Functional analysis of three topoisomerases that regulate DNA supercoiling levels inChlamydia

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Abstract

Chlamydiais a medically important bacterium that infects eukaryotic cells. Temporal expression of chlamydial genes during the intracellular infection is proposed to be regulated by changes in DNA supercoiling levels. To understand how chlamydial supercoiling levels are regulated, we purified and analyzed three putativeChlamydia trachomatistopoisomerases. As predicted by sequence homology, CT189/190 are the two subunits of DNA gyrase, whereas CT643 is a topoisomerase I. CT660/661 have been predicted to form a second DNA gyrase, but the reconstitute holoenzyme decatenated and relaxed DNA, indicating that the proteins are subunits of topoisomerase IV. Promoter analysis showed that each topoisomerase is transcribed from its own operon by the major chlamydial RNA polymerase. Surprisingly, all three topoisomerase promoters had higher activity from a more supercoiled DNA template. This supercoiling-responsivesness is consistent with negative feedback control of topoisomerase I and topoisomerase IV expression, which is typical of other bacteria. However, activation of the chlamydial gyrase promoter by increased supercoiling is unorthodox compared with the relaxation-induced transcription of gyrase in other bacteria. We present a model in which supercoiling levels during the intracellular chlamydial developmental cycle are regulated by unusual positive feedback control of the gyrase promoter and the temporal expression of three topoisomerases.

In this study, we show thatChlamydia trachomatisencodes three functional DNA topoisomerase, enzymes that modulate DNA supercoiling levels. The promoter for each topoisomerase was upregulated by increased supercoiling, which indicates an unprecedented positive feedback control of the chlamydial gyrase promoter. Our findings support a model in which DNA supercoiling levels during the intracellular chlamydial infection are regulated by unusual features of the promoters for the topoisomerase genes, including their temporal expression and feedback control.

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