133 Mutational analysis of the promoter and 5’UTR of the fljB flagellin in Salmonella

Faculty Mentors: Fabienne Chevance and Kelly T. Hughes (School for Biological Sciences)

Salmonella enterica subspecies contribute significant public health concern in the United States, causing roughly 1.4 million infections and nearly 600 deaths annually. Many Salmonella enterica, including S. Typhimurium and S. Typhi, are motile pathogens and their virulence and survival are dependent on the presence of surface motility organelles termed flagella. The bacterial flagellum is comprised of three main structural components: the basal body, the hook, and the filament. A Salmonella bacterium expresses, on average, four peritrichous flagella per cell. Each flagellum constitutes one percent of the total cellular protein production. The biosynthesis of these structures is tightly regulated. A single bacterial flagellar filament is composed of approximately 20,000 flagellin protein subunits encoded by either the fliC or fljB genes. Most of the flagellum (the hook-filament) is located extracellularly, and structural components need to be secreted from the cytosol through the growing organelle where they self-assemble at the structure’s tip. The translocation of these proteins is mediated by a Type III secretion system embedded at the base of the flagellum. Previous work has suggested that the 5’-untranslated region (5’UTR) of the flagellar genes contains crucial regulatory signals responsible for efficient protein production during flagellum assembly. In the present study, we aim to dissect the regulatory signals within the 5’UTR of the filament gene fljB with targeted doped mutagenesis via the targeted gene editing process of lambda-Red recombineering. Base substitutions that are defective in transcription identified the fljB promoter region that is recognized by RNA polymerase for transcription. Our results also identified several distinct predicted stem-loop 2° structures in the 5’UTR region of fljB mRNA. Analysis of motile revertants from a fljB mutant pool revealed possible alternative RNA stem-loop structures in the 5’UTR that affect ribosome binding needed for fljB mRNA translation. The outcome of the study intends to understand the unexplored aspects of Salmonella flagellar regulation, offering insights into the intricate mechanisms governing virulence and host survivability. Understanding these regulatory signals at the molecular level holds potential implications for developing targeted drug strategies, including novel therapeutic interventions or vaccines, in order to mitigate Salmonella infections.