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J Bacteriol. 2019 Sep 9. pii: JB.00366-19. doi: 10.1128/JB.00366-19. [Epub ahead of print]

Development of transposon mutagenesis for Chlamydia muridarum.

Author information

1
Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, USA.
2
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA.
3
Department of Biomedical Sciences, Oregon State University, Corvallis, Oregon, USA.
4
Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, USA khybiske@uw.edu.

Abstract

Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Herein, we report the development of the Himar C9 transposon system for C. muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (cam) resistant, GFP-expressing C. muridarum transposon mutants. The majority of mutants contained single transposon insertions spread throughout the entire C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD, were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum. The generation of a complete library of C. muridarum mutants will ultimately enable a comprehensive identification of the functional genetic requirements for Chlamydia infection in vivo.Importance Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization for the ∼1000 genes encoded by chlamydial genomes. Here we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted species of Chlamydia that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis. We demonstrate the generation of an initial library of 33 mutants, containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.

PMID:
31501283
DOI:
10.1128/JB.00366-19

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