The dnaK operon of Bacillus subtilis is heptacistronic

Abstract

In 1992, we described the cloning and sequencing of the dnaK locus of Bacillus subtilis which, together with transcriptional studies, implied a tetracistronic structure of the operon consisting of the genes hrcA, grpE, dnaK, and dnaJ. We have repeated the Northern blot analysis, this time using riboprobes instead of oligonucleotides, and have detected a heat-inducible 8-kb transcript, suggesting the existence of additional heat shock genes downstream of dnaJ. Cloning and sequencing of that region revealed the existence of three novel heat shock genes named orf35, orf28, and orf50, extending the tetra- into a heptacistronic operon. This is now the largest dnaK operon to be described to date. The three new genes are transcribed as a part of the entire dnaK operon (8.0-kb heptacistronic heat-inducible transcript) and as part of a suboperon starting at an internal vegetative promoter immediately upstream of dnaJ (4.3-kb tetracistronic non-heat-inducible transcript). In addition, the Northern blot analysis detected several processing products of these two primary transcripts. To demonstrate the existence of the internal promoter, a DNA fragment containing this putative promoter structure was inserted upstream of a promoterless bgaB gene, resulting in the synthesis of beta-galactosidase. Challenging this transcriptional fusion with various stress factors did not result in the activation of this promoter. To assign a biological function to the three novel genes, they have each been inactivated by the insertion of a cat cassette. All of the mutants were viable, and furthermore, these genes are (i) not essential for growth at high temperatures, (ii) not involved in the regulation of the heat shock response, and (iii) sporulation proficient. Blocking transcription of the suboperon from the upstream heat-inducible promoter did not impair growth and viability at high temperatures.