Regulation of tetracycline accumulation in Bacillus subtilis bearing B. subtilis plasmid pNS1981

Abstract Accumulation of tetracycline (Tc) into Bacillus subtilis was studied by two methods, one involving a fluorescence assay and the other an absolute determination of accumulated drug. B. subtilis GSY908 harboring B. subtilis plasmid pNS1981 and the plasmidless host strain accumulated equivalent amounts of Tc. Prior exposure of the plasmid-harboring cells to subinhibitory concentration of Tc resulted in marked decrease in accumulation of the drug, indicating that pNS1981-determined Tc resistance is inducible. Of interest was the fact that the amount of Tc accumulated by the Tc-induced plasmid-harboring cells is increased somewhat by the addition of carboxylcyanide- m -chlorophenyl hydrazone (CCCP), an uncoupling reagent. This seems to show that energy-dependent accelerated Tc efflux is involved in decreased Tc accumulation.     

Tetracyclines: antibiotic action,uptake, and resistance mechanisms

Tetracyclines probably penetrate bacterial cells by passive diffusion and inhibit bacterial growth by interfering with protein synthesis or by destroying the membrane. A growing number of various bacterial species acquire resistance to the bacteriostatic activity of tetracycline. The two widespread mechanisms of bacterial resistance do not destroy tetracycline: one is mediated by efflux pumps, the other involves an EF-G-like protein that confers ribosome protection. Oxidative destruction of tetracycline has been found in a few species. Several efflux transporters, including multidrug-resistance pumps and tetracycline-specific exporters, confer bacterial resistance against tetracycline. Single amino acids of these carrier proteins important for tetracycline transport and substrate specificity have been identified, allowing the mechanism of tetracycline transport to begin to emerge. Received: 19 January 1996 / Accepted: 1 March 1996     

Trypanosoma brucei 29-13 strain is inducible in but not permissive for the tsetse fly vector

Using green fluorescent protein as a reporter, we have shown that the strain 29-13 of Trypanosoma brucei, widely used for inducible down-regulation of mRNA, is inducible in, but not permissive for the tsetse flies Glossina palpalis gambiensis and Glossina morsitans morsitans. Within two weeks post-infection, 42% males and females of teneral and non-teneral tsetse flies harboured intestinal infections, yet not a single infection progressed into the salivary glands.     

An improved procedure for expression and purification of ribosomal protection protein Tet(O) for high-resolution structural studies

Tetracycline (Tc) is a broad spectrum antibiotic that binds to the A site of the bacterial ribosome inhibiting delivery of aminoacyl-tRNA to the A site for productive protein biosynthesis. Tet(O) is in a class of the ribosomal protection proteins (RPPs) found in many pathogenic bacteria, that dislodges Tc from the A site of 70S ribosome to restore polypeptide elongation and confer Tc resistance to the bacteria. Considerable difficulty has been encountered in overexpressing and purifying Tet(O) from various Escherichia coli strains using lambdaPI, tac or T7 promoters. Here we report molecular cloning, overexpression of His-tagged Tet(O) in E. coli, an improved purification procedure and initial biochemical and biophysical characterization of His-tagged Tet(O).     

一株受四环素及其衍生物诱导表达的Tet-on鼻咽癌细胞系

Ｔｅｔ－ｏｎ基因表达系统是新近发展的一种真核生物体外表达系统．该系统利用四环素及其衍生物对所感兴趣基因进行诱导表达．这种诱导表达具有严密、高效、可控性强、表达泄露小等优点．对于研究一些致死基因及毒性强的基因在细胞或体内的表达提供了极好的工具．利用Ｔｅｔ－ｏｎ基因表达系统,构建了一株鼻咽癌细胞系,该细胞系的建立为进一步研究一些鼻咽癌发病相关基因,如ＥＢ病毒潜伏膜蛋白基因、瘤基因、抑瘤基因等与鼻咽癌的相关性,提供了理想的细胞模型．     

Hydrixyl radical generation by the tetracycline antibiotics with free radical damage to DNA, lipids and carbohydrate in the presence of iron and copper salts

Tetracycline antibiotics caused the degradation of carbohydrate in the presence of a ferric salt at pH 7.4. This degradation appeared to involve hydroxyl radicals since the damage was substantially reduced by the presence of catalase, superoxide dismutase, scavengers of the hydroxyl radical and metal chelators. Similarly, the tetracycline antibiotics in the presence of a ferric salt greatly stimulated the peroxidation of liposomal membranes. This damage, which did not implicate the hydroxyl radical, was significantly reduced by the addition of chain-breaking antioxidants and metal chelators. Only copper salts in the presence of tetracycline antibiotics, however, caused substantial damage to linear duplex DNA. Studies with inhibitors suggested that damage to DNA did involve hydroxyl radicals.     

Deciphering and engineering of the final step halogenase for improved chlortetracycline biosynthesis in industrial Streptomyces aureofaciens

Chlortetracycline (CTC) is an important member from antibiotics tetracycline (TC) family, which inhibits protein synthesis in bacteria and is widely involved in clinical therapy, animal feeds and aquaculture. Previous works have reported intricately the biosynthesis of CTC from the intermediates in random mutants of Streptomyces aureofaciens and the crucial chlorination remained unclear. We have developed the genetic manipulation in an industrial producer, in which about 15.0 g/l CTC predominated along with 1.2 g/l TC, and discovered that chlorination by ctcP (an FADH₂-dependent halogenase gene) is the last inefficient step during CTC biosynthesis. Firstly, the ΔctcP strain accumulated about 18.9 g/l “clean” TC without KBr addition and abolished the production of CTC. Subsequently, CtcP was identified to exhibit a substrate stereo-specificity to absolute TC (4S) rather than TC (4R), with low k_cat of 0.51±0.01 min⁻¹, while it could halogenate several TC analogs. Accordingly, we devised a strategy for overexpression of ctcP in S. aureofaciens and improved CTC production to a final titer of 25.9 g/l. We anticipate that our work will provide a biotechnological potential of enzymatic evolution and strain engineering towards new TC derivatives in microorganisms.     

Synthetic riboswitches — A tool comes of age

Within the last decade, it has become obvious that RNA plays an important role in regulating gene expression. This has led to a plethora of approaches aiming at exploiting the outstanding chemical properties of RNA to develop synthetic RNA regulators for conditional gene expression systems. Consequently, many different regulators have been developed to act on various stages of gene expression. They can be engineered to respond to almost any ligand of choice and are, therefore, of great interest for applications in synthetic biology. This review presents an overview of such engineered riboswitches, discusses their applicability and points out recent trends in their development. This article is part of a Special Issue entitled: Riboswitches.