Cloning, molecular characterization, and transcriptional analysis of dnaK operon in a methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726

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 deduced 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 nucleotide 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 σ³²-type heat shock promoter. The putative promoter worked under both normal and heat shock conditions in E. coli. The nature of the nucleotidc sequence in the second half of the -35 region played a critical role during transcription. 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.