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While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature-sensitive\n            <jats:italic>dnaE<\/jats:italic>\n            mutant (the\n            <jats:italic>\n              dnaE\n              <jats:sup>ts<\/jats:sup>\n            <\/jats:italic>\n            mutant) that exhibits an \u223c100-fold reduction in genome copy number at the nonpermissive temperature (40\u00b0C) but replicates similarly to the parent at the permissive temperature of 37\u00b0C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in the\n            <jats:italic>\n              dnaE\n              <jats:sup>ts<\/jats:sup>\n            <\/jats:italic>\n            mutant at 40\u00b0C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. The\n            <jats:italic>\n              dnaE\n              <jats:sup>ts<\/jats:sup>\n            <\/jats:italic>\n            mutant formed fewer and smaller pathogenic vacuoles (inclusions) at 40\u00b0C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernible peptidoglycan and polymerized MreB, the major cell division-organizing protein in\n            <jats:italic>Chlamydia<\/jats:italic>\n            responsible for nascent peptidoglycan biosynthesis. We also found that the absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion were reversed when the\n            <jats:italic>\n              dnaE\n              <jats:sup>ts<\/jats:sup>\n            <\/jats:italic>\n            mutant was shifted from 40\u00b0C to 37\u00b0C early in infection, and intragenic suppressor mutations in\n            <jats:italic>\n              dnaE\n              <jats:sup>ts<\/jats:sup>\n            <\/jats:italic>\n            also restored genome replication and inclusion expansion in the\n            <jats:italic>\n              dnaE\n              <jats:sup>ts<\/jats:sup>\n            <\/jats:italic>\n            mutant at 40\u00b0C. Overall, our results show that genome replication in\n            <jats:named-content content-type=\"genus-species\">C. trachomatis<\/jats:named-content>\n            is required for inclusion expansion, septum formation, and the transition between the microbe\u2019s replicative and infectious forms.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and noninfectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen\u2019s infectious cycle has not been characterized. We show that genome replication is dispensable for EB-to-RB conversion but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in\n            <jats:italic>Chlamydia<\/jats:italic>\n            that facilitate the ordering of events in the microbe\u2019s biphasic life cycle. Our findings suggest that\n            <jats:italic>Chlamydia<\/jats:italic>\n            utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment.\n          <\/jats:p>","DOI":"10.1128\/jb.00630-20","type":"journal-article","created":{"date-parts":[[2021,1,8]],"date-time":"2021-01-08T14:43:02Z","timestamp":1610116982000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":6,"title":["Genome Copy Number Regulates Inclusion Expansion, Septation, and Infectious Developmental Form Conversion in Chlamydia trachomatis"],"prefix":"10.1128","volume":"203","author":[{"given":"Julie A.","family":"Brothwell","sequence":"first","affiliation":[{"name":"Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, 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