Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.     

Characteristics of thermostable pullulanase from Bacillus stearothermophilus and the nucleotide sequence of the gene

Thermostable pullulanase was purified to homogeneity on sodium dodecyl sulfate-polyacrylamide gel from the culture supernatant of Bacillus stearothermophilus TRS128. However, multiformity of the pullulanase was suggested by activity staining on a pullulan-reactive red plate. The thermostability of the enzyme was tested. In the presence of Ca²⁺, the optimum temperature of the pullulanase was 75°C, and nearly 100% of the enzyme activity was retained even after treatment at 68°C for 60 min. Since the thermostable pullulanase gene (pulT) has been cloned, the nucleotide sequence was determined. Although the DNA sequence revealed only one large open reading frame, two possible pairs of SD sequence and initiation codon were found in the frame. To analyze the regulatory region, several mutations (deletion, insertion and substitution of nucleotides) were introduced in the flanking region of pulT, using site-directed mutagenesis. A putative promoter, SD sequence and initiation codon were inferred. The pulT gene was composed of 1974 bases and 658 amino acid residues (molecular weight 75,375). The deduced amino acid sequence of the thermostable pullulanase exhibited a fairly low homology with that of the thermolabile pullulanase from Klebsiella aerogenes. However, four consensus sequences containing catalytic and/or substrate binding sites for amylolytic enzymes were also found in the thermostable pullulanase and the thermolabile enzyme.     

Cloning and nucleotide sequence of the highly thermostable neutral protease gene from Bacillus stearothermophilus

The gene (nprM) for the highly thermostable neutral protease of Bacillus stearothermophilus MK232 was cloned in Bacillus subtilis using pTB53 as a vector. The nucleotide sequence of nprM and its flanking regions was determined. The DNA sequence revealed only one large open reading frame, composed of 1656 base pairs and 552 amino acid residues. A Shine-Dalgarno (SD) sequence was found 12 bases upstream from the translation start site (ATG). A possible promotor sequence (TTTTCC for the -35 region and TATTGT for the -10 region), which was nearly identical to the promoter for another thermostable neutral protease gene, nprT, was also found about 40 bases upstream of the SD sequence. The deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first five amino acids of purified extracellular protease completely matched residues 237-241 of the open reading frame. This suggests that the enzyme is translated as a large polypeptide containing a pre-pro structure as is known for other neutral proteases. The amino acid sequence of the extracellular form of this protease (316 amino acids, molecular mass 34,266 Da) was identical to that of the thermostable neutral protease (thermolysin) from Bacillus thermoproteolyticus except for two amino acid substitutions (Asp37 to Asn37 and Glu119 to Gln119). The G + C content of the coding region of nprM was 42 mol%, while that of the third letter of the codons was lower (36 mol%). This extremely low content is an exceptional case for genes from thermophiles. When the protease genes, nprM and nprT, were cloned on pTB53 in B. subtilis, the expression of nprM was about 20 times higher than that of nprT. The reason for the difference between the two systems is discussed.     

Nucleotide sequence of the neopullulanase gene from Bacillus stearothermophilus

The gene (nplT) for a new type of pullulan-hydrolysing enzyme, neopullulanase, from Bacillus stearothermophilus TRS40 was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1764 bases and 588 amino acid residues (Mr 69144). Although the thermostable neopullulanase contained eight cysteine residues, they did not provide conformational stability by disulphide bonds. A comparison was made of the amino acid sequences of alpha-amylase, neopullulanase, isoamylase, pullulanase and cyclodextrin glucanotransferase. All the enzymes examined contained four highly conserved regions which probably constitute the active centres of the enzymes. The amino acid residues required for the specificity of neopullulanase are compared with those of alpha-amylase and other amylolytic enzymes.     

Cloning and nucleotide sequence of Bacillus stearothermophilus pyruvate carboxylase

A gene for prokaryotic pyruvate carboxylase (PC) was cloned from Bacillus stearothermophilus. It has an open reading frame of 3441 base pairs which can code for a protein of 128 353 Da. Not only the molecular size and domain organization but also the deduced amino acid sequence of B. stearothermophilus PC are similar to those of eukaryotic PCs.     

Characterization of the relA/spoT gene from Bacillus stearothermophilus

By use of degenerate primers, we amplified a fragment of a relA/spoT homologous gene from Bacillus stearothermophilus. Chromosomal walking enabled us to sequence the entire gene and its flanking regions. The primary sequence of the gene product is 78% identical to the RelA/SpoT homologue of Bacillus subtilis and both gene loci share a similar genetic organization. The B. stearothermophilus rel gene was analyzed in vivo by heterologous expression in the B. subtilis relA deletion strain TW30, and is shown to complement the growth defects of TW30. The recombinant RelBst protein was detected by Western immunoanalysis, and synthesizes guanosine-3'-diphosphate-5'-(tri)diphosphate ((p)ppGpp) after amino acid stress and carbon starvation. These in vivo data, the genetic organization, and the primary structure compared to other RelA/SpoT homologues provide circumstantial evidence that the identified gene encodes the only (p)ppGpp synthetase in B. stearothermophilus presumed to serve also as (p)ppGpp hydrolase.     

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.     

Characterization of an extracellular protease inhibitor of Bacillus brevis HPD31 and nucleotide sequence of the corresponding gene.

A novel proteinaceous protease inhibitor was isolated from the culture supernatant of Bacillus brevis HPD31. The protease inhibitor of B. brevis (designated BbrPI) was produced extracellularly in multiple forms having at least three different molecular weights. One of them, BbrPI-a, was purified to near homogeneity and only showed inhibitory activity toward serine proteases, such as trypsin, chymotrypsin, and subtilisin. BbrPI was presumed to form a trypsin-inhibitor complex in a molar ratio of 1:1. The inhibitor was found to be heat resistant at neutral and acidic pHs. The gene coding for BbrPI was cloned into Escherichia coli, and its nucleotide sequence was determined. The sequence suggested that BbrPI is produced with a signal peptide of 24 amino acid residues. The amino acid sequence of the protein deduced from the DNA sequence contained the amino acid sequences of amino termini of the inhibitors, a, b, and c, and their putative precursor determined chemically. The molecular weight of the precursor was about 33,000, and the molecular weights of inhibitors a, b, and c were about 22,000, 23,500, and 24,000, respectively. It is presumed that the secreted precursor protein, which is probably inactive, is cleaved by protease into several active protease inhibitor molecules. BbrPI shows no significant homology to the protease inhibitors described previously and is unique in not having any cysteine residues in its molecule.     

Complete nucleotide sequence of pSTK1, a cryptic plasmid from Bacillus stearothermophilus TK015

Summary The complete nucleotide sequence of pSTK1, a cryptic plasmid isolated from B. stearothermophilus TK015, has been determined. pSTK1 has been shown to be 1883 bp in length and contain three open reading frames (ORFs), one of which has a helix-turn-helix motif typical of DNA-binding proteins. Also identified was a region that can form an extensive secondary structure, which would show a high degree of similarity to palA, an origin for minus strand elongation in rolling circle replication.     

Cloning and nucleotide sequence of the gene coding for a subunit of the respiratory NADH dehydrogenase from Bacillus stearothermophilus

The putative gene coding for a subunit of the respiratory NADH dehydrogenase from Bacillus stearothermophilus was cloned in Escherichia coli and the nucleotide sequence was determined. A large open reading frame (ORF1) was recognized, which was composed of 879 bp corresponding to 293 amino acids and a molecular weight of 33,600. Possible promoter and Shine-Dalgarno sequences were found upstream from the initiation codon. The deduced amino acid sequence of the gene was homologous to the NADH dehydrogenase of Paramecium aurelia.     

Nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene.

The nucleotide sequence of the Bacillus stearothermophilus alpha-amylase gene and its flanking regions was determined. An open reading frame was found, comprising a total of 1,647 base pairs (549 amino acids) and starting from a GUG codon as methionine. It was shown by NH2-terminal amino acid sequence analysis that the extracellular amylase consisted of 515 amino acid residues, which corresponded to a molecular weight of 58,779. Thus the NH2-terminal portion of the gene encodes 34 amino acid residues as a signal peptide. The amino acid sequence deduced from the alpha-amylase gene was fairly homologous (61%) with that of another thermostable amylase from Bacillus amyloliquefaciens.     

Cloning and characterization of the HPr kinase/phosphorylase gene from Bacillus stearothermophilus No. 236

The Bacillus stearothermophilus no. 236 gene encoding the bifunctional enzyme HprK/P, the key regulator of carbon catabolite repression/activation (CCR/CCA) in most Gram-positive bacteria, was cloned and the (His)(6)-tagged gene product was characterized in detail. The nucleotide sequence of the hprK/P gene corresponded to an open reading frame of 951 bp that encoded a polypeptide of 316 amino acid residues with a calculated molecular mass of 35,458 Da. The deduced amino acid sequence of the B. stearothermophilus no. 236 HprK/P showed 64.5% identity with the B. subtilis enzyme, allowing us to identify two highly conserved motifs, the nucleotide binding P-loop (Walker motif A) and the HprK/P family signature sequence in the C-terminal half of the protein. Furthermore, complementation experiments showed that the cloned hprK/P gene product was functionally active in the B. subtilis cells. The purified (His)(6)-tagged B. stearothermophilus no. 236 HprK/P migrated on SDS-PAGE gel as a single species with a molecular mass of about 36 kDa, and behaved in gel filtration like a hexameric protein. The recombinant protein catalyzes the pyrophosphate (PPi)-dependent (highest activity at pH 7.0 and 40 degrees C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45 degrees C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40 degrees C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg(2+), was an indispensable cofactor for the three activities of the HprK/P. Fructose-1,6-bisphosphate (FBP) was observed to stimulate ATP-dependent kinase activity, while inorganic phosophate (Pi) inhibited ATP-dependent kinase activity. Mutations in the Walker motif A simultaneously abolished both types of kinase and phosphorylase activities. On the other hand, the conserved signature residues were confirmed to be involved in the PPi-dependent kinase and phosphorylase reactions.     

嗜热脂肪芽孢杆菌DNA解链蛋白1的纯化及性质

以嗜热脂肪芽孢杆菌为材料,通过ＰｏｌｙｍｉｎＰ沉淀,硫酸铵分级及Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ,ＤＥＡＥ纤维素,磷酸纤维素,ＦＰＬＣ ＭｏｎｏＱ,ＦＰＬＣ Ｓｕｐｅｒｏｓｅ１２等柱层析,得到部分纯化的ＤＮＡ解链蛋白１。ＢｓｔＨ１具有依赖ＤＮＡ和Ｍｇ＾２＋的ＡＴＰ酶活力,不同类型的核酸对ＢｓｔＨ１的ＡＴＰ酶活力的促进作用不同。     

Nucleotide sequence and promoter region for the neutral protease gene from Bacillus stearothermophilus.

The thermostable neutral protease gene nprT of Bacillus stearothermophilus was sequenced. The DNA sequence revealed only one large open reading frame, composed of 1,644 bases and 548 amino acid residues. A Shine-Dalgarno sequence was found 9 bases upstream from the translation start site (ATG), and the deduced amino acid sequence contained a signal sequence in its amino-terminal region. The sequence of the first 14 amino acids of purified extracellular protease completely matched that deduced from the DNA sequence starting at GTC (Val), 687 bases (229 amino acids) downstream from ATG. This suggests that the protease is translated as a longer polypeptide. The amino acid sequence of the extracellular form of this protease (319 amino acids) was highly homologous to that of the thermostable neutral protease from Bacillus thermoproteolyticus but less homologous to the thermolabile neutral protease from Bacillus subtilis. A promoter region determined by S1 nuclease mapping (TTTTCC for the -35 region and TATTTT for the -10 region) was different from the conserved promoter sequences recognized by the known or factors in bacilli. However, it was very homologous to the promoter sequence of the spo0B gene from B. subtilis. The guanine-plus-cytosine content of the coding region of the nprT gene was 58 mol%, while that of the third letter of the codons was much higher (72 mol%).     

Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis.

Summary The structural gene for the Bacillus stearothermophilus glycogen branching enzyme (glgB) was cloned in Escherichia coli. Nucleotide sequence analysis revealed a 1917 nucleotide open reading frame (ORF) encoding a protein with an M_r of 74787 showing extensive similarity to other bacterial branching enzymes, but with a shorter N-terminal region. A second ORF of 951 nucleotides encoding a 36971 Da protein started upstream of the glgB gene. The N-terminus of the ORF2 gene product had similarity to the Alcaligenes eutrophus czcD gene, which is involved in cobalt-zinc-cadmium resistance. The B. stearothermophilus glgB gene was preceded by a sequence with extensive similarity to promoters recognized by Bacillus subtilis RNA polymerase containing sigma factor H (E - ^H). The glgB promoter was utilized in B. subtilis exclusively in the stationary phase, and only transcribed at low levels in B. subtilis spoOH, indicating that sigma factor H was essential for the expression of the glgB gene in B. subtilis. In an expression vector, the B. stearothermophilus glgB gene directed the synthesis of a thermostable branching enzyme in E. coli as well as in B. subtilis, with optimal branching activity at 53° C.     

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.     

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.