Transcription of DNA transfer genes is a prerequisite for conjugative DNA transfer of F-like plasmids. Transfer gene expression is sensed by the donor cell and is regulated by a complex network of plasmid- and host-encoded factors. In this study we analyzed the effect of induction of the heat shock regulon on transfer gene expression and DNA transfer in Escherichia coli. Raising the growth temperature from 22 degrees C to 43 degrees C transiently reduced transfer gene expression to undetectable levels and reduced conjugative transfer by 2 to 3 orders of magnitude. In contrast, when host cells carried the temperature-sensitive groEL44 allele, heat shock-mediated repression was alleviated. These data implied that the chaperonin GroEL was involved in negative regulation after heat shock. Investigation of the role of GroEL in this regulatory process revealed that, in groEL(Ts) cells, TraJ, the plasmid-encoded master activator of type IV secretion (T4S) system genes, was less susceptible to proteolysis and had a prolonged half-life compared to isogenic wild-type E. coli cells. This result suggested a direct role for GroEL in proteolysis of TraJ, down-regulation of T4S system gene expression, and conjugation after heat shock. Strong support for this novel role for GroEL in regulation of bacterial conjugation was the finding that GroEL specifically interacted with TraJ in vivo. Our results further suggested that in wild-type cells this interaction was followed by rapid degradation of TraJ whereas in groEL(Ts) cells TraJ remained trapped in the temperature-sensitive GroEL protein and thus was not amenable to proteolysis.