Nelson, David E.Brothwell, Julie AnnBauer, Margaret E.Gilk, Stacey D.Sullivan, William J., Jr.2017-05-112018-05-122016-12-07https://hdl.handle.net/1805/12509http://dx.doi.org/10.7912/C2/1748Indiana University-Purdue University Indianapolis (IUPUI)Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease. Chlamydia spp. are all obligate intracellular organisms that undergo a biphasic developmental cycle within a vacuole termed the inclusion. Infectious, non metabolically active elementary bodies (EBs) are endocytosed and differentiate into non infectious, metabolically active reticulate bodies (RBs) before re-differentiating back into EBs. The chlamydial factors that mediate these differentiation events are mostly unknown. Comparative genomics revealed that Chlamydia spp. have small, highly conserved genomes, suggesting that many of their genes may be essential. Genetic manipulation strategies for Chlamydia spp. are in their infancy, and most of these cannot be used to inactivate essential genes. We generated a clonal ethyl methanesulfonate (EMS)-mutagenized C. trachomatis library and screened it for temperature sensitive (TS) mutants that produced fewer inclusions at either 32°C or 40°C compared to 37°C. Because EMS mutagenesis elicited multiple mutations in most of the library isolates, we also developed a novel lateral gene transfer strategy for mapping mutations linked to TS phenotypes. We identified TS alleles of genes that are essential in other bacteria and that are involved in diverse biological processes including DNA replication, protein synthesis, carbohydrate metabolism, fatty acid biosynthesis, and energy generation, as well as in highly conserved chlamydial hypothetical genes. TS DNA polymerase (dnaEts) and glutamyl-tRNA synthestase (gltXts) mutants were characterized further. Both the dnaEts and gltXts mutants failed to replicate their genomes at 40°C but exhibited unique signs of stress. Chlamydial DNA replication begins by 12 hpi and protein synthesis begins by 2 hpi. However, inclusion expansion and replication of both of the mutants could be rescued by shifting to them to 37°C prior to mid-late development. Since gltXts is likely unable to produce aminoacyl-tRNAs at 40°C, our observation suggests that de novo chlamydial translation uses a pre-existing pool of aminoacyl-tRNA in EBs. Genetic suppressor analysis indicated that the inability of the dnaEts mutant to replicate its genome at 40°C might be linked to an inability of mutant DnaE to bind the DNA template. The tools and mutants we have identified will be invaluable assets for investigating many essential aspects of chlamydial biology.en-USChlamydiaEssential genesGeneticsMutagenesisDissertation10.7912/C2CP5N