Chlamydia trachomatis is the most common cause of bacterial sexually transmitted infection. It produces an unusual intracellular infection in which a vegetative form, called the reticulate body (RB), replicates and then converts into an elementary body (EB), which is the infectious form. Here we use quantitative three-dimensional electron microscopy (3D EM) to show that C. trachomatis RBs divide by binary fission and undergo a sixfold reduction in size as the population expands. Conversion only occurs after at least six rounds of replication, and correlates with smaller RB size. These results suggest that RBs only convert into EBs below a size threshold, reached by repeatedly dividing before doubling in size. A stochastic mathematical model shows how replication-dependent RB size reduction produces delayed and asynchronous conversion, which are hallmarks of the Chlamydia developmental cycle. Our findings support a model in which RB size controls the timing of RB-to-EB conversion without the need for an external signal.

Bacteria in the genus Chlamydia are major human pathogens that cause an intracellular infection. A chlamydial protease, CPAF, has been proposed as an important virulence factor that cleaves or degrades at least 16 host proteins, thereby altering multiple cellular processes. We examined 11 published CPAF substrates and found that there was no detectable proteolysis when CPAF activity was inhibited during cell processing. We show that the reported proteolysis of these putative CPAF substrates was due to enzymatic activity in cell lysates rather than in intact cells. Nevertheless, Chlamydia-infected cells displayed Chlamydia-host interactions, such as Golgi reorganization, apoptosis resistance, and host cytoskeletal remodeling, that have been attributed to CPAF-dependent proteolysis of host proteins. Our findings suggest that other mechanisms may be responsible for these Chlamydia-host interactions, and raise concerns about all published CPAF substrates and the proposed roles of CPAF in chlamydial pathogenesis.

Chlamydia trachomatis RNA polymerase was partially purified by heparin-agarose chromatography and used in conjunction with a plasmid-borne G-less cassette template to characterize the C. trachomatis rRNA P1 promoter in vitro. Stepwise mutational analysis revealed that sequences in the ؊10, ؊25, and ؊35 regions are necessary for promoter activity, but no sequence upstream of position ؊40 is required. Partially purified C. trachomatis RNA polymerase and purified Escherichia coli holoenzyme exhibited some differences in promoter specificity.Chlamydia trachomatis is a gram-negative obligate intracellular pathogen with a complex life cycle characterized by two serial morphologic forms (reviewed in references 26 and 34). Stage-specific expression has been demonstrated in early (38) and late (1-3, 8, 14, 19, 28) periods of the developmental cycle, but the molecular basis of this gene regulation is not understood. Direct genetic studies of promoter structures have not been feasible; transient transformation of chlamydiae has been described (35), but a practical DNA-mediated transformation system does not exist.In the absence of a genetic system, several approaches have been taken to identify and characterize chlamydial promoters. The transcription initiation sites of several abundant transcripts have been identified, and their upstream regions have been examined for putative promoter sequences. By inspection, appropriately spaced canonical Escherichia coli 70 -type promoter sequences are present in a few of these upstream regions. These include the C. trachomatis plasmid countertranscript (PCT) (15), envA P1 (13, 22), and the dnaK operon of the C. trachomatis mouse pneumonitis biovar (11). Others, including the hctB, ltuA, and ltuB genes, have 70 promoterlike sequences at the putative Ϫ10 region only. Interestingly, these three genes are all transcribed late in the developmental cycle (14). However, many chlamydial genes are not preceded by 70 -like promoter sequences (i.e., there is no more than a 50% match), and no consensus chlamydial promoter sequence is apparent (8). Explanations for this sequence diversity include the possibilities that (i) some of the presumptive chlamydial transcription initiation sites are instead processing sites, (ii) the putative promoters belong to more than one class of promoters, and (iii) there is latitude in the promoter specificity of C. trachomatis A RNA polymerase (RNAP). The subunits of C. trachomatis RNAP have been cloned and sequenced (9,10,18,21).A , a C. trachomatis 70 homolog (9, 21), is the only sigma factor identified in chlamydiae to date.A and 70 share striking amino acid sequence conservation in subregions 2.4 and 4.2 (9, 21) of 70 , including the specific amino acid residues which have been shown in E. coli to be involved in promoter recognition at the Ϫ10 and Ϫ35 positions (reviewed in reference 5). This conservation of amino acid residues involved in promoter recognition contrasts with the observed variability in the DNA sequences of the putative C. trachomatis promote...

Tryptophan is an essential amino acid that is required for normal development in Chlamydia species, and tryptophan metabolism has been implicated in chlamydial persistence and tissue tropism. The ability to synthesize tryptophan is not universal among the Chlamydiaceae, but species that have a predicted tryptophan biosynthetic pathway also encode an ortholog of TrpR, a regulator of tryptophan metabolism in many gram-negative bacteria. We show that in Chlamydia trachomatis serovar D, TrpR regulates its own gene and trpB and trpA, the genes for the two subunits of tryptophan synthase. These three genes form an operon that is transcribed by the major form of chlamydial RNA polymerase. TrpR acts as a tryptophan-dependent aporepressor that binds specifically to operator sequences upstream of the trpRBA operon. We also found that TrpR repressed in vitro transcription of trpRBA in a promoter-specific manner, and the level of repression was dependent upon the concentrations of TrpR and tryptophan. Our findings provide a mechanism for chlamydiae to sense changes in tryptophan levels and to respond by modulating expression of the tryptophan biosynthesis genes, and we present a unified model that shows how C. trachomatis can combine transcriptional repression and attenuation to regulate intrachlamydial tryptophan levels. In the face of host defense mechanisms that limit tryptophan availability from the infected cell, the ability to maintain homeostatic control of intrachlamydial tryptophan levels is likely to play an important role in chlamydial pathogenesis.

HrcA is a regulator of bacterial heat shock gene expression that binds to a cis-acting DNA element called CIRCE. It has been proposed that HrcA and CIRCE function as a repressor-operator pair. We have purified recombinant HrcA from the pathogenic bacterium Chlamydia trachomatis and have shown that it is a DNAbinding protein that functions as a negative regulator of transcription. HrcA bound specifically to the CIRCE element in a concentration-dependent manner. HrcA repressed the in vitro transcription of a chlamydial heat shock promoter, and this repression was promoter specific. HrcA-mediated repression appears to be dependent on the topological state of the promoter, as repression on a supercoiled promoter template was greater than that on a linearized template. These results provide direct support for the role of HrcA as a transcriptional repressor in bacteria. This is the first report of the in vitro reconstitution of transcriptional regulation in Chlamydia.