# Investigation of small RNA expression in single cells

> **NIH NIH FI2** · U.S. NATIONAL INST/CHILD HLTH/HUMAN DEV · 2024 · —

## Abstract

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.

## Key facts

- **NIH application ID:** 10937302
- **Project number:** 1FI2GM154649-01
- **Recipient organization:** U.S. NATIONAL INST/CHILD HLTH/HUMAN DEV
- **Principal Investigator:** Dennis Zhu
- **Activity code:** FI2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** —
- **Award type:** 1
- **Project period:** 2024-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10937302

## Citation

> US National Institutes of Health, RePORTER application 10937302, Investigation of small RNA expression in single cells (1FI2GM154649-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10937302. Licensed CC0.

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