Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus aquaticus YT-1 and the deduced amino acid sequence of the enzyme

The gene for L-lactate dehydrogenase (LDH) from Thermus aquaticus YT-1 was cloned in Escherichia coli, using the Thermus caldophilus LDH gene as a hybridization probe, and its complete nucleotide sequence was determined. The LDH gene comprised 930 base pairs, starting with a GTG initiation codon. Its sequence had high homology (85.8% identity) with the LDH gene of T. caldophilus. The G + C content of the T. aquaticus gene was 70.9%, higher than that of the chromosomal DNA (67.4%). In particular, that in the third position of the codons used was 91.0%, similar to the T. caldophilus gene. The primary structure of T. aquaticus LDH was deduced from the nucleotide sequence of the LDH gene. It comprises 310 amino acid residues, as does T. caldophilus LDH, and its molecular mass was calculated to be 33,210 daltons. The amino acid sequence of the T. aquaticus LDH had 87.1% identity with that of the T. caldophilus LDH. At 23 positions, the respective residues differed in charge and polarity. These differences must be related to the differences in kinetic properties between the two enzymes. The constructed plasmid overproduced the T. aquaticus LDH in E. coli.     

The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G2

The complete nucleotide sequence of the Pseudomonas chromosomal gene coding for the enzyme carboxypeptidase G2 (CPG2) has been determined. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that of randomly derived peptide fragments and by N-terminal sequencing of the purified protein. The gene has been shown to code for a 22 amino acid signal peptide at its N-terminus which closely resembles the signal peptides of other secreted proteins. An alternative 36 amino acid signal peptide which may function in Pseudomonas has also been identified. The codon utilisation of the gene is influenced by the high G + C (67.2%) content of the DNA and exhibits a 92.8% preference for codons ending in G or C. This unusual codon preference may contribute to the generally observed weak expression of Pseudomonas genes in Escherichia coli. A region of DNA upstream of the structural gene has also been sequenced and a ribosome binding site and two putative promoter sequences identified.     

Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8.

The gene coding for isocitrate dehydrogenase of an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. This gene consists of a single open reading frame of 1,485 bp preceded by a Shine-Dalgarno ribosome binding site. Promoter- and terminatorlike sequences were detected upstream and downstream of the open reading frame, respectively. The G + C content of the coding region was 65.6%, and that of the third nucleotide of the codons was 90.3%. On the basis of the deduced amino acid sequence, the Mr of the monomeric enzyme was calculated as 54,189, an Mr which is similar to that of the purified protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison of the amino acid sequence of the T. thermophilus enzyme with that of the Escherichia coli enzyme showed (i) a 37% overall similarity; (ii) the conservation of the Ser residue, which is known to be phosphorylated in the E. coli enzyme, and of the surrounding sequence; and (iii) the presence of 141 extra residues at the C terminus of the T. thermophilus enzyme. T. thermophilus isocitrate dehydrogenase showed a high sequence homology (33% of the amino acid sequence is identical) to isopropylmalate dehydrogenase from the same organism and was suggested to have evolved from a common ancestral enzyme.     

Molecular cloning, nucleotide sequence and expression of the tufB gene encoding elongation factor Tu from Thermus thermophilus HB8

The tufB gene encoding elongation factor Tu (EF-Tu) of Thermus thermophilus HB8 was cloned and expressed. Compared with the known tufA gene of T. thermophilus, nucleotide differences were found at 10 positions out of 1221 nucleotides, and amino acid substitutions were found at 4 positions out of 406 amino acids. The tufB product was 70.9% homologous to the corresponding sequence of the tufB product of E. coli. The G+C content of the third base of the codon in the tufB gene was 84.8% and G was especially preferred in this position.     

Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8.

The gene coding for isocitrate dehydrogenase of an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. This gene consists of a single open reading frame of 1,485 bp preceded by a Shine-Dalgarno ribosome binding site. Promoter- and terminatorlike sequences were detected upstream and downstream of the open reading frame, respectively. The G + C content of the coding region was 65.6%, and that of the third nucleotide of the codons was 90.3%. On the basis of the deduced amino acid sequence, the Mr of the monomeric enzyme was calculated as 54,189, an Mr which is similar to that of the purified protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison of the amino acid sequence of the T. thermophilus enzyme with that of the Escherichia coli enzyme showed (i) a 37% overall similarity; (ii) the conservation of the Ser residue, which is known to be phosphorylated in the E. coli enzyme, and of the surrounding sequence; and (iii) the presence of 141 extra residues at the C terminus of the T. thermophilus enzyme. T. thermophilus isocitrate dehydrogenase showed a high sequence homology (33% of the amino acid sequence is identical) to isopropylmalate dehydrogenase from the same organism and was suggested to have evolved from a common ancestral enzyme.     

Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase gene (leuB) from an extreme thermophile, Thermus aquaticus YT-1

A gene (leuB) coding for 3-isopropylmalate dehydrogenase [EC 1.1.1.85] from an extreme thermophile, Thermus aquaticus YT-1 was cloned in Escherichia coli and the nucleotide sequence was determined. It contains an open reading frame of 1,035 bp encoding 344 amino acid residues. The homology with that from T. thermophilus HB8 is 87.0% in nucleotide and 91.3% in amino acid sequences. No overlapped gene was found in the present leuB gene, in contrast to the previous prediction that Thermus leuD gene is overlapped with leuB [Croft et al. (1987) Mol. Gen. Genet. 210, 490-497]. Substitutions in the primary structure which are unique for the thermophile sequences are discussed in relation to the unusual stability of the thermophile dehydrogenase based on amino acid sequence comparison of 9 microorganisms including thermophiles and mesophiles.     

Nucleotide sequence of the gene for alkaline phosphatase of Thermus caldophilus GK24 and characteristics of the deduced primary structure of the enzyme

The gene encoding Thermus caldophilus GK24 (Tca) alkaline phosphatase was cloned into Escherichia coli. The primary structure of Tca alkaline phosphatase was deduced from its nucleotide sequence. The Tca alkaline phosphatase precursor, including the signal peptide sequence, was comprised of 501 amino acid residues. Its molecular mass was determined to be 54? omitted?760 Da. On the alignment of the amino acid sequence, Tca alkaline phosphatase showed sequence homology with the microbial alkaline phosphatases, 20% identity with E. coli alkaline phosphatase and 22% Bacillus subtilis (Bsu) alkaline phosphatases. High sequence identity was observed in the regions containing the Ser-102 residue of the active site, the zinc and magnesium binding sites of E. coli alkaline phosphatase. Comparison of Tca alkaline phosphatase and E. coli alkaline phosphatase structures suggests that the reduced activity of the Tca alkaline phosphatase, in the presence of zinc, is directly involved in some of the different metal binding sites. Heat-stable Tca alkaline phosphatase activity was detected in E. coli YK537, harboring pJRAP.     

Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.     

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.     

Nucleotide sequence of the gene for aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1 and characteristics of the deduced primary structure of the enzyme

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extreme thermophile [Matsuzawa, H., Hamaoki, M. & Ohta, T. (1983) Agric. Biol. Chem. 47, 25-28]. The gene encoding aqualysin I was cloned into Escherichia coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequence, agreed with the NH2-terminal sequence previously reported and the determined amino acid sequences, including the COOH-terminal sequence, of the tryptic peptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28,350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin-type serine proteases, and 43% identity with proteinase K, 37-39% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. The nucleotide sequence of the cloned DNA (1105 nucleotides) revealed that it contains the entire gene encoding aqualysin I and one open reading frame without a translational stop codon. Therefore, aqualysin I was considered to be produced as a large precursor, which contains a NH2-terminal portion, the protease and a COOH-terminal portion. The G + C content of the coding region for aqualysin I was 64.6%, which is lower than those of other Thermus genes (68-74%). The codon usage in the aqualysin I gene was rather random in comparison with that in other Thermus genes.     

Molecular cloning and sequence determination of the tuf gene coding for the elongation factor Tu of Thermus thermophilus HB8

The tuf gene, which encodes the elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, and its flanking regions were cloned and sequenced. The gene encoding EF-G was found upstream of the 5' end of the tuf gene. The tuf gene of T. thermophilus HB8 had a very high G + C content and 84.5% of the third base in codon usage was either G or C. The deduced primary structure of the EF-Tu was composed of 405 amino acid residues with a Mr = 44658. A comparison of the amino acid sequence of EF-Tu from T. thermophilus HB8 with those of Escherichia coli and Saccharomyces cerevisiae mitochondria showed a very high sequence homology (65-70%). Two Cys residues out of the three found in E. coli EF-Tu had been replaced with Val in T. thermophilus HB8 EF-Tu. An extra amino acid sequence of ten residues, consisting predominantly of basic amino acids (Met-182-Gly-191), which does not occur in EF-Tu of E. coli, was found in T. thermophilus HB8.     

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.     

Glutamyl-tRNA synthetase from Thermus thermophilus HB8. Molecular cloning of the gltX gene and crystallization of the overproduced protein.

The gene for the Glu-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8, was isolated using a synthetic oligonucleotide probe coding for the N-terminal amino acid sequence of Glu-tRNA synthetase. Nucleotide-sequence analysis revealed an open reading frame coding for a protein composed of 468 amino acid residues (Mr 53,901). Codon usage in the T. thermophilus Glu-tRNA synthetase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G + C content in the third position of the codons was as high as 94%. In contrast, the amino acid sequence of T. thermophilus Glu-tRNA synthetase showed high similarity with bacterial Glu-tRNA synthetases (35-45% identity); the sequences of the binding sites for ATP and for the 3' terminus of tRNA(Glu) are highly conserved. The Glu-tRNA synthetase gene was efficiently expressed in Escherichia coli under the control of the tac promoter. The recombinant T. thermophilus Glu-tRNA synthetase was extremely thermostable and was purified to homogeneity by heat treatment and three-step column chromatography. Single crystals of T. thermophilus Glu-tRNA synthetase were obtained from poly(ethylene glycol) 6000 solution by a vapor-diffusion technique. The crystals diffract X-rays beyond 0.35 nm. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters of a = 8.64 nm, b = 8.86 nm and c = 8.49 nm.     

Cloning and nucleotide sequences of the mdh and sucD genes from Thermus aquaticus B

A 3 kb DNA fragment containing the gene (mdh) encoding malate dehydrogenase (MDH) from the thermophile Thermus aquaticus B was cloned in Escherichia coli and its nucleotide sequence determined. Comparative analysis showed the nucleotide sequence to be very closely related to that determined for the Thermus flavus mdh gene and flanking regions, with no differences between the predicted amino acid sequences of the MDHs. A proximal open reading frame, identified as the sucD gene, and the mdh gene may be parts of the same operon in T. aquaticus B. Expression of the T. aquaticus B mdh gene in E. coli was found to be at a relatively low level. A simple method for purification of thermostable MDH from the E. coli clone containing the T. aquaticus B mdh gene is presented.     

Nucleotide sequence of the phosphoglycerate kinase gene from the extreme thermophile Thermus thermophilus. Comparison of the deduced amino acid sequence with that of the mesophilic yeast phosphoglycerate kinase.

Using oligonucleotide probes derived from amino acid sequencing information, the structural gene for phosphoglycerate kinase from the extreme thermophile, Thermus thermophilus, was cloned in Escherichia coli and its complete nucleotide sequence determined. The gene consists of an open reading frame corresponding to a protein of 390 amino acid residues (calculated Mr 41,791) with an extreme bias for G or C (93.1%) in the codon third base position. Comparison of the deduced amino acid sequence with that of the corresponding mesophilic yeast enzyme indicated a number of significant differences. These are discussed in terms of the unusual codon bias and their possible role in enhanced protein thermal stability.     

Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2

Metapyrocatechase which catalyzes the oxygenative ring cleavage of catechol to form alpha-hydroxymuconic epsilon-semialdehyde is encoded by the xylE gene on the TOL plasmid of Pseudomonas putida mt-2. We have cloned the xylE region in Escherichia coli and determined the nucleotide sequence of the DNA fragment of 985 base pairs around the gene. The fragment included only one open translational frame of sufficient length to accommodate the enzyme. The predicted amino acid sequence consisted of 307 residues, and its NH2- and COOH-terminal sequences were in perfect agreement with those of the enzyme recently determined (Nakai, C., Hori, K., Kagamiyama, H., Nakazawa, T., and Nozaki, M. (1983) J. Biol. Chem. 258, 2916-2922). A mutant plasmid was isolated which did not direct the synthesis of the active enzyme. This plasmid had a DNA region corresponding to the NH2-terminal two-thirds of the polypeptide. From the deduced amino acid sequence, the secondary structure was predicted. Around 10 base pairs upstream from the initiator codon for metapyrocatechase, there was a base sequence which was complementary to the 3'-end of 16 S rRNAs from both E.coli and Pseudomonas aeruginosa. A preferential usage of C- and G-terminated codons was found in the coding region xylE, which contributed to the relatively high G + C content (57%) of this gene.