Testing the effect of a wbbI knockout on T4 bacteriophage resistance in Escherichia coli K-12

Abstract

Since its isolation, Escherichia coli K-12 has been used as a model organism and has become a common laboratory strain. In the outer membrane of E. coli, the O16 antigen is attached to the core sugars in lipopolysaccharide. The O16 antigen is synthesized by the WbbL, WbbK, WbbJ, and WbbI proteins encoded by the rfb cluster. Previous studies have shown that strains that possess a functional wbbL and express the O16 antigen demonstrate resistance to T4 bacteriophage, while strains that do not express the O16 antigen due to a disrupted wbbL gene are susceptible. However, the mechanism of this resistance remains unknown. Knowing that WbbI catalyzes the linkage of the distal sugar, D-galactofuranose, to the O16 antigen, the goal of this study was to determine the effect of a wbbI knockout on T4 bacteriophage resistance. Previous mechanisms have proposed that T4 tails attach to D-galactofuranose of the O16 antigen, which hinders the attachment of the T4 tails to the OmpC receptor. Thus, we hypothesized that the absence of D-galactofuranose on the O16 antigen as a result of a wbbI knockout would decrease resistance to T4, since attachment is no longer hindered by the O16 antigen. To investigate our hypothesis, we cloned wbbL back into JW2019-1, which contains a wbbI knockout as well as a disrupted wbbL gene, such that it expressed the truncated O16 antigen. We tested the wbbI knockout strain for T4 bacteriophage susceptibility via a stab assay. However, because our controls did not demonstrate the expected resistance phenotype to T4 bacteriophage, we were unable to determine the effect of a wbbI knockout on T4 bacteriophage resistance.