Overexpression of the cat-86 gene is associated with thermosensitivity in Bacillus subtilis

Bacillus subtilis harboring the cat-86 constitutive plasmid pPL708C2 with a n ochre mutation at the 9th codon (terc 9) was sensitive to chloramphenicol (Cm-s) and exhibited relative thermostability when heated at 47 degrees C, Reversion to chloramphenicol resistance (Cm-r) occurred at a frequency of 5.4 x 10(-8). All of the plasmid Cm-r revertants tested were thermosensitiv e, Similarly, wild-type pPL708C2 present in B. subtilis also rendered the b acterium thermosensitive. When a nonsense mutation is introduced at codon 1 41, however, this terc 141 variant of pPL708C2 failed to thermosensitize B. subtilis, Another variant of pPL708C2 that produces intact yet catalytical ly inactive CAT-86 has both His-16 and His-17 at the active site replaced b y Pro. Nevertheless, cells of B, subtilis carrying this variant were thermo sensitive, Plasmid-free and pPL708C2-bearing strains did not exhibit differ ences in major heat shock proteins. Electron micrographs revealed a threefo ld increase of inclusion bodies present in a strain harboring pPL708C2 when compared with those in an isogenic plasmid-free strain.