PROJECT SUMMARY The foodborne pathogen Salmonella is an important model organism for understanding genetic regulation and bacterial pathogenesis. A requisite for Salmonella to cause disease is the direct injection of effector proteins into host cells via a Type Three Secretion System (T3SS) encoded on Salmonella Pathogenicity Island 1 (SPI1). This critical virulence factor is controlled in response to a plethora of environmental and regulatory signals that dictate expression of the system at the proper time and place in the host. Our long- term goal is to understand overall signal integration that allows this precise regulation. The SPI1 regulatory circuit is controlled by three AraC-like regulators, HilD, HilC, and RtsA, which act in a complex feed-forward regulatory loop to control expression of hilA, encoding the direct regulator of the SPI1 structural genes. Much of the regulatory input is integrated at the level of HilD, including at hilD mRNA translation or stability. The hilD mRNA has an unusual 300 nucleotide 3’ untranslated region (UTR) that acts as an independent module to confer instability to the mRNA. A primary hypothesis is that the hilD 3’ UTR serves as a critical node for integration of regulatory signals. Preliminary data show that mRNA stability is regulated by a novel mechanism involving interaction between Rho-mediated transcriptional termination at the 3’ UTR and RNase E-dependent degradation. Moreover, these activities are independently controlled by sRNAs. The first aim of this proposal is to identify sRNAs and cis-acting sites that regulate via the hilD 3’ UTR. Interacting sRNAs will be identified using an unbiased molecular technique, with base pairing confirmed by mutagenesis. Deletion analysis will identify the site of Rho action in the 3’ UTR. The resulting hilD mRNAs with mutations in sRNA binding sites or Rho-utilization site provide tools for further mechanistic analyses. The second aim is to characterize the mechanism of post-transcriptional regulation via the hilD 3' UTR. The roles of Rho, RNase E, and the small RNAs in the creation and/or processing of the 3’ ends in the hilD 3’ UTR will be monitored using tagging and deep sequence analysis. In vitro transcription will more precisely define the action of Rho in creating terminated hilD transcripts. The interactions of these factors will reveal the mechanistic details of this novel regulation. The SP1 T3SS regulatory circuit serves as a paradigm for understanding the integration of host environmental signals to control a complex virulence phenotype. Analysis of this system is critical to our understanding of this important pathogen.