Topological promoter coupling in Escherichia coli: delta topA-dependent activation of the leu-500 promoter on a plasmid

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Abstract
The leu-500 promoter of Salmonella typhimurium is activated in topA mutants. We have previously shown that this promoter can be activated on circular plasmids in a manner that depends on transcription and translation of the tetracycline resistance gene tetA and insertion of its product into the cell membrane. We have suggested that in the absence of enzymatic relaxation by topoisomerase I, the local domain of transcription-induced DNA supercoiling reaches a steady-state level that leads to the activation of the leu-500 promoter. In the present paper, we have shown that the leu-500 promoter may also be activated in Escherichia coli. Comparison of the closely related pair of E. coli strains DM800 (delta topA) and SD108 (topA+) shows that the activation is dependent on the presence of a null mutation in topA. We have also shown that activation of the plasmid-borne leu-500 promoter depends, as in S. typhimurium, on the function of an adjacent tetA gene, suggesting that membrane anchorage of the TetA peptide prevents dissipation of transcription-induced supercoiling by superhelical diffusion. The activity of the leu-500 promoter is boosted by placing a divergent tac promoter on the side opposite to tetA. The topoisomer distributions of these plasmids extracted from the cell have been analyzed. We find that when the parent plasmid pLEU500Tc, containing the leu-500 promoter upstream of the complete tetA gene, is extracted from E. coli DM800 (delta topA), the distribution of linking numbers is bimodal. There is a fraction with a lower level of supercoiling (mean linking difference approximately -0.05) that is constant for all plasmids extracted from either delta topA or topA+ cells. In addition, we observe a second fraction with highly negatively supercoiled DNA (mean linking difference approximately -0.09) only in DNA extracted from delta topA cells. The proportion of the oversupercoiled fraction correlates with the activity of the leu-500 promoter: it is strongly reduced when the tetA promoter is deleted or when translation of TetA is prematurely terminated, while it is increased when the strong tac promoter is present in cis. We suggest that this oversupercoiled fraction represents the proportion of plasmid molecules active in tetA transcription and that it is this supercoiling that activates the leu-500 promoter.