ABSTRACT Phenotypic heterogeneity refers to the ability of genetically identical cells to display differences in phenotypic behavior. This phenomenon can be observed in the expression of flagella and the extracellular amyloid fiber curli in Escherichia coli. In planktonic culture, exponentially growing E. coli express flagella in stochastic transcriptional bursts while curli are expressed in a bimodal “on-or-off” pattern in stationary phase E. coli cells. Heterogeneous expression of flagella and curli allow populations of E. coli to employ a bet-hedging strategy to simultaneously explore their environment, attach to surfaces, and protect themselves against host immune recognition or sudden environmental changes. Despite the functional importance of heterogeneity, the regulatory mechanisms underlying this process are not well characterized. The cascades controlling flagella and curli biosynthesis are both subject to regulation by numerous small RNAs (sRNAs), which form crosstalk pathways and feedback loops within these pathways. Both network structures have been shown to potentiate phenotypic heterogeneity between cells. sRNAs are involved in regulating many phenotypically heterogeneous processes in E. coli but the single-cell variability in sRNA expression has never been investigated. We hypothesize that sRNA-mediated crosstalk pathways and regulatory feedback loops contribute to cell-to-cell variability in flagella and curli expression in E. coli. To test this hypothesis, we will disrupt these networks by generating null mutants of McaS, a sRNA providing crosstalk between flagella and curli biosynthesis, and FliX, a sRNA forming a feedback loop in flagella synthesis, and examining the distribution of flagella and curli biosynthesis across single cells using flow cytometry. We will directly examine the single-cell expression pattern of McaS and FliX using fluorescence in situ hybridization (FISH) combined with flow cytometry. Additionally, we will globally profile the expression of sRNAs within individual cells in E. coli using bacterial single-cell RNA-sequencing. The results from this proposal will provide the first direct measurement of sRNA expression in E. coli at the single-cell level. Understanding the molecular mechanisms that give rise to phenotypic heterogeneity can inform strategies to disrupt processes such as antibiotic tolerant persister cell formation in bacterial pathogens.