An Autonomously Replicating Transforming Vector for Sulfolobus solfataricus

A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E. coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker. The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C.     

Small multicopy, non-integrative shuttle vectors based on the plasmid pRN1 for Sulfolobus acidocaldarius and Sulfolobus solfataricus, model organisms of the (cren-)archaea

The extreme thermoacidophiles of the genus Sulfolobus are among the best-studied archaea but have lacked small, reliable plasmid vectors, which have proven extremely useful for manipulating and analyzing genes in other microorganisms. Here we report the successful construction of a series of Sulfolobus–Escherichia coli shuttle vectors based on the small multicopy plasmid pRN1 from Sulfolobus islandicus. Selection in suitable uracil auxotrophs is provided through inclusion of pyrEF genes in the plasmid. The shuttle vectors do not integrate into the genome and do not rearrange. The plasmids allow functional overexpression of genes, as could be demonstrated for the β-glycosidase (lacS) gene of S. solfataricus. In addition, we demonstrate that this β-glycosidase gene could function as selectable marker in S. solfataricus. The shuttle plasmids differ in their interruption sites within pRN1 and allowed us to delineate functionally important regions of pRN1. The orf56/orf904 operon appears to be essential for pRN1 replication, in contrast interruption of the highly conserved orf80/plrA gene is tolerated. The new vector system promises to facilitate genetic studies of Sulfolobus and to have biotechnological uses, such as the overexpression or optimization of thermophilic enzymes that are not readily performed in mesophilic hosts.     

Peptidyl-tRNA hydrolase from Sulfolobus solfataricus

An enzyme capable of liberating functional tRNA^Lys from Escherichia coli diacetyl-lysyl-tRNA^Lys was purified from the archae Sulfolobus solfataricus. Contrasting with the specificity of peptidyl- tRNA hydrolase (PTH) from E.coli, the S.solfataricus enzyme readily accepts E.coli formyl-methionyl-tRNA^fMet as a substrate. N-terminal sequencing of this enzyme identifies a gene that has homologs in the whole archaeal kingdom. Involvement of this gene (SS00175) in the recycling of peptidyl-tRNA is supported by its capacity to complement an E.coli strain lacking PTH activity. The archaeal gene, the product of which appears markedly different from bacterial PTHs, also has homologs in all the available eukaryal genomes. Since most of the eukaryotes already display a bacterial-like PTH gene, this observation suggests the occurrence in many eukaryotes of two distinct PTH activities, either of a bacterial or of an archaeal type. Indeed, the bacterial- and archaeal-like genes encoding the two full-length PTHs of Saccharomyces cerevisiae, YHR189w and YBL057c, respectively, can each rescue the growth of an E.coli strain lacking endogeneous PTH. In vitro assays confirm that the two enzymes ensure the recycling of tRNA^Lys from diacetyl-lysyl-tRNA^Lys. Finally, the growth of yeast cells in which either YHR189w or YBL057c has been disrupted was compared under various culture conditions. Evidence is presented that YHR189w, the gene encoding a bacterial-like PTH, should be involved in mitochondrial function.     

CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus

The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)-mediated virus defense represents an adaptive immune system in many bacteria and archaea. Small CRISPR RNAs cause cleavage of complementary invading nucleic acids in conjunction with an associated protein or a protein complex. Here, we show CRISPR-mediated cleavage of mRNA from an invading virus in the hyperthermophilic archaeon Sulfolobus solfataricus. More than 40% of the targeted mRNA could be cleaved, as demonstrated by quantitative polymerase chain reaction. Cleavage of the mRNA was visualized by northern analyses and cleavage sites were mapped. In vitro, the same substrates were cleaved by the purified CRISPR-associated CMR complex from Sulfolobus solfataricus. The in vivo system was also re-programmed to knock down mRNA of a selected chromosomal gene (β-galactosidase) using an artificial miniCRISPR locus. With a single complementary spacer, ∼50% reduction of the targeted mRNA and of corresponding intracellular protein activity was achieved. Our results demonstrate in vivo cleavage of mRNA in a prokaryote mediated by small RNAs (i.e. analogous to RNA interference in eukaryotes) and the re-programming of the system to silence specific genes of interest.     

Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome

The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during the past years, systems have been developed for Thermococcus kodakarensis and two Sulfolobus species, S. acidocaldarius and derivatives of S. solfataricus 98/2. Here we describe an optimization of the method for integration of exogenous DNA into S. solfataricus PBL 2025, an S. solfataricus 98/2 derivative, based on lactose auxotrophy that now allows for routine gene inactivation.     

Development of a simvastatin selection marker for a hyperthermophilic acidophile, Sulfolobus islandicus

We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P(sac7d)-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P(sac7d)-hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His(6)-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEF and hmg) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.     

A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: cloning, overexpression and properties

A new gene from the hyperthermophilic archaeon Sulfolobus solfataricus MT4, coding for a putative protein reported to show sequence identity with the phosphotriesterase-related protein family (PHP), was cloned by means of the polymerase chain reaction from the S. solfataricus genomic DNA. In order to analyse the biochemical properties of the protein an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble form at 5 mg/l of E. coli culture, was purified to homogeneity and characterized. In contrast with its mesophilic E. coli counterpart that was devoid of any tested activity, the S. solfataricus enzyme was demonstrated to have a low paraoxonase activity. This activity was dependent from metal cations with Co²⁺, Mg²⁺ and Ni²⁺ being the most effective and was thermophilic and thermostable. The enzyme was inactivated with EDTA and o-phenantroline. A reported inhibitor for Pseudomonas putida phosphotriesterase (PTE) had no effect on the S. solfataricus paraoxonase. The importance of a stable paraoxonase for detoxification of chemical warfare agents and agricultural pesticides will be discussed.     

Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius

Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli–Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266 bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2–0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30–50 h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169 bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1 mg L⁻¹ culture. In addition we also determined the promoter strength of several constitutive promoters.     

Evolution of the family of pRN plasmids and their integrase-mediated insertion into the chromosome of the crenarchaeon Sulfolobus solfataricus

Plasmid pHEN7 from Sulfolobus islandicus was sequenced (7.83 kb) and shown to belong to the archaeal pRN family, which includes plasmids pRN1, pRN2, pSSVx and pDL10 that share a large conserved sequence region. pHEN7 is most closely related to pRN1 in this conserved region. It also shares a large variant region containing several homologous genes with pDL10, which is absent from the other plasmids. The variant region is flanked by the sequence motif TTAGAATGGGGATTC and similar duplicated motifs occur in plasmids pRN1 and pRN2, separated by a few bases. It is inferred that recombination at these sites produces the main genetic variability in the plasmid family. The conserved region of the plasmid, and duplicated copies of the motif, are also present in the genome of Sulfolobus solfataricus P2. Moreover, they are bordered by a partitioned integrase gene (int) and by a 45 bp perfect direct repeat corresponding to the downstream half of a tRNA(Val) gene. The integrase and the direct repeat are highly similar in sequence to the integrase and the chromosomal integration site (att), respectively, of the SSV1 virus, which integrates into the chromosome of Sulfolobus shibatae. Recombination at the att repeats in S. solfataricus would produce a novel plasmid, pXQ1, which carries both an intact integrase gene and a single integration site (att). This strongly suggests that the same mechanism of site-specific integration at a tRNA gene is used for both viruses and plasmids in Sulfolobus.     

Small Abundant DNA Binding Proteins from the Thermoacidophilic Archaeon Sulfolobus shibatae Constrain Negative DNA Supercoils

Major DNA binding proteins, designated Ssh7, were purified from the thermoacidophilic archaeon Sulfolobus shibatae. The Ssh7 proteins have an apparent molecular mass of 6.5 kDa and are similar to the 7-kDa DNA binding proteins from Sulfolobus acidocaldarius and Sulfolobus solfataricus in N-terminal amino acid sequence. The proteins constitute about 4.8% of the cellular protein. Upon binding to DNA, the Ssh7 proteins constrain negative supercoils. At the tested Ssh7/DNA mass ratios (0 to 1.65), one negative supercoil was taken up by approximately 20 Ssh7 molecules. Our results, together with the observation that the viral DNA isolated from S. shibatae is relaxed, suggest that regions of free DNA in the S. shibatae genome, if present, are highly positively supercoiled.     

Unexpectedly broad target recognition of the CRISPR-mediated virus defence system in the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus carries an extensive array of clustered regularly interspaced short palindromic repeats (CRISPR) systems able to mediate DNA degradation of invading genetic elements when complementarity to the small CRISPR-derived (cr)RNAs is given. Studying virus defence in vivo with recombinant viral variants, we demonstrate here that an unexpectedly high number of mutations are tolerated between the CRISPR-derived guide RNAs (crRNAs) and their target sequences (protospacer). Up to 15 mismatches in the crRNA still led to ∼50% of DNA degradation, when these mutations were outside the ‘seed’ region. More than 15 mutations were necessary to fully abolished interference. Different from other CRISPR systems investigated in vivo, mutations outside the protospacer region indicated no need for a protospacer adjacent motif sequence to confer DNA interference. However, complementarity of only 3 nucleotides between the repeat-derived 5′ handle of the crRNA and nucleotides adjacent to the protospacer enabled self-recognition, i.e. protection of the host locus. Our findings show commonalities and differences among the various CRISPR-mediated defence systems and suggest that they should not merely be perceived as a ‘first-barrier-defence system’ but may be considered to have a broader mechanism that allows host cells to cope with viruses keeping them at reduced levels.     

Appendage-Mediated Surface Adherence of Sulfolobus solfataricus

Attachment of microorganisms to surfaces is a prerequisite for colonization and biofilm formation. The hyperthermophilic crenarchaeote Sulfolobus solfataricus was able to attach to a variety of surfaces, such as glass, mica, pyrite, and carbon-coated gold grids. Deletion mutant analysis showed that for initial attachment the presence of flagella and pili is essential. Attached cells produced extracellular polysaccharides containing mannose, galactose, and N-acetylglucosamine. Genes possibly involved in the production of the extracellular polysaccharides were identified.In microbiology, organisms are isolated from their natural habitats and typically cultivated in the laboratory as planktonic species. Though this method has been essential for understanding the concept of life, it remains unclear how microbial ecosystems operate. For bacteria, it is well known that they are able to form large cellular communities with highly complex cellular interactions and symbioses between different microbial or eukaryotic species. Biofilm formation is an essential component of such communities, and studies have shown that bacteria within biofilms are physiologically different from planktonic ones (20, 21). They can exhibit extensive networks of pili on their surfaces and produce and secrete extracellular polysaccharides (EPS), their growth rate is decreased, and cells are much more resistant to physical stresses and antibiotics (19).The study of surface colonization and cellular communities of archaea is crucial for understanding their ecological properties. The only detailed study showed that the hyperthermophilic organism Archaeoglobus fulgidus produced biofilms when challenged with heavy metals and pentachlorophenol (10). Pyrococcus furiosus was able to adhere to different surfaces, such as mica and carbon-coated gold grids, and cells were connected via cable-like bundles of flagella (12). Methanopyrus kandleri was shown to adhere to glass, but P. furiosus could colonize only by attaching to M. kandleri cells, using flagella and direct cell contacts (16).Here we report on the function of cell surface appendages in initial attachment to surfaces of archaea, using directed gene inactivation mutants. The crenarchaeote Sulfolobus solfataricus P2 is a thermoacidophile which grows optimally at 80°C and pH values of 2 to 4 (22). S. solfataricus possesses cell surface structures such as flagella and UV-induced pili (1, 2). The flagellum operon of S. solfataricus encodes, in addition to the structural subunit FlaB, four proteins of unknown function, the ATPase FlaI, and the only integral membrane protein, FlaJ. Previously, we isolated a ΔflaJ mutant which was nonflagellated and had lost its ability for surface motility on Gelrite plates (17). Recently, we described UV-inducible pili in S. solfataricus that directed cellular aggregation after UV stress (8). Deletion of the central ATPase UpsE, responsible for pilus assembly, rendered cells devoid of pili and defective in cellular aggregation after UV treatment (8). In this study, wild-type cells and deletion strains were tested for the ability to attach to a variety of surfaces and the formed structures and extracellular material were analyzed.     

The genetic element pSSVx of the extremely thermophilic crenarchaeon Sulfolobus is a hybrid between a plasmid and a virus

A new Sulfolobus islandicus strain, REY15/4, harboured both a novel fusellovirus, SSV2, and a small plasmid, pSSVx. The plasmid spread in S. solfataricus P1 together with the virus after infection with either the supernatant of a culture of REY15/4 or purified virus. Spreading of the plasmid required co-transfection with either SSV2 or the related SSV1 as helpers. Virus purified from REY15/4 constituted a mixture of two sizes of particles, one with the dimensions of a normal fusellovirus and the other smaller. Cloned SSV2 produced only the larger particles and only SSV2 DNA, indicating that the smaller particles contained pSSVx packaged into capsids made up of SSV2 components. The 5.7 kb genome of pSSVx revealed regions of high sequence similarity to the cryptic Sulfolobales plasmids pRN1, pRN2 and pDL10. Thus, pSSVx belongs to the family of pRN plasmids that share a highly conserved region, which probably constitutes the minimal replicon. They also contain a variable region showing no sequence similarity. In pSSVx, this region contains three open reading frames (ORFs), two of which are juxtapositioned and show high sequence similarity to a tandem of ORFs in fusellovirus genomes. Neither pRN1 nor pRN2, which lack this tandem, spread in the presence of the fuselloviruses, which implies that the sequences of these ORFs enable pSSVx to use the packaging system of the viral helpers for spreading.     

Cloning and overexpression in Escherichia coli of the gene encoding citrate synthase from the hyperthermophilic Archaeon Sulfolobus solfataricus

The citrate synthase (CS) gene from the hyperthermophilic Archaeon Sulfolobus solfataricus has been cloned and sequenced. The gene encodes a polypeptide of 378 amino acids with a calculated polypeptide molecular mass of 42 679. High-level expression was achieved in Escherichia coli and the recombinant citrate synthase was purified to homogeneity using a heat step and dye-ligand affinity chromatography. This procedure yielded approximately 26 mg of pure CS per liter of culture, with a specific activity of 41 U/mg. The enzyme exhibited a half-life of 8 min at 95°C. A homology-modelled structure of the S. solfataricus CS has been generated using the crystal structure of the enzyme from the thermoacidophilic Archaeon Thermoplasma acidophilum with which it displays 58% sequence identity. The modelled structure is discussed with respect to the thermostability properties of the enzyme. Received: August 10, 1997 / Accepted: October 23, 1997     

A plasmid in the archaebacterium Sulfolobus acidocaldarius

A plasmid of mol. wt. ~9 × 10⁶ has been isolated from the archaebacterium Sulfolobus acidocaldarius strain B12. Plasmid production is induced by u.v. radiation. A copy of the plasmid is probably carried by the chromosome, integrated at a specific site. The entire plasmid, and also restriction fragments of it, has been cloned into Escherichia coli plasmid vectors, and the cleavage sites on the plasmid DNA of three restriction endonucleases have been mapped.     

A novel thermostable arylesterase from the archaeon Sulfolobus solfataricus P1: 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°C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90°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 (C₄) to palmitate (C₁₆), 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·μM⁻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.     

The Exopolyphosphatase Gene from Sulfolobus solfataricus: Characterization of the First Gene Found To Be Involved in Polyphosphate Metabolism in Archaea

Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Although the presence of polyP in members of the domain Archaea has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain. We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (ppx) from thermophilic Sulfolobus solfataricus. The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa). The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60°C. The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs. In contrast, informatic analysis revealed that the deduced amino acid sequence of S. solfataricus PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs. To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the Archaea.     

Enhanced Production of β-D-glycosidase and α-L-arabinofuranosidase in Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> in Fed-batch Culture for the Biotransformation of Ginseng Leaf Extract to Ginsenoside Compound K

Ginsenoside compound K is an essential ingredient in nutritional supplements, cosmetics, and traditional medicines. However, cultivation for the production of enzymes involved in ginsenoside biotransformation has not been attempted in a fermenter. The host strain Escherichia coli ER2566 and the constitutive pHCE vector were selected for the efficient production of β-D-glycosidase, and expression medium composition to produce Sulfolobus solfataricus β-glycosidase expressed in E. coli was optimized in flask and batch cultures. The total activity of β-Dglycosidase in fed-batch culture using a fermenter increased 14-fold before optimization. S. solfataricus β-D-glycosidase and Thermotoga petrophila α-L-arabinofuranosidase were produced in a fed-batch culture. These two enzymes completely converted protopanaxadiol-type ginsenosides in ginseng leaf extract obtained from discarded ginseng leaves as a renewable substrate to compound K. The effective bioprocess for compound K production developed here will contribute to the industrial biological production of compound K.