Defective O-Antigen Polymerization intolAandpalMutants ofEscherichia coliin Response to Extracytoplasmic Stress

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Abstract
We have previously shown that the TolA protein is required for the correct surface expression of theEscherichia coliO7 antigen lipopolysaccharide (LPS). In this work, ΔtolAand Δpalmutants ofE. coliK-12 W3110 were transformed with pMF19 (encoding a rhamnosyltransferase that reconstitutes the expression of O16-specific LPS), pWQ5 (encoding theKlebsiella pneumoniaeO1 LPS gene cluster), or pWQ802 (encoding the genes necessary for the synthesis ofSalmonella entericaO:54). Both ΔtolAand Δpalmutants exhibited reduced surface expression of O16 LPS as compared to parental W3110, but no significant differences were observed in the expression ofK. pneumoniaeO1 LPS andS. entericaO:54 LPS. Therefore, TolA and Pal are required for the correct surface expression of O antigens that are assembled in awzy(polymerase)-dependent manner (like those ofE. coliO7 and O16) but not for O antigens assembled bywzy-independent pathways (likeK. pneumoniaeO1 andS. entericaO:54). Furthermore, we show that the reduced surface expression of O16 LPS in ΔtolAand Δpalmutants was associated with a partial defect in O-antigen polymerization and it was corrected by complementation with intacttolAandpalgenes, respectively. Using derivatives of W3110ΔtolAand W3110ΔpalcontaininglacZreporter fusions tofkpAanddegP, we also demonstrate that the RpoE-mediated extracytoplasmic stress response is upregulated in these mutants. Moreover, an altered O16 polymerization was also detected under conditions that stimulate RpoE-mediated extracytoplasmic stress responses intol+andpal+genetic backgrounds. A Wzy derivative with an epitope tag at the C-terminal end of the protein was stable in all the mutants, ruling out stress-mediated proteolysis of Wzy. We conclude that the absence of TolA and Pal elicits a sustained extracytoplasmic stress response that in turn reduces O-antigen polymerization but does not affect the stability of the Wzy O-antigen polymerase.

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