An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus

A Thermus thermophilus host strain of which the leuB gene was totally deleted was constructed from a delta pyrE strain by a two step method. First, the leuB gene was replaced with the pyrE gene. Second, the inserted pyrE gene was deleted by using 5-fluoroorotic acid. A plasmid vector with the leuB marker was constructed and the plasmid complemented the leuB deficiency of the host. When the leuB gene from Escherichia coli and its derivative encoding a stabilized enzyme were expressed with the host-vector system, their growth temperature reflected the stability of the enzyme. These results suggest that the gene replacement deletion method using the pyrE gene is useful for the construction of a reliable plasmid vector system and it can be applied to the selection of stabilized enzymes.     

极端嗜热菌Thermus thermophilus HB8中天冬氨酸转氨酶在大肠杆菌中的表达、纯化及酶学性质研究

为获得具有热稳定性的天冬氨酸转氨酶,从极端嗜热细菌Thermus thermophilus HB8中克隆得到天冬氨酸转氨酶基因aspC,并在大肠杆菌BL21(DE3)和Rosetta(DE3)中进行表达,发现在Rosetta(DE3)中具有较高的表达量。重组酶的最适反应pH是7.0,37℃下在pH8～10的缓冲液中保温1h酶活几乎不改变。重组酶反应的最适温度为75℃,酶活稳定的温度范围为25～55℃。重组酶在65℃时半衰期为3.5h,75℃时为2.5h。重组酶的KmKG为7.559mmol/L,VmaxKG为0.086mmol/(L·min),KmAsp为2.031mmol/L,VmaxAsp为0·024mmol/(L·min)。Ca2 、Fe3 、Mn2 等金属离子对酶活性有微弱抑制作用。     

A plasmid vector for an extreme thermophile, Thermus thermophilus

The host-vector system for an extreme thermophile, Thermus thermophilus HB27, was developed. The host strain has a mutation in tryptophan synthetase gene (trpB), and the mutation was determined to be a missense mutation by DNA sequence analysis. A Thermus-E. coli shuttle vector pYK109 was constructed. pYK109 consists of Thermus cryptic plasmid pTT8, tryptophan synthetase gene (trpB) of Thermus T2 and E. coli plasmid vector pUC13. pYK109 transformed T. thermophilus HB27 trpB5 to Trp+ at a frequency of 10(6) transformants per microgram DNA.     

Highly efficient production of enzymes of an extreme thermophile, Thermus thermophilus: A practical method to overexpress GC-rich genes in Escherichia coli

The GC-rich leuB gene (coding for 3-isopropylmalate dehydrogenase) of Thermus thermophilus is scarcely expressed in Escherichia coli, unless a leader open reading frame (ORF) is provided. We conducted experiments on nonexpressible plasmids and obtained a modified plasmid showing greatly enhanced expression: the degree of expression from the plasmid was higher than that from any other plasmid so far constructed. Sequence analysis of the plasmid showed that a 258-bp leader ORF overlapped with the initiation codon of leuB was newly formed as a consequence of the insertion of a 0.5-kb BamHI fragment derived from the E. coli chromosome. The degree of expression from the plasmid was further improved by shortening the leader ORF to 36 bp without changing the overlapping portion, and the flanking sequence between the promoter and the leader ORF was removed. The expression in E. coli of the pfk1 gene (coding for phosphofructokinase) of T. thermophilus was improved by the construction of a structure similar to that which enhanced the expression of the leuB gene. Based on the results, a practical method for the overexpression of GC-rich genes in E. coli is proposed. Received: November 26, 1996 / Accepted: May 17, 1997     

Application of N-terminally Truncated DNA Polymerase from Thermus thermophilus ({Delta}Tth Polymerase) to DNA Sequencing and Polymerase Chain Reactions: Comparative Study of {Delta}Tth and Wild-Type Tth Polymerases

N-Terminally truncated DNA polymerase from Thermus thermophilus(Tth polymerase) lacking 5'-3' exonuclease activity was usedfor DNA sequencing and polymerase chain reaction (PCR). In contrastto the high background of the sequencing ladder observed withthe wild-type Tth polymerase, Tth polymerase gave readable sequencingpatterns which extend up to more than 500 bases from the primersite on cycle sequencing and automated sequencing. The Tth polymerasewas used for the standard and mutagenic PCR, and net amplificationof the DNA and the mutations accumulated during PCR were analyzed.Under mutagenic PCR, the mutation rates were 7.0 x 10^–4(Tth) and 8.3 x 10^–4 (Tth) per nucleotide per cycle ofamplification, which were 4–9 times higher than the ratesunder standard PCR.     

Close proximity of phosphorylation sites to ligand in the phosphoproteome of the extreme thermophile Thermus thermophilus HB8

We performed phosphoproteome analysis of proteins from the extremely thermophilic Gram‐negative eubacterium Thermus thermophilus HB8 using gel‐free mass spectrometric method. We identified 52 phosphopeptides from 48 proteins and determined 46 phosphorylation sites: 30 on serine, 12 on threonine, and 4 on tyrosine. The identified phosphoproteins are known to be involved in a wide variety of cellular processes. To help elucidate the functional roles of these phosphorylation events, we mapped the phosphorylation sites on the known tertiary structures of the respective proteins. In all, we succeeded in mapping 46 sites (approximately 88%) on the corresponding structures. Most of the phosphorylation sites were found to be located on loops and terminal regions of the secondary structures. Surprisingly, 28 of these sites were situated at or near the active site of the enzyme. In particular, 18 sites were within 4 Å of the ligand, including substrate or cofactor. Such structural locations suggest direct effects of the phosphorylation on the binding of ligand in addition to inducing a conformational change. Interestingly, 19 of these 28 phosphorylation sites were situated near the phosphate moiety of a substrate or cofactor. In oligomeric proteins, 5 phosphorylation sites were found at the subunit interface. Based on these results, we propose a regulatory mechanism that involves Ser/Thr/Tyr phosphorylation in T. thermophilus HB8.     

Cloning and Nucleotide Sequence of the Pullulanase Gene of Thermus thermophilus HB8 and Production of the Enzyme in Escherichia coli

A 3.4-kb SphI fragment carrying the pullulanase gene of Thermus thermophilus HB8 was cloned. Based on the nucleotide sequence of it and the flanking region analyzed by direct sequencing of the inverse PCR product, an expression vector was constructed. The E. coli cells harboring the plasmid produced an about 80-kDa protein having pullulanase activity, the optimum temperature of which was 70°C.     

Expression,purification and characterization of recombinant protein tyrosine phosphatase from Thermus thermophilus HB27

The low-molecular-weight protein tyrosine phospha- tases （PTPase） exist ubiquitously in prokaryotes and eukaryotes and play important roles in the regulation of physiological activities. We report here the expression, purification and characterization of an active and soluble PTPase from Thermus thermophilus HB27 in Escherichia coli. This PTPase has an optimum pH range of 2.8-4.8 when using p-nitrophenyl phosphate as the substrate. The thermal inactivation results indicate a high thermal stability of this enzyme, with the optimum temperature of 75℃ for activity. It can be activated by Mn^2＋, Mg^2＋, Ca^2＋, Ba^2＋, and Ni^2＋, but inhibited by Zn^2＋, Cu^2＋, Cl^-, and SO^2-. These results suggest that this heat-resistant PTPase may play important roles in vivo in the adaptation of the microorganism to extreme temperatures and specific nutritional conditions.     

Studies on interdomain interaction of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus, by constructing chimeric enzymes

In our previous study, we showed that a chimeric isopropylmalate dehydrogenase, 2T2M6T, between an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, isopropylmalate dehydrogenases (the name roughly denotes the primary structure; the first 20% from the N-terminal is coded by the thermophile leuB gene, next 20% by mesophile, and the rest by the thermophile gene) denatured in two steps with a stable intermediate, suggesting that in the chimera some of the interdomain interaction was lost by amino acid substitutions in the "2M" part. To identify the residues involved in the interdomain interactions, the first and the second halves of the 2M part of the chimera were substituted with the corresponding sequence of the thermophile enzyme. Both chimeras, 3T1M6T and 2T1M7T, apparently showed one transition in the thermal denaturation without any stable intermediate state, suggesting that the cooperativity of the conformational stability was at least partly restored by the substitutions. The present study also suggested involvement of one or more basic residues in the unusual stability of the thermophile enzyme. Received: September 29, 1998 / Accepted: June 25, 1999     

Overexpression of genes of an extreme thermophile Thermus thermophilus, in Escherichia coli cells.

The 3-isopropylmalate dehydrogenase gene from an extreme thermophile, Thermus thermophilus, was not expressed in Escherichia coli unless a palindromic structure around the ribosome binding site was eliminated or a leader open reading frame was introduced into the upstream flanking region of the gene. This report suggests a way to increase the expression of this gene, with a high G+C content, in E. coli.     

Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases from an extreme thermophile Thermus thermophilus HB8: protein structure and Zn2+ binding

Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases have been purified from an extreme thermophile, Thermus thermophilus HB8. Valyl-tRNA and isoleucyl-tRNA synthetases are found to be monomer proteins (Mr 108000 and 129000, respectively), while methionyl-tRNA synthetase is a dimer protein (Mr 150000). These enzymes are very similar with respect to amino acid compositions and alpha-helix contents as estimated by circular dichroism analyses. Furthermore, two Zn2+ are tightly bound to each of these synthetases. These data suggest that valyl-tRNA and isoleucyl-tRNA synthetases consist of two domains, each corresponding to the subunit of methionyl-tRNA synthetase.     

A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2)

An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.     

Purification and characterization of the Thermus thermophilus HB8 RecX protein

The RecA protein plays a central role in homologous recombination by promoting strand exchange between ssDNA and homologous dsDNA. Since RecA alone can advance this reaction in vitro, it is widely used in gene manipulation techniques. The RecX protein downregulates the function of RecA, indicating that it could be used as an inhibitor to control the activities of RecA in vitro. In this study, the RecX protein of the hyper-thermophilic bacterium Thermus thermophilus (ttRecX) was over-expressed in Escherichia coli and purified by heat treatment and several column chromatography steps. Size-exclusion chromatography indicated that purified ttRecX exists as a monomer in solution. Circular dichroism measurements indicated that the alpha-helical content of ttRecX is 54% and that it is stable up to 80 degrees C at neutral pH. In addition, ttRecX inhibited the DNA-dependent ATPase activity of the T. thermophilus RecA protein (ttRecA). The stable ttRecX may be applicable for variety of techniques using the ttRecA reaction.     

Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent

We examined a single, non-spore-forming, aerobic, thermophilic strain that was isolated from a deep-sea hydrothermal vent in the Guaymas Basin at a depth of 2000 m and initially placed in a phenetic group with Thermus scotoductus (X-1). We identified this deep-sea isolate as a new strain belonging to Thermus thermophilus using several parameters. DNA–DNA hybridization under stringent conditions showed 74% similarity between the deep-sea isolate and T. thermophilus HB-8^T (T = type strain). Phenotypic characteristics, such as the utilization of carbon sources, hydrolysis of different compounds, and antibiotic sensitivity were identical in the two strains. The polar lipids composition showed that strain Gy1211 belonged to the genus Thermus. The fatty acids composition indicated that this strain was related to the marine T. thermophilus strain isolated from the Azores. The new isolate T. thermophilus strain Gy1211 grew optimally at 75°C, pH 8.0, and 2% NaCl. A hydrostatic pressure of 20 MPa, similar to the in situ hydrostatic pressure of the deep-sea vent from which the strain was isolated, had no effect on growth. Strain HB-8^T, however, showed slower growth under these conditions. Received: November 26, 1997 / Accepted: May 20, 1999     

Screening of stable proteins in an extreme thermophile, Thermus thermophilus

The leuB gene codes for 3-isopropylmalate dehydrogenase of the leucine biosynthetic pathway in an extreme thermophile, Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature-sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucine was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.     

Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60°C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.     

Thermus thermophilus HB8葡萄糖异构酶在大肠杆菌中表达

为了增加高热稳定性的葡萄糖异构酶的得率,采用PCR技术扩增得到Thermus thermophilusHB8葡萄糖异构酶基因xylA,连接到表达载体pET-22b( )上,获得重组质粒pET-22b( )-xylA。将重组质粒转化到大肠杆菌Rosetta(DE3)中,经IPTG诱导后,通过半胱氨酸-咔唑法测葡萄糖异构酶酶活。重组菌经诱导培养,SDS-PAGE电泳结果显示出明显的分子量约为44 kD特异性蛋白质条带,比酶活约为18.562 U/mg,比野生型菌株提高了2倍。     

Cocrystallization of Lysyl–tRNA synthetase from Thermus thermophilus with its cognate tRNAlys and with Escherichia coli tRNAlys

Lysyl-tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNA^lYS or Escherichia coli tRNA^lys using ammonium sulfate as precipitant. The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18–22% ammonium sulfate in 50 mM Tris-maleate buffer at pH 7.5. Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 Å, c = 119 Å) and diffract to at least 2.7 Å resolution. However the homocomplex is of space group P42₁2 and the heterocomplex of space group I422. © 1995 Wiley-Liss, Inc.