Altruistic feeding and cell-cell signaling during bacterial differentiation actively enhance phenotypic heterogeneity.

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation non-uniformly to secure against the possibility that favorable growth conditions, which puts sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early utilize a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay non-sporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.

Charged Amino Acids Contribute to ZorO Toxicity.

Chromosomally encoded toxin-antitoxin systems have been increasingly identified and characterized across bacterial species over the past two decades. Overproduction of the toxin gene results in cell growth stasis or death for the producing cell, but co-expression of its antitoxin can repress the toxic effects. For the subcategory of type I toxin-antitoxin systems, many of the described toxin genes encode a small, hydrophobic protein with several charged residues distributed across the sequence of the toxic protein. Though these charged residues are hypothesized to be critical for the toxic effects of the protein, they have not been studied broadly across different type I toxins. Herein, we mutated codons encoding charged residues in the type I toxin zorO, from the zor-orz toxin-antitoxin system, to determine their impacts on growth inhibition, membrane depolarization, ATP depletion, and the localization of this small protein. The non-toxic variants of ZorO accumulated both in the membrane and cytoplasm, indicating that membrane localization alone is not sufficient for its toxicity. While mutation of a charged residue could result in altered toxicity, this was dependent not only on the position of the amino acid within the protein but also on the residue to which it was converted, suggesting a complex role of charged residues in ZorO-mediated toxicity. A previous study indicated that additional copies of the zor-orz system improved growth in aminoglycosides: within, we note that this improved growth is independent of ZorO toxicity. By increasing the copy number of the zorO gene fused with a FLAG-tag, we were able to detect the protein expressed from its native promoter elements: an important step for future studies of toxin expression and function.