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Three structural genes that consist of a dnaK operon in a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 were cloned and characterized. The genes were clustered in the transcription order grpE-dnaK-dnaJ. The cloned grpE, dnaK, and dnaJ genes had open-reading frames of 474, 1,926, and 1,116 nucleotides, coding for proteins with calculated molecular masses of 17,390, 69,761, and 41,050, respectively. The overall identities in the de-duced amino acid sequences of GrpE, DnaK, and DnaJ with those of the Escherichia coli homologs were 45.2, 74.5, and 61.2%, respectively. Northern blot analyses with grpE-, dnaK-, and dnaJ-specific probes revealed that the three genes are co-transcribed as a 4.0-kb mRNA. A primer extension analysis revealed that the transcription of the dnaK operon started at the nucleo-tide A that is located 28 bp upstream of the grpE start codon. The transcription start site was preceded by a putative promoter region [5¢-CCCCGCTTGAA(13-bp)CCCCAATTT-3¢], which is highly homologous to the consensus sequences of the E. coli s32-type heat shock promoter. The putative promoter worked under both normal and heat shock conditions in E. coli. The nature of the nucleotide sequence in the second half of the -35 region played a critical role during transcrip-tion. The heat shock mRNA was maximally produced at about 10 min after transfer of the Methylovorus sp. strain SS1 from 30 to 42°C. The dnaK operon was also induced by ethanol, hydrogen peroxide, and NaCl shocks. The cloned dnaK operon complemented the E. coli dnaK mutant.