FIG. 4.

Identified HilA box in the hilA promoter is important for hilA regulation. A and B. hilA reporter gene fusion assays. A single-base-pair substitution was introduced into the promoter sequence of hilA present in pLS31 by site-directed mutagenesis, giving rise to pCMPG5401 (hilA*-lacZY). The lacZY fusion strains in either a wild-type (SL1344) (45) or hilA deletion background (VV302) (7) were grown under derepressing conditions (i.e., high osmolarity [10 g/liter NaCl], oxygen-limiting) (56) (A) and repressing conditions (low osmolarity [0 g/liter NaCl], aeration) (B) and assayed for β-galactosidase activity (71). Values are expressed in Miller units and represent the mean of eight independent experiments. Miller unit values of strains containing the vector pRW50 (58) were zero (data not shown). Error bars indicate standard deviations. C. HilA⁺ extract alters the gel mobility of the hilA promoter DNA fragment. Gel mobility shift assays were performed with the hilA (lanes 1 to 6) and hilA* (lanes 7 and 8) probe as outlined in Materials and Methods. Lanes 1, 5, and 7 are controls showing the migration of the probe in the absence of any added protein. Lanes 2, 6, and 8 contain 33 μg of HilA⁺ extract. Lane 3 contains 33 μg of the HilA⁻ extract. Lane 4 contains 100 ng unlabeled hilA promoter fragment as a specific competitor.