M.BstF5I-4, the forth DNA methyltransferase of BstF5I restriction-modification system from Bacillus stearothermophilus F5

The fourth DNA-methyltransferase of the BstF5I restriction-modification (RM) system from Bacillus stearothermophilus F5 (M.BstF5I-4) was discovered, which modifies the adenine residue within the upper strand of the recognition site 5'-GGATG-3'/5'-CATCC-3'. Thus, unlike other known RM systems, the BstF5I RM system comprises four genes encoding DNA-methyltransferases, three of which possess the same substrate specificity and methylate adenine within the 5'-GGATG sequence. The English version of the paper.     

Substrate specificity and biochemical properties of M3.BstF5I DNA methyltransferase from the BstF5I restriction-modification system

Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of λ phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I has nearly fourfold lower turnover number (0.24 min⁻¹) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one. In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally modify the noncanonical 5′-GGATC-3′ DNA sequence with a rate more than one order of magnitude lower than the methylation rate of the canonical 5′-GGATG-3′ recognition site.     

Multiplicity of site-specific DNA methyltransferases of theBstF5I restriction-modification system

A fragment located downstream of the genes for DNA methyltransferases ofBacillus stearothermophilus F5 (M.BstF5I-1 and M.BstF5I-2) was sequenced. The fragment contains a gene for another methylase, M.BstF5I-3, structurally and functionally similar to the N-terminal domain of M.FokI. Thus, in contrast to other restriction-modification systems, theBstF5I system includes three methylases, two being homologous to the individual M.FokI domains.     

Cold lability and dissociation of the F1-ATPase from Bacillus stearothermophilus

Effects of inadequate vitamin E (E) and/or selenium (Se) nutrition on the activities of cytochrome P-450 mixed function oxidase system (heme hydroperoxidase, p-nitroanisole O-demethylase), and epoxide hydrolase have been investigated. Heme hydroperoxidase activity of liver and lung microsomes was significantly decreased in E deficiency. In the liver, Se deficiency resulted in a significant increase in hydroperoxidase activity. In contrast to the peroxidase activity, liver demethylase activity was only marginally affected in $\text{E}\text{Se}$ deficiency states. However, kidney demethylase activity was increased two fold in Se deficient states. Liver microsomal epoxide hydrolase activity was significantly increased in both E and Se deficiency states.     

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。     

Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1

A thermophilic Bacillus stearothermophilus F1 that produced an extremely thermostable alkaline protease was isolated from decomposed oil palm branches. The isolated protease was purified to homogeneity by heat treatment, ultrafiltration and gel filtration chromatography with a 128-fold increase in specific activity and 75% recovery. The protease, which is a serine-type enzyme, has a relative molecular mass of 33 500 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis but only 20 000 by gel-filtration chromatography. The enzyme was optimally active at pH 9.0 and was stable for 24 h at 70° C and in the pH range from 8.0 to 10.0. It was capable of hydrolysing many soluble and insoluble protein substrates but no esterase activity was detected. The enzyme activity was markedly inhibited by Co²⁺ and Hg²⁺, whereas Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺ and Sr²⁺ had little or no inhibitory effect. However, Mn²⁺ strongly activated the protease activity. The protease exhibited a high degree of thermostability [t _1/2 (85° C) = 4 h, (90° C) = 25 min]. The stability at higher temperatures (85° C and above) was shown to be dependent on the presence of Ca²⁺. Correspondence to: A. B. Salleh     

Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus.

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.     

Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis.

A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance [Tcr]), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C. The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline. Bacillus subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.     

Kinetics and inhibition of cyclomaltodextrinase from alkalophilic Bacillus sp. I-5

The cyclomaltodextrinase from alkalophilic Bacillus sp. I-5 (CDase I-5) was expressed in Escherichia coli and the purified enzyme was used for characterization of the enzyme action. The hydrolysis products were monitored by both HPLC and high-performance ion chromatography analysis that enable the kinetic analysis of the cyclomaltodextrin (CD)-degrading reaction. Analysis of the kinetics of cyclomaltodextrin hydrolysis by CDase I-5 indicated that ring-opening of the cyclomaltodextrin was the major limiting step and that CDase I-5 preferentially degraded the linear maltodextrin chain by removing the maltose unit. The substrate binding affinity of the enzyme was almost same for those of cyclomaltodextrins while the rate of ring-opening was the fastest for cyclomaltoheptaose. Acarbose and methyl 6-amino-6-deoxy-alpha-d-glucopyranoside were relatively strong competitive inhibitors with K(i) values of 1.24 x 10(-3) and 8.44 x 10(-1) mM, respectively. Both inhibitors are likely to inhibit the ring-opening step of the CD degradation reaction.     

A pulse-radiolysis study of the manganese-containing superoxide dismutase from Bacillus stearothermophilus.

In the preceding paper the mechanism of catalysis of the manganese-containing superoxide dismutase from Bacillus stearothermophilus was shown to involve a 'fast cycle' and a 'slow cycle' [McAdam, Fox, Lavelle & Fielden, 1977 (Biochem. J. 165, 71-79)]. Further properties of the enzyme was considered in the present paper. Pulse-radiolysis studies, under conditions of low substrate concentration to (i.e. when the fast cycle predominates), showed that enzyme activity decreases as pH increases (6.5-10.2). Activity was unaffected by the addition of H2O2 or NaN3 but slightly decreased by KCN. Both H2O2 and the reducing radical anion CO2-- caused a decrease in A480 of the native enzyme. The rate of the fast catalytic cycle was independent of temperature (5-55 degrees C), and as temperature increases the slow cycle becomes relatively more important. Arrhenius parameters of the rate contants were estimated. The possible identity of the various forms of the enzyme is considered.     

Role of DNA minor groove interactions in substrate recognition by the M.SinI and M.EcoRII DNA (cytosine-5) methyltransferases

The SinI and EcoRII DNA methyltransferases recognize sequences (GG^A/_TCC and CC^A/_TGG, respectively), which are characterized by an ^A/_T ambiguity. Recognition of the A·T and T·A base pair was studied by in vitro methyltransferase assays using oligonucleotide substrates containing a hypoxanthine·C base pair in the central position of the recognition sequence. Both enzymes methylated the substituted oligonucleotide with an efficiency that was comparable to methylation of the canonical substrate. These observations indicate that M.SinI and M.EcoRII discriminate between their canonical recognition site and the site containing a G·C or a C·G base pair in the center of the recognition sequence (GG^G/_CCC and CC^G/_CGG, respectively) by interaction(s) in the DNA minor groove. M.SinI mutants displaying a decreased capacity to discriminate between the GG^A/_TCC and GG^G/_CCC sequences were isolated by random mutagenesis and selection for the relaxed specificity phenotype. These mutations led to amino acid substitutions outside the variable region, previously thought to be the sole determinant of sequence specificity. These observations indicate that ^A/_T versus ^G/_C discrimination is mediated by interactions between the large domain of the methyltransferase and the minor groove surface of the DNA.     

Cloning and Expression of Thermostable α-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis

The structural gene for a thermostable α-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more α-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the α-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the α-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80°C for 60 min.     

Structure of the arginine repressor from Bacillus stearothermophilus.

The arginine repressor (ArgR) is a hexameric DNA-binding protein that plays a multifunctional role in the bacterial cell. Here, we present the 2.5 A structure of apo-ArgR from Bacillus stearothermophilus and the 2.2 A structure of the hexameric ArgR oligomerization domain with bound arginine. This first view of intact ArgR reveals an approximately 32-symmetric hexamer of identical subunits, with six DNA-binding domains surrounding a central oligomeric core. The difference in quaternary organization of subunits in the arginine-bound and apo forms provides a possible explanation for poor operator binding by apo-ArgR and for high affinity binding in the presence of arginine.     

Characterization of a restriction-modification system of the thermotolerant methylotroph Bacillus methanolicus.

We report the isolation of a restriction endonuclease, BmeTI, an isoschizomer of BclI, that recognizes the DNA sequence 5' TGATCA 3'. We also report that BmeTI sites are modified to TGm6ATCA. These findings provide the basis for devising strategies to prevent BmeTI restriction of any DNA introduced into Bacillus methanolicus.     

Partial purification and characterization of type I DNA topoisomerase from Bacillus stearothermophilus

Type I DNA topoisomerase was partially purified from Bacillus stearothermophilus by ammonium sulfate precipitation and column chromatographies on phosphocellulose, DEAE-cellulose and heparin-agarose. On heparin-agarose chromatography, topoisomerase I activity was separated into three fractions (designated Fractions A, B, and C). Each fraction was further subjected to gel filtration on Sephacryl S-200. From electrophoretic analysis on polyacrylamide gel, Fraction A was found to contain two enzyme species having molecular weights of 110,000 and 100,000, and Fraction B one enzyme species with a molecular weight of 80,000. The molecular weight of the enzyme in Fraction C was estimated to be around 150,000 by gel filtration. The enzymes in Fractions A and B exhibited little activity in the presence of Mg2+, while the activity was increased remarkably by NaCl with Mg2+. No activity was observed in the presence of NaCl alone. The enzyme in Fraction C required only Mg2+ for full activity. With Fraction A, the topoisomerase I-induced cleavage sites on tetracycline-resistant plasmid pNS1 (2.55 megadaltons) were mapped. Fraction A cleaved the DNA at ten specific sites. These sites were compared to those of the Haemophilus gallinarum enzyme, which have already been mapped (Shishido et al. (1983) Biochem. Biophys. Acta 740, 108). The results showed that there is a remarkably coincidence between the cleavage sites induced by the B. stearothermophilus and H. gallinarum enzymes.