Extreme Thermophilic Bacterium Thermus Thermophilus Research Articles

Integral caa3-Cytochrome c Oxidase from Thermus thermophilus: Purification and Crystallization.

Cytochrome c oxidase is a respiratory enzyme catalyzing the energy-conserving reduction of molecular oxygen to water-a fundamental biological process of cell respiration. The first crystal structures of the type A cytochrome c oxidases, bovine heart and Paracoccus denitrificans cytochrome c oxidases, were published in 1995 and contributed immensely to the understanding of the enzyme's mechanism of action. The senior author's research focus was directed toward understanding the structure and function of the type B cytochrome c oxidases, ba3-oxidase and type A2 caa3-oxidase, both from the extreme thermophilic bacterium Thermus thermophilus. While the ba3-oxidase structure was published in 2000 and functional characterization is well-documented in the literature, we recently successfully solved the structure of the caa3-nature made enzyme-substrate complex. This chapter is dedicated to the purification and crystallization process of caa3-cytochrome c oxidase.

Reconstitution of Functionally Active Thermus thermophilus 30S Ribosomal Subunit from Ribosomal 16S RNA and Ribosomal Proteins.

In vitro reconstitution systems of ribosomal subunits from free ribosomal RNA and ribosomal proteins are helpful tool for studies on the structure, function and assembly of ribosome. Using this system mutant or modified ribosomal proteins or ribosomal RNA can be incorporated into ribosomal subunits for studying ribosome structure and function. Developing the protocol for reconstitution of 30S subunits from an extreme thermophilic bacterium Thermus thermophilus can be beneficial especially for structural studies, as proteins and nucleic acids from this organism are very stable and crystallize easier than those from mesophilic organisms.

Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.

Thermus thermophilus encodes an archaeal-like fructose-1,6-bisphosphatase: Purification of native and recombinant protein for structural studies

Fructose-1,6-bisphosphatase is one of the key enzymes of the gluconeogenic pathway. It catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. Fructose-1,6-bisphosphatase from the extreme thermophilic bacterium Thermus thermophilus has been purified by crystallisation approach. The final well-shaped crystals have been obtained using vapour diffusion sitting-drops in the presence of PEG 400 as the precipitating agent. The initially obtained native twinned crystals diffracted up to 1.2 Å resolution. Untwinned crystals used for data collection, however, were grown in the presence of thiomersal. They diffract to 1.8 Å resolution and belong to the space groups I422 with cell dimensions (i) a = b = 108.8 Å, c = 336.3 Å showing two molecules in the asymmetric unit, and (ii) a = b = 113.7 Å, c = 151.0 Å with one molecule in the asymmetric unit. The crystal structure has been solved by single anomalous dispersion using a 1.9 Å resolution. For further biochemical and biophysical investigations recombinant fructose-1,6-bisphosphatase has been produced in Escherichia coli. Both native (dissolved crystals) and recombinant material have been characterised by SDS–PAGE, N-terminal sequencing and MALDI-MS.

Biochemical and regulatory properties of a respiratory island encoded by a conjugative plasmid in the extreme thermophile Thermus thermophilus

In the present paper, we summarize the current knowledge on the first step of the denitrification pathway in the ancestral extreme thermophilic bacterium Thermus thermophilus. In this organism, nitrate respiration is performed by a mobilizable respiratory island that encodes a new type of respiratory NADH dehydrogenase as electron donor, a tetrameric membrane nitrate reductase as final electron acceptor, two nitrate/nitrite transporters and the transcription factors required for their expression in response to nitrate and anoxia.

Hyperalkaline and thermostable phosphatase in Thermus thermophilus.

The phosphatases existing in the extreme thermophilic bacterium Thermus thermophilus have been studied. Utilizing ion exchange, hydrophobic, pseudoaffinity, and affinity chromatography, a number of distinct phosphatase activities were identified. At least four phosphatases, with optimum pH ranging between 5.0 and 11.5, were assayed with p-nitrophenylphosphate, and two with optimum pH between 7.0 and 11.0, with 32P-casein as substrate. The authors have focused on the hyperalkaline phosphatase and have tried to purify and characterize it. This hyperalkaline phosphatase reaches a maximal level at the stationary phase of the growth, and is co-purified with alkaline phosphatase with optimum pH of 10.2. The enzymes present a relative mol wt of 65 and 58 kDa, respectively, as judged by SDS-PAGE and Sephadex G-150 column, and possess similar properties, indicating that they are isoforms. These enzymes barely function in the presence of tartrate, and are inhibited by EDTA, pyrophosphate, and molybdate. Among the metals tested, Hg2+ appeared as the strongest inhibitor of the hyperalkaline phosphatase. The two enzymes are thermostable and, upon treatment at 90 degrees C for 10 min, 75% of their activity remains. The physiological role and function of these phosphatases need further investigation.

Genes and enzymes of the acetyl cycle of arginine biosynthesis in the extreme thermophilic bacterium Thermus thermophilus HB27.

An arginine biosynthetic gene cluster, argC-argJ, of the extreme thermophilic bacterium Thermus thermophilus HB27 was isolated by heterologous complementation of an Escherichia coli acetylornithinase mutant. The recombinant plasmid (pTHM1) conferred ornithine acetyltransferase activity to the E. coli host, implying that T. thermophilus uses the energetically more economic pathway for the deacetylation of acetylornithine. pTHM1 was, however, unable to complement an E. coli argA mutant and no acetylglutamate synthase activity could be detected in E. coli argA cells containing pTHM1. The T. thermophilus argJ-encoded enzyme is thus monofunctional and is unable to use acetyl-CoA to acetylate glutamate (contrary to the Bacillus stearothermophilus homologue). Alignment of several ornithine acetyltransferase amino acid sequences showed no obvious pattern that could account for this difference; however, the monofunctional enzymes proved to have shorter N-termini. Sequence analysis of the pTHM1 3.2 kb insert revealed the presence of the argC gene (encoding N-acetylglutamate-5-semialdehyde dehydrogenase) upstream of the argJ gene. Alignment of several N-acetylglutamate-5-semialdehyde dehydrogenase amino acid sequences allowed identification of two strongly conserved putative motifs for cofactor binding: a putative FAD-binding site and a motif reminiscent of the NADPH-binding fingerprint. The relationship between the amino acid content of both enzymes and thermostability is discussed and an effect of the GC content bias is indicated. Transcription of both the argC and argJ genes appeared to be vector-dependent. The argJ-encoded enzyme activity was twofold repressed by arginine in the native host and was inhibited by ornithine. Both upstream of the argC gene and downstream of the argJ gene an ORF with unknown function was found, indicating that the organization of the arginine biosynthetic genes in T. thermophilus is new.

Identification and purification of translation initiation factor 2 (IF2) from Thermus thermophilus.

Translation initiation factor 2 (IF2) is one of three protein factors required for initiation of protein synthesis in eubacteria. The protein is responsible for binding the initiator RNA to the ribosomal P site. IF2 is a member of the GTP GDP-binding protein superfamily. In the extreme thermophilic bacterium Thermus thermophilus, IF2 was identified as a 66-kDa protein by affinity labeling and immunoblotting. The protein was purified to homogeneity. The specific activity indicates a stoichiometric IF2-mediated binding of formylmethionine-tRNA to 70S ribosomes. The N-terminal amino acid sequences of the intact protein and of two proteolytic fragments of 25 kDa and 40 kDa were determined. Comparison with other bacterial IF2 sequences indicates a similar domain architecture in all bacterial IF2 proteins.

Overexpression and Purification of Thermus thermophilus Elongation Factors G, Tu, and Ts from Escherichia coli

The translation elongation factors G (EF-G), Tu (EF-Tu), and Ts (EF-Ts) from the extreme thermophilic bacterium Thermus thermophilus were overproduced in Escherichia coli. The fus gene coding for EF-G; and the tufA gene coding for EF-Tu were expressed under the control of a tac promoter, whereas EF-Ts was overproduced with the T7 RNA polymerase system. A detailed description for the purification of the three elongation factors from E. coli is presented, EF-G and EF-Tu are isolated by Q-Sepharose FF chromatography, heat treatment at 65 or 60°C, respectively, and Sephacryl S200 gel permeation chromatography. For the purification of EF-Ts, a heat denaturation step is followed by DEAF-cellulose chromatography and a cation exchange EMD-SO−3 650 column, The overproduced factors show the same properties as those purified from T. thermophilus. As the crystal structures of T. thermophilus EF-Tu and EF-Cf have been solved recently, many questions concerning the function of particular residues or domains arise, which may be best addressed by studying the in vitro behavior and structure of altered recombinant constructs, The methods presented here should facilitate such studies.

Phenylalanyl-tRNA synthetase from Thermus thermophilus can attach two molecules of phenylalanine to tRNA Phe

Phenylalanyl-tRNA synthetase from the extreme thermophilic bacterium Thermus thermophilus can incorporate more than one molecule of phenylalanine into the tRNA Phe. It is shown that the ‘hyperaminoncylated’ tRNAP Phe is the bis-2′,3′- O-phenylalanyl-tRNA Phe, and its formation is typical for the thermophilic enzyme but does not occur for E. coli phenylalanyl-tRNA synthetase under the same conditions.

Purification and crystallization of components of the protein-synthesizing system from Thermus thermophilus

An extreme thermophilic bacterium Thermus thermophilus has been chosen as a source for the isolation of components of the protein-synthesizing system to investigate their structures by X-ray crystallographic methods. The scheme of simultaneous isolation of ribosomes, tRNA, three elongation factors, several aminoacyl-tRNA synthetases and several enzymes has been developed. Methods of purification of ribosomes and individual ribosomal proteins without denaturation were elaborated. Crystals of the elongation factor G, the 70S ribosome, the 30S ribosomal subunit, six ribosomal proteins and three aminoacyl-tRNA synthetases have been obtained. Structural investigations of EF-G and the 70S ribosome are underway.

The primary structure of DNA binding protein II from the extreme thermophilic bacterium Thermus thermophilus

The primary structure of DNA binding protein II (DNA bp II) from the extreme thermophilic bacterium Thermus thermophilus has been established by combination of manual and automated techniques. The protein has 95 residues and a molecular mass of 11843. Comparison of the primary structure with the known sequence data of DNA bp II from Clostridium pasteurineum, Baccillus stearothermophilus, Escherichia coli, Rhizobium meliloti, Anabena, Thermoplasma acidophilum, Pseudomonas aeruginosa and Bacillus caldolyticus reveals a clear homology among these small basic proteins. In particular, two short sequences in the middle and C-terminal part of the proteins (residues N-Gly-Phe-Gly-X-Phe and Pro-X-Thr at positions 46–51 and 63–65, respectively) are completely conserved.

Proteins of the Thermus thermophilus ribosome Purification of several individual proteins and crystallization of protein TL7

The procedure of selective removal of eight proteins from the 50 S ribosomal subunit of the extreme thermophilic bacterium Thermus thermophilus has been developed based on extraction at 60°C in the presence of 0.5 M or 1 M NH 4Cl and 50% ethanol. CM-Sepharose CL column chromatography of the protein mixture under non-denaturing conditions yielded five proteins with a purity of 95% or higher. Crystals of one of these proteins, namely TL7 (probably an analog of L6 protein from the Escherichia coli ribosome) have been obtained using the ‘hanging drop’ method with ammonium sulphate as a precipitant.

Ser-tRNAs from bovine mitochondrion form ternary complexes with bacterial elongation factor Tu and GTP.

Transfer ribonucleic acids were isolated from mitochondria of bovine heart and aminoacylated in vitro by a crude mitochondrial enzyme. Ser-tRNASerUCN and Ser-tRNASerAGY were isolated and characterized by partial sequencing. Although these tRNAs possess unique structural features not found in any bacterial tRNA, they form a ternary complex with elongation factor from the extreme thermophilic bacterium Thermus thermophilus and GTP.