TitleInterrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination.
Publication TypeJournal Article
Year of Publication2016
AuthorsBrothwell, JA, Muramatsu, MK, Toh, E, Rockey, DD, Putman, TE, Barta, ML, P Hefty, S, Suchland, RJ, Nelson, DE
JournalJ Bacteriol
Date Published2016 08 01
KeywordsAlleles, Chlamydia trachomatis, Gene Expression Regulation, Bacterial, Genome, Bacterial, Genotype, HeLa Cells, Humans, Mutation, Recombination, Genetic, Temperature

UNLABELLED: 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.

IMPORTANCE: 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.

Alternate JournalJ Bacteriol
PubMed ID27246568
PubMed Central IDPMC4944222
Grant ListP20 GM113117 / GM / NIGMS NIH HHS / United States
T32 AI007637 / AI / NIAID NIH HHS / United States
R01 AI116706 / AI / NIAID NIH HHS / United States
R56 AI099278 / AI / NIAID NIH HHS / United States
R01 AI099278 / AI / NIAID NIH HHS / United States