Plasmid-mediated complementation of wza restores erythromycin susceptibility in Escherichia coli K30 strain CWG281

SUMMARY Group 1 capsules are recognized as virulence determinants in bacterial pathogens of humans and animals. They enhance virulence by conferring resistance to phagocytosis and increasing adherence to host tissues. The assembly and transport of type 1 capsular polysaccharide in Escherichia coli K30 is mediated through the Wzy-dependent pathway. This pathway involves: Wza, an outer membrane channel; Wzb, a cytosolic phosphatase; and Wzc, an autokinase in the inner membrane. Previous research has shown that deletion of Wza confers resistance to erythromycin. It has been suggested that the route of entry of erythromycin is partially dependent on the Wza channel. In this study, we hypothesize that plasmid-mediated complementation of wza will restore erythromycin sensitivity in an E. coli wza knockout mutant. The wza gene was previously cloned into an arabinose-inducible pBAD24 vector and transformed into the wza knockout mutant. Using a modified arabinose-antibiotic disc diffusion assay, 1% arabinose was observed to induce optimal pBAD24-wza expression without impairing cell growth and appropriately evaluate erythromycin sensitivity in the wza knockout mutant. We found that, when induced, pBAD24-wza transformants showed higher erythromycin sensitivity compared to the control strains without the plasmid. Our findings indicate that plasmid-mediated complementation of wza gene restores erythromycin sensitivity in the wza knockout mutant strain.