Identification of an Mlc-regulated hilE promoter. Primer extension analysis was performed using total RNA from SL1344 (lane 1: wild type), SR1304 (lane 2: mlc− mutant), SR1304 harboring pUC19 (lane 3) and SR1304 harboring pKB (lane 4; Mlc over-expression), all of which were grown to exponential phase in LB medium without shaking. Aliquots of the RNA (30 μg) were subjected to primer extension analysis, and a sequence ladder was generated using the same end-labeled primer that was used for the primer extension analysis. (A) The effects of the mlc mutation on the P1 and P2 promoters were examined using the hilE3 primer (see Table 2). (B) The transcriptional start site of the new promoter (P3), which is modulated by the mlc mutation, was identified using the hilE5 primer (see Table 2). (C) The start site (+1) is indicated by an arrow, and the promoter elements that resemble the consensus −10 and −35 sites are boxed.