# Molecular basis of bacterial spatial structure formation by a post-transcriptional regulator

> **NIH NIH R15** · DUQUESNE UNIVERSITY · 2020 · $414,000

## Abstract

PROJECT SUMMARY/ABSTRACT
Although we have a great understanding on the physiological importance of the biofilm matrix as a whole, we
know relatively little about the mechanistic role of the individual components and how they interact to form
structures at the micron-scale. Here, we utilize a simple model system that naturally produces striking structures
to dissect the individual and collective roles of extracellular secretions in structure formation. Aging colonies of
Pseudomonas fluorescens produce spatially isolated patches exclusively through mutations that reduce or
remove the function of a post-transcriptional repressor (RsmE). RsmE homologs are widely spread among
bacteria and prevent the translation of cognate mRNA to regulate diverse traits, including the production of
extracellular secretions and biofilm formation. At the micron-scale, clonal aggregates of rsmE mutants expand
space by pushing away and preventing the encroachment of the parent cells, dramatically reducing the local
density within the clonal patch. Importantly, rsmE mutants neither show growth advantage in pure culture nor
produce toxic compounds, and the evolutionary advantage requires the physical presence of the parent cells.
We thus hypothesize that RsmE-regulated secretions function collectively to form spatial structures that solve
the problem of crowding in a densely populated colony. To address this, we will: (1) identify the components of
the RsmE-secretome through transposon mutagenesis, mass spectrometry, and RIP-seq to disrupt and identify
secretion genes that prevent/reduce patch formation; mass spectrometry to directly identify the secretions; RIP-
seq to identify mRNA that bind directly to RsmE; (2) characterize the functional role of RsmE-secretions in
structure formation through competitions and confocal microscopy; (3) identify the key amino acid residues of
RsmE that define its functional specificity by competing naturally derived missense mutants against one another,
RIP-seq and microscopy of to assess the loss of mRNA-specificity and spatial consequences, respectively, that
are associated with substitutions in the key residues, and RIP-seq to identify mRNA that bind exclusively to
RsmE and those that bind promiscuously to two distinct paralogs of RsmE that are highly conserved in amino
acid sequence. At the completion of this project, we will demonstrate (1) how a change to a single nucleotide
could drive complex multicellular behaviors that are not predictable based on the attributes of the individuals in
isolation, (2) how extracellular secretions function individually and collectively at the micron-scale to capture
space and optimal positioning, and (3) how RsmE manifests specificity to change the general perception that
Rsm-paralogs are functionally redundant. Particularly, the convergence of these individual outcomes onto spatial
structures will represent a unique and substantial contribution to advance the field. We will also demonstrate the
pow...

## Key facts

- **NIH application ID:** 9965029
- **Project number:** 1R15GM132856-01A1
- **Recipient organization:** DUQUESNE UNIVERSITY
- **Principal Investigator:** Wook Kim
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $414,000
- **Award type:** 1
- **Project period:** 2020-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9965029

## Citation

> US National Institutes of Health, RePORTER application 9965029, Molecular basis of bacterial spatial structure formation by a post-transcriptional regulator (1R15GM132856-01A1). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/9965029. Licensed CC0.

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