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Related Concept Videos

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection11:50

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We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro...
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This is an adapted method for identifying candidate insect colonization factors in a Burkholderia beneficial symbiont. The beetle host is infected with a random mutant library generated via transposon mutagenesis, and library complexity after colonization is compared to a control grown in vitro.
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Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant...
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Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during...
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Related Experiment Video

Updated: Jan 20, 2026

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection
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Insertion Pool Sequencing for Insertional Mutant Analysis in Complex Host-Microbe Interactions.

Simon Uhse1, Florian G Pflug2, Arndt von Haeseler2,3

  • 1Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna, Austria.

Current Protocols in Plant Biology
|September 4, 2019
PubMed
Summary

We developed insertion pool sequencing (iPool-Seq) to analyze microbial gene function in host tissues. This method overcomes challenges in studying underrepresented species, enabling direct analysis of colonized environments.

Keywords:
Ustilago maydisfungal genomicsmaizeplant genomicsplant-microbe interactionsunique molecular identifiersvirulence factors

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Last Updated: Jan 20, 2026

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Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles
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Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Published on: August 12, 2021

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Area of Science:

  • Microbiology
  • Genetics
  • Plant Pathology

Background:

  • Insertional mutant libraries are crucial for deciphering microbial gene function through negative depletion screens.
  • Analyzing microbial species that colonize host tissues presents a significant challenge due to underrepresentation.
  • Studying host-microbe interactions requires robust methods for analyzing microbial gene function within the host environment.

Purpose of the Study:

  • To develop a novel method for the direct analysis of microbial gene function within colonized host tissues.
  • To overcome the bottleneck of underrepresentation of microbial species in host colonization studies.
  • To provide detailed protocols for studying the interaction between Ustilago maydis and its host, maize.

Main Methods:

  • Development of insertion pool sequencing (iPool-Seq) for high specificity analysis of insertional mutant cassettes.
  • Establishment of detailed protocols for host infection, including maize and Ustilago maydis.
  • Optimization of genomic DNA extraction from colonized tissues.
  • Development of protocols for library preparation and bioinformatic data analysis.

Main Results:

  • iPool-Seq enables direct analysis of microbial gene function in colonized host tissue.
  • The developed protocols facilitate the study of Ustilago maydis-maize interactions.
  • The methodology is adaptable for various host-microbe interaction systems.

Conclusions:

  • iPool-Seq is an effective tool for overcoming challenges in studying microbial gene function in host-associated microbes.
  • The provided protocols offer a comprehensive framework for host-microbe interaction research.
  • This approach enhances the understanding of microbial roles in colonized environments.