Overexpression of the cat-86 Gene Is Associated with Thermosensitivity in Bacillus subtilis

Abstract.

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