Flagellar Biosynthesis in E.coli Is Regulated by a Cascade of Stochastic Pulses

Abstract

Genetically identical populations of bacteria can exhibit significant phenotypic heterogeneity even in the absence of environmental variations. In bacteria such as Bacillus and Salmonella, flagellar motility has been found to be one such phenotype. By contrast, in Escherichia coli, flagella are traditionally thought to be synthesized continuously and homogeneously throughout exponential growth. In this system, flagella synthesis is regulated by a three-tiered transcriptional cascade: First, a master regulator FlhDC (Class 1) regulates the expression of genes that encode the flagellar basal body (Class 2). One Class 2 gene encodes the alternative sigma factor FliA which then regulates the transcription of Class 3 genes encoding the filament and chemotaxis machinery. Here, I examined the transcriptional dynamics of flagellar genes in individual cells by a combination of fluorescent protein readouts and time-lapse microscopy. Unexpectedly, flagellar gene expression within a single cell fluctuated dramatically over time in stochastic pulses. I discovered that this behavior was obscured in many laboratory strains due to a previously described mutation. Genes within the same class pulsed synchronously; however, pulses ranged from more to less frequent in Classes 1, 2 and 3 respectively. In turn, I investigated how pulses in gene expression were generated and propagated through the flagellar cascade. At both steps of the cascade, only a subset of upstream fluctuations (or pulses) activated downstream genes. Analyzing the “dose-response” relationships revealed a tri-phasic response: a “repressed”, an “ultrasensitive”, and a “proportional” phase. I propose how this behavior might arise from a “molecular titration model” and propose YdiV and FlgM, post-translational regulators of FlhD and FliA, respectively, to be critical regulators. My work also excludes some proposed molecules as regulators of pulsing and suggests other molecules as potential secondary modulators of basic regulatory cascade. These results contrast stochastic pulses in E. coli with previous examples of flagellar heterogeneity in Salmonella. In summary, this dissertation describes the discovery of stochastic pulsing in E. coli flagellar synthesis. My work offers mechanistic insight into how these pulses propagate across the transcriptional cascade and establishes flagellar synthesis in E. coli as a new model system for the study of phenotypic heterogeneity.