Abstract
Isogenic populations of cells exhibit phenotypic variability that has specific physiological consequences. For example, individual bacteria can differ in their sensitivity to an antibiotic, but whether this variability is regulated or an unavoidable consequence of stochastic fluctuations is unclear. We observed that a bacterial stress response gene, the (p)ppGpp synthetase sasA, exhibits high levels of extrinsic noise in expression, suggestive of a regulatory process. We traced this variability to the convergence of two signaling systems that together control an event largely unexplored in bacteria, the multisite phosphorylation of a transcription factor. We found that this regulatory intersection is crucial for controlling the appearance of outliers, rare cells with unusually high levels of sasA expression. Additionally, by examining the full distributions of gene expression we calculated the importance of multisite phosphorylation in setting the relative abundance of cells with a given a level of SasA. We then created a predictive model for the probability of a given cell surviving antibiotic treatment as a function of sasA expression. Therefore, our data show that multisite phosphorylation can be used to strongly regulate bacterial physiology and sensitivity to antibiotic treatment.
Significance Statement Cells possess many signaling pathways for sensing and responding to their environment. Although these pathways are commonly characterized individually, signals between separate pathways are often integrated. One way that pathways can intersect is through multisite phosphorylation controlling the activity of a common target, where each phosphorylation is contributed by a separate signaling system. Bacteria are an ideal place to study the effects of these intersections on gene expression since the number of such intersections is comparatively small, and subsequent gene regulation relatively simple. Here we show that signal integration through multisite phosphorylation is important for setting the frequency of bacterial cells with increased antibiotic tolerance by controlling the heterogeneity, or noise, in gene expression across the population.
