Three temporal classes of gene expression during the Chlamydia trachomatisdevelopmental cycle

The obligate intracellular bacterium Chlamydia trachomatis has a unique dev elopmental cycle that involves functionally and morphologically distinct ce ll types adapted for extracellular survival and intracellular multiplicatio n. Infection is initiated by an environmentally resistant cell type called an elementary body (EB). Over the first several hours of infection, EBs dif ferentiate into a larger replicative form, termed the reticulate body (RB). Late in the infectious process, RBs asynchronously begin to differentiate back to EBs, which accumulate within the lumen of the inclusion until relea sed from the host cell for subsequent rounds of infection. In an effort to characterize temporal gene expression in relation to the chlamydial develop mental cycle, we have used quantitative-competitive polymerase chain reacti on (QC-PCR) and reverse transcription (RT)-PCR techniques. These analyses d emonstrate that C. trachomatis double their DNA content every 2-3 h, with s ynthesis beginning between 2 and 4 h after infection. We determined the ons et of transcription of specific temporal classes of developmentally express ed genes. RT-PCR analysis was performed on several genes encoding key enzym es or components of essential biochemical pathways and functions. This comp arison encompassed approximately 8% of open reading frames on the C. tracho matis genome. In analysis of total RNA samples harvested at 2, 6, 12 and 20 h after infection, using conditions under which a single chlamydial transc ript per infected cell is detected, three major temporal classes of gene ex pression were resolved. Initiation of transcription appears to occur in thr ee temporal classes which we have operationally defined as: early, which ar e detected by 2 h after infection during the germination of EBs to RBs; mid -cycle, which appear between 6 and 12 h after infection and represent trans cripts expressed during the growth and multiplication of RBs; or late, whic h appear between 12 and 20 h after infection and represent those genes tran scribed during the terminal differentiation of RBs to EBs. Collectively, th e data suggest that chlamydial early gene functions are weighted toward ini tiation of macromolecular synthesis and the establishment of their intracel lular niche by modification of the inclusion membrane. Surprisingly, repres entative enzymes of intermediary metabolism and structural proteins do not appear to be transcribed until 10-12 h after infection; coinciding with the onset of observed binary fission of RBs. Late gene functions appear to be predominately those associated with the terminal differentiation of RBs bac k to EBs.