Bacterial small RNAs (sRNAs) are short transcripts that play critical roles in post-transcriptional gene regulation. In the obligate intracellular pathogen Chlamydia trachomatis, however, sRNAs are poorly understood because genetic tools in Chlamydia have only recently become available. As such, functional studies of two chlamydial sRNAs were limited to an E. coli heterologous system. A major issue with this approach is that Chlamydia lacks the important sRNA chaperone protein, Hfq, that is found in E. coli. The heterologous system is also not suitable for studying the function of a sRNA in the Chlamydia developmental cycle, which involves conversion between the infectious (EB) and the replicative (RB) forms within a eukaryotic host cell.

With recent advances in Chlamydia genetics, we have developed an inducible sRNA overexpression system in C. trachomatis. Utilizing our approach, we conducted a genetic screen and identified 4 previously uncharacterized sRNAs (i.e. CtrR3, CtrR7, CtrR0332, and CTIG684) whose individual overexpression reduced the production of infectious chlamydial progeny. We then determined that overexpression of CtrR3 and CtrR0332 both blocked RB-to-EB conversion, whereas CtrR7 overexpression hindered RB replication. To demonstrate how our system can be used to find mRNA targets of a chlamydial sRNA, we took a multi-step approach to identify YtgB and CTL0389 as targets of CtrR3. We also showed the versatility of this genetic approach by applying it to develop a novel sRNA-mediated conditional knockdown system in C. trachomatis.

This is the first study to analyze sRNAs in Chlamydia and to experimentally demonstrate that chlamydial sRNAs can regulate gene expression. Our genetic approach for studying sRNAs and for identifying mRNA targets can be readily applied to elucidate the function of other chlamydial sRNAs. In addition, our knockdown system can be utilized to investigate the function of essential genes in C. trachomatis. Overall, the work described in this dissertation has significantly advanced our understanding of sRNAs and post-transcriptional gene regulation in C. trachomatis.

Some, but not all, Chlamydia spp. are predicted to encode a homolog of ArgR, a master regulatory molecule that modulates arginine biosynthesis and catabolism in bacteria in response to intracellular arginine levels. While genes for arginine biosynthesis are apparently missing in Chlamydia, a putative arginine transport system encoded by glnP, glnQ, and artJ is present. We found that recombinant Chlamydia pneumoniae ArgR functions as an arginine-dependent aporepressor that bound specifically to operator sequences upstream of the glnPQ operon. ArgR was able to repress transcription in a promoter-specific manner that was dependent on the concentration of the corepressor L-arginine. We were able to locate ArgR operators upstream of glnPQ in C. pneumoniae and Chlamydophila caviae but not Chlamydia trachomatis, which corresponded to the predicted presence or absence of ArgR in these chlamydial species. Our findings indicate that only some members of the family Chlamydiaceae have an arginine-responsive mechanism of gene regulation that is predicted to control arginine uptake from the host cell. This is the first study to directly demonstrate a species-specific mechanism of transcriptional regulation in Chlamydia.

HrcA is a transcriptional repressor that regulates stress response genes in many bacteria by binding to the CIRCE operator. We have previously shown that HrcA regulates the promoter for the dnaK heat shock operon in Chlamydia. Here we demonstrate that HrcA represses a second heat shock promoter that controls the expression of groES and groEL, two other major chlamydial heat shock genes. The CIRCE element of C. trachomatis groEL is the most divergent of known bacterial CIRCE elements, and HrcA had a decreased ability to bind to this nonconsensus operator and repress transcription. We demonstrate that the CIRCE element is necessary and sufficient for transcriptional regulation by chlamydial HrcA and that the inverted repeats of CIRCE are the binding sites for HrcA. Addition of a CIRCE element upstream of a non-heat-shock promoter allowed this promoter to be repressed by HrcA, showing in principle that a chlamydial promoter can be genetically modified to be inducible. These results demonstrate that HrcA is the regulator of the major chlamydial heat shock operons, and we infer that the mechanism of the heat shock response in Chlamydia is derepression. However, derepression is likely to involve more than a direct effect of increased temperature as we found that HrcA binding to CIRCE and HrcA-mediated repression were not altered at temperatures that induce the heat shock response.

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.