title: Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination.
abstract: Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle.
  
  
  
  Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms.
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Brothwell
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Muramatsu
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Toh
authors.affiliation: Department of Biomedical Sciences, Oregon State University, Corvallis, Oregon, USA.
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Rockey
authors.affiliation: Department of Biomedical Sciences, Oregon State University, Corvallis, Oregon, USA.
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Putman
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Barta
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA.
authors.forename: P Scott
authors.initials: PS
authors.lastname: Hefty
authors.affiliation: Division of Allergy and Infectious Disease, Department of Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Suchland
authors.affiliation: Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA nelsonde@indiana.edu.
authors.forename: David E
authors.initials: DE
authors.lastname: Nelson
mesh_terms: D000483:Alleles; D002692:Chlamydia trachomatis / Q000235:genetics / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D016680:Genome, Bacterial; D005838:Genotype; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D011995:Recombination, Genetic; D013696:Temperature