Figure 5. EtpY121F retained phosphatase activity but failed to support capsule formation.

(A) Proteins were extracted from wild-type EPEC, the Δetp::kan mutant, or mutant complemented with plasmids derived from pEtp (pCNY506) and expressing different Etp variants with C-terminal 6His tags. The levels of Etk, phosphorylated Etk, recombinant Etp, and phosphorylated Etp were determined by Western blot analysis using anti-Etk, anti-6His, and anti-PY antibodies. The strain and the complementing mutant of Etp are indicated above the blots and the antibodies used in the immunoblots are indicated on the left side. The levels of Etk dephosphorylation and Etp autodephosphorylation are indicated below the blots. (B) Capsule polysaccharide was extracted and purified from the same strains presented in (A). Two-fold serial dilutions of purified capsule were dotted on PVDF and developed with anti-O127 antibody. The identity of the strain and complementing plasmid are indicated above the blot and direction of capsule polysaccharide dilution is indicated at the right side of the blot.