Unraveling the phenotype of colicin cytoplasmic import (cim) mutants

Abstract

Colicins are a type of bacterial toxin produced by Escherichia coli to kill E. coli and closely related bacteria. In contrast to the protein secretion pathway, colicins are released into the extracellular environment due to lysis of the cell. The colicins then enter the target bacterial cell by binding to a surface receptor (ex. BtuB) and translocating through an outer membrane channel (ex. OmpF) which is facilitated by the interaction with the Tol proteins. Once inside the target cell, pore-forming colicins form membrane channels in the inner membrane whereas enzymatic colicins enter the cytoplasm to degrade the DNA or RNA. The mechanism used by enzymatic colicins to enter the cytoplasm is still unknown and is the focus of my project. Cim (cytoplasmic import) mutants were previously discovered in the Benedik lab. These mutants are resistant to enzymatic colicins (E2 and E3) but sensitive to pore forming colicins (A and E1). It was determined that this phenotype was due to a single nucleotide substitution resulting in an amino acid change in tolB. The focus of my project was to understand the phenotype of the cim mutants so that I could gain more insight regarding the import of enzymatic colicins. To do this I constructed colicin hybrids by swapping the T-R domains with the A-I domains of colicins A, E1, and E2. With these hybrids I was able to test whether an enzymatic colicin that was coupled with a pore-forming colicin would be functional or not. The lack of activity in these hybrids may be due to a lack of essential recognition elements used by enzymatic colicins and not possessed by pore-forming colicins.