Dynamic heterogeneity in an E. coli stress response regulon mediates gene activation and antimicrobial peptide tolerance

The bacterial stress response is an intricately regulated system that plays a critical role in cellular resistance to drug treatment. The complexity of this response is further complicated by cell-to-cell heterogeneity in the expression of bacterial stress response genes. These genes are often organized into networks comprising one or more transcriptional regulators that control expression of a suite of downstream genes. While the expression heterogeneity of many of these upstream regulators has been characterized, the way in which this variability affects the larger downstream stress response remains hard to predict, prompting two key questions. First, how does heterogeneity and expression noise in stress response regulators propagate to the diverse downstream genes in their regulons? Second, when expression levels vary, how do upstream and downstream genes act together to protect cells from stress? To address these questions, we focus on the transcription factor PhoP, a critical virulence regulator which coordinates pathogenicity in several gram-negative species. We use optogenetic stimulation to precisely control PhoP expression levels and utilize information theory to examine how variations in PhoP affect the downstream activation of genes in the PhoP regulon. We find that these downstream genes exhibit differences in mean expression level, sensitivity to increasing levels of PhoP, and signal transmission reliability. These response functions can also vary between individual cells, increasing heterogeneity in the population. We tie these variations to cell survival when bacteria are exposed to a clinically-relevant antimicrobial peptide, showing that while high expression of the PhoP-regulon gene pmrD provides a protective effect against Polymyxin B, cell survival chances are best determined by integrated dynamic PhoP and pmrD expression levels. Overall, we demonstrate that cell-to-cell and temporal heterogeneous expression of a stress response regulator can have clear consequences for enabling bacteria to survive stress.