Cells are capable of responding to stimuli extremely rapidly, on timescales of seconds or less 
. In some situations, however, cells respond to stimuli only after extended delays of multiple cell cycles. A classic example occurs in the developing mammalian nervous system, where, in the presence of appropriate signaling molecules, precursor cells will proliferate for up to eight cell generations before differentiating into oligodendrocytes 
. Although many aspects of the system remain unclear, oligodendrocyte differentiation is similarly delayed in vivo and in cell culture, suggesting a cell-autonomous “timer” mechanism. Another example is the mid-blastula transition in developing Xenopus embryos, which occurs after 12 cell cycles of proliferation 
. In both cases, the deferral of differentiation enables a period of proliferation preceding commitment to new fates.
In bacteria, non-cell-autonomous strategies for deferring responses are well known. For example, in the marine bioluminescent bacterium Vibrio fischeri
, cells use quorum sensing mechanisms to defer light production until the population reaches a critical density 
. Similarly, Bacillus subtilis
can defer sporulation through cannibalism 
, a response triggered by cell-cell signaling at high cell density, in which one subpopulation of cells lyses another, releasing nutrients that sustain growth.
Although there has been much work on circuit architectures that speed response times 
, fewer studies have addressed cell-autonomous deferral mechanisms. Cell autonomous deferral requires the cell to keep track of the total time or number of division events since the appearance of the stimulus. It has remained unclear whether and how individual bacterial cells can achieve this functionality using genetic circuit components. The key problem is that as the cell grows and divides, its components dilute out. This dilution process sets an effective upper limit to the typical timescale over which the concentration of a protein responds to a step change in its production rate 
. For example, a step change in the rate of production of a stable protein causes the concentration of that protein to exponentially approach its new steady-state value with a timescale of one cell cycle 
. Thus, most gene circuits tend to relax to new steady states over timescales close to, or faster than, that of the cell cycle. Alternatively, genetic circuits can give rise to long deferral times in some cells through occasional stochastic switching between metastable states. While such systems can be tuned to generate long mean intervals between switching events, without cascades of multiple states, these mechanisms cannot generate well-defined, unimodal distributions of deferral times across a population 
sporulation provides an ideal model system to address this problem. Sporulation is a canonical microbial stress response behavior, during which cells respond to stress by differentiating into an environmentally resistant spore. Sporulation is a terminal differentiation decision, and its initiation is regulated by a well-characterized genetic circuit whose dynamics can be analyzed in individual cell lineages 
. This circuit, in response to diverse environmental and metabolic signals 
, controls the activation of the master regulator Spo0A through transcriptional regulation and phosphorylation 
. High levels of phosphorylated Spo0A (Spo0AP
) are sufficient to induce sporulation 
. However, under some conditions, Spo0AP
levels increase gradually over multiple cell cycles, allowing cells to proliferate prior to differentiation. The ability to defer sporulation while proliferating could provide a fitness advantage to cells by increasing their numbers relative to immediate sporulators (), although it could also impose a cost to cells that do not sporulate in time to survive extreme conditions. During the deferral period, cells may also explore other fates, such as biofilm formation, which are known to occur at intermediate levels of Spo0AP
Pulsed Spo0A activity dynamics occur during the deferral of sporulation initiation in B. subtilis.
The genetic circuitry controlling Spo0A activation includes multiple types of interactions (). Histidine kinases such as KinA, KinB, and others autophosphorylate and transfer phosphates through a phosphorelay consisting of Spo0F and Spo0B to Spo0A 
. Phosphatases reduce the total level of Spo0AP
. For example, Spo0E directly dephosphorylates Spo0AP
, while rap
phosphatases drain phosphates from the phosphorelay through Spo0F 
. The system also includes extensive transcriptional regulation. Spo0AP
regulates its own transcription as well as that of spo0F
. It also regulates many other genes, including global regulators such as AbrB 
. Finally, Spo0AP
also indirectly regulates its own activity by activating kinase expression 
. These transcriptional interactions typically occur at much longer timescales than the fast phosphotransfer reactions of the phosphorelay. Nevertheless, it remains unclear whether and how this circuit facilitates deferred differentiation.
Here, using time-lapse fluorescence microscopy of individual cells, we show that under some conditions B. subtilis cells defer sporulation for multiple cell cycles through a predominantly cell-autonomous mechanism. We observed a progressively increasing series of pulses of Spo0A phosphorylation during deferral. Manipulation of circuit interactions revealed that pulse growth and regulated deferral both required positive feedback on kinase expression. These results suggest that B. subtilis uses a pulsed positive feedback loop to gradually “ratchet up” Spo0AP activity pulses over multiple cell cycles in order to defer sporulation. Finally, mathematical modeling of this mechanism further suggests that pulsing could enable a “polyphasic” feedback mechanism, in which different parts of the overall positive feedback loop are active at different times, facilitating regulation of deferral. This may be a general strategy that cells can use to enable regulation of timescales much longer than the cell cycle.