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A Chemical Mutagenesis Approach to Identify Virulence Determinants in the Obligate Intracellular Pathogen Chlamydia trachomatis

  • Bidong Nguyen
  • Raphael ValdiviaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1197)

Abstract

Our understanding of how most microbes “work” is hindered by the lack of molecular genetic and recombinant DNA tools to manipulate their genomes. We devised an approach to perform genetic analysis in one such microbe, the obligate intracellular bacterial pathogen Chlamydia trachomatis. Comprehensive libraries of clone-purified mutants with distinct plaque morphologies were generated through chemical mutagenesis. Whole-genome sequencing (WGS) was then employed to identify the underlying genetic lesions and to draw correlations between mutated gene(s) and a common phenotype. Taking advantage of the ability of Chlamydia to exchange DNA in co-infection settings, we then generated recombinant strains after co-infection of mammalian cells with mutant and wild type bacteria. In this manner, causal relationships between genotypes and phenotypes were established. The pairing of chemically induced gene variation and WGS to establish correlative genotype–phenotype associations should be broadly applicable to a large list of medically and environmentally important microorganisms currently not amenable to genetic analysis.

Key words

Chlamydia Genetic analysis Whole genome sequencing Chemical mutagenesis Genetic mapping 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Molecular Genetics and MicrobiologyDuke University Medical CenterDurhamUSA

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