FIG. 4.
Effect of single mutations on transcription activation and binding by ComK. (A) β-Galactosidase assays were performed to determine the effects of single-base-pair mutations on transcription activation by ComK in vivo, using different K-box-comG promoter-lacZ fusion constructs located on low-copy-number plasmids in B. subtilis. For all mutants, β-galactosidase (Betagal) activities are represented as percentages of the level of activity determined for the perfect K-box in the same experiment. The figure depicts the activities measured after 4 h of stationary growth. Error bars representing the standard deviation are included for all single-base-pair mutations. (A and B) P indicates the perfect K-box. Numbers 1 to 4 under “b1A” or “b1T” indicate the position that is replaced by a G in the A or T stretch of AT-box 1 (b1; the right AT-box, in which the A's and T's are named Ax or Tx) of the perfect K-box, used as the control in this experiment. (B) Details are the same as described for panel A, but in this case, the K-box-comG promoter-lacZ fusion constructs used were integrated into the amyE locus on the chromosome of B. subtilis. The A2-G mutant is missing, because the required mutation was not obtained. (C and D) To determine the effect of single-base-pair mutations on DNA binding by ComK, EMSAs were performed. ComK shifts for the mutant K-boxes were quantified and are represented as percentages of the shifts for the perfect K-box at different ComK concentrations. The standard deviation determined for the average over multiple experiments are depicted for each mutant. Quantifications are shown for the binding reaction with 0.07 μM ComK. For each gel in panel D, results for a blank (−) and three samples with ComK concentrations increasing in twofold increments from 0.018 to 0.07 μM are shown (indicated by the wedges). Perfect, the perfect K-box; Ax-G, A-G substitution at position x in the A-stretch of AT-box 1 (right AT-box); Tx-G, T-G substitution at position x in the T stretch of AT-box 1.