# Investigating regulators of bacterial chromosome organization

> **NIH NIH R35** · UNIVERSITY OF WASHINGTON · 2024 · $441,250

## Abstract

PROJECT SUMMARY
Chromosomes must be highly packaged to fit with cells. Modern tools like 3D chromosome capture allow for
visualization of chromosome organization but are limited in describing gene-level packaging and cannot reveal
the mechanisms that underlie packaging. Understanding these mechanisms is critical to understand genome
function and to identify chromosome vulnerabilities as targets for future therapeutics. We propose to attack two
major challenges limiting our understanding of chromosome organization in bacteria: understanding
supercoiling and structuring protein function. First, supercoiling represents the DNA winding about itself, with
“positive” supercoiling (+SC) describing an overwound structure, which is refractory to DNA duplex melting.
+SC is generated as a byproduct of replication and must be removed by topoisomerase enzymes or replication
will cease, leading to cell death. Prior to my work, a lack of tools to map +SC and a lack of knowledge of
topoisomerase regulators meant that our cellular understanding of +SC was limited. Second, bacterial
chromosomes are structured by nucleoid-associated proteins (NAPs). NAPs are a group of sequence-diverse,
functionally heterogeneous, and poorly understood proteins. Our discoveries in Caulobacter crescentus of an
essential NAP and +SC regulator called GapR has empowered us to begin untangling chromosome
packaging. We initially showed that GapR binds +SC in a sequence-independent manner to stimulate
topoisomerase activity and promote replication. We then developed GapR into technology that allows us to
“see” where +SC occurs. While GapR homologs are ubiquitous in ⍺-proteobacteria, analogous NAPs that
regulate topoisomerase activity in other bacteria have not been identified. Our preliminary data suggests that
bacterial viruses (phages) encode GapR homologs that hijack bacterial GapR to promote infection, uncovering
a beneficial role for phage hijacking of bacterial NAPs during infection. We have also developed technology to
capture topoisomerase regulators in bacteria. In this proposal, we will 1) elucidate GapR mechanism as a
model for understanding how bacteria and phage control +SC to proliferate, 2) mine bacteria and phage
genomes to characterize additional topoisomerase regulators, and 3) examine NAP hijacking during phage
infection. These projects will describe fundamental paradigms of bacterial chromosome organization and
improve our understanding of phage-bacterial warfare. Further, topoisomerases are important antibacterial
targets while phages are potential antibiotic alternatives, thus the findings of this proposal will identify
vulnerabilities in bacterial chromosome regulation as future antibiotic targets and improve our understanding of
phage infection for phage therapy.

## Key facts

- **NIH application ID:** 10937922
- **Project number:** 1R35GM154727-01
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Monica S. Guo
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $441,250
- **Award type:** 1
- **Project period:** 2024-08-01 → 2029-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10937922

## Citation

> US National Institutes of Health, RePORTER application 10937922, Investigating regulators of bacterial chromosome organization (1R35GM154727-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10937922. Licensed CC0.

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