Chlamydial rRNAOperons: GeneOrganization andIdentification of Putative TandemPromoters

We isolated andcharacterized therRNA operonsofmurineChlamydia trachomatis. Byexhaustively screening a library ofchlamydial DNAandbyblot hybridization ofgenomic DNA,we showedthatthere are onlytworRNAoperonsinC.trachomatis. S1nuclease protection andprimer extension analysis wereusedto map the5'and3'endsofthemature16Sand23Stranscripts inbothrRNAcistrons and,additionally, to demonstrate thelackofintervening sequencesinthese genes.The5'endsofthepresumed primaryrRNA transcript werelocated andfound tooriginate attwotandem sites separated by100basepairs. Thetwotandem chlamydial rDNA transcripts werenotdifferentially regulated. Their products werecoordinately expressed and weredetectable asearlyas9hpostinfection. However, theupstream transcript was only10%asabundant as thedownstream transcript. Thesequencessurrounding thetranscription initiation sites borelittle homology witheachother orwiththeclassic Escherichia coli -10and-35promoter sequences.Thisfinding suggests that chlamydial transcription signals may differ fromthose ofpreviously studied procaryotes. Chlamydiae areobligate intracellular eubacterial parasites ofmammalian cells andareimportant humanpathogens. Theyarethemajorcauseofsexually transmitted diseases andpreventable infertility intheUnitedStates andthe leading causeofpreventable blindness (trachoma) inless developed countries. Chlamydiae display a complex life cycle involving thesequential alternation oftwodifferent morphologic forms, theelementary body(EB)andthe reticulate body.TheEB,theextracellular formofthe organism, isinfectious, although itismetabolically inactive. Entry ofEBsinto susceptible cells initiates anintracellular growth anddifferentiation cycle whichproceeds according toastrict temporal program. Thisdevelopmental cycle has beendescribed morphologically andtoa limited extent biochemically (for areview, seereferences 1and17), but little isknownabout thesignals that trigger these differentiation events orthemolecular mechanisms that govern their operation. By analogy withotherprocaryotes, suchas Myxococcus spp.(9,20)andBacillus spp.(8,14),the chlamydial developmental program islikely toinvolve the constitutive expression ofsomegenesandthedevelopmentally regulated expression ofothers. Characterization of these geneclasses andidentification oftheir cis- andtransacting regulatory elements arefundamental tounderstanding theprogramming ofthechlamydial life cycle. Tobegin ourstudy ofthemolecular control ofthechlamydial life cycle, wechosetostudy therRNAgenes ofthe mousepneumonitis strain (MoPn)ofChlamydia trachomatis. Thischoice wasmadeforseveral reasons. Transcripts of these genes arelikely tobeamongthemostabundant inthe cell, whichmakesitrelatively straightforward toclone them. Sequence comparisons ofpromoters fronm these andother constitutive chlamydial genes will helptodefine theprimary structure ofchlamydial promoters. Inaddition, rRNAgenes inother procaryotes havebeenextensively characterized, permitting ready sequence comparison between species (for areview, seereference 11). Lastly, these rRNAgenesare transcribed veryefficiently; thus, their strong promoters