FIG 4.

Continued replication in cells with constitutive transcription by T7 RNAP next to oriC. (Left panels) (A) Schematic of the oriC region showing the genes mioC and gidA as well as RNAP (purple) for WT. Transcription of gidA (black arrow) by RNAP normally introduces negative supercoils (−sc) into oriC. (B to E) Schematic of oriC showing the location and direction of the engineered T7 promoter with transcript produced by T7 RNAP shown as an orange arrow. (Right panels) Flow cytometry profiles of strains from panels A to E grown in M9-glycerol plus Casamino Acids. IPTG (1 mM) was added to induce T7 RNAP, and 100 nM aTc was added to induce RelA′ for 90 min. Only cells with a T7 promoter facing away from oriC continue replication in the presence of ppGpp. (F) Representative immunoblot for DnaA in all strains tested above with or without 1 mM IPTG and 100 nM aTc to induce T7 RNAP and RelA′ as in panels A to E. The control is a nonspecific band in the DnaA antibody.