A sequence of 412bp, spanning the terminal half of the grpE and the proximal portion of the dnaK-homologues in Bacillus subtilis, was amplified with PCR technology. This fragment was cloned into pJH101, an Escherichia coli plasmid, and transformed into B. subtilis strain YB886. Several chloramphenicol-resistant colonies were obtained from this transformation. The integration of the plasmid into the B. subtilis chromosome was verified by restriction endonuclease analysis and Southern hybridization. Strain BUL101, a chloramphenicol-resistant transformant, lacked the DnaK-homologue as demonstrated by two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. BUL101 grew at slower rates than parental cells at both 37-degrees-C and 48-degrees-C, produced abnormal cell shapes at 48-degrees-C, and was unable to grow at 51-degrees-C. The 412bp fragment did not exhibit detectable promoter activity.