Figure 4.

Experimental observation confirm that 0A~P pulsing depends on kinA-0F chromosomal arrangement and transcriptional feedback from 0A~P to 0F negative feedback. A–E Chromosomal arrangements of 0F and kinA in Wildtype B. subtilis (A), Trans-0F^gltA (B), Trans-0F^amyE (C), Trans-kinA^gltA (D) and Trans-kinA^amyE (E) strains. Blue and orange bars mark strains that have transient kinA:0F imbalance and negative feedback respectively. F–J Measurements of P_0A-yfp activity in single cells during starvation. 0A~P pulses seen in Wildtype B. subtilis cells (F) are abolished by the translocation of 0F to the gltA locus in the Trans-0F^gltA strain (G) but not by the translocation of 0F to the amyE locus in the Trans-0F^amyE strain (H). 0A~P pulses are also abolished by the translocation of kinA to the amyE locus in the Trans-kinA^gltA strain (J) but not by the translocation of kinA to the gltA locus in the Trans-0F^gltA strain (I). Note that P_0A-yfp promoter activity level does not increase in the Trans-0F^gltA strain. Vertical dashed lines indicate cell divisions. K–O Chromosomal arrangements of 0F and kinA in iTrans-0F (K), i0F^amyE (L, N) and i0F^gltA strains (M, O). Note that both i0F strains lack the 0A~P-0F negative feedback. P–T Measurements of P_0A-yfp activity in single cells during starvation. Addition of an IPTG inducible copy of 0F near the origin in the iTrans-0F strain (P) recovers the transient kinA:0F imbalance lost in Trans-0F^gltA strain and rescues the 0A~P pulses seen in wildtype B. subtilis cells. 0A activity pulsing is greatly decreased in the i0F^amyE (Q, S) and i0F^gltA strains (R, T) which lack the negative feedback. 0A activity in the i0F^amyE (Q) strain fluctuates due to transient changes in kinA:P_hsp-0F but does not pulse (Figure S5). If 0F expression is low (at 5μM IPTG; Q, R), both i0F strains accumulate high 0A activity levels. High level expression of 0F (at 20μM IPTG; S, T) blocks 0A activation in both i0F strains similar to Trans-0F^gltA.