Date of Award

6-2020

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Dr. Brian Tripp

Second Advisor

Dr. Silvia Rossbach

Third Advisor

Dr. Pamela Hoppe

Keywords

Salmonella typhimurium, flagella, motility, hyper-coiling, site-directed mutagenesis

Access Setting

Masters Thesis-Open Access

Abstract

Bacterial flagella are comprised of ~20,000 repeating subunits called flagellin and are synthesized by transporting unfolded flagellin monomers through the central channel of the flagellar filament to the distal end where they are folded and incorporated into the growing structure. The transport mechanism is predicted to be facilitated by a hydrophilic coating of amino acid side chains on the inside wall of the central channel that repels the mostly hydrophobic surface of the unfolded flagellin monomer. The lack of interaction between the flagellin monomer and the central channel wall is thought to allow for the passive diffusion of protein monomers. In this study, the necessity of the hydrophilic lining of the central channel wall was investigated by site-directed mutagenesis of amino acids that are predicted to line the central channel, called “channel-lining residues”, which consist of Gln 484, Asn 488, Ser 491 and Arg 494. Single and double mutants were created that substituted each channel-lining residue with a different amino acid, primarily alanine. Characterization of the mutants suggested that channel lining residues may not be explicitly necessary for flagellin monomer transport. However, mutants were found to influence other aspects of the flagellar filament such as length, morphology and stability. It is likely that the channel-lining residues are multifunctional, serving many functional roles and not just the transport facilitating role that has been predicted.

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