Thermoregulation of virB transcription in Shigella flexneri by sensing of changes in local DNA superhelicity

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Abstract
Transcription of the virB gene, a transcriptional regulator of invasion genes on the large plasmid of Shigella flexneri, is strictly regulated by growth temperature; when bacteria are grown at 37 degrees C, virB transcription is highly activated, while at 30 degrees C the level of virB transcription decreases to less than 5% of that at 37 degrees C. Transcription from the virB promoter is activated by VirF, which is encoded on the same plasmid, in a DNA superhelicity-dependent manner (T. Tobe, M. Yoshikawa, T. Mizuno, and C. Sasakawa, J. Bacteriol. 175:6142-6149, 1993). Here we provide evidence supporting the involvement of negative superhelicity in the thermoregulation of virB transcription. A local negatively supercoiled domain in the virB promoter region was created by activating a divergent transcription from the T7 RNA polymerase-dependent promoter, phi 10, which was placed upstream of the virB promoter in the opposite orientation. Transcription from the virB promoter was activated even at 30 degrees C by induction of divergent transcription. Levels of virB transcription correlated with levels of expressed T7 RNA polymerase. Transcriptional activation of virB by the system depended completely upon VirF function. The level of virB transcription achieved by introducing a negatively supercoiled domain was enough to give rise to expression of invasion capacity at 30 degrees C. These results indicated that the repression of virB transcription at 30 degrees C was caused by a reduction in negative superhelicity around the virB promoter region at 30 degrees C.