Expression of the virB gene, the transcriptional regulator for the invasion genes encoded by the large plasmid of Shigella flexneri, is temperature-regulated. virB transcription is under the control of VirF and H-NS, which act as positive and negative regulators, respectively, and is highly responsive to changes in DNA superhelicity. To further investigate the molecular mechanisms underlying the thermoregulation of virB transcription, a mutant which expressed an invasion phenotype at both 30 degrees C and 37 degrees C was isolated using miniTn10-kan (miniKAN) random insertion mutagenesis. The insertion site was mapped to the rho gene, and resulted in the addition of 11 amino acids to the C-terminus of the Rho protein. Consequently, decreased transcription termination activity at a rho-dependent terminator, lambda tL1, was observed. In the rho mutant, both the transcription of virB and expression of invasion genes were activated at 30 degrees C and were less responsive to changes in temperature. The deregulation of virB expression by the mutation was dependent upon the virB promoter, since the effects of the mutation on virB transcription were abolished when its promoter region was replaced by the tac promoter. Temperature-responsive changes in DNA topology, as determined by linking numbers of a reporter plasmid, showed that changes in DNA superhelicity in the rho mutant were smaller than that in the wild type. Furthermore, when the mutant was grown in medium containing novobiocin, an inhibitor of DNA gyrase, virB transcription at 30 degrees C as well as at 37 degrees C was greatly diminished. These results indicated that Rho protein could have a profound effect on topological temperature-dependent changes in DNA structure, thus contributing to thermoregulation of virB transcription.