Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of Chlamydia.
Gene inactivation by transposon insertion or allelic exchange is a powerful approach to probe gene function. Unfortunately, many microbes, including Chlamydia, are not amenable to routine molecular genetic manipulations. Here we describe an arrayed library of chemically induced mutants of the genetically intransigent pathogen Chlamydia trachomatis, in which all mutations have been identified by whole-genome sequencing, providing a platform for reverse genetic applications. An analysis of possible loss-of-function mutations in the collection uncovered plasticity in the central metabolic properties of this obligate intracellular pathogen. We also describe the use of the library in a forward genetic screen that identified InaC as a bacterial factor that binds host ARF and 14-3-3 proteins and modulates F-actin assembly and Golgi redistribution around the pathogenic vacuole. This work provides a robust platform for reverse and forward genetic approaches in Chlamydia and should serve as a valuable resource to the community.
Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, 268 CARL Building, Box 3054, Durham, NC 27710, USA.
Marcela
M
Kokes
Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, 268 CARL Building, Box 3054, Durham, NC 27710, USA.
Joe Dan
JD
Dunn
Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, 268 CARL Building, Box 3054, Durham, NC 27710, USA; Department of Biostatistics and Bioinformatics, Duke University Medical Center, 2424 Erwin Road, Suite 1102 Hock Plaza, Box 2721, Durham, NC 27710, USA.
Joshua A
JA
Granek
Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, 268 CARL Building, Box 3054, Durham, NC 27710, USA.
Bidong D
BD
Nguyen
Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, 268 CARL Building, Box 3054, Durham, NC 27710, USA.
Jeffrey R
JR
Barker
Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, 268 CARL Building, Box 3054, Durham, NC 27710, USA. Electronic address: raphael.valdivia@duke.edu.
Raphael H
RH
Valdivia
Department of Molecular Genetics and Microbiology and Center for the Genomics of Microbial Systems, Duke University Medical Center, 268 CARL Building, Box 3054, Durham, NC 27710, USA. Electronic address: robert.bastidas@duke.edu.
Robert J
RJ
Bastidas
D002692:Chlamydia trachomatis / Q000235:genetics*; D005819:Genetic Markers; D005820:Genetic Testing; D005827:Genetics, Microbial / Q000379:methods*; D016680:Genome, Bacterial; D008967:Molecular Biology / Q000379:methods*; D016296:Mutagenesis; D009154:Mutation; D059386:Reverse Genetics / Q000379:methods*; D017422:Sequence Analysis, DNA
D005819:Genetic Markers