Transcriptional studies have demonstrated that the dnaK gene of Streptomyces coelicolor A3(2) is contained within a 4.3 kb operon. The operon is transcribed from a single (transiently) heat-inducible promoter, dnaKp, that resembles the typical vegetative (sigma 70-recognized) eubacterial consensus promoter sequence. dnaK transcription was found to be heat-inducible at all stages of development in surface-grown cultures. In addition, at the normal growth temperature of 30 degrees C, dnaK transcript levels were shown to vary at different stages of development, being more abundant in young germinating cultures and in mycelium undergoing sporogenesis. The nucleotide sequence of the dnaK operon has been completed, revealing the gene organization 5'dnaK-grpE-dnaJ orfX. orfX represents a novel heat-shock gene. Its predicted product displays high similarity to the GlnR repressor proteins of Bacillus spp. and to the MerR family of eubacterial transcriptional regulators. The S. coelicolor OrfX protein has been over-produced in Escherichia coli, and DNA-binding experiments indicate that it interacts specifically with the dnaKp region, binding to three partially related inverted repeat sequences; they are centered at -75, -49 and +4, respectively, relative to the transcription start site of the operon. These results suggest that OrfX plays a direct role in the regulation of the dnaK operon.