Comparative analysis of ribosomal protein L5 sequences from bacteria of the genus Thermus.

The genes for the ribosomal 5S rRNA binding protein L5 have been cloned from three extremely thermophilic eubacteria, Thermus flavus, Thermus thermophilus HB8 and Thermus aquaticus (Jahn et al, submitted). Genes for protein L5 from the three Thermus strains display 95% G/C in third positions of codons. Amino acid sequences deduced from the DNA sequence were shown to be identical for T flavus and T thermophilus, although the corresponding DNA sequences differed by two T to C transitions in the T thermophilus gene. Protein L5 sequences from T flavus and T thermophilus are 95% homologous to L5 from T aquaticus and 56.5% homologous to the corresponding E coli sequence. The lowest degrees of homology were found between the T flavus/T thermophilus L5 proteins and those of yeast L16 (27.5%), Halobacterium marismortui (34.0%) and Methanococcus vannielii (36.6%). From sequence comparison it becomes clear that thermostability of Thermus L5 proteins is achieved by an increase in hydrophobic interactions and/or by restriction of steric flexibility due to the introduction of amino acids with branched aliphatic side chains such as leucine. Alignment of the nine protein sequences equivalent to Thermus L5 proteins led to identification of a conserved internal segment, rich in acidic amino acids, which shows homology to subsequences of E coli L18 and L25. The occurrence of conserved sequence elements in 5S rRNA binding proteins and ribosomal proteins in general is discussed in terms of evolution and function.     

Development of a gene expression vector for Thermus thermophilus based on the promoter of the respiratory nitrate reductase

A specific expression system for Thermus spp. is described. Plasmid pMKE1 contains replicative origins for Escherichia coli and Thermus spp., a selection gene encoding a thermostable resistance to kanamycin, and a 720 bp DNA region containing the promoter (Pnar), and the regulatory sequences of the respiratory nitrate reductase operon of Thermus thermophilus HB8. Two genes, encoding a thermophilic beta-galactosidase and an alkaline phosphatase were cloned in pMKE1 as cytoplasmic and periplasmic reporters, respectively. The expression of the reporters was specifically induced by the combined action of nitrate and anoxia in facultative anaerobic derivatives of T. thermophilus HB27 to which the gene cluster for nitrate respiration was transferred by conjugation. Overexpressions in the range of approximately 200-fold were obtained for the cytoplasmic reporter, whereas that of the periplasmic reporter was limited to approximately 20-fold, with respect to their intrinsic respective activities.     

Thermus thermophilus TMY isolated from silica scale taken from a geothermal power plant

Aims:  To identify an extreme thermophile, strain TMY, isolated from silica scale from the geothermal electric power plant and to examine microdiversity of Thermus thermophilus strains.
Materials and Results:  The isolated strain TMY was identified by morphological, biochemical and physiological tests. Phylogenetic comparison of the strain and other Thermus strains with 16S rDNA analysis, RAPD and ERIC-PCR fingerprinting were performed. Strain TMY was closely related to strain which was isolated from a hot spring in New Zealand and shown to belong to the Japanese Thermus cluster. However, there were considerable genetic differences between strain TMY and other Thermus species using DNA fingerprinting.
Conclusions:  Based on morphological, physiological and genetic properties, strain TMY could be a strain of T. thermophilus . The distinct properties of strain TMY suggest that microdiversity of T. thermophilus strains should be considered.
Significance and Impact of the Study:  The results of this study have demonstrated genetic diversity within T. thermophilus strains, which were previously masked by an almost identical 16S rDNA sequence. RAPD and ERIC-PCR could be potential methods for distinguishing between Thermus strains.     

Ribosomal protein S1 from Thermus thermophilus: its detection,identification and overproduction

Ribosomal protein S1 has been identified in Thermus thermophilus ribosomes. The gene of ribosomal protein S1 from Thermus thermophilus has been cloned and overexpressed in Escherichia coli. A procedure for purification of the protein has been developed.     

Atomic structures at last: the ribosome in 2000

Last year, atomic structures of the 50S ribosomal subunit from Haloarcula marismortui and of the 30S ribosomal subunit from Thermus thermophilus were published. A year before that, a 7.8 A resolution electron density map of the 70S ribosome from T. thermophilus appeared. This information is revolutionizing our understanding of protein synthesis.     

Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases.

The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermophilus. Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.     

Thermus thermophilus ribosomes for crystallographic studies.

Three-dimensional crystals of the 70S ribosomes, the 70S ribosome-mRNA-tRNA complex, the 30S ribosomal subunits, several ribosomal proteins, the elongation factor G and threonyl- and seryl-tRNA synthetases from a Gram-negative extreme thermophilic bacterium, Thermus thermophilus, have been obtained at our institute. X-ray and neutronographic data from the 70S ribosome crystals have been collected up to 18 A and 60 A, respectively. Two-dimensional crystalline sheets of the 70S ribosomes have been studied by electron microscopy. Structural studies of crystals of 2 ribosomal proteins, L1 and S6, elongation factor G and threonyl- and seryl-tRNA synthetases are also in progress. At present, Thermus thermophilus seems to be the most suitable microorganism to isolate ribosomes and their constituents for crystallographic studies.     

Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus

We have isolated spontaneous streptomycin-resistant, streptomycin-dependent and streptomycin-pseudo-dependent mutants of the thermophilic bacterium Thermus thermophilus IB-21. All mutant phenotypes were found to result from single amino acid substitutions located in the rpsL gene encoding ribosomal protein S12. Spontaneous suppressors of streptomycin dependence were also readily isolated. Thermus rpsL mutations were found to be very similar to rpsL mutations identified in mesophilic organisms. This similarity affords greater confidence in the utility of the crystal structures of Thermus ribosomes to interpret biochemical and genetic data obtained with Escherichia coli ribosomes. In the X-ray crystal structure of the T. thermophilus HB8 30 S subunit, the mutated residues are located in close proximity to one another and to helices 18, 27 and 44 of 16 S rRNA. X-ray crystallographic analysis of ribosomes from streptomycin-resistant, streptomycin-pseudo-dependent and streptomycin-dependent mutants described here is expected to reveal fundamental insights into the mechanism of tRNA selection, translocation, and conformational dynamics of the ribosome.     

Spontaneous Erythromycin Resistance Mutation in a 23S rRNA Gene, rrlA, of the Extreme Thermophile Thermus thermophilus IB-21

Spontaneous, erythromycin-resistant mutants of Thermus thermophilus IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons. The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism. This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes of Thermus.     

Effects of streptomycin resistance mutations on posttranslational modification of ribosomal protein S12

Ribosomal protein S12 contains a highly conserved aspartic acid residue that is posttranslationally beta-methylthiolated. Using mass spectrometry, we have determined the modification states of several S12 mutants of Thermus thermophilus and conclude that beta-methylthiolation is not a determinant of the streptomycin phenotype.     

Structure of the L1 protuberance in the ribosome

The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1-rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.     

High-level overproduction of <Emphasis Type="Italic">Thermus</Emphasis> enzymes in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Biotechnology needs to explore the capacity of different organisms to overproduce proteins of interest at low cost. In this paper, we show that Streptomyces lividans is a suitable host for the expression of Thermus thermophilus genes and report the overproduction of the corresponding proteins. This capacity was corroborated after cloning the genes corresponding to an alkaline phosphatase (a periplasmic enzyme in T. thermophilus) and that corresponding to a beta-glycosidase (an intracellular enzyme) in Escherichia coli and in S. lividans. Comparison of the production in both hosts revealed that the expression of active protein achieved in S. lividans was much higher than in E. coli, especially in the case of the periplasmic enzyme. In fact, the native signal peptide of the T. thermophilus phosphatase was functional in S. lividans, being processed at the same peptide bond in both organisms, allowing the overproduction and secretion of this protein to the S. lividans culture supernatant. As in E. coli, the thermostability of the expressed proteins allowed a huge purification factor upon thermal denaturation and precipitation of the host proteins. We conclude that S. lividans is a very efficient and industry-friendly host for the expression of thermophilic proteins from Thermus spp.     

Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA

We present a detailed analysis of the protein structures in the 30 S ribosomal subunit from Thermus thermophilus, and their interactions with 16 S RNA based on a crystal structure at 3.05 A resolution. With 20 different polypeptide chains, the 30 S subunit adds significantly to our data base of RNA structure and protein-RNA interactions. In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA component and are involved in stabilizing RNA tertiary structure. Many ribosomal proteins share similar alpha+beta sandwich folds, but we show that the topology of this domain varies considerably, as do the ways in which the proteins interact with RNA. Analysis of the protein-RNA interactions in the context of ribosomal assembly shows that the primary binders are globular proteins that bind at RNA multihelix junctions, whereas proteins with long extensions assemble later. We attempt to correlate the structure with a large body of biochemical and genetic data on the 30 S subunit.     

Study of the binding of the S7 protein with 16S rRNA fragment 926-986/1219-1393 as a key step in the assembly of the small subunit of prokaryotic ribosomes

Both structural and thermodynamic studies are necessary to understand the ribosome assembly. An initial step was made in studying the interaction between a 16S rRNA fragment and S7, a key protein in assembling the prokaryotic ribosome small subunit. The apparent dissociation constant was obtained for complexes of recombinant Escherichia coli and Thermus thermophilus S7 with a fragment of the 3' domain of the E. coli 16S rRNA. Both proteins showed a high rRNA-binding activity, which was not observed earlier. Since RNA and proteins are conformationally labile, their folding must be considered to correctly describe the RNA-protein interactions.     

Proteolysis of ribosomal protein S1 from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Thermus thermophilus</Emphasis> leads to formation of two different fragments

As a result of limited tryptic proteolysis of S1 ribosomal protein (molecular mass 60 kD) from Thermus thermophilus, 25 N-terminal amino acid residues and 71 C-terminal amino acid residues are split off and a stable high-molecular-weight fragment with molecular mass of 49 kD is formed that retains RNA-binding properties and is capable of interacting with 30S ribosomal subunit. Earlier, application of a similar procedure for the formation of a fragment of S1 protein from Escherichia coli resulted in splitting of 171 N-terminal amino acid residues with the formation of a 41.3 kD fragment that possesses RNA-binding properties only. Thus, in spite of high homology between E. coli and T. thermophilus proteins, the proteolysis leads to the formation of two different fragments, which points, in our opinion, to the fact of significant differences between their structures.     

Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27

Horizontal gene transfer has been a major force for genome plasticity over evolutionary history, and is largely responsible for fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Recently, by performing a genome-wide mutagenesis approach with Thermus thermophilus HB27, we identified the first genes in a thermophilic bacterium for the uptake of free DNA, a process called natural transformation. Here, we present the first data on the biochemistry and bioenergetics of the DNA transport process in this thermophile. We report that linear and circular plasmid DNA are equally well taken up with a high maximal velocity of 1.5 microg DNA.(mg protein)(-1).min(-1), demonstrating an extremely efficient binding and uptake rate of 40 kb.s(-1).cell(-1). Uncouplers and ATPase inhibitors immediately inhibited DNA uptake, providing clear evidence that DNA translocation in HB27 is an energy-dependent process. DNA uptake studies with genomic DNA of Bacteria, Archaea and Eukarya revealed that Thermus thermophilus HB27 takes up DNA from members of all three domains of life. We propose that the extraordinary broad substrate specificity of the highly efficient Thermus thermophilus HB27 DNA uptake system may contribute significantly to thermoadaptation of Thermus thermophilus HB27 and to interdomain DNA transfer in hot environments.     

Rapid and sensitive amino-acid sequencing of cloning Thermus thermophilus HB8 ferredoxin by proteomics

Recombinant holo Thermus thermophilus [7Fe-8S] ferredoxin was synthesized by cloning from Thermus thermophilus HB8 gene. A specific sequence (Pro-His-Val-Ile) at the N-terminus of the recombinant ferredoxin was determined by a rapid and highly sensitive mass spectral method using a novel Ru(II) Edman reagent, [(tpy)Ru(tpy-C6H4-NCS)](PF6)2 (tpy=terpyridine). The formation of the recombinant holoTtFd was established by the characteristic absorptions and CD extrema as [7Fe-8S] ferredoxin. The catalytic electron-transfer reactivity of the [7Fe-8S] ferredoxin between ferredoxin-NADP+ reductase and cytochrome c was recognized.     

The genome sequence of the extreme thermophile Thermus thermophilus

Thermus thermophilus HB27 is an extremely thermophilic, halotolerant bacterium, which was originally isolated from a natural thermal environment in Japan. This organism has considerable biotechnological potential; many thermostable proteins isolated from members of the genus Thermus are indispensable in research and in industrial applications. We present here the complete genome sequence of T. thermophilus HB27, the first for the genus Thermus. The genome consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27. The 2,218 identified putative genes were compared to those of the closest relative sequenced so far, the mesophilic bacterium Deinococcus radiodurans. Both organisms share a similar set of proteins, although their genomes lack extensive synteny. Many new genes of potential interest for biotechnological applications were found in T. thermophilus HB27. Candidates include various proteases and key enzymes of other fundamental biological processes such as DNA replication, DNA repair and RNA maturation.     

Isolation of Thermus strains from hot composts (60 to 80 degrees C).

High numbers (10(7) to 10(10) cells per g [dry weight]) of heterotrophic, gram-negative, rod-shaped, non-sporeforming, aerobic, thermophilic bacteria related to the genus Thermus were isolated from thermogenic composts at temperatures between 65 and 82 degrees C. These bacteria were present in different types of wastes (garden and kitchen wastes and sewage sludge) and in all the industrial composting systems studied (open-air windows, boxes with automated turning and aeration, and closed bioreactors with aeration). Isolates grew fast on a rich complex medium at temperatures between 40 and 80 degrees C, with optimum growth between 65 and 75 degrees C. Nutritional characteristics, total protein profiles, DNA-DNA hybridization (except strain JT4), and restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs (16S rDNAs) showed that Thermus strains isolated from hot composts were closely related to Thermus thermophilus HB8. These newly isolated T. thermophilus strains have probably adapted to the conditions in the hot-compost ecosystem. Heterotrophic, ovalspore-forming, thermophilic bacilli were also isolated from hot composts, but none of the isolates was able to grow at temperatures above 70 degrees C. This is the first report of hot composts as habitats for a high number of thermophilic bacteria related to the genus Thermus. Our study suggests that Thermus strains play an important role in organic-matter degradation during the thermogenic phase (65 to 80 degrees C) of the composting process.     

Comparative structural analysis of eubacterial 5S rRNA by oxidation of adenines in the N-1 position.

Adenines in free 5S rRNA from Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus have been oxidized at their N-1 position using monoperphthalic acid. The determination of the number of adenine 1-N-oxides was on the basis of UV spectroscopic data of the intact molecule. Identification of the most readily accessible nucleotides by sequencing gel analysis reveals that they are located in conserved positions within loops, exposed hairpin loops and single-base bulge loops. Implications for the structure and function of 5S rRNA will be discussed on the basis of this comparative analysis.