THE DNAK OPERON OF BACILLUS-SUBTILIS IS HEPTACISTRONIC

In 1992, we described the cloning and sequencing of the dnaK locus of Bacillus subtilis which, together with transcriptional studies, implie d a tetracistronic structure of the operon consisting of the genes hrc A, grpE, dnaK, and dnaJ, We have repeated the Northern blot analysis, this time using riboprobes instead of oligonucleotides, and hare detec ted a heat-inducible 8-kb transcript, suggesting the existence of addi tional heat shock genes downstream of dnaJ. Cloning and sequencing of that region revealed the existence of three novel heat shock genes nam ed 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 b ope ron (8.0-kb heptacistronic heat-inducible transcript) and as part of a suboperon starting at an internal vegetative promoter immediately ups tream of dnaJ (4.3-kb tetracistronic non-heat-inducible transcript), I n addition, the Northern blot analysis detected several processing pro ducts of these two primary transcripts, To demonstrate the existence o f the internal promoter, a DNA fragment containing this putative promo ter structure was inserted upstream of a promoterless bgaB gene, resul ting in the synthesis of beta-galactosidase. Challenging this transcri ptional fusion with various stress factors did not result in the activ ation of this promoter, To assign a biological function to the three n ovel genes, they have each been inactivated by the insertion of a cat cassette, All of the mutants mere viable, and furthermore, these genes are (i) not essential for growth at high temperatures, (ii) not invol ved in the regulation of the heat shack response, and (iii) sporulatio n proficient, Blocking transcription of the suboperon from the upstrea m heat-inducible promoter did not impair growth and viability at high temperatures.