The assembly of the bacterial flagellum in Salmonella is achieved via a flagellar type III secretion system (fT3SS), translocating flagellar proteins from the cytoplasm through the center of the growing organelle where they self-assemble at the distal end of a growing structure. Upon completion of an intermediate assembly structure (the hook-basal body or HBB), the fT3SS undergoes a secretion substrate specificity switch from early assembly proteins (HBB subunits) secretion to late substrate proteins (filament subunit) secretion. We previously isolated a spontaneous mutation in FliQ (G32D) that enables late substrate (FlgM-bla) secretion prior to HBB assembly. FliQ is a core component of the fT3SS. To explore how FliQ partakes in the flagellar specificity switch, FliQ was subjected to targeted doped oligo mutagenesis in order to identify other residues in FliQ that would cause a similar effect as the FliQ G32D mutant. We were able to isolate such a critical residue in at amino acid 48 (T48M) in FliQ. Subsequent targeted randomized (NNN) mutagenesis of amino acid codons 32 and 48 of FliQ revealed amino acid substitutions with similar secretion properties as the FliQ G32D and T48M alleles that are able to switch flagellar specificity prior to HBB assembly plus other substitutions that were defective in secretion. We therefore can conclude that both the G32 and T48 amino acid residues of FliQ are crucial to the function of FliQ and to the secretion-specificity switch. To test whether FliQ mutants lose or retain the ability to facilitate early protein secretion, we will construct a fusion product of β-lactamase protein lacking its Sec-dependent secretion signal (Bla) to the C-terminus of FlgE, a hook subunit protein (early secretion-substrate), as a reporter for detecting early flagellar protein secretion. In addition, all fliQ mutants will be complemented with wild type fliQ to characterize their dominant or recessive features. This in-depth research is a continuation of my UROP project, and this study should add its own unique contribution to the current understanding of the flagellar secretion-substrate specificity switch.