Surface expression of O-specific lipopolysaccharide in Escherichia coli requires the function of the TolA protein

We investigated the involvement of Tol proteins in the surface expression o f lipopolysaccharide (LPS). tolQ, -R, -A and -B mutants of Escherichia coli K-12, which do not form a complete LPS-containing O antigen, were transfor med with the O7(+) cosmid pJHCV32. The tolA and tolQ mutants showed reduced O7 LPS expression compared with the respective isogenic parent strains. No changes in O7 LPS expression were found in the other tol mutants. The O7-d eficient phenotype in the tolQ and tolA mutants was complemented with a pla smid encoding the tolQRA operon, but not with a similar plasmid containing a frameshift mutation inactivating tolA. Therefore, the reduction in O7 LPS was attributed to the lack of a functional tolA gene, caused either by a d irect mutation of this gene or by a polar effect on tolA gene expression ex erted by the tolQ mutation. Reduced surface expression of O7 LPS was not ca used by changes in lipid A-core structure or downregulation of the O7 LPS p romoter. However, an abnormal accumulation of radiolabelled mannose was det ected in the plasma membrane. As mannose is a sugar unique to the O7 subuni t, this result suggested the presence of accumulated O7 LPS biosynthesis in termediates. Attempts to construct a tolA mutant in the E. coli O7 wild-typ e strain VW187 were unsuccessful, suggesting that this mutation is lethal. In contrast, a polar tolQ mutation affecting tolA expression in VW187 cause d slow growth rate and serum sensitivity in addition to reduced O7 LPS prod uction. VW187 tolQ cells showed an elongated morphology and became permeabl e to the membrane-impermeable dye propidium iodide. All these phenotypes we re corrected upon complementation with cloned tol genes but were not restor ed by complementation with the tolQRA operon containing the frameshift muta tion in tolA. Our results demonstrate that the TolA protein plays a critica l role in the surface expression of O antigen subunits by an as yet unchara cterized involvement in the processing of O antigen.