# High-Resolution Mapping of Colonization Factors in a Model Microbiome > **NIH NIH F32** · UNIVERSITY OF WISCONSIN-MADISON · 2021 · $68,562 ## Abstract PROJECT SUMMARY Animals form stable and reproducible associations with specific microbes that are essential for health. However, the molecular mechanisms that result in the selection and retention of the appropriate beneficial symbionts are understudied. Here we use the beneficial symbiosis between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri as a model system to study the molecular dialogue that occurs at the microbe-host interface to ensure specificity in the interaction. Using a global transposon insertion sequencing approach (INSeq/Tn-seq) our lab previously identified 344 putative novel colonization factors. Pilot validation of a subset of these candidates showed that approximately half are true colonization factors. This approach identified factors ranging across various molecular functions, including amino acid metabolism, motility, biofilm formation, and signal transduction, as well as a number of so-called hypothetical proteins. Various copper exporters were also identified as validated colonization factors, suggesting that copper is playing a role at the microbe-host interface in this marine system. Further study of the identified factors required clean deletions, but we were limited in obtaining these due to the complexity of the random libraries generated via transposon mutagenesis and the time-consuming process of plasmid-based allelic exchange. To address these obstacles, I developed an updated mutagenesis approach to quickly generate barcoded in-frame gene deletions in V. fischeri. Barcode sequencing (BarSeq) can then be used to track population dynamics and perform high- throughput competition experiments with defined populations of deletion mutants. The overall goal of this proposal is to characterize the molecular pathways and functions that are responsible for the establishment of the specific and reproducible beneficial symbiosis between V. fischeri and its squid host. To this end, I used the method I developed to generate 48 barcoded deletions, including several copper exporters. In Aim 1, I will continue using this approach to generate barcoded deletions of all 344 novel putative colonization factors. In addition, barcoded mutants will be used in Aim 1 to perform high-throughput competitive squid colonization experiments using BarSeq to track population dynamics and determine colonization efficiency for each mutant strain. This approach will be used to define stages of host colonization with high resolution. Preliminary experiments have validated additional copper export mutants as true colonization factors. In Aim 2, I will use a combination of BarSeq and traditional biochemical and molecular biology techniques to characterize the role that copper has during squid colonization by V. fischeri. Completion of these aims will generate a valuable deletion library resource that will be used to characterize the molecular factors that determine specificity during establishment of the... ## Key facts - **NIH application ID:** 10309044 - **Project number:** 1F32GM140673-01A1 - **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON - **Principal Investigator:** Hector Luis Burgos - **Activity code:** F32 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2021 - **Award amount:** $68,562 - **Award type:** 1 - **Project period:** 2021-12-01 → 2022-11-30 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10309044 ## Citation > US National Institutes of Health, RePORTER application 10309044, High-Resolution Mapping of Colonization Factors in a Model Microbiome (1F32GM140673-01A1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10309044. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*