Elevated unconstrained supercoiling of plasmid DNA generated by transcription and translation of the tetracycline resistance gene in eubacteria

Abstract

Our previous studies have indicated that the leu-500 promoter of Salmonella typhimurium is activated by local supercoiling arising from the transcription of a divergent promoter (Chen et al., 1992). For this to occur on a plasmid, we have shown that the transcribing RNA polymerase must be anchored to the cell membrane by transcription, translation, and export of the tetA gene and that the cell background must be topA. In this study we have used (AT)n reporter sequences to analyze changes in unconstrained supercoiling of plasmid DNA under the circumstances in which the leu-500 promoter becomes activated. (AT)n sequences undergo a structural transition to a cruciform at a threshold level of negative supercoiling that is determined by the length of the tract, and this can be detected in the cellular DNA by in situ chemical probing. These studies have shown that there is elevated unconstrained supercoiling in tetA-carrying plasmids in either Escherichia coli or S. typhimurium cells in exponential growth. This oversupercoiling depends on the function of the tetA gene in cis and the delta topA cell background. These are exactly the conditions that lead to the activation of the leu-500 promoter, supporting the proposed mechanism for the suppression of the leu-500 mutation by topA. Use of (AT)n sequences of different lengths has permitted us to estimate the extent of oversupercoiling. When the tetA gene was initiated using the strong tac promoter, we were able to detect increased unconstrained DNA supercoiling even in topA+ E. coli cells.