Fig. 1.

EilR repressor-regulated promoter engineering. a Alignment of intergenic regions that separate the genes encoding efflux pumps and their cognate repressors in gamma-proteobacteria homologous to the E. lignolyticus eilAR locus. All intergenic regions contain two repressor-binding sites eilO₁ and eilO₂, from which the 24-bp consensus operator (eilO_c) motif emerges. The asterisks indicate the base pairs conserved in all operators. Predicted promoter hexamers are shown for the repressor genes (−35_r, −10_r) and for the efflux pumps (−35_e, −10_e). Each colored box represents a nucleotide: A (green); T (magenta); G (yellow); C (blue). b Electrophoretic mobility shift binding assays of purified EilR with the full-length native E. lignolyticus operators (eilO₁, eilO₂), the full consensus operator (eilO_c), half consensus operator (½ eilO_c) and random DNA. Molar ratios of the 21.6 kDa EilR monomer and duplex DNA are indicated. c A library of randomized E. coli consensus promoter boxes fused with a truncated consensus operator generated the biosensor P_EilO1t, into which an additional full-length eilOc was placed at the transcriptional start site to yield P_JEx1. Immediate-early coliphage-promoters P_D/E20 (P_JExD) and P_H207 (P_JExH1, P_JExH2) from phage T5, P_L (P_JExL) from phage lambda, and P_A1 (P_JExA1, P_JExA2) from phage T7, were reorganized by placing a truncated eilOc into the spacer region, partially overlapping the −35 or the −10 hexamers, followed by addition of a full-length consensus operator at the transcriptional start site. An arrow indicates the transcriptional start site. Colors of the nucleotides belonging to the eilO_c -operator are highlighted and the promoter −35 and −10 hexamers are boxed