Extended Data Fig. 9: Spatially and temporally resolved transcriptional activity.
a. Time-resolved RT-qPCR of mRNA from an eyfp gene under the control of the CtrA-regulated P350 promoter integrated at four loci of the chromosome (L1, L3, L7, and L8, cf. Figure 3). Shown is mean ± s.d.; n = 3. Below plot: Replication times of these loci during the cell cycle, and cell cycle schematic of loci/dividing cells. Chromosome: black ovals. Red areas indicate polar PopZ microdomain, green tone indicates CtrA~P levels. Transcription is normalized to the five-minutes time point of L1. b. Time-resolved RT-qPCR of eyfp expressed from PxylX::eyfp at chromosomal loci L1 and L8 demonstrates a copy number effect. The observed increase at L1 and L8 represents the effect of chromosome replication, as xylose concentration and promotor activity are not expected to vary over time. Shown is mean ± s.d.; n = 3. c. eyfp transcription measured in cells at the 90 minute cell cycle time point at L1, L3, L7, and L8 in five genetic backgrounds. In the first three backgrounds, eyfp expression is controlled by the CtrA~P-regulated P350 promoter in the presence of: native wildtype ctrA (green), the phosphomimetic ctrA(D51E) driven by PxylX in a delta ctrA background (black), or ctrA driven by PxylX in a delta ctrA background (red). In the remaining backgrounds, eyfp expression is driven by the CtrA~P-regulated promoter PpilA in the presence of native wildtype ctrA (blue), and the xylose-driven promoter PxylX in the presence of xylose (gray). The wildtype ctrA, the phosphomimetic ctrA D51E, and eyfp driven by PxylX (green, black, and gray) are a subset of the data presented in Fig. 3b. The position-specific transcriptional output present for the CtrA~P-regulated P350 promoter in native conditions is lost when the sole CtrA copy is xylose-induced phosphomimetic CtrA(D51E) (black) but is recovered with xylose-inducible wildtype ctrA (red). Shown is mean + s.d.; n = 3. d. Simulated CtrA~P distribution profiles and their effect on transcription from P350. (left), possible CtrA~P profiles simulated with altered binding coefficient value between ChpT and PopZ at the new pole (cf. Extended Data Fig. 7a). Profile 4 (light brown) reflects wildtype parameters (Fig. 3a). (right) calculated transcript levels from P350 for each CtrA~P profile (Methods). Profile 4 was generated using wild-type parameters and best matched the experimental data (black, Fig. 3b).
