Group A colicins are proteinaceous bacteriocins encoded by plasmids that exploit the cellular envelope protein TolA to translocate the cell wall barrier and cellular envelope of the bacterium Escherichia coli. These colicins offer protocols for studying certain protein-protein interactions involved in such membrane transport functions. Previous experimentations suggest the carboxyl-terminal domain of TolA protein contains specific amino acid binding regions required for the translocation of group A colicins into E. coli. The amino acid sequence of this domain varies between E. coli and other gram-negative bacterial species. It has been suggested that this diversity could be utilized to identify specific TolA residues involved in the binding of colicins. Additionally, other Enterobacteriaceae species’ TolA genes were cloned into a plasmid that is regulated by the monosaccharide sugar, arabinose. This plasmid was transformed into an E. coli strain that lacked a functional tolA gene. The plasmid was expressed in this strain. Homologous complementation of this phenotype lacking tolA expression has been demonstrated through resistance to the bile acid deoxycholate. The expression of this complemented phenotype through cloned ΔtolA genes demonstrates expression and translocation of TolA protein through the outer membrane into the periplasmic space. Bacterial strains possessing these constructs and functional TolA were tested for sensitivity against two group a colicins: A and N. Two such species, Chronobacter muytjensii and Yersinia enterocolitica complements the phenotype lacking functional TolA with their own encoded TolA proteins. These functional proteins grant sensitivity to various group A colicins, apart from colicin N. The TolA proteins of C. muytjensii, E. coli, Y. enterocolitica and the corresponding genetic sequences can be contrasted to highlight site of differing residues. These sites of genetic sequence divergence in E. coli, C. muytjensii, and Y. enterocolitica can be altered through mutagenesis to identify sequence domains required for colicin N binding to TolA, and thus import into the corresponding bacterium.
Dr. Raymond Larsen
First Advisor Department
Dr. Neocles Leontis
Second Advisor Department
Ferrante, Monica, "Identification of a TolA Protein Binding Site for Bacterial Toxins" (2018). Honors Projects. 348.