Ratiometric population sensing by a pump-probe signaling system in Bacillus subtilis.

Communication by means of diffusible signaling molecules facilitates higher-level organization of cellular populations. Gram-positive bacteria frequently use signaling peptides, which are either detected at the cell surface or 'probed' by intracellular receptors after being pumped into the cytoplasm. While the former type is used to monitor cell density, the functions of pump-probe networks are less clear. Here we show that pump-probe networks can, in principle, perform different tasks and mediate quorum-sensing, chronometric and ratiometric control. We characterize the properties of the prototypical PhrA-RapA system in Bacillus subtilis using FRET. We find that changes in extracellular PhrA concentrations are tracked rather poorly; instead, cells accumulate and strongly amplify the signal in a dose-dependent manner. This suggests that the PhrA-RapA system, and others like it, have evolved to sense changes in the composition of heterogeneous populations and infer the fraction of signal-producing cells in a mixed population to coordinate cellular behaviors.

Phenotypic memory in Bacillus subtilis links dormancy entry and exit by a spore quantity-quality tradeoff.

Some bacteria, such as Bacillus subtilis, withstand starvation by forming dormant spores that revive when nutrients become available. Although sporulation and spore revival jointly determine survival in fluctuating environments, the relationship between them has been unclear. Here we show that these two processes are linked by a phenotypic "memory" that arises from a carry-over of molecules from the vegetative cell into the spore. By imaging life histories of individual B. subtilis cells using fluorescent reporters, we demonstrate that sporulation timing controls nutrient-induced spore revival. Alanine dehydrogenase contributes to spore memory and controls alanine-induced outgrowth, thereby coupling a spore's revival capacity to the gene expression and growth history of its progenitors. A theoretical analysis, and experiments with signaling mutants exhibiting altered sporulation timing, support the hypothesis that such an intrinsically generated memory leads to a tradeoff between spore quantity and spore quality, which could drive the emergence of complex microbial traits.

Ectopic integration vectors for generating fluorescent promoter fusions in Bacillus subtilis with minimal dark noise.

Fluorescent protein promoter reporters are important tools that are widely used for diverse purposes in microbiology, systems biology and synthetic biology and considerable engineering efforts are still geared at improving the sensitivity of the reporter systems. Here we focus on dark noise, i.e. the signal that is generated by the empty vector control. We quantitatively characterize the dark noise of a few common bacterial reporter systems by single cell microscopy. All benchmarked reporter systems generated significant amounts of dark noise that exceed the cellular autofluorescence to different extents. We then reengineered a multicolor set of fluorescent ectopic integration vectors for Bacillus subtilis by introducing a terminator immediately upstream of the promoter insertion site, resulting in an up to 2.7-fold reduction of noise levels. The sensitivity and dynamic range of the new high-performance pXFP_Star reporter system is only limited by cellular autofluorescence. Moreover, based on studies of the rapE promoter of B. subtilis we show that the new pXFP_Star reporter system reliably reports on the weak activity of the rapE promoter whereas the original reporter system fails because of transcriptional interference. Since the pXFP_Star reporter system properly isolates the promoter from spurious transcripts, it is a particularly suitable tool for quantitative characterization of weak promoters in B. subtilis.