Negative regulation of heat shock genes by the HrcA repressor protein in Chlamydia trachomatis

This body of work is focused on defining the mechanisms of transcriptional regulation of heat shock genes in the pathogenic bacterium Chlamydia trachomatis. Understanding the regulation of the heat shock genes is significant because the major heat shock proteins of C. trachomatis , GroEL and DnaK, contribute to the development of Chlamydia -related disease. Our work has shown that binding of the transcriptional repressor protein HrcA to an operator sequence called CIRCE is necessary and sufficient for the transcriptional repression of the groE and dnaK heat shock operons. This work required the in vitro reconstitution of regulated transcription, which had not previously been reported for Chlamydia. We found that the CIRCE sequence varies between chlamydial species and that the level of HrcA-mediated transcriptional repression correlates with the degree of CIRCE sequence conservation. We infer from these results that the CIRCE sequence is used to set the non-stress levels of heat shock gene expression and that there are species-specific differences in these basal expression levels.; We have also identified multiple response mechanisms to cellular stress that result in release of HrcA-mediated transcriptional repression. HrcA-mediated transcriptional repression is regulated by interactions with GroEL, a heat shock protein whose transcription is itself regulated by HrcA. GroEL enhances transcriptional repression by HrcA, and native GroEL and GroES were purified together with HrcA from chlamydiae grown in vivo. Other chlamydial heat shock proteins do not appear to affect HrcA activity, indicating a specific role for a GroEL auto-regulatory feedback loop. HrcA activity also appears to be regulated by a unique carboxyl-terminal tail not found in other bacterial HrcA homologs. The Chlamydia-specific carboxyl-terminal domain inhibited HrcA function and was subject to post-transcriptional regulation. Truncated HrcA lacking the carboxyl-terminal tail was highly active while the full-length HrcA or heterodimers of the two forms were inactive in transcriptional repression. We propose that the full-length HrcA can function as a dominant negative to modulate activity. These studies are the first functional analysis of a transcriptional repressor in Chlamydia and provide valuable insight into the regulation of a process significant to chlamydial pathogenesis.