Characterization of the DNA gyrase from the thermoacidophilic archaeon Thermoplasma acidophilum

Thermoplasma acidophilum is sensitive to the antibiotic drug novobiocin, which inhibits DNA gyrase. We characterized DNA gyrases from T. acidophilum strains in vitro. The DNA gyrase from a novobiocin-resistant strain and an engineered mutant were less sensitive to novobiocin. The novobiocin-resistant gyrase genes might serve as T. acidophilum genetic markers.     

Flagellation and swimming motility of Thermoplasma acidophilum.

Electron microscopy of thin sections of Thermoplasma acidophilum confirmed previous observations of the absence of a typical cell wall in this organism. Negatively stained specimens revealed the almost consistent occurrence in both strains examined of monotrichously arranged flagella, about 9 micrometer long, which describe a sinuous curve with a wavelength of 1.5 to 2.0 micrometer and an amplitude of 0.33 to 0.59 micrometer. Motility of T. acidophilum could be demonstrated microscopically by microcinematography and macroscopically. The theoretical implications of the demonstration of functioning flagella in a wall-defective organism are discussed in the light of current theories of the mechanism of flagellar motility and from a taxonomic point of view.     

Phylogenetic affinities between eukaryotic cells and a thermophilic mycoplasma

Thermoplasma acidophilum, a thermophilic mycoplasma, has several unusual features suggesting a possible relationship to eukaryotic cells. One feature is a histone-like protein that is associated with the DNA, condensing it into subunits similar to those in eukaryotic chromatin. A second feature is an association of cytoplasmic proteins that resembles eukaryotic actin and myosin. These two components are widely distributed in different groups of eukaryotic cells, but are typically lacking in prokaryotic cells. Furthermore, T. acidophilum lacks cytochromes and respires by enzymes that apparently are not coupled to oxidative phosphorylation. This primitive type of respiration resembles that of microbodies, another feature which is represented in the cytoplasm of all groups of eukaryotic cells. Furthermore, since T. acidophilum lacks a cell wall and appears to have a primitive correlate of endocytosis, it would appear to be mechanically capable of acquiring a symbiotic mitochondrion. Thus, our observations are consistent with the symbiotic hypothesis for the origin of eukaryotic cells. We suggest that an organism similar to T. acidophilum was the host cell for the original symbiosis, becoming the nucleus and cytoplasm of modern eukaryotic cells.     

Structure of thermoplasmaquinone from Thermoplasma acidophilum

Abstract The structure of the methyl-substituted menaquinone (designated thermoplasmaquinone) from the extremely thermophilic and acidophilic archaebacterium Thermoplasma acidophilum was investigated by mass spectrometry and proton nuclear magnetic resonance spectrometry. The results of the present study show that the novel quinone from T. acidophilum corresponds to 2,[5 or 8]-dimethyl-3-heptaprenyl-1,4-naphthoquinone.     

Structure of a putative lipoate protein ligase from Thermoplasma acidophilum and the mechanism of target selection for post-translational modification

Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in Escherichia coli. The recombinant protein, a monomer of molecular mass 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 A resolution. The protein was found to fall into the alpha/beta class and to be structurally homologous to the catalytic domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA. Lipoic acid in LplA was bound in the same position as biotin in BirA. The structure of the T.acidophilum LplA and limited proteolysis of E.coli LplA together highlighted some key features of the post-translational modification. A loop comprising residues 71-79 in the T.acidophilum ligase is proposed as interacting with the dithiolane ring of lipoic acid and discriminating against the entry of biotin. A second loop comprising residues 179-193 was disordered in the T.acidophilum structure; tryptic cleavage of the corresponding loop in the E.coli LplA under non-denaturing conditions rendered the enzyme catalytically inactive, emphasizing its importance. The putative LplA of T.acidophilum lacks a C-terminal domain found in its counterparts in E.coli (Gram-negative) or Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears to have structural homology to the additional domain in the E.coli and S.pneumoniae enzymes was detected alongside the structural gene encoding the putative LplA in the T.acidophilum genome. It is likely that this protein is required to confer activity on the LplA as currently purified, one protein perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and the other transferring the lipoyl group from it to the specific lysine residue in the target protein.     

Characterization of the precursor of tetraether lipid biosynthesis in the thermoacidophilic archaeon<Emphasis Type="Italic"> Thermoplasma acidophilum</Emphasis>

Polar lipid biosynthesis in the thermoacidophilic archaeon Thermoplasma acidophilum was analyzed using terbinafine, an inhibitor of tetraether lipid biosynthesis. Cells of T. acidophilum were labeled with [(14)C]mevalonic acid, and their lipids were extracted and analyzed by two-dimensional thin-layer chromatography. Lipids labeled with [(14)C]mevalonic acid, [(14)C]glycerol, and [(32)P]orthophosphoric acid were extracted and hydrolyzed under different conditions to determine the structure of polar lipids. The polar lipids were estimated to be archaetidylglycerol, glycerophosphatidylcaldarchaetidylglycerol, caldarchaetidylglycerol, and beta- l-gulopyranosylcaldarchaetidylglycerol, the main polar lipid of T. acidophilum. Pulse and chase experiments with terbinafine revealed that one tetraether lipid molecule is synthesized by head-to-head condensation of two molecules of archaetidylglycerol and that a sugar group of tetraether phosphoglycolipid is expected to attach to the tetraether lipid core after head-to-head condensation in T. acidophilum. A precursor accumulated in the presence of terbinafine with a fast-atom-bombardment mass spectrometry peak m/z 806 was compatible with archaetidylglycerol. The relative height of the peak m/z 806 decreased after removal of the inhibitor. The results suggest that most of the precursor, archaetidylglycerol, is in fully saturated form.     

嗜酸异养菌对自养菌Acidithiobacillus ferrooxidans金属离子抗性和生物浸出的影响

【目的】了解嗜酸异养菌在诸如酸性矿坑水(AMD)和生物浸出体系等极端酸性环境中对浸矿微生物产生的影响。【方法】研究由嗜酸异养菌Acidiphilium acidophilum和自养菌Acidithiobacillus ferrooxidans经长期驯化后形成的共培养体系分别在Cd2+、Cu2+、Ni2+和Mg2+胁迫下的稳定性;并将此共培养体系应用于黄铁矿和低品位黄铜矿的生物浸出实验。【结果】在上述4种金属离子分别存在的条件下,异养菌Aph.acidophilum均能促进At.ferrooxidans对亚铁的氧化,提高其对能源利用的效率。共培养体系中的异养菌Aph.acidophilum使At.ferrooxidans对Cu2+的最大耐受浓度(MTC)由2.0 g/L提高到5.0 g/L,而且共培养的细胞数量与2.0 g/L Cu2+条件下生长的At.ferrooxidans纯培养相似。另外,共培养中的At.ferrooxidans对Mg2+的MTC也由12.0 g/L提高到17.0 g/L。生物浸出实验中嗜酸异养菌Aph.acidophilum促进了At.ferrooxidans对黄铁矿样品的浸出,浸出率较其纯培养提高了22.7%;但在含铁量较低的低品位黄铜矿浸出体系中共培养和At.ferrooxidans纯培养的浸出率均低于33%。在加入2.0 g/L Fe2+的低品位黄铜矿浸出体系中,共培养和At.ferrooxidans纯培养的浸出率均得到提高,分别达到52.22%和41.27%。【结论】以上结果表明,Aph.acidophilum与At.ferrooxidans共培养在一定的环境胁迫下仍能保持其稳定性并完成各自的生态功能,并且嗜酸异养菌Aph.acidophilum适合在含铁量较高的浸出体系中与铁氧化细菌共同作用来提高生物浸出的效率。     

Ultrastructure of lipopolysaccharide isolated from Thermoplasma acidophilum.

The fine structure of lipopolysaccharide isolated from Thermoplasma acidophilum was examined by electron microscopy. Negative staining of the lipopolysaccharide revealed long, ribbon-like structures with some branching. The average width of the lipopolysaccharide ribbons was 5 nm. Treatment of the lipopolysaccharide with 0.5% sodium dodecyl sulfate resulted in the dissociation of the ribbon-like structures to spherical- and vesicular-shaped particles and some short, rodlike structures. Results suggest that the lipopolysaccharide from T. acidophilum is morphologically similar to lipopolysaccharide isolated from gram-negative bacteria.     

Effects of pH and temperature on the composition of polar lipids in Thermoplasma acidophilum HO-62

Thermoplasma acidophilum HO-62 was grown at different pHs and temperatures, and its polar lipid compositions were determined. Although the number of cyclopentane rings in the caldarchaeol moiety increased when T. acidophilum was cultured at high temperature, the number decreased at low pHs. Glycolipids, phosphoglycolipids, and phospholipids were analyzed by high-performance liquid chromatography with an evaporative light-scattering detector. The amount of caldarchaeol with more than two sugar units on one side increased under low-pH and high-temperature conditions. The amounts of glycolipids increased and those of phosphoglycolipids decreased under these conditions. The proton permeability of the liposomes obtained from the phosphoglycolipids that contained two or more sugar units was lower than that of the liposomes obtained from the phosphoglycolipids that contained one sugar unit. From these results, we propose the hypothesis that T. acidophilum adapts to low pHs and high temperatures by extending sugar chains on their cell surfaces, as well as by varying the number of cyclopentane rings.     

Biosynthesis of archaeal membrane lipids: digeranylgeranylglycerophospholipid reductase of the thermoacidophilic archaeon Thermoplasma acidophilum

The basic core structure of archaeal membrane lipids is 2,3-di-O-phytanyl-sn-glyceryl phosphate (archaetidic acid), which is formed by the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate. The reductase activity for the key enzyme in membrane lipid biosynthesis, 2,3-digeranylgeranylglycerophospholipid reductase, was detected in a cell free extract of the thermoacidophilic archaeon Thermoplasma acidophilum. The reduction activity was found in the membrane fraction, and FAD and NADH were required for the activity. The reductase was purified from a cell free extract by ultracentrifugation and four chromatographic steps. The purified enzyme showed a single band at ca. 45 kDa on SDS-PAGE, and catalyzed the formation of archaetidic acid from 2,3-di-O-geranylgeranylglyceryl phosphate. Furthermore, the enzyme also catalyzed the reduction of 2,3-di-O-geranylgeranylglyceryl phosphate analogues such as 2,3-di-O-phytyl-sn-glyceryl phosphate, 3-O-(2,3-di-O-phytyl-sn-glycero-phospho)-sn-glycerol and 2,3-di-O-phytyl-sn-glycero-phosphoethanolamine. The N-terminal 20 amino acid sequence of the purified enzyme was determined and was found to be identical to the sequence encoded by the Ta0516m gene of the T. acidophilum genome. The present study clearly demonstrates that 2,3-digeranylgeranylglycerophospholipid reductase is a membrane associated protein and that the hydrogenation of each double bond of 2,3-digeranylgeranylglycerophospholipids is catalyzed by a single enzyme.     

Crystal structure of an archaeal actin homolog

Prokaryotic homologs of the eukaryotic structural protein actin, such as MreB and ParM, have been implicated in determination of bacterial cell shape, and in the segregation of genomic and plasmid DNA. In contrast to these bacterial actin homologs, little is known about the archaeal counterparts. As a first step, we expressed a predicted actin homolog of the thermophilic archaeon Thermoplasma acidophilum, Ta0583, and determined its crystal structure at 2.1A resolution. Ta0583 is expressed as a soluble protein in T.acidophilum and is an active ATPase at physiological temperature. In vitro, Ta0583 forms sheets with spacings resembling the crystal lattice, indicating an inherent propensity to form filamentous structures. The fold of Ta0583 contains the core structure of actin and clearly belongs to the actin/Hsp70 superfamily of ATPases. Ta0583 is approximately equidistant from actin and MreB on the structural level, and combines features from both eubacterial actin homologs, MreB and ParM. The structure of Ta0583 co-crystallized with ADP indicates that the nucleotide binds at the interface between the subdomains of Ta0583 in a manner similar to that of actin. However, the conformation of the nucleotide observed in complex with Ta0583 clearly differs from that in complex with actin, but closely resembles the conformation of ParM-bound nucleotide. On the basis of sequence and structural homology, we suggest that Ta0583 derives from a ParM-like actin homolog that was once encoded by a plasmid and was transferred into a common ancestor of Thermoplasma and Ferroplasma. Intriguingly, both genera are characterized by the lack of a cell wall, and therefore Ta0583 could have a function in cellular organization.     

Reduction of ferric iron by acidophilic heterotrophic bacteria: evidence for constitutive and inducible enzyme systems in Acidiphilium spp

AIMS: To compare the abilities of two obligately acidophilic heterotrophic bacteria, Acidiphilium acidophilum and Acidiphilium SJH, to reduce ferric iron to ferrous when grown under different culture conditions. METHODS AND RESULTS: Bacteria were grown in batch culture, under different aeration status, and in the presence of either ferrous or ferric iron. The specific rates of ferric iron reduction by fermenter-grown Acidiphilium SJH were unaffected by dissolved oxygen (DO) concentrations, while iron reduction by A. acidophilum was highly dependent on DO concentrations in the growth media. The ionic form of iron present (ferrous or ferric) had a minimal effect on the abilities of harvested cells to reduce ferric iron. Whole cell protein profiles of Acidiphilium SJH were very similar, regardless of the DO status of the growth medium, while additional proteins were present in A. acidophilum grown microaerobically compared with aerobically-grown cells. CONCLUSIONS: The dissimilatory reduction of ferric iron is constitutive in Acidiphilium SJH while it is inducible in A. acidophilum. SIGNIFICANCE AND IMPACT OF THE STUDY: Ferric iron reduction by Acidiphilium spp. may occur in oxygen-containing as well as anoxic acidic environments. This will detract from the effectiveness of bioremediation systems where removal of iron from polluted waters is mediated via oxidation and precipitation of the metal.     

Identification and characterization of Thermoplasma acidophilum glyceraldehyde dehydrogenase: a new class of NADP+-specific aldehyde dehydrogenase

Thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus solfataricus are known to metabolize D-glucose via the nED (non-phosphorylated Entner-Doudoroff) pathway. In the present study, we identified and characterized a glyceraldehyde dehydrogenase involved in the downstream portion of the nED pathway. This glyceraldehyde dehydrogenase was purified from T. acidophilum cell extracts by sequential chromatography on DEAE-Sepharose, Q-Sepharose, Phenyl-Sepharose and Affi-Gel Blue columns. SDS/PAGE of the purified enzyme showed a molecular mass of approx. 53 kDa, whereas the molecular mass of the native protein was 215 kDa, indicating that glyceraldehyde dehydrogenase is a tetrameric protein. By MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight MS) peptide fingerprinting of the purified protein, it was found that the gene product of Ta0809 in the T. acidophilum genome database corresponds to the purified glyceraldehyde dehydrogenase. The native enzyme showed the highest activity towards glyceraldehyde, but no activity towards aliphatic or aromatic aldehydes, and no activity when NAD+ was substituted for NADP+. Analysis of the amino acid sequence and enzyme inhibition studies indicated that this glyceraldehyde dehydrogenase belongs to the ALDH (aldehyde dehydrogenase) superfamily. BLAST searches showed that homologues of the Ta0809 protein are not present in the Sulfolobus genome. Possible differences between T. acidophilum (Euryarchaeota) and S. solfataricus (Crenarchaeaota) in terms of the glycolytic pathway are thus expected.     

Thermoplasma acidophilum: intracellular pH and potassium concentration.

Thermoplasma acidophilum is a free-living thermophilic mycoplasma. Although the organism lacks a cell wall, it can grow in medium as dilute as 66 mosM. The intracellular K+ concentration can be as low as 17 mM, but varies according to the osmolality of the culture medium. The internal pH can be measured by taking advantage of the fact that T. acidophilum undergoes lysis when the pH is adjusted to neutrality. Thus, by appropriate analysis of titration curves, it is possible to conclude that the internal pH is near 5.5. This result was confirmed by a second type of experiment in which the internal pH was analyzed by rupturing the cells in a French Pressure Cell.     

Stereochemistry of reduction in digeranylgeranylglycerophospholipid reductase involved in the biosynthesis of archaeal membrane lipids from Thermoplasma acidophilum

The basic core structure of archaeal membrane lipids is 2,3-di-O-phytanylglyceryl phosphate, which is formed by reduction of 2,3-di-O-geranylgeranylglyceryl phosphate. This reaction is the final committed step in the biosynthesis of archaeal membrane lipids and is catalyzed by digeranylgeranylglycerophospholipid reductase (DGGGPL reductase). The putative DGGGPL reductase gene (Ta0516m) of Thermoplasma acidophilum was cloned and expressed. The purified recombinant enzyme appeared to catalyze the formation of 2,3-di-O-phytanylglyceryl phosphate from 2,3-di-O-geranylgeranylglyceryl phosphate, which confirmed that the Ta0516m gene of T. acidophilum encodes DGGGPL reductase. The stereospecificity in reduction of 2,3-di-O-phytylglyceryl phosphate by the recombinant reductase appeared to take place through addition of hydrogen in a syn manner by analyzing the enzyme reaction product by NMR spectroscopy.     

Differential features of ribosomes and of poly(U)-programmed cell-free systems derived from sulphur-dependent archaebacterial species

The properties of poly(U)-directed cell-free systems developed from the sulphur-dependent, thermophilic archaebacteria Desulfurococcus mobilis, Thermoproteus tenax, Sulfolobus solfataricus, Thermococcus celer and Thermoplasma acidophilum have been compared. All systems are truly thermophilic in requiring incubation at temperatures close to the physiological optimum for cell growth. Under optimized conditions the error frequency in tRNA selection is less than 0.4% at 80 degrees C, and synthetic efficiencies (Phe residues polymerized per ribosome in 40 min) span from 4 for Tp. tenax, to 10 for Tc. celer, to 20-25 for D. mobilis and T. acidophilum and to 40 for S. solfataricus. According to requirements for polypeptide synthesis and to degree of stability of the ribosomal subunits' association, sulphur-dependent thermophiles cluster into two groups. Group I organisms (D. mobilis, Tp. tenax, S. solfataricus) harbour 70-S monomers composed of weakly associated subunits, whose poly(Phe)-synthesizing capacity is totally dependent on added spermine while being drastically inhibited by monovalent cations. Group II organisms (Tc. celer and T. acidophilum) contain 70-S particles composed of tightly bonded subunits, whose synthetic capacity is independent of spermine while being totally dependent on monovalent cations. Spermine promotes poly(Phe) synthesis on ribosomes of group I organisms by converting the peptidyltransferase center into an active conformation, while monovalent cations are inhibitory by preventing the interaction between the free ribosomal subunits. The closeness between Tc. celer and T. acidophilum ribosomes provides new insight on the phylogenetic placement of Thermococcaceae.     

The nucleotide sequence of the 5S rRNA from the archaebacterium Thermoplasma acidophilum.

The complete nucleotide sequence of the 5S ribosomal RNA isolated from the archaebacterium Thermoplasma acidophilum has been determined. The sequence is: pG GCAACGGUCAUAGCAGCAGGGAAACACCAGAUCCCAUUCCGAACUCGACGGUUAAGCCUGCUGCGUAUUGCGUUGUACU GUAUGCCGCGAGGGUACGGGAAGCGCAAUAUGCUGUUACCAC(U)OH. The homology with the 55 rRNA from another archaebacterial species, Halobacterium cutirubrum, is only 60.6% and other 55 rRNAs are even less homologous. Examination of the potential for forming secondary structure is revealing. T. acidophilum does not conform to the usual models employed for either procaryotic or eucaryotic 5S rRNAs. Instead this 5S rRNA has a mixture of the characteristic features of each. On the whole this 5S rRNA does however appear more eucaryotic than eubacterial. These results give further support to the notion that the archaebacteria represent an extremely early divergence among entities with procaryotic organization.     

Expression of functional Thermoplasma acidophilum proteasomes in Escherichia coli.

The two genes encoding the constituent subunits of the Thermoplasma acidophilum proteasome were expressed in Escherichia coli yielding fully assembled molecules as shown by electron microscopy. The recombinant proteasomes were purified to homogeneity and were shown to have proteolytic activity indistinguishable from proteasomes isolated from T. acidophilum.     

The plasmids found in isolates of the acidothermophilic archaebacterium Thermoplasma acidophilum

Abstract Plasmids were detected in isolates of an acidothermophilic archaebacterium Thermoplasma acidophilum . One of the plasmids, pTA1, was characterized. The plasmid was a circular DNA of 15.2 kbp. A physical map was constructed using three restriction endonucleases. A copy number of this plasmid was estimated to be 7–13 per cell. The homologous sequence was not found in the chromosomal DNA of the host cell.