Partial Purification and Characterization of Thermostable Acid Phosphatase from Thermoacidophilic Archaeon Sulfolobus acidocaldarius

Thermostable acid phosphatase (APase) from thermoacidophilic archaeon Sulfolobus acidocaldarius was isolated, partially purified, and characterized. The optimum pH and temperature of the enzyme for p-nitrophenylphosphate (pNPP) as a substrate were 5.0 and 70°C, respectively. The apparent K _m value was 1.9 mM. This APase showed a native molecular mass of 20 kDa on a gel filtration chromatography. Of the APase activity, 60% remained after 60 min of heat treatment at 75°C. To confirm whether the APase is active in the monomeric form, we attempted to elute the enzyme from SDS-polyacrylamide gels with Disk electrophoresis apparatus and renature the enzyme. The APase activity was recovered up to 50% in the 14- to 35-kDa range, and maximum around 25 kDa. These results suggest that this APase is monomeric protein. Received: 8 July 1999 / Accepted: 9 August 1999     

Novel Medium-Chain Prenyl Diphosphate Synthase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

Two open reading frames which encode the homologues of (all-E) prenyl diphosphate synthase are found in the whole-genome sequence of Sulfolobus solfataricus, a thermoacidophilic archaeon. It has been suggested that one is a geranylgeranyl diphosphate synthase gene, but the specificity and biological significance of the enzyme encoded by the other have remained unclear. Thus, we isolated the latter by the PCR method, expressed the enzyme in Escherichia coli cells, purified it, and characterized it. The archaeal enzyme, 281 amino acids long, is highly thermostable and requires Mg(2+) and Triton X-100 for full activity. It catalyzes consecutive E-type condensations of isopentenyl diphosphate with an allylic substrate such as geranylgeranyl diphosphate and yields the medium-chain product hexaprenyl diphosphate. Despite such product specificity, phylogenetic analysis revealed that the archaeal medium-chain prenyl diphosphate synthase is distantly related to the other medium- and long-chain enzymes but is closely related to eucaryal short-chain enzymes.     

A Protein Related to Eucaryal and Bacterial DNA-Motor Proteins in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius

We have isolated a new gene encoding a putative 103-kDa protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius. Analysis of the deduced amino-acid sequence shows an extended central domain, predicted to form coiled-coil structures, and two terminal domains that display purine NTPase motifs. These features are reminiscent of mechanochemical motor proteins which use the energy of ATP hydrolysis to move specific cellular components. Comparative analysis of the amino-acid sequence of the terminal domains and predicted structural organization of this putative purine NTPase show that it is related both to eucaryal proteins from the ``SMC family' involved in the condensation of chromosomes and to several bacterial and eucaryal proteins involved in DNA recombination/repair. Further analyses revealed that these proteins are all members of the so called ``UvrA-related NTP-binding proteins superfamily' and form a large subgroup of motor-like NTPases involved in different DNA processing mechanisms. The presence of such protein in Archaea, Bacteria, and Eucarya suggests an early origin of DNA-motor proteins that could have emerged and diversified by domain shuffling. Received: 29 June 1996 / Accepted: 28 February 1997     

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.     

Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh

Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-prone motifs in Sulfolobus chromosomes and used them to measure the efficiency and accuracy of bypass in normal and dbh⁻ strains of Sulfolobus acidocaldarius. Oligonucleotide-mediated transformation allowed low levels of abasic-site bypass to be observed in S. acidocaldarius and demonstrated that the local sequence context affected bypass specificity; in addition, most erroneous TLS did not require Dbh function. Applying the technique to another common lesion, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), revealed an antimutagenic role of Dbh. The efficiency and accuracy of replication past 8-oxo-dG was higher in the presence of Dbh, and up to 90% of the Dbh-dependent events inserted dC. A third set of assays, based on phenotypic reversion, showed no effect of Dbh function on spontaneous −1 frameshifts in mononucleotide tracts in vivo, despite the extremely frequent slippage at these motifs documented in vitro. Taken together, the results indicate that a primary genetic role of Dbh is to avoid mutations at 8-oxo-dG that occur when other Sulfolobus enzymes replicate past this lesion. The genetic evidence that Dbh is recruited to 8-oxo-dG raises questions regarding the mechanism of recruitment, since Sulfolobus spp. have eukaryotic-like replisomes but no ubiquitin.     

Population ecology of Sulfolobus acidocaldarius

Ecology of Sulfolobus acidocaldarius was studied in situ by the use of the immunofluorescence and immunodiffusion techniques. The fluorescent antibodies (FA) prepared against four strains of Sulfolobus were highly reactive against their homologous antigens. Two of the FA's were strain specific and the other two exhibited reciprocal corssreactions against each other's antigens, but immunodiffusion patterns showed that the two strains were not identical. The growth of a serologically distinct isolate in a hot spring was measured by immunofluorescence staining of immersion slides. On glass immersion slides Sulfolobus grew and formed colonies with a mean-doubling time of approximately 36 h. Immunofluorescence was applied to study the geographical distribution of two serologically different strains and to establish population composition of individual springs. One strain was found in all sites studied, and most springs contained more than one serologic type. Immunodiffusion was capable of detecting specific Sulfolobus antigens in hot springs which contained a high population of FA-reactive cells.     

Expression, Isolation, and Crystallization of the Catalytic Domain of CopB, a Putative Copper Transporting ATPase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.     

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

Cell cycle regulation in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius

The regulation and co-ordination of the cell cycle of the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius was investigated with antibiotics. We provide evidence for a core regulation involving alternating rounds of chromosome replication and genome segregation. In contrast, multiple rounds of replication of the chromosome could occur in the absence of an intervening cell division event. Inhibition of the elongation stage of chromosome replication resulted in cell division arrest, indicating that pathways similar to checkpoint mechanisms in eukaryotes, and the SOS system of bacteria, also exist in archaea. Several antibiotics induced cell cycle arrest in the G2 stage. Analysis of the run-out kinetics of chromosome replication during the treatments allowed estimation of the minimal rate of replication fork movement in vivo to 250 bp s-1. An efficient method for the production of synchronized Sulfolobus populations by transient daunomycin treatment is presented, providing opportunities for studies of cell cycle-specific events. Possible targets for the antibiotics are discussed, including topoisomerases and protein glycosylation.     

Characterization of the RNase P RNA of Sulfolobus acidocaldarius.

RNase P is the ribonucleoprotein enzyme that cleaves precursor sequences from the 5' ends of pre-tRNAs. In Bacteria, the RNA subunit is the catalytic moiety. Eucaryal and archaeal RNase P activities copurify with RNAs, which have not been shown to be catalytic. We report here the analysis of the RNase P RNA from the thermoacidophilic archaeon Sulfolobus acidocaldarius. The holoenzyme was highly purified, and extracted RNA was used to identify the RNase P RNA gene. The nucleotide sequence of the gene was determined, and a secondary structure is proposed. The RNA was not observed to be catalytic by itself, but it nevertheless is similar in sequence and structure to bacterial RNase P RNA. The marked similarity of the RNase P RNA from S. acidocaldarius and that from Haloferax volcanii, the other known archael RNase P RNA, supports the coherence of Archaea as a phylogenetic domain.     

Microbial oxidation of dibenzothiophene by the thermophilic organism Sulfolobus acidocaldarius

The refractory organic sulfur compound dibenzothiophene (DBT) has been oxidized by the thermophilic, sulfur oxidizing organism Sulfolobus acidocaldarius. Sulfate ions were released into the medium as the oxidation product. The kinetics of this oxidation have been investigated on the basis of sulfate released as a result of oxidation. Dibenzothiophene was found to be inhibitory to the organisms for initial concentrations over 500 mg/L. The organism may prove to be capable of oxidizing thiophene compounds present in oil refinery wastewater, coal, and crude oil.     

Genetic responses of the thermophilic archaeon Sulfolobus acidocaldarius to short-wavelength UV light.

The archaea which populate geothermal environments are adapted to conditions that should greatly destabilize the primary structure of DNA, yet the basic biological aspects of DNA damage and repair remain unexplored for this group of prokaryotes. We used auxotrophic mutants of the extremely thermoacidophilic archaeon Sulfolobus acidocaldarius to assess genetic and physiological effects of a well-characterized DNA-damaging agent, short-wavelength UV light. Simple genetic assays enabled quantitative dose-response relationships to be determined and correlated for survival, phenotypic reversion, and the formation of genetic recombinants. Dose-response relationships were also determined for survival and phenotypic reversion of the corresponding Escherichia coli auxotrophs with the same equipment and procedures. The results showed S. acidocaldarius to be about twice as UV sensitive as E. coli and to be equally UV mutable on a surviving-cell basis. Furthermore, UV irradiation significantly increased the frequency of recombinants recovered from genetic-exchange assays of S. acidocaldarius. The observed UV effects were due to the short-wavelength (i.e., UV-C) portion of the spectrum and were effectively reversed by subsequent illumination of S. acidocaldarius cells with visible light (photoreactivation). Thus, the observed responses are probably initiated by the formation of pyrimidine dimers in the S. acidocaldarius chromosome. To our knowledge, these results provide the first evidence of error-prone DNA repair and genetic recombination induced by DNA damage in an archaeon from geothermal habitats.     

Three-dimensional arrangement of the cell wall protein of Sulfolobus acidocaldarius

The three-dimensional structure of the S-layer that surrounds the bacterium Sulfolobus acidocaldarius is described in detail. Pieces of the S-layer, which are two-dimensional crystals with p6 symmetry, have been studied by crystallographic analysis of electron micrographs of tilted specimens. In the density map, each asymmetric unit appears to consist of several domains connected by strong hinges. On the basis of the ragged appearance of the structure at torn edges, we now suggest that the single species of polypeptide is in a highly extended conformation, with much overlap between different molecules, and show how such molecules might be weaved together to produce the morphological domains. We show that the closed surface lattice of the intact cell wall contains 5-fold and 7-fold vertices and show how the subunit structure appears to be well suited to form 5-fold and 7-fold symmetric rings at these points in place of the 6-fold rings of the hexagonal lattice.     

A restriction endonuclease SuaI from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

A type II restriction endonuclease (SuaI) has been isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspRI. It does not cut S. acidocaldarius DNA, as the recognition sequence GGCC in this DNA contains modified nucleotide(s). The enzyme is most active at 60-70 degrees C and is highly thermostable.     

Purification of glycerol dialkyl nonitol tetraether from Sulfolobus acidocaldarius

A modified procedure for extraction and purification of hydrolyzed archaebacterial lipids is described. Lipids were extracted from Sulfolobus acidocaldarius using a Soxhlet extraction procedure followed by trichloroacetic acid solvent-extraction of the residue. The yield of total extractable material by this protocol was 14% which, after a two-phase wash, yielded 10% lipid. Modifications to the published steps for purifying the subsequently hydrolyzed lipids were developed to purify glycerol dialkyl nonitol tetraether (GDNT). The nearly colorless final macrocyclic product was characterized by TLC, IR, NMR, and mass spectrometry.