Activation and silencing of leu-500 promoter by transcription-induced DNA supercoiling in the Salmonella chromosome

The notion that transcription can generate supercoils in the DNA template l argely stems from work with small circular plasmids. In the present work, w e tested this model in the bacterial chromosome using a supercoiling-sensit ive promoter as a functional sensor of superhelicity changes. The leu-500 p romoter of Salmonella typhimurium is a mutant and inactive variant of the l eucine operon promoter that regains activity if negative DNA supercoiling r ises above normal levels, typically as a result of mutations affecting DNA topoisomerase I (topA mutants). Activation of the leu-500 promoter was anal ysed in topA mutant cells harbouring transcriptionally inducible tet or cat gene cassettes inserted in the region upstream from the leu operon, Some i nsertions inhibited leu-500 promoter activation in the absence of inducer. This effect is dramatic in the interval between 1.7 kb and 0.6 kb from the leu operon, suggesting that the insertions physically interfere with the me chanism responsible for activation. Superimposed on these effects, transcri ption of the inserted gene stimulated or inhibited leu-500 promoter activit y depending on whether this gene was oriented divergently from the leu oper on or in the same direction respectively. Interestingly, transcription-medi ated inhibition of leu-500 promoter was observed with inserts as far as 5 k b from the leu operon, and it could be relieved by the introduction of a st rong gyrase site between the inserted element and the leu-500 promoter. The se results are consistent with the idea that transcriptionally generated po sitive and negative supercoils can diffuse along chromosomal DNA and, depen ding on their topological sign, elicit opposite responses from the leu-500 promoter.