Extreme Thermophile Thermus Thermophilus Research Articles

Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry

A protocol has been developed that allows protein identifications using available DNA-based or protein sequences from a reference strain of a bacterial species to be extended to bacterial strains for which no prior DNA-based or protein sequence information exists. The protocol is predicated on careful isolation of a specific sub-cellular group of proteins. In this study, ribosomal proteins were chosen due to their high relative abundance and similarity in copy number per cell. After isolation of ribosomal proteins, MALDI-MS is used to acquire accurate protein molecular weights. An iterative comparison of reference protein molecular weights and identities is made to the resulting data, allowing for the straightforward identification of ribosomal proteins from any non-reference strains. This approach can reveal differences between proteins at the amino acid or post-translational level. The protocol was developed, validated and applied to ribosomal proteins from three strains of the extreme thermophile Thermus thermophilus. This approach revealed that nearly 60% of the ribosomal proteins from all three strains are identical. The extension of protein identification to additional bacterial strains can be useful in phylogenetic studies as well as in biomarker identification.

N1-Aminopropylagmatine, a New Polyamine Produced as a Key Intermediate in Polyamine Biosynthesis of an Extreme Thermophile, Thermus thermophilus

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase = agmatine ureohydrolase) accumulated N1-aminopropylagmatine (N8-amidino-1,8-diamino-4-azaoctane, N8-amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N1-aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N1-Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N1-Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N1-Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N1-aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N1-aminopropylagmatine to spermidine but does not act on agmatine.

A Mutation in the Decoding Center of Thermus thermophilus 16S rRNA Suggests a Novel Mechanism of Streptomycin Resistance

A spontaneous kanamycin resistance and capreomycin resistance mutation, A1408G, in the decoding center of 16S rRNA, was identified in the extreme thermophile Thermus thermophilus. Unexpectedly, this mutation also confers resistance to streptomycin. We propose a novel mechanism of streptomycin resistance by which A1408G influences conformational changes in 16S rRNA during tRNA selection.

Testing the conservation of the translational machinery over evolution in diverse environments: assaying Thermus thermophilus ribosomes and initiation factors in a coupled transcription-translation system from Escherichia coli

Ribosomes from the extreme thermophile Thermus thermophilus are capable of translation in a coupled transcription-translation system derived from Escherichia coli. At 45 degrees C, T.thermophilus ribosomes translate at approximately 25-30% of the maximal rate of E.coli ribosomes, and synthesize full-length protein. T.thermophilus and E.coli subunits can be combined to effect translation, with the spectrum of proteins produced depending upon the source of the 30S subunit. In this system, T.thermophilus ribosomes function in concert with E.coli translational factors and tRNAs, with elongation and release factors being supplied from the E.coli extract, and purified initiation factors (IFs) being added exogenously. Cloned and purified T.thermophilus IF1, IF2 and IF3 supported the synthesis of the same products in vitro as the E.coli factors, although the relative levels of some polypeptides were factor dependent. We conclude that, at least between these two phylogenetically distant species, translational factor function and subunit-subunit interactions are conserved. This functional compatibility is remarkable given the extreme and highly divergent environments to which these species have adapted.

A New Type of NADH Dehydrogenase Specific for Nitrate Respiration in the Extreme Thermophile Thermus thermophilus

A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor. Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfur-containing subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs). The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank. Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants. A fusion to a thermophilic reporter gene was used for the first time in Thermus spp. to show that expression of nrc required the presence of nitrate and anoxic conditions. Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced. Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen. The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system. Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins. Interactions were also detected between NrcN and NrcF. Inactivation of nrcF produced solubilization of NrcN, but not of NrcD. These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH.

Ribosomal protein S15 represses its own translation via adaptation of an rRNA-like fold within its mRNA.

The 16S rRNA-binding ribosomal protein S15 is a key component in the assembly of the small ribosomal subunit in bacteria. We have shown that S15 from the extreme thermophile Thermus thermophilus represses the translation of its own mRNA in vitro, by interacting with the leader segment of its mRNA. The S15 mRNA-binding site was characterized by footprinting experiments, deletion analysis and site-directed mutagenesis. S15 binding triggers a conformational rearrangement of its mRNA into a fold that mimics the conserved three-way junction of the S15 rRNA-binding site. This conformational change masks the ribosome entry site, as demonstrated by direct competition between the ribosomal subunit and S15 for mRNA binding. A comparison of the T.thermophilus and Escherichia coli regulation systems reveals that the two regulatory mRNA targets do not share any similarity and that the mechanisms of translational inhibition are different. Our results highlight an astonishing plasticity of mRNA in its ability to adapt to evolutionary constraints, that contrasts with the extreme conservation of the rRNA-binding site.

Efficient trans-cleavage by the Schistosoma mansoni SMalpha1 hammerhead ribozyme in the extreme thermophile Thermus thermophilus

The catalytic hammerhead structure has been found in association with repetitive DNA from several animals, including salamanders, crickets and schistosomes, and functions to process in cis the long multimer transcripts into monomer RNA in vivo. The cellular role of these repetitive elements and their transcripts is unknown. Moreover, none of these natural hammerheads have been shown to trans-cleave a host mRNA in vivo. We analyzed the cis- and trans-cleavage properties of the hammerhead ribozyme associated with the SMalpha DNA family from the human parasite Schistosoma mansoni. The efficiency of trans-cleavage of a target RNA in vitro was affected mainly by both the temperature-dependent chemical step and the ribozyme-product dissociation step. The optimal temperature for trans-cleavage was 70 degrees C. This result was confirmed when both the SMalpha1 ribozyme and the target RNA were expressed in the extreme thermophile Thermus thermophilus. Moreover, SMalpha1 RNA showed a remarkable thermostability, equal or superior to that of the most stable RNAs in this species, suggesting that SMalpha1 RNA has been selected for stability. Computer analysis predicts that the monomer and multimer transcripts fold into highly compact secondary structures, which may explain their exceptional stability in vivo.

Regulation of GTPases in the bacterial translation machinery.

Several GTPases participate in bacterial protein biosynthesis. Initiation factor 2 controls the formation of the ribosomal initiation complex and places initiator fMet-tRNAfMet in the ribosomal P-site. Elongation factors Tu and G are responsible for codon-specific binding of the aminoacyl-tRNA to the A-site, and peptidyl-tRNA to the P-site, respectively, during the elongation phase of protein biosynthesis. Release factor 3, a GTPase which is not ubiquitous, is involved in termination and release of the nascent polypeptide. Other translation factors, including initiation factors 1 and 3, elongation factor Ts, release factors 1 and 2, and ribosomal release factor do not belong to the family of GTP/GDP binding proteins. The guanosine nucleotide binding domains of the GTPases involved in translation are structurally related to the Galpha subunit of heterotrimeric G proteins and to the proteins of the Ras family. We have identified and sequenced all genes coding for translation factors in the extreme thermophile Thermus thermophilus. The proteins were overproduced in Escherichia coli, purified, biochemically characterised and used for crystallisation and structural analysis. Further biochemical investigations were aimed at gaining insight into the molecular mechanism underlying the regulation of the GTPase activity of the translation factors, and to elucidate the role of their ribosomal binding sites in this process.

A peculiarity of the reaction of tRNA aminoacylation catalyzed by phenylalanyl-tRNA synthetase from the extreme thermophile Thermus thermophilus

It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation reaction catalyzed by Thermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two bound phenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quite low: the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. The thermophilic synthetase can aminoacylate twice not only T. thermophilus tRNA Phe but also Escherichia coli tRNA Phe and E. coli tRNA Phe transcript, indicating that the presence of modified nucleotides is not necessary for tRNA Phe overcharging. Bisphenylalanyl-tRNA is stable in acidic solution, but it decomposes in alkaline medium yielding finally tRNA and free phenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate as from monophenylalanyl-tRNA. In the presence of the enzyme the rate of bisphenylalanyl-tRNA deacylation increases. Aminoacylated tRNA Phe isolated from T. thermophilus living cells was observed to contain no detectable bisphenylalanyl-tRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed.

Cloning and sequence analysis of fumarase and superoxide dismutase genes from an extreme thermophile Thermus thermophilus HB27

Class II fumarase ( fumC) and Mn-containing superoxide dismutase ( sodA) genes were cloned and sequenced from Thermus thermophilus HB27. The fumC and sodA genes existed in tandem. Comparison of the DNA and amino acid sequences of the funC-sodA regions between T. thermophilus HB27 and Thermus aquaticus YT1 revealed that coding regions showed high homology but noncoding regions were less homologous between the two strains. The deduced amino acid sequences of T. thermophilus fumC and sodA genes showed 58.2% and 53.2% identities to those of the corresponding Escherichia coli genes, respectively. The T. thermophilus fumC gene was expressed in E. coli and the heat-resistant fumarase activity was detected at 70°C.

Crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile, Bacillus coagulans: two strategies for thermostabilization of protein structures.

The crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans (BcIPMDH) has been determined by the X-ray method. BcIPMDH is a dimeric enzyme composed of two identical subunits, each of which takes an open alpha/beta structure with 11 alpha-helices and 14 beta-strands. The polypeptide is folded into two domains. The first domain is composed of residues 1-101 and 257-356, and the second domain, of residues 102-256. The latter domains of the two subunits are associated with one another by a dyad axis to make the dimer, locally forming a beta-sheet and a four-helix bundle. As compared with the structure of the enzyme from the extreme thermophile Thermus thermophilus (TtIPMDH), a new short beta-sheet (residues 329-330 and 340-341) absent in TtIPMDH is formed by the insertion of 5 residues in BcIPMDH. In terms of determinants for thermostabilization, both consistent and inconsistent changes were found between the two enzymes. The regions including inconsistent changes are formed by different usages of the determinants for stabilizing the loops at different levels. Those in BcIPMDH contain some structural redundancies in length of amino acid sequence and flexibility of residues, which seem to be unnecessary for the enzymatic reaction. Such redundancies are also found in the primary structure of the enzyme of the mesophile Bacillus subtilis, but these parts are more stabilized in BcIPMDH by hydrogen bonds and salt bridges. On the other hand, TtIPMDH is stabilized by reducing such redundant parts. This contrast suggests that different strategies may be preferred for thermostabilization, depending on temperature.

Ornithine carbamoyltransferase from the extreme thermophile Thermus thermophilus--analysis of the gene and characterisation of the protein.

The ornithine carbamoyltransferase (OTC) gene from Thermus thermophilus was cloned from a lambda-ZAP genomic library. An ORF of 903 bp was found coding for a protein of Mr 33,200. The coding region has a very high overall G+C content of 68.0%. T. thermophilus OTC displays 38-48% amino acid identity with other OTC, the most closely related proteins being OTC from the archaeon Pyrococcus furiosus and from Bacillus subtilis. The enzyme was expressed in Escherichia coli and purified to homogeneity using a thermoshock followed by affinity chromatography on delta-N-phosphonoacetyl-L-ornithine-Sepharose. The native enzyme has an Mr of about 110,000, suggesting a trimeric structure, as for most anabolic OTC from various organisms. T. thermophilus OTC exhibits Michaelis-Menten kinetics for carbamoyl phosphate and ornithine with a Km(app) of 0.10 mM for both substrates. The pH optimum was dependent on ornithine concentration with an optimum at pH 8 for ornithine concentrations around Km values. Higher concentrations shift the optimum towards lower pH. The optimal temperature was above 65 degrees C and the activation energy 39.1 kJ/mol. The enzyme is highly thermostable. In the presence of its substrates the half-life time was several hours at 85 degrees C. Ionic and hydrophobic interactions contribute to the stability. The expression of T. thermophilus OTC was negatively regulated by arginine.

Crystallization and structural studies of components of the protein-synthesizing system from Thermus thermophilus

A long-term program on crystallization and structural studies of the protein synthesis machinery components from an extreme thermophile Thermus thermophilus was set up at the Institute of Protein Research (Russia) about 15 years ago. These studies have recently revealed the structures of elongation factor G, aspartyl-tRNA synthetase and ribosomal proteins S6 and L1. Different components of the protein synthesis machinery from T.thermophilus are also being investigated in other groups and many important results have been obtained recently. Here we describe only some special problems on crystal handling and non-isomorphism that have been overcome during structural studies of EF-G and ribosomal proteins in our group. This paper presents also new data on the crystallization of ribosomal proteins S7, S8, S15, L22 and leucyl-tRNA synthetase from T.thermophilus.

Crystallization of Thermus thermophilus histidyl-tRNA synthetase and its complex with tRNAHis.

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNAHis. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2(1)2(1)2 with cell parameters a = 171.3 A, b = 214.7 A, c = 49.3 A, alpha = beta = gamma = 90 degrees. A complete data set to a resolution of 2.7A with an Rmerge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNAHis shows that the crystals are tetragonal with cell parameters a = b = 232 A, c = 559 A, alpha = beta = gamma = 90 degrees and diffract to about 4.5 A resolution.

Threonyl-tRNA synthetase from Thermus thermophilus: Purification and some structural and kinetic properties

Threonyl-tRNA synthetase (ThrRS) has been isolated from an extreme thermophile Thermus thermophilus strain HB8. The enzyme was purified to electrophoretic homogeneity by combinations of column chromatographies on DEAE-Sepharose, S-Sepharose, ACA-44 Ultrogel and HA-Ultrogel. Seventeen mg of purified enzyme were obtained from 1 kg of biomass. In parallel, purified aspartyl- and phenylalanyl-tRNA synthetases were obtained. The purified ThrRS is composed of two identical subunits with a molecular mass of about 77 000 (virtually the same as E coli ThrRS). The N-terminal sequence has been determined. The homology between the first 45 amino acid residues of ThrRS from T thermophilus and E coli is about 29%. A comparative study of tRNA Thr charging by ThrRS from E coli and T thermophilus reveals a similar efficiency of the reaction in both homologous systems. This efficiency remains unchanged for aminoacylation of tRNA Thr from T thermophilus by the heterologous ThrRS from E coli, but decreases 700 times for aminoacylation of E coli tRNA Thr by ThrRS from T thermophilus.

S-layer protein from Thermus thermophilus HB8 assembles into porin-like structures.

The cells of the extreme thermophile Thermus thermophilus are surrounded by a regular layer (S-layer) built up by a protein with an apparent molecular mass of 100 kDa (P100). From purified membrane fractions, three different class of two-dimensional crystals can be obtained by following alternative extractive procedures. One of these crystals, with p6 symmetry, clearly represents the native S-layer detected by freeze etching on whole cells, while the other two, showing p2 and p3 symmetries respectively, closely resemble aggregates of bacterial porins. We demonstrate here by limited proteolysis and Western blotting the surprising fact that the protein component of the three crystals is the P100 protein. Our biochemical data also show how this protein forms Ca(2+)-stabilized trimers in each crystal, which support the structural analysis that points to p3 units as the common structural block in all of them, and again resembles the situation found in bacterial porins.

Determination of tRNA Phe recognition nucleotides for phenylalanyl-tRNA synthetase from Thermus thermophilus

The tRNA Phe recognition nucleotides for phenylalanyl-tRNA synthetase from an extreme thermophile Thermus thermophilus were investigated. Using yeast tRNA Phe T7 transcripts with various point mutations it was shown that four recognition points (G34, A35, A36 from anticodon and A73 from acceptor stem) are important for aminoacylation at 37°C. In the case of the 73rd discriminator base A→U, but not A→C, substitution suppresses aminoacylation. Position 20, which proved to be essential for all phenylalanyl-tRNA synthetases so far studies, is not involved in the recognition of tRNA Phe by the T thermophilus enzyme.

Crystallization of the seryl-tRNA synthetase-tRNA Ser complex from Thermus thermophilus

The complex between seryl-tRNA synthetase and its cognate tRNA from the extreme thermophile Thermus thermophilus has been crystallized from ammonium sulphate solutions. Two different tetragonal crystal forms have been characterized, both diffracting to about 6 Å using synchrotron radiation. One form grows as large bipyramids and has cell dimensions a = b = 127 A ̊ , c = 467 A ̊ , and the second form occurs as long, thin square prisms with cell dimensions a = b = 101 A ̊ , c = 471 A ̊ . Analysis of washed and dissolved crystals demonstrates the presence of both protein and tRNA.

Crystals of intact elongation factor Tu from Thermus thermophilus diffracting to high resolution

The intact elongation factor Tu from the extreme thermophile Thermus thermophilus has been crystalized as a complex with the GTP analogue guanosine-5′-(β-γ-imido)triphosphate. The crystals are very stable in the X-ray beam and diffract to 1·9 Å resolution. They exhibit space group C2, with a = 150·(36) A ̊ , b = 99·6(3) A ̊ , c = 40·1(1) A ̊ , β = 95·4(2)°, and contain one elongation factor Tu molecule per asymmetric unit.

Crystals of threonyl-tRNA synthetase from Thermus thermophilus: Preliminary crystallographic data

Crystals have been obtained of threonyl-tRNA synthetase from the extreme thermophile Thermus thermophilus using sodium formate as a precipitant. The crystals are very stable and diffract to at least 2.4 Å. The crystals belong to space group P2 12 12 1 with cell parameters a = 61.4 A ̊ , b = 156.1 A ̊ , c = 177.3 A ̊ .