CLONING, NUCLEOTIDE-SEQUENCE, AND REGULATORY ANALYSIS OF THE NITROSOMONAS-EUROPAEA DNAK GENE

The dnaK gene of an ammonia-oxidizing bacterium, Nitrosomonas europaea , was cloned and sequenced, It was found that the dnaK gene product wa s highly homologous to previously analyzed dnaK gene products from oth er organisms at the amino acid level, Two partial open reading frames located upstream and downstream of the dnaK gene were also found and i dentified as grpE and dnaJ genes, respectively, by the predicted amino acid homology of their gene products to other bacterial GrpE and DnaJ proteins, Transcription of the dnaK gene was strongly induced by a he at shock from 30 to 37 degrees C, An analysis of the expression of the dnaK gene fused to the lacZ translational reporter gene also showed e ightfold increase in beta-galactosidase activity after the heat shock induction, Heat-inducible transcription start sites of the dnaK gene, revealed by primer extension analysis, were located 16 and 17 nucleoti des upstream from the translational start codon of the dnaK gene, and the predicted promoter sequence showed a homology to the consensus seq uence of sigma(32)-dependent heat shock promoters of gram-negative bac teria, The upstream region of the dnaK gene did not contain the invert ed repeat structure that was involved in the regulation of the heat sh ock gene of several gram-negative and gram-positive bacteria, Therefor e, we conclude that the heat shock regulatory mechanism of the N. euro paea dnaK gene may be similar to the sigma(32)-dependent mechanism obs erved in other gram-negative bacteria.