Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA).

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillus stearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragment containing the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open reading frame encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B. stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a large amount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B. stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the amino acid sequence to the catalase-peroxidase of E. coli.     

Gene cloning and sequence determination of leucine dehydrogenase from Bacillus stearothermophilus and structural comparison with other NAD(P)+-dependent dehydrogenases

The gene for leucine dehydrogenase (EC 1.4.1.9) from Bacillus stearothermophilus was cloned and expressed in Escherichia coli. The selection for the cloned gene was based upon activity staining of the replica printed E. coli cells. A transformant showing high leucine dehydrogenase activity was found to carry an about 9 kilobase pair plasmid, which contained 4.6 kilobase pairs of B. stearothermophilus DNA. The nucleotide sequence including the 1287 base pair coding region of the leucine dehydrogenase gene was determined by the dideoxy chain termination method. The translated amino acid sequence was confirmed by automated Edman degradation of several peptide fragments produced from the purified enzyme by trypsin digestion. The polypeptide contained 429 amino acid residues corresponding to the subunit (Mr 49,000) of the hexameric enzyme. Comparison of the amino acid sequence of leucine dehydrogenase with those of other pyridine nucleotide dependent oxidoreductases registered in a protein data bank revealed significant sequence similarity, particularly between leucine and glutamate dehydrogenases, in the regions containing the coenzyme binding domain and certain specific residues with catalytic importance.     

Nucleotide sequence of the phosphofructokinase gene from Bacillus stearothermophilus and comparison with the homologous Escherichia coli gene

The gene (Bs-pfk) for phosphofructokinase (PFK) from Bacillus stearothermophilus has been cloned and sequenced. The deduced amino acid sequence is nearly identical to the sequence which was previously determined by peptide analysis. The elevated G + C content of Bs-pfk relative to the homologous Ec-pfkA from Escherichia coli is consistent with previous observations concerning genes from thermophilic prokaryotes. A significant degree of homology exists when the deduced amino acid sequence of B. stearothermophilus PFK is compared with the corrected sequences of rabbit muscle PFK or E. coli PFK-1. The cloning and sequencing of Bs-pfk completes the first step toward using site-specific mutagenesis to investigate the structure-function relationships for this allosteric enzyme.     

Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus caldophilus GK24 and the deduced amino-acid sequence of the enzyme

The gene for L-lactate dehydrogenase (LDH) (EC 1.1.1.27) of Thermus caldophilus GK24 was cloned in Escherichia coli using synthetic oligonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of the LDH was deduced from the nucleotide sequence. The deduced amino acid sequence agreed with the NH2-terminal and COOH-terminal sequences previously reported and the determined amino acid sequences of the peptides obtained from trypsin-digested T. caldophilus LDH. The LDH comprised 310 amino acid residues and its molecular mass was determined to be 32,808. On alignment of the whole amino acid sequences, the T. caldophilus LDH showed about 40% identity with the Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle LDHs. The T. caldophilus LDH gene was expressed with the E. coli lac promoter in E. coli, which resulted in the production of the thermophilic LDH. The gene for the T. caldophilus LDH showed more than 40% identity with those for the human and mouse muscle LDHs on alignment of the whole nucleotide sequences. The G + C content of the coding region for the T. caldophilus LDH was 74.1%, which was higher than that of the chromosomal DNA (67.2%). The G + C contents in the first, second and third positions of the codons used were 77.7%, 48.1% and 95.5% respectively. The high G + C content in the third base caused extremely non-random codon usage in the LDH gene. About half (48.7%) the codons in the LDH gene started with G, and hence there were relatively high contents of Val, Ala, Glu and Gly in the LDH. The contents of Pro, Arg, Ala and Gly, which have high G + C contents in their codons, were also high. Rare codons with U or A as the third base were sometimes used to avoid the TCGA sequence, the recognition site for the restriction endonuclease, TaqI. Two TCGA sequences were found only in the sequence of CTCGAG (XhoI site) in the sequenced region of the T. caldophilus DNA. There were three segments with similar sequences in the two 5' non-coding regions, probably the promoter and ribosome-binding regions, of the genes for the T. caldophilus LDH and the Thermus thermophilus 3-isopropylmalate dehydrogenase.     

The rabbit muscle phosphofructokinase gene: cDNA cloning and sequencing

A partial cDNA for rabbit muscle phosphofructokinase (Rm-PFK) has been cloned and sequenced. The nucleotide sequence of the cDNA agrees with the previously determined Rm-PFK genomic sequence. In addition, the amino acid sequence deduced from the cDNA is nearly identical to the Rm-PFK sequence previously determined by peptide analysis. A significant degree of homology exists when the amino acid sequence of Rm-PFK is compared with the sequences of Bacillus stearothermophilus PFK or Escherichia coli PFK-1. The cloning and sequencing of the PFK cDNA fragment represents significant progress toward the long term goal of using site-specific mutagenesis to investigate the structure-function relationships in this allosteric enzyme.     

Nucleotide sequence and linkage map position of the secX gene in maize chloroplast and evidence that it encodes a protein belonging to the 50S ribosomal subunit

The nucleotide sequence of the segment of maize chloroplast DNA lying between the map coordinate positions 32.59 and 32.98 Kb and containing the secX gene has been determined. The derived amino acid sequence of maize chloroplast secX is 95%, 87% and 62% identical to the corresponding derived amino acid sequences from two plant chloroplasts and Escherichia coli, respectively. It is also 70% identical to the experimentally determined amino acid sequence of a protein isolated from Bacillus stearothermophilus ribosomes. Separation of the 50S ribosomal subunit proteins of E. coli by reversed phase HPLC gave a peak which contained pure secX protein, as determined by N-terminal amino acid sequencing. Spinach chloroplast 50S subunit proteins separated by HPLC also gave a peak corresponding to pure secX protein. From these results we conclude that the secX gene in E. coli and in plant chloroplasts encodes a small (37-38 amino acid residues) ribosomal protein belonging to the 50S subunit. The same conclusion has been reached recently by A. Wada with respect to E. coli secX. In agreement with Wada, we name the secX protein L36. Its chloroplast gene is designated rpL36.     

Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfkA, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene has been subcloned into a high-copy-number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 X 10(3)-nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using 'reverse genetics'.     

Glutamyl-tRNA synthetases of Bacillus subtilis 168T and of Bacillus stearothermophilus. Cloning and sequencing of the gltX genes and comparison with other aminoacyl-tRNA synthetases

The glutamyl-tRNA synthetase (GluRS) of Bacillus subtilis 168T aminoacylates with glutamate its homologous tRNA(Glu) and tRNA(Gln) in vivo and Escherichia coli tRNA(1Gln) in vitro (Lapointe, J., Duplain, L., and Proulx, M. (1986) J. Bacteriol. 165, 88-93). The gltX gene encoding this enzyme was cloned and sequenced. It encodes a protein of 483 amino acids with a Mr of 55,671. Alignment of the amino acid sequences of four bacterial GluRSs (from B. subtilis, Bacillus stearothermophilus, E. coli, and Rhizobium meliloti) gives 20% identity and reveals the presence of several short highly conserved motifs in the first two thirds of these proteins. Conserved motifs are found at corresponding positions in several other aminoacyl-tRNA synthetases. The only sequence similarity between the GluRSs of these Bacillus species and the E. coli glutaminyl-tRNA synthetase (GlnRS), which has no counterpart in the E. coli GluRS, is in a segment of 30 amino acids in the last third of these synthetases. In the three-dimensional structure of the E. coli tRNA(Gln).GlnRS.ATP complex, this conserved peptide is near the anticodon of tRNA(Gln) (Rould, M. A., Perona, J. J., S?ll, D., and Steitz, T. A. (1989) Science 246, 1135-1142), suggesting that this region is involved in the specific interactions between these enzymes and the anticodon regions of their tRNA substrates.     

On the DNA binding protein II from Bacillus stearothermophilus. II. The amino acid sequence and its relation to those of homologous proteins from other prokaryotes

The complete amino acid sequence of DNA binding protein II from Bacillus stearothermophilus has been determined. The protein contains 90 amino acid residues and has a calculated Mr of 9716. The sequence is compared to homologous molecules from Escherichia coli, Thermoplasma acidophilum, and Pseudomonas aeruginosa (where only a partial sequence is available). The B. stearothermophilus molecule has 58% and 59% residues identical with the two forms of the E. coli protein and 32% with the T. acidophilum protein. There are totally conserved residues at positions 46-48 and 61-65 with an intervening cluster of basic amino acids in all four proteins.     

Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites

The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B. stearothermophilus was determined. The NH2-terminal amino acid sequence analysis of the B. stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids). The B. stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E. coli. The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation. Comparison of the amino acid sequence inferred from the B. stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions.     

Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli.

The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined. The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) beta-galactosidase. The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus beta-galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these beta-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ beta-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.     

Nucleotide sequences of genes encoding heat-stable and heat-labile glyceraldehyde-3-phosphate dehydrogenases; amino acid sequence and protein thermostability

The structural gene (gapST) encoding glyceraldehyde-3-phosphate dehydrogenase (GPDH; EC 1.2.1.12) from Bacillus stearothermophilus has been cloned in Escherichia coli using plasmid pBR322 as a vector; the homologous gene (gapCO) from Bacillus coagulans was cloned from a phage lambda library. Expression of the cloned gap genes revealed that, as in the wild-type (wt) organisms, the GPDH from B. stearothermophilus (GPDH-ST) was intrinsically heat stable (hs) and that from B. coagulans (GPDH-CO) heat labile (hl). The cloned gap genes were sequenced and the deduced amino acid (aa) sequences were found to be highly conserved (91.6% homology), despite the large difference in thermostability between these two enzymes. Of the 28 aa which differ between the two proteins, most of which occur in the middle third of the monomeric subunit, 5 aa involve replacement of alanine in the hl GPDH-CO, by proline in the hs GPDH-ST, and are especially interesting in terms of their potential contributions to thermostability. Conservation at the DNA level is equally dramatic, with the two gap genes exhibiting 93.3% nucleotide sequence homology. These highly expressed genes exhibit an equivalent codon bias, which more closely resembles that of highly expressed E. coli genes, than that of B. stearothermophilus genes whether highly or weakly expressed.     

Complete nucleotide sequence of a gene coding for heat- and pH-stable alpha-amylase of Bacillus licheniformis: comparison of the amino acid sequences of three bacterial liquefying alpha-amylases deduced from the DNA sequences

The gene coding for the heat-stable and pH-stable alpha-amylase of Bacillus licheniformis 584 (ATCC 27811) was cloned in Escherichia coli and the nucleotide sequence of a DNA fragment of 1,948 base pairs containing the entire amylase gene was determined. As inferred from the DNA sequence, the B. licheniformis alpha-amylase had a signal peptide of 29 amino acid residues and the mature enzyme comprised 483 amino acid residues, giving a molecular weight of 55,200. The amino acid sequence of B. licheniformis alpha-amylase showed 65.4% and 80.3% homology with those of heat-stable Bacillus stearothermophilus alpha-amylase and relatively heat-unstable Bacillus amyloliquefaciens alpha-amylase, respectively. Nevertheless, several regions of the alpha-amylases appeared to be clearly distinct from one another when their hydropathy profiles were compared.