Effect of Site-directed Mutations on Flagellin Transport in Salmonella Typhimurium
Love, Chloe R.
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Bacterial flagella are a self-assembling organelle which enable motility. The flagellar filament is composed of a single protein, flagellin, and forms a long tube-like structure with a narrow channel at the center. Flagellin monomers diffuse down this channel and self-assemble at the tip of the filament. Four amino acids line this channel, including positively-charged arginine. To determine the role of the positive charge, polarity, and identity of the terminal arginine in the export of flagellin and general motility, we performed site-directed mutagenesis, replacing arginine with glutamate, aspartic acid, and tryptophan and creating three mutant strains of Salmonella typhimurium. SDS-PAGE gel electrophoresis showed that export of flagellin occurs in all mutant strains, and motility assays indicated limited swimming ability for both glutamate and aspartic acid mutants. Analysis of tracking data indicated that the average speed of one of the aspartic acid strains was greater than that of pTH890-FLiC, the positive control, while another aspartic acid mutant showed no significant difference in average speed. The glutamate strain was found to have a significantly slower movement speed than the control. The tryptophan mutant was completely nonmotile and did not swim on motility plates or in growth media when observed under a microscope. The results suggest that size and lack of charge may be important factors in channel-lining amino acids, and that these characteristics may have an impact on filament assembly and motility.