Rolf Menzel

DNA supercoiling and bacterial adaptation: thermotolerance and thermoresistance.

When bacterial cells are shifted to higher temperatures their degree of DNA supercoiling changes. Topoisomerases are involved in bacterial adaptation to environmental changes requiring rapid shifts in gene expression. This role in heat shock has been elucidated by genetic studies on the Escherichia coli topA gene and its sigma 32-dependent promoter, P1. Other studies have shown that certain gyrA mutants have increased thermoresistance.

EFFECT OF THE DELETION OF THE SIGMA 32-DEPENDENT PROMOTER (P1) OF THE ESCHERICHIA COLI TOPOISOMERASE I GENE ON THERMOTOLERANCE

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli. The P1 promoter of topA has previously been shown to be a σ32‐dependent heat‐shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is >10‐fold more sensitive to heat treatment (52°C) than the wild type. After brief treatment at 42°C, wild‐type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52°C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100‐fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat‐survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat‐inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse‐labelling pattern of proteins at 42°C (displayed on SDS–polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.

The creation of a camptothecin-sensitive Escherichia coli based on the expression of the human topoisomerase I

The synthesis of the human topoisomerase I (Topo I) in Escherichia coli (Ec) results in phenotypes consistent with the appearance of a DNA-relaxing activity. High-level expression was problematic and recloning the gene in a reduced-copy-number plasmid under more stringently regulated control produced a stable plasmid. An Ec strain with an inducible sensitivity to the eukaryotic Topo I poison, camptothecin (CPT), was constructed by introducing a mutation (imp4312) known to enhance the permeability of Ec to a wide variety of compounds. We are able to infer that CPT sensitivity in Ec involves DNA damage by noting elevated sensitivity in a repair-deficient recA strain and by observing the induction of a sulA::lac fusion following exposure to the drug.

Microbial resistance: novel screens for a contemporary problem.

Historically, natural products have been the source of a large variety of antibacterial agents. In the 1980s, no additional useful antibacterial agents were discovered, leading to the belief that most useful chemotypes from natural product sources had already been discovered. At this time, advances in biotechnology made it feasible to produce sufficient enzyme to set up cell‐free screens. Chemical compound libraries and combinatorial synthesis became the source of chemical diversity for the screens. In spite of these efforts, very few new antibacterial agents have been discovered in the last decade. At Small Molecule Therapeutics, Inc., we have developed phenotype‐based screens that take advantage of the natural physiology and biochemistry of the target enzymes. We have developed a screen to identify bacterial DNA gyrase and topoisomerase IV poisons. The “hits” identified in this screen are being characterized further. A second screen has also been developed against bacterial topoisomerase 1 in which compounds that cause DNA damage through their interaction with bacterial topoisomerase 1 have been identified. Three of the compounds identified in the screen inhibit DNA relaxation mediated by bacterial topoisomerase 1, induce DNA cleavage, are noncytotoxic at >10μM, and have MICs of 4.0 μg/mL against Staphylococcus aureus.