Interactions of thiophenes and acidophilic,thermophilic bacteria

The growth and oxygen consumption of a variety of thermophilic, acidophilic bacteria in the presence of thiophene-2-carboxylate (T2C) and dibenzothiophene (DBT) have been determined. T2C was extremely toxic to the acidophiles in comparison with neutrophiles, but appeared to be degraded by a heterotrophicSulfolobus- like thermophile. DBT proved to be unstable at high temperatures, even in the absence of bacteria, and was not a substrate for the thermophiles.     

Expression and Characterization of a Thermostable Acyl-peptide Releasing Enzyme ST0779 from Sulfolobus tokodaii

Acyl-peptide releasing enzyme(AARE) belongs to a serine peptidase family and catalyzes the NH₂-terminal hydrolysis of N^α-acylpeptides to release N^α-acylated amino acids. ORF0779(ORF=open reading frame) from thermophilic archaea Sulfolobus tokodaii(ST0779) was cloned and expressed in E. coli BL21 and the expressed protein was identified as a thermostable AARE. The target protein could be optimally overexpressed in E. coli at 30 ℃ for 8 h with 0.1 mmol/L isopropyl β-dthiogalactoside(IPTG). The crude enzyme was heated at 70 ℃ for 30 min, and then the target protein could account for above 40% of the total protein. The purification fold was 27 and the enzyme showed both esterase activity and peptidase activity. The optimal temperature and pH for ST0779 were 70 ℃ and 8.0 when Ac-Ala₃ was used as substrate. The half-life of the enzyme(0.2 mg/mL) at 90 ℃ was about 16 h, indicating that the enzyme exhibits a favorable thermostability. The activity of ST0779 could still remain over 85% after being treated at 25 ℃ in different buffers with pH range from 6.0 to10.0 for 24 h, which indicates ST0779 is stable in neutral or slight alkali environment. Under neutral or slightly alkali conditions, the enzyme exhibits really high catalytic efficiency against acyl-peptide, and the optimal substrate is Ac-Ala₃. Most metal ions have no inhibition effect on the activity of ST0779, while 4% activity of ST0779 is inhibited in the presence of K⁺. This enzyme was supposed to be applied in the analysis of protein sequencing and the synthesis of small peptides.     

Discontinuous reversed-phase high performance liquid chromatography increases load capacity of analytical columns. Separation of ribosomal proteins from the archaebacteriumSulfolobus acidocaldarius

Summary Since ribosomes are a fundamental feature component of all organisms, they present a good model for studying evolutionary diversity. We investigated the ribosomal proteins of the archaebacteriumSulfolobus acidocaldarius, which contains slightly more ribosomal proteins thanEscherichia coli. While the ribosomal proteins of most organisms contain a high proportion of lysine and arginine residues, these basic amino acids are particularly prevelent in the thermaacidophile organismSulfolobus, a possible reason for poor separations of total ribosomal proteins ofS. acidocaldarius by single column HPLC. To solve this complex separation problem, we developed the discontinuous reversed-phase HPLC (Disc RPC) method. Discontinuus chromatography combines at least two different stationary phases in a sequence of increasing retention times for the elution of proteins. Unlike other multi-column techniques, all of the injected sample passes through this discontinous stationary phase, which is used in place of single columns, thus permitting separations to be carried out without the need any changes in the HPLC system. Here we present Disc RPC separations of 30S ribosomal proteins fromS. acidocaldarius with a variety of stationary phases of different legnths and diameters. Each of five Disc RPC categories presented in this paper has some special application possibilities when used in protein HPLC.     

Biochemical Characterization of Translesion Synthesis by Sulfolobus acidocaldarius DNA Polymerases

To study the DNA synthesis mechanism of Sulfolobus acidocaldarius, a thermophilic species from Crenarchaeota, two DNA polymerases of B family(polB1 and polB3), and one DNA polymerase of Y family(polIV) were recombinantly expressed, purified and biochemically characterized. Both DNA polymerases polB1(Saci_1537) and polB3(Saci_0074) possessed DNA polymerase and 3' to 5' exonuclease activities; however, both the activities of B3 were very inefficient in vitro. The polIV(Saci_0554) was a polymerase, not an exonuclease. The activities of all the three DNA polymerases were dependent on divalent metal ions Mn²⁺ and Mg²⁺. They showed the highest activity at pH values ranging from 8.0 to 9.5. Their activities were inhibited by KCl with high concentration. The optimal reaction temperatures for the three DNA polymerases were between 60 and 70℃. Deaminated bases dU and dI on DNA template strongly hindered primer extension by the two DNA polymerases of B family, not by the DNA polymerase of Y family. DNA polymerase of Y Family bypassed the two AP site analogues dSpacer and propane on template more easily than DNA polymerases of B family. Our results suggest that the three DNA polymerases coordinate to fulfill various DNA synthesis in Sulfolobus acidocaldarius cell.     

A fast method for quantitative proteomics based on a combination between two-dimensional electrophoresis and 15N-metabolic labelling

We provide a method for accurate protein quantitation that uses two-dimensional (2-D) gel electrophoresis for protein separation, but does not require extensive statistical analysis of staining intensities on gels. Instead, accurate quantitation occurs on the mass spectrometer (MAS) on multiple peptides to provide statistical evidence. In an example study, Sulfolobus solfataricus cells were grown on the carbon sources glucose, fructose and glutamate. The glucose phenotype (reference) was grown on (15)N-enriched medium. Next, the glutamate and the fructose phenotypes are mixed with the reference and two 2-D gels are created. Staining intensities of gel spots in this case are used for initial, semiquantitative assessment of differential expression. On this basis, spots are selected for accurate quantitation on the MAS. A number of differentially expressed proteins were found, for example: a (25.2 +/- 8.2)-fold upregulation of isocitrate lyase and a (7.14 +/- 0.82)-fold downregulation of glucose dehydrogenase on glutamate compared to glucose. With this protocol, intergel and interlaboratory comparisons are facilitated, since the light and heavy versions of a protein are equally affected by variations in sample preparation and buffer composition. Because the statistical evidence is gathered on the MAS, the need to run vast numbers of gels is removed.     

Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high‐resolution shotgun lipidomics

The molecular structures, biosynthetic pathways and physiological functions of membrane lipids produced by organisms in the domain Archaea are poorly characterized as compared with that of counterparts in Bacteria and Eukaryota. Here we report on the use of high‐resolution shotgun lipidomics to characterize, for the first time, the lipid complement of the archaeon Sulfolobus islandicus. To support the identification of lipids in S. islandicus, we first compiled a database of ether lipid species previously ascribed to Archaea. Next, we analyzed the lipid complement of S. islandicus by high‐resolution Fourier transform mass spectrometry using an ion trap‐orbitrap mass spectrometer. This analysis identified five clusters of molecular ions that matched ether lipids in the database with sub‐ppm mass accuracy. To structurally characterize and validate the identities of the potential lipid species, we performed structural analysis using multistage activation on the ion trap‐orbitrap instrument as well as tandem mass analysis using a quadrupole time‐of‐flight machine. Our analysis identified four ether lipid species previously reported in Archaea, and one ether lipid species that had not been described before. This uncharacterized lipid species features two head group structures composed of a trisaccharide residue carrying an uncommon sulfono group (?SO₃) and an inositol phosphate group. Both head groups are linked to a glycerol dialkyl glycerol tetraether core structure having isoprenoid chains with a total of 80 carbon atoms and 4 cyclopentane moieties. The shotgun lipidomics approach deployed here defines a novel workflow for exploratory lipid profiling of Archaea. Copyright © 2015 John Wiley & Sons, Ltd.     

The use of a thermostable signature amidase in the resolution of the bicyclic synthon (rac)-γ-lactam

The resolution of the bicyclic synthon (rac)-γ-lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) is an important step in the synthesis of a group of chemotherapuetic agents known as carbocyclic nucleosides. The archaeon Sulfolobus solfataricus MT4 produces a thermostable γ-lactamase that has a high sequence homology to the signature amidase family of enzymes. It shows similar inhibition patterns of amidases towards benzonitrile, phenylmethylsulfonyl fluoride and heavy metals such as Hg²⁺, and is activated by thiol reagents. The enzyme selectively cleaves the (+)-enantiomer from a racemic mix of γ-lactam. It also exhibits general amidase activity by cleaving linear and branched aliphatic and aromatic amides. The enzyme catalyses the synthesis of benzoic hydrazide from benzamide preferentially to benzamide cleavage in the presence of excess hydrazine. This enzyme has potential for use in industrial biotransformations in the production of both carbocyclic nucleosides and hydrazides.     

Facile distinction of neutral and acidic tetraether lipids in archaea membrane by halogen atom adduct ions in electrospray ionization mass spectrometry

Calditocaldarchaeol (neutral tetraether lipid) from Sulfolobus acidocaldarius (acidothermophilic archaea) and intact total lipid from the thermoacidophilic archaea Sulfolobus sp. was examined by electrospray ionization time-of-flight mass spectrometry in the negative-ion mode using high resolution. When the sample was injected as a solution in a 3:1 mixture of methanol (MeOH) and chloroform (CHCl(3)) using an infusion system, the total ether lipid afforded molecular-related ions as [M - H](-) for acidic polar lipids containing a phosphoric or sulfuric group, and as [M + Cl](-) ion for neutral glycolipids. The attachment of chloride was confirmed by the observation of [M + Br](-) ion, instead of [M + Cl](-) ion, when a 3:1 mixture of MeOH and CHBr(3) was used in place of MeOH-CHCl(3) as the solvent. The composition of tetraether neutral glycolipids that are different from each other only in the number of five-membered rings in the isoprenoid chain was determined on the basis of the isotope-resolved mass spectrum of [M + Cl](-) ions. As for acidic tetraether lipids, molecular-related ions [M - H](-)) were not observed when the 3:1 MeOH-CHBr(3) mixture was used as the solvent. These results together afforded a facile method of distinguishing neutral from acidic tetraether lipids in intact total lipids of acidothermophilic archaea. This method was applied to determine the difference of the number of five-membered rings in isoprenyl chains of neutral tetraether glycolipids yielded by the Sulfolobus sp. grown at different temperatures. Discrimination of neutral tetraether glycolipids from acidic tetraether lipids in the total lipids obtained from Thermoplasma sp. was also achieved by this method.     

Separation of 50 S ribosomal proteins from sulfolobus acidocaldarius by discontinuous reversed-phase chromatography

Summary The total protein of the 50S ribosomal subunit (TP50) from the archaebacterium Sulfolobus acidocaldarius was prefractionated by discontinuous reversed-phase HPLC with several column combinations. The purity of the eluted fractions was tested by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional PAGE (2D-PAGE). With regard to the load capacity and selectivity, best results were obtained with a semi-preparative three-column combination (total length 67.5cm). Twelve A₂₃₀ units of TP50 were separated into 35 fractions, 19 of which contained nearly pure proteins. When the sample load was increased to 176A₂₃₀ units (109mg), 13 of 28 collected fractions still contained nearly pure proteins. The selectivity of a semi-preparative short-column combination (total length 12cm) was similar to that of the semi-preparative column combination, and separation time could be reduced to one third of that required for the longer column combination. The load capacity of the short-column combination was lower than that of the semi-preparative column combination.