ANALYSIS OF AN INSERTIONAL OPERATOR MUTATION (GNTOI) THAT AFFECTS THEEXPRESSION LEVEL OF THE BACILLUS-SUBTILIS GNT OPERON, AND CHARACTERIZATION OF GNTOI SUPPRESSOR MUTATIONS

The Bacillus subtilis gnt operon is negatively regulated via interacti on of the gnt repressor (GntR) with an operator upstream of gntR, whic h is antagonized by gluconate. An 8 bp insertional operator mutation ( gntOi) of the gnt operon was constructed which affected the expression level of this operon. Two suppressors of this gnt0i mutation, exhibit ing normal expression, were also isolated; one involved a threonine su bstitution for the Ala-48 residue (gntR48T) within the helix-turn-heli x DNA-binding motif of GntR, and the other an adenine substitution for the guanine at nucleotide -4 within the gnt0i operator (gntOiM4A) (1 is the transcription initiation site). The gntR48T mutation by itsel f rendered the gnt operon partially constitutive. When the gntR43L mut ation, which renders the gnt operon fully constitutive, was introduced into the gntOi or gntOiM4A mutant, the operator mutations were found not to affect the promoter activity of the gnt operon. These in vivo r esults indicate that the gnt0i mutation affects the operator interacti on with GntR, causing a low expression level even in the presence of g luconate. In vitro gel retardation and DNase I footprint analyses demo nstrated that even when gluconate was present, GntR still bound to the gnt0i operator region.