Cloning, expression, and characterization of cis-polyprenyl diphosphate synthase from the thermoacidophilic archaeon Sulfolobus acidocaldarius

cis-polyprenyl diphosphate synthases are involved in the biosynthesis of the glycosyl carrier lipid in most organisms. However, only little is known about this enzyme of archaea. In this report, we isolated the gene of cis-polyprenyl diphosphate synthase from a thermoacidophilic archaeon, Sulfolobus acidocaldarius, and characterized the recombinant enzyme.     

Distribution of reverse gyrase in representative species of eubacteria and archaebacteria

Reverse gyrase is a topoisomerase which positively supercoils closed circular plasmid DNA. Reverse gyrase activity is restricted to the thermoacidophilic group of archaebacteria. Thermophilic methanogens and eubacteria and all mesophilic organisms screened had no reverse gyrase activity. The result supports the deep phylogenetic divergence in archaebacterial evolution.     

Uncultured archaea dominate in the thermal groundwater of Uzon Caldera,Kamchatka

The thermoacidophilic microbial community inhabiting the groundwater with pH 4.0 and temperature 50°C at the East Thermal Field of Uzon Caldera, Kamchatka, was examined using pyrosequencing of the V3 region of the 16S rRNA gene. Bacteria comprise about 30% of microorganisms and are represented primarily by aerobic lithoautotrophs using the energy sources of volcanic origin—thermoacidophilic methanotrophs of the phylum Verrucomicrobia and Acidithiobacillus spp. oxidising metals and reduced sulfur compounds. More than 70% of microbial population in this habitat were represented by archaea, in majority affiliated with “uncultured” lineages. The most numerous group (39% of all archaea) represented a novel division in the phylum Euryarchaeota related to the order Thermoplasmatales. Another abundant group (33% of all archaea) was related to MCG1 lineage of the phylum Crenarchaeota, originally detected in the Yellowstone hot spring as the environmental clone pJP89. The organisms belonging to these two groups are widely spread in hydrothermal environments worldwide. These data indicate an important environmental role of these two archaeal groups and should stimulate the investigation of their metabolism by cultivation or metagenomic approaches.     

Fusion-type lycopene beta-cyclase from a thermoacidophilic archaeon Sulfolobus solfataricus

Examination of the sequence of a hypothetical gene with an unknown function included in the carotenogenic gene cluster in the genome of a thermoacidophilic archaeon Sulfolobus solfataricus led to the prediction that the gene encodes a novel-type lycopene beta-cyclase, whose N- and C-terminal halves are homologous to the subunits of the bacterial heterodimeric enzymes. The recombinant expression of the gene in lycopene-producing Escherichia coli resulted in the accumulation of beta-carotene in the cells, which verifies the function of the gene. Homologues of the archaeal lycopene beta-cyclase from various organisms such as bacteria, archaea, and fungi have been reported. Although their primary structures are clearly specific to the biological taxa, a phylogenetic analysis revealed the unexpected complicity of the evolutional route of these enzymes.     

Comparative sequence analyses on the 16S rRNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for creation of a new genus, Alicyclobacillus gen. nov.

Comparative 16S rRNA (rDNA) sequence analyses performed on the thermophilic Bacillus species Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus revealed that these organisms are sufficiently different from the traditional Bacillus species to warrant reclassification in a new genus, Alicyclobacillus gen. nov. An analysis of 16S rRNA sequences established that these three thermoacidophiles cluster in a group that differs markedly from both the obligately thermophilic organisms Bacillus stearothermophilus and the facultatively thermophilic organism Bacillus coagulans, as well as many other common mesophilic and thermophilic Bacillus species. The thermoacidophilic Bacillus species B. acidocaldarius, B. acidoterrestris, and B. cycloheptanicus also are unique in that they possess omega-alicylic fatty acid as the major natural membranous lipid component, which is a rare phenotype that has not been found in any other Bacillus species characterized to date. This phenotype, along with the 16S rRNA sequence data, suggests that these thermoacidophiles are biochemically and genetically unique and supports the proposal that they should be reclassified in the new genus Alicyclobacillus.     

Molecular biology of extremophiles: recent progress on the hyperthermophilic archaeon Sulfolobus

Extremophiles are microorganisms that flourish in habitats of extreme temperature, pH, salinity, or pressure. All extreme environments are dominated by microorganisms belonging to Archaea, the third domain of life, evolutionary distinct from Bacteria and Eucarya. Over the past few years the biology of extremophilic Archaea has stimulated a lot of interest, aimed at understanding at molecular level the adaptation to their life conditions, as well as their evolutionary relationships to other organisms. Here, we review recent insights in the molecular biology of thermoacidophilic Archaea of the genus Sulfolobus, which has been used as a model system for biochemical, structural, and genetic studies in Archaea and extremophiles in general. With the recent completion of the genome sequence of Sulfolobus solfataricus it is expected that these organisms will contribute new discoveries in the near future. This revised version was published online in August 2006 with corrections to the Cover Date.     

Signal peptides of secreted proteins of the archaeon Sulfolobus solfataricus: a genomic survey

Analysis of the recently completed genome sequence of the thermoacidophilic archaeon Sulfolobus solfataricus reveals that about 4.2% of its proteome consists of putative secretory proteins with signal peptides. This includes members of the four major classes of signal peptides: secretory signal peptides, twin-arginine signal peptides, possible lipoprotein precursors, and type IV pilin signal peptides. The latter group is surprisingly large compared to the size of the groups in other organisms and seems to be used predominately for a subset of extracellular substrate-binding proteins.     

Distribution of spermine in bacilli and lactic acid bacteria

Obligate moderately thermophilic bacilli and obligate moderately thermoacidophilic bacilli contained spermine as the major polyamine in addition to putrescine and spermidine. The identity of spermine was confirmed by thin-layer chromatography and high-performance liquid chromatography before and after treatment with putrescine oxidase. Using these methods, thermospermine and spermine can be separated; thermospermine was not present in these organisms. On the other hand, various facultative thermophiles and mesophilic strains of the genus Bacillus, including alkalophiles and halophiles, lack spermine and other tetraamines. No spermine was detected in several strains of mesophilic or facultative slightly thermophilic lactic acid bacteria, Lactobacillus and Streptococcus.     

Archaebacterial ATPase: studies on subunit composition and quaternary structure of the F1-analogous ATPase from Sulfolobus acidocaldarius

A modified procedure for the purification of soluble ATPase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius is described. In addition to (alpha) 65 and (beta) 51 kDa polypeptides, further subunits gamma * (20 kDa) and delta * (12 kDa) are demonstrated to be components of the enzyme, exhibiting a total molecular mass of 380 kDa. Molecular electron microscopic images of the native enzyme indicate a quaternary structure probably formed by the gamma *, delta *-complex as a central mass surrounded by a pseudohexagon of the peripherally arranged larger alpha and beta subunits. As can be derived from both molecular mass and electron microscopy data, the archaebacterial Sulfolobus-ATPase emerges to exist as an alpha 3 beta 3-quaternary structure with respect to the larger subunits. This is normally found in typical F1-ATPases of eubacterial and eukaryotic organisms. Therefore it is postulated that F1- and F0F1-ATPases, respectively, can occur ubiquitously in all urkingdoms of organisms as functional units of energy-transducing membranes.     

Aminoacylation in Sulfolobus acidocaldarius and in methanogenic and halophilic archaebacteria

Abstract Phenylalanyl-tRNA synthetase (PRS) from the sulphur-metabolizing thermoacidophilic archaebacterium Sulfolobus acidocaldarius has been purified 150-fold using different chromatographic steps. The enzyme has a M _r of 270 000 and exhibits considerable thermostability in a temperature range up to 90°C with optimal activity at 70°C. Conservation of antigenic determinants could not be detected by antibodies against various PRS of all primary kingdoms. As a further means to detect traits of phylogenetic relationship, the cross-species reactivity between PRS and tRNAs of organisms from the three branches of archaebacteria and from all primary kingdoms reveals the group character of all 3 branches of the archaebacterial domain, the sulphur-metabolizing, methanogenic and halophilic archaebacteria.     

Purification and properties of NADH dehydrogenase from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

An NADH dehydrogenase was purified to electrophoretical homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75 degrees C. A 2,100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95,000, consisting of two 50,000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2,6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.     

Insensitivity of archaebacterial ribosomes to protein synthesis inhibitors. Evolutionary implications.

The effect on Sulfolobus solfataricus (an extremely thermoacidophilic archaebacterium) of selected inhibitors affecting reactions of the polypeptide elongation cycle has been tested by using poly(U) and poly(UG) directed cell-free systems. The results reveal a unique pattern of antibiotic sensitivity of Sulfolobus ribosomes with an inhibitory effect observed for only three of 60 compounds tested. Through comparison with suitable eubacterial and eukaryotic cell-free systems the insensitivity of Sulfolobus ribosomes to most inhibitors of protein synthesis appears to reflect a phylogenetic distinction of ribosome structure, rather than the high temperature conditions of the Sulfolobus assay system. In this respect ribosomes of thermoacidophilic archaebacteria differ not only from their eubacterial and eukaryotic counterparts, but also from ribosomes of archaebacteria belonging to the methanogenic-halophilic branch of the 'third' kingdom. The evolutionary implications of these findings are discussed.     

Continuous culture as a tool for investigating the growth physiology of heterotrophic hyperthermophiles and extreme thermoacidophiles

Although there is great scientific and technological interest in examining the physiology and bioenergetics of microorganisms from extreme environments, difficulties encountered in their cultivation and lack of genetic systems hampers the investigation of these issues. As such, we have adapted methods for continuous cultivation of mesophilic organisms to extremes of temperature and pH to study extremophiles. Since the risk for contamination of extremophilic continuous cultures is relatively small, long-term, steady state experiments investigating physiological response to culture perturbations are possible. Experiments along these lines have provided insights into the significance of specific enzymes in the metabolism of particular substrates, in addition to providing a better understanding of stress response and unusual physiological characteristics of hyperthermophilic and extremely thermoacidophilic microorganisms. Several examples are provided here, including the thermal stress response of Metallosphaera sedula (T(opt) 74 °C) growing at pH 2.0, and the response of the heterotrophic hyperthermophiles Pyrococcus furiosus (T(opt) 98 °C), Thermococcus litoralis (T(opt) 88 °C) and T. maritima (T(opt) 80 °C) to changes in growth medium. Also discussed will be how the same experimental systems have been used to study exopolysaccharide production and biofilm formation by hyperthermophilic heterotrophs and facilitated the estimation of bioenergetic parameters for these organisms under a variety of growth conditions. Continuous culture, used in conjunction with genome sequence information, two-dimensional gel electrophoresis and differential gene expression, can provide important insights into the metabolism of high temperature extremophiles.     

Uracil-DNA glycosylase of Thermoplasma acidophilum directs long-patch base excision repair, which is promoted by deoxynucleoside triphosphates and ATP/ADP, into short-patch repair

Hydrolytic deamination of cytosine to uracil in DNA is increased in organisms adapted to high temperatures. Hitherto, the uracil base excision repair (BER) pathway has only been described in two archaeons, the crenarchaeon Pyrobaculum aerophilum and the euryarchaeon Archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. In the former the apurinic/apyrimidinic (AP) site intermediate is removed by the sequential action of a 5'-acting AP endonuclease and a 5'-deoxyribose phosphate lyase, whereas in the latter the AP site is primarily removed by a 3'-acting AP lyase, followed by a 3'-phosphodiesterase. We describe here uracil BER by a cell extract of the thermoacidophilic euryarchaeon Thermoplasma acidophilum, which prefers a similar short-patch repair mode as A. fulgidus. Importantly, T. acidophilumcell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ～15 nucleotides. Supplementation of recombinant uracil-DNA glycosylase (rTaUDG; ORF Ta0477) increased the formation of short-patch at the expense of long-patch repair intermediates, and additional supplementation of recombinant DNA ligase (rTalig; Ta1148) greatly enhanced repair product formation. TaUDG seems to recruit AP-incising and -excising functions to prepare for rapid single-nucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER.     

Inhibitory effects of high pressure and heat on Alicyclobacillus acidoterrestris spores in apple juice

The effectiveness of combined high pressure and heat treatment for reducing spore levels of Alicyclobacillus acidoterrestris, a thermoacidophilic spore-forming bacterium, in commercial pasteurized apple juice was investigated. Spores suspended in apple juice were successfully destroyed by combining high pressure with a mild or high temperature (45, 71, or 90 degrees C).     

Membrane-bound ATPase of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius

The membranes of Sulfolobus, a thermoacidophilic archaebacterium showed two types of ATP hydrolyzing activity. One was that of a neutral ATPase at an optimum pH around 6.5. This enzyme was activated by 10 mM sulfate with a shift of optimum pH to 5. In these respects, the enzyme was similar to membrane-bound ATPase of Thermoplasma, another thermoacidophilic archaebacterium, reported by Searcy and Whatley [1982) Zbl. Bakt. Hyg., I. Abt. Orig. C3, 245-257). The enzyme hydrolyzed ATP and other NTPs, but not ADP or AMP. It was highly thermostable, but irreversibly inactivated in 0.1 M HCl. The other activity was that of an acidic apyrase at an optimum pH around 2.5. This enzyme was extremely stable toward high temperature and acid and inhibited by sulfate. Both of these ATP hydrolyzing enzymes were resistant to N,N'-dicyclohexylcarbodiimide (DCCD), azide, oligomycin, N'-ethylmaleimide, p-chloromercuribenzoate, orthovanadate, or ouabain. Sulfolobus ATPases differ from F1 and other transport ATPases so far described.     

A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.     

Archaebacterial elongation factor Tu insensitive to pulvomycin and kirromycin

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.     

Total reconstitution of active large ribosomal subunits of the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The large ribosomal subunit of the extremely thermoacidophilic archaebacterium Sulfolobus solfataricus has been reconstituted from the completely dissociated RNA and proteins by a two-step incubation procedure at high temperatures. Successful reconstitution requires a preliminary incubation of the ribosomal components for 45 min at 65 degrees C, followed by a second heat-treatment at 80 degrees C for 60 min. Structural reassembly depends upon high concentrations of K+ (300-400 mM) and Mg2+ (20-40 mM) ions. In addition, complete recovery of subunit function stringently requires the presence of a polyamine, thermine (or spermine). The reconstituted archaebacterial subunits are essentially indistinguishable from the native ones by a number of structural and functional criteria.