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Development of Transposon Mutagenesis for Chlamydia muridarum
Functional genetic analysis of has been a challenge due to the historical genetic intractability of , although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for , a mouse-adapted species that...
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Published in: | Journal of bacteriology 2019-12, Vol.201 (23), p.1 |
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Main Authors: | , , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Functional genetic analysis of
has been a challenge due to the historical genetic intractability of
, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for
, a mouse-adapted
species that is widely used in
infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing
transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the
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 the insertions. Four mutants with transposon insertions in
,
,
, and
were investigated further for
and
phenotypes, including growth, inclusion morphology, and attachment to host cells. The
mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3
mutants,
was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for
The generation of a complete library of
mutants will ultimately enable comprehensive identification of the functional genetic requirements for
infection
Historical issues with genetic manipulation of
have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for
, a mouse-adapted
species that is widely used for
investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for
We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized
phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins. |
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ISSN: | 0021-9193 1098-5530 |
DOI: | 10.1128/JB.00366-19 |