Figure 3.
Induction kinetics of pvuIICR operon. (A) System used. To allow stable titration with C.PvuII, its positive feedback loop was broken by placing the pvuIIC gene under control of the arabinose-inducible ParaBAD promoter (top), while fusing the lacZ reporter gene (β-galactosidase) to the promoter that normally controls pvuIIC and is regulated by C.PvuII (PpvuIICR, middle; including both PCR1 and PCR2). The strain background was E. coli TOP10. The tet gene (tetracycline resistance) is on the same plasmid as lacZ, and was used to normalize gene expression in some experiments. The C.PvuII binding sites are shown at the bottom, with ovals representing C.PvuII homodimers. Some experiments use a non-repressing variant, in which C-box 2B (bottom) is altered (AGTC → GATC). For references, see text. (B) C.PvuII levels. Results of C.PvuII measurements from western blots of cell extracts. C.PvuII was detected by a polyclonal primary antiserum, with final readout via luminescence densitometry. Means of triplicates, ±SE, are shown. The loading normalization was to BCCP, a naturally biotinylated E. coli protein. See Methods for details.