Cloning of a tellurite resistance determinant from Bacillus stearothermophilus V in Escherichia coli

A potassium tellurite-resistance determinant was isolated from Bacillus stearothermophilus V and cloned in Escherichia coli. Transformed cells formed black colonies when grown on solid media containing permissive tellurite concentrations. The resistance determinant was contained in a B. stearothermophilus V chromosomal DNA fragment of 7 kb.     

Overproduction and single-step purification of Bacillus stearothermophilus peroxidase in Escherichia coli

Summary The cloned peroxidase gene from Bacillus stearothermophilus was highly expressed in Escherichia coli. Using the high copy number plasmid which is temperature-sensitive and its own strong promoter, this thermostable peroxidase was produced at 28% of the total cell proteins when the cells were grown at 42°C. The enzyme could be easily purified from E. coli by heat treatment and single-column Sephadex G-200 chromatography. From a 200 ml culture, 30 mg of purified enzyme was obtained. The peroxidase produced by E. coli showed a thermostability, haem type and content identical with those of the peroxidase produced by B. stearothermophilus.Offprint requests to: H. Okada     

Expression of the ubiE Gene of Geobacillus stearothermophilus V in Escherichia coli K-12 Mediates the Evolution of Selenium Compounds into the Headspace of Selenite- and Selenate-Amended Cultures

The ubiE gene of Geobacillus stearothermophilus V, with its own promoter, was cloned and introduced into Escherichia coli. The cloned gene complemented the ubiE gene deficiency of E. coli AN70. In addition, the expression of this gene in E. coli JM109 resulted in the evolution of volatile selenium compounds when these cells were grown in selenite- or selenate-amended media. These compounds were dimethyl selenide and dimethyl diselenide.     

Bacillus stearothermophilus plasmid pSTK1 replicon is functional in Escherichia coli

Summary A kanamycin-resistant plasmid possessing a thermostable replicon derived from Bacillus stearothermophilus cryptic plasmid pSTK1 was constructed. The plasmid could transform not only B. stearothermophilus and Bacillus subtilis, but also Gram-negative Escherichia coli. The behavior of the plasmid in the hosts was examined. The plasmid was stably maintained even at 67°C in B. stearothermophilus without selective pressure. During the plasmid replication, single-stranded DNA (ssDNA) intermediates were found in E. coli, while these were not found in B. subtilis.     

Thermal denaturation and loss of viability in Escherichia coli and Bacillus stearothermophilus

When Escherichia coli was heated at 10°C/min in a differential scanning calorimeter, the onset of irreversible thermal denaturation occurred at 51°C, about 5°C above the maximum growth temperature. The temperature at which death rate was maximal (63°C) coincided with the thermogram peak caused by denaturation of the 30S ribosomal subunit. The maximum death rate in vegetative cells of Bacillus stearothermophilus occurred at the higher temperature of 71°C which also coincided with the leading edge of the main thermogram peak.     

The BaeSR Two-Component Regulatory System Mediates Resistance to Condensed Tannins in Escherichia coli

The gene expression profiles of Escherichia coli strains grown anaerobically with or without Acacia mearnsii (black wattle) extract were compared to identify tannin resistance strategies. The cell envelope stress protein gene spy and the multidrug transporter-encoding operon mdtABCD, both under the control of the BaeSR two-component regulatory system, were significantly up-regulated in the presence of tannins. BaeSR mutants were more tannin sensitive than their wild-type counterparts.     

Resistance of Thermus spp. to Potassium Tellurite

Two members of the genus Thermus were examined for their resistance to toxic inorganic compounds. They both proved to be fairly resistant to tellurite and selenite and to many other heavy metal salts. Cell extracts of Thermus thermophilus HB8 and of T. flavus AT-62 catalyze the reduction of K₂TeO₃ in a reaction which is dependent on NADH oxidation.     

Correlation between 30S ribosomal proteins of Bacillus stearothermophilus and Escherichia coli

Summary The 30S ribosomal proteins from Bacillus stearothermophilus strains 799 and 10 were purified and correlated with those from E. coli by comparing their two-dimensional electrophoretic mobility, immunological cross-reaction, molecular weight, amino acid composition and partial amino acid sequence. A high degree of similarity was observed among the proteins from these taxonomically distant bacterial species.Paper No. 82 on Ribosomal proteins-preceding paper is by J. Horne and V. A. Erdmann, FEBS Letters, in press.N.R.C.C. No. 13514.     

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

The structural gene for a thermostable alpha-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 alpha-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the alpha-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 alpha-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80 degrees C for 60 min.     

xyLE基因在嗜热脂肪芽孢杆菌中的表达

以嗜热脂肪芽孢杆菌CU21的表达型质粒pFDC11为载体,插入来自恶臭假单孢菌的编码邻苯二酚2,3-双加氧酶(CatO_2ase)的xylE基因,构建成重组质粒pFDX1转化入CU21受体,在48℃培养时得到了表达产物,表明常温细菌的xylE基因可以在高温菌启动子的带动下在高温菌宿主中表达。采用提高培养温度后选择卡那毒素抗性突变的方法,得到了在55℃及6O℃的CatO_2ase表达量明显提高的变异菌株CU21-161。本文同时报道一种用完整细胞悬浮液测定CatO_2ase活力的方法。     

Binding oligonucleotides to Escherichia coli and Bacillus stearothermophilus 5 S RNA.

Binding complementary tri- and tetranucleotides to Escherichia coli A19 and Bacillus stearothermophilus 799 5 S RNAs permitted identification of single-stranded regions in these RNAs. Sequences around positions 10, 30, 60, 70, 85 and 95 are in a single-stranded conformation in both 5 S RNAs. It is concluded that the overall structure of bacterial 5 S RNA has been conserved during evolution. Two types of structural conservation have been observed at specific sites of the 5 S RNA: firstly, nucleotide sequence and single strandedness and secondly, single strandedness only. The oligonucleotide binding data for E. coli 5 S RNA are in general agreement with a previous study (Lewis and Doty, 1970) and do not support fully any proposed structural model.     

Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.

Summary E. coli [³²P]-labelled 5S RNA was complexed with E. coli and B. stearothermophilus 50S ribosomal proteins. Limited T₁ RNase digestion of each complex yielded three major fragments which were analysed for their sequences and rebinding of proteins. The primary binding sites for the E. coli binding proteins were determined to be sequences 18 to 57 for E-L5, 58 to 100 for E-L18 and 101 to 116 for E-L25. Rebinding experiments of purified E. coli proteins to the 5S RNA fragments led to the conclusion that E-L5 and E-L25 have secondary binding sites in the section 58 to 100, the primary binding site for E-L18. Since B. stearothermophilus proteins B-L5 and BL22 were found to interact with sequences 18 to 57 and 58 to 100 it was established that the thermophile proteins recognize and interact with RNA sequences similar to those of E. coli. Comparison of the E. coli 5S RNA sequence with those of other prokaryotic 5S RNAs reveals that the ribosomal proteins interact with the most conserved sections of the RNA.Paper number 12 on structure and function of 5S RNA.Preceding paper: Wrede, P. and Erdmann, V.A. Proc. Natl. Acad. Sci. USA 74, 2706–2709 (1977)     

Sequence homologies between the tryptophanyl tRNA synthetases of Bacillus stearothermophilus and Escherichia coli.

Subcultures of ovaries and testis of the crab Carcinus maenas have been performed in the presence of L-[Me-¹⁴C]methionine. Introduction in the medium of a chromatographically-purified liposoluble fraction from the androgenic glands of the same animal inhibits the biological methylation of the tRNA of the ovaries by 62%. The inhibition of methylation of five individual bases varies from 45% to 84%. No inhibition of tRNA methylation is observed under the same conditions with testis subcultures.     

Molecular cloning of promoter-containing fragments from Bacillus stearothermophilus and their expression in Escherichia coli and Bacillus subtilis

Abstract Bacillus stearothermophilus DNA fragments containing a promoter were isolated in Escherichia coli using a shuttle promoter-probe vector. The molecular sizes of the isolated fragments ranged from 0.78 to 10 kb. The 0.78 and 1.1 kb fragments were selected and examined in some detail for promoter activity in both E. coli and Bacillus subtilis by analysis of expression of erythromycin-resistance (Em^r) and β-galactosidase. The results showed that the two fragments exhibit a high promoter activity in both bacteria. In vitro promoter activity of the 1.1 kb fragment was also shown by RNA syntheses catalyzed by RNA polymerases prepared from E. coli, B. subtilis and B. stearothermophilus .     

Properties of cysK mutants of Escherichia coli K12.

Triazole and azaserine resistant mutants of E. coli K12 affecting cysK gene coding for O-acetylserine sulphydrylase were isolated. The cysK gene in E. coli is located in the same region of chromosome as the cycK gene in Salmonella typhimurium. All azaserine and some triazole resistant mutants require cysteine for growth at a normal rate. The cysK mutants have reduced sulphate uptake. Stability and transfer by conjugation of triazole resistant phenotype were checked. Differences in sulphate metabolism between closely related organisms E. coli and S. typhimurium are discussed.     

The crystallization of ribosomal proteins from the 50 S subunit of the Escherichia coli and Bacillus stearothermophilus ribosome

Several individual intact ribosomal proteins purified from bacterial sources under mild conditions have been crystallized. A number of these are suitable candidates for three-dimensional structural studies by x-ray diffraction techniques. Data collection to 3 A resolution for one of these proteins is in progress.