CLONING, SEQUENCING, AND EXPRESSION OF DNAK-OPERON PROTEINS FROM THE THERMOPHILIC BACTERIUM THERMUS-THERMOPHILUS

We propose that the dnaK operon of Thermus thermophilus HB8 is compose d of three functionally linked genes: dnaK, grpE, and dnaJ. The dnaK a nd dnaJ gene products are most closely related to their cyanobacterial homologs. The DnaK protein sequence places T. thermophilus in the pla stid Hsp70 subfamily. In contrast, the grpE translated sequence is mos t similar to GrpE from Clostridium acetobutylicum, a Gram-positive ana erobic bacterium. A single promoter region, with homology to the Esche richia coli consensus promoter sequences recognized by the sigma(70) a nd sigma(32) transcription factors, precedes the postulated operon. Th is promoter is heat-shock inducible. The dnaK mRNA level increased mor e than 30 times upon 10 min of heat shock (from 70 degrees C to 85 deg rees C). A strong transcription terminating sequence was found between the dnaK and grpE genes. The individual genes were cloned into pET ex pression vectors and the thermophilic proteins were overproduced at hi gh levels in E. coli and purified to homogeneity. The recombinant T. t hermophilus DnaK protein was shown to have a weak ATP-hydrolytic activ ity, with an optimum at 90 degrees C. The ATPase was stimulated by the presence of GrpE and DnaJ. Another open reading frame, coding for Clp B heat-shock protein, was found downstream of the dnaK operon. (C) 199 7 Published by Elsevier Science B.V.