAs from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, which play important roles in prokaryotic microbial defense systems. Archaea represent a unique life form next to Bacteria and Eukarya, and our results may provide a useful resource for further in-depth study on the regulation and evolution of smRNAs in this special organism.

Transcriptome-wide discovery of circular RNAs in Archaea

Circular RNA forms had been described in all domains of life. Such RNAs were shown to have diverse biological functions, including roles in the life cycle of viral and viroid genomes, and in maturation of permuted tRNA genes. Despite their potentially important biological roles, discovery of circular RNAs has so far been mostly serendipitous. We have developed circRNA-seq, a combined experimental/computational approach that enriches for circular RNAs and allows profiling their prevalence in a whole-genome, unbiased manner. Application of this approach to the archaeon Sulfolobus solfataricus P2 revealed multiple circular transcripts, a subset of which was further validated independently. The identified circular RNAs included expected forms, such as excised tRNA introns and rRNA processing intermediates, but were also enriched with non-coding RNAs, including C/D box RNAs and RNase P, as well as circular RNAs of unknown function. Many of the identified circles were conserved in Sulfolobus acidocaldarius, further supporting their functional significance. Our results suggest that circular RNAs, and particularly circular non-coding RNAs, are more prevalent in archaea than previously recognized, and might have yet unidentified biological roles. Our study establishes a specific and sensitive approach for identification of circular RNAs using RNA-seq, and can readily be applied to other organisms.

Resonance assignments of a putative PilT N-terminus domain protein SSO1118 from hyperthermophilic archaeon Sulfolobus solfataricus P2

PilT N-terminus (PIN) domains exist broadly in all three kingdoms of life, but the functions are not clear for most of them. Archaea species often encode multiple PIN domain-containing proteins, and the signaling and stress response roles have been proposed for these proteins. Some PIN domain proteins possess nuclease activities, which were proposed to be important in toxin-antitoxin stress response, nonsense-mediated mRNA decay, or RNA interference. SSO1118 from hyperthermophilic archaeon Sulfolobus solfataricus P2 is a putative PIN domain protein with low homology to other known PIN domain proteins. Here we report the NMR resonance assignments of SSO1118 for further structural determination and functional studies. The secondary structures predicted from the assigned chemical shifts consist with those of archaeal PIN domain proteins.

A novel dienelactone hydrolase from the thermoacidophilic archaeon Sulfolobus solfataricus P1: Purification, characterization, and expression

Background Dienelactone hydrolases catalyze the hydrolysis of dienelactone to maleylacetate, which play a key role for the microbial degradation of chloroaromatics via chlorocatechols. Here, a thermostable dienelactone hydrolase from thermoacidophilic archaeon Sulfolobus solfataricus P1 was the first purified and characterized and then expressed in Escherichia coli. Methods The enzyme was purified by using several column chromatographys and characterized by determining the enzyme activity using p-nitrophenyl caprylate and dienelactones. In addition, the amino acids related to the catalytic mechanism were examined by site-directed mutagenesis using the identified gene. Results The enzyme, approximately 29 kDa monomeric, showed the maximal activity at 74 °C and pH 5.0, respectively. The enzyme displayed remarkable thermostability: it retained approximately 50% of its activity after 50 h of incubation at 90 °C, and showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme displayed substrate specificities toward trans-dienelactone, not cis-isomer, and also carboxylesterase activity toward p-nitrophenyl esters ranging from butyrate (C 4) to laurate (C 12). The k cat/ K m ratios for trans-dienelactone and p-nitrophenyl caprylate (C 8), the best substrate, were 92.5 and 54.7 s −1 μM −1, respectively. Conclusions The enzyme is a typical dienelactone hydrolase belonging to α/β hydrolase family and containing a catalytic triad composed of Cys151, Asp198, and His229 in the active site. General significance The enzyme is the first characterized archaeal dienelactone hydrolase.

Quantitative Proteomic Analysis of Sulfolobus solfataricus Membrane Proteins

A quantitative proteomic analysis of the membrane of the archaeon Sulfolobus solfataricus P2 using iTRAQ was successfully demonstrated in this technical note. The estimated number of membrane proteins of this organism is 883 (predicted based on Gravy score), corresponding to 30% of the total number of proteins. Using a modified iTRAQ protocol for membrane protein analysis, of the 284 proteins detected, 246 proteins were identified as membrane proteins, while using an original iTRAQ protocol, 147 proteins were detected with only 133 proteins being identified as membrane proteins. Furthermore, 97.2% of proteins identified in the modified protocol contained more than 2 distinct peptides compared to the original workflow. The successful application of this modified protocol offers a potential technique for quantitatively analyzing membrane-associated proteomes of organisms in the archaeal kingdom. The combination of 3 different iTRAQ experiments resulted in the detection of 395 proteins (>or=2 distinct peptides) of which 373 had predicted membrane properties. Approximately 20% of the quantified proteins were observed to exhibit >or=1.5-fold differential expression at temperatures well below the optimum for growth.

Crystallization and preliminary X-ray characterization of a PaaX-like protein fromSulfolobus solfataricusP2

PaaX is a global regulator of the phenylacetyl-coenzyme A catabolon that adjusts the expression of different operons to that of the paa-encoded central pathway. In this study, the PaaX-like protein from the hyperthermophilic archaeon Sulfolobus solfataricus P2 was successfully crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant. Diffraction data were obtained to a resolution of 3.0 A using synchrotron radiation at the Photon Factory. The crystal belonged to space group P321, with unit-cell parameters a = 86.4, b = 86.4, c = 105.5 A.

A Novel Thermostable Arylesterase from the ArchaeonSulfolobus solfataricusP1: Purification, Characterization, and Expression

A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 94 degrees C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90 degrees C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin and p-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C(4)) to palmitate (C(16)), but also paraoxonase activity toward organophosphates such as p-nitrophenylphosphate, paraoxon, and methylparaoxon. The k(cat)/K(m) ratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s(-1) microM(-1), respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase from Acinetobacter sp. strain ADP1. Furthermore, we successfully expressed active recombinant S. solfataricus arylesterase in Escherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.

Oligomeric and functional properties of a debranching enzyme (TreX) from the archaeon Sulfolobus solfataricus P2

A gene, treX, encoding a debranching enzyme previously cloned from the trehalose biosynthesis gene cluster of Sulfolobus solfataricus P2 was expressed in Escherichia coli as a His-tagged protein and the biochemical properties were studied. The specific activity of the S. solfataricus debranching enzyme (TreX) was highest at 75°C and pH 5.5. The enzyme exhibited hydrolysing activity toward α-1,6-glycosidic linkages of amylopectin, glycogen, pullulan, and other branched substrates, and glycogen was the preferred substrate. TreX has a high specificity for hydrolysis of maltohexaosyl α-1,6-β-cyclodextrin, indicating the high preference for side chains consisting of 6 glucose residues or more. The enzyme also exhibited 4-α-sulfoxide-glucan transferase activity, catalysing transfer of α-1,4-glucan oligosaccharides from one chain to another. Dimethyl sulfoxide (10%, v/v) increased the hydrolytic activity of TreX. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation revealed that the enzyme exists mostly as a dimer at pH 7.0, and as a mixture of dimers and tetramers at pH 5.5. Interestingly, TreX existed as a tetramer in the presence of DMSO at pH 5.5–6.5. The tetramer showed a 4-fold higher catalytic efficiency than the dimer. The enzyme catalysed not only intermolecular trans-glycosylation of malto-oligosaccharides (disproportionation) to produce linear α-1,4-glucans, but also intramolecular trans-glycosylation of glycogen. The results presented in this study indicated that TreX may be associated with glycogen metabolism by selective cleavage of the outer side chain.

Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 °C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 °C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C 4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C 8) with K m and k cat values of 71 μM and 14,700 s −1, respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.

Sir2 and the Acetyltransferase, Pat, Regulate the Archaeal Chromatin Protein, Alba

The DNA binding affinity of Alba, a chromatin protein of the archaeon Sulfolobus solfataricus P2, is regulated by acetylation of lysine 16. Here we identify an acetyltransferase that specifically acetylates Alba on this residue. The effect of acetylation is to lower the affinity of Alba for DNA. Remarkably, the acetyltransferase is conserved not only in archaea but also in bacteria where it appears to play a role in metabolic regulation. Therefore, our data suggest that S. solfataricus has co-opted this bacterial regulatory system to generate a rudimentary form of chromatin regulation.