FIG. 3.

The Walker A motif of ImpL_M is required for Hcp secretion. (A) Alignment of the GXXXXGKT/S Walker A motifs conserved in selected T6SS and T4SSb IcmF family proteins, indicated by protein name and accession number, in several gram-negative bacteria, with the conserved amino acid residues highlighted. The arrowheads indicate the amino acids targeted for mutagenesis, and the underlining indicates the Walker A motif targeted for deletion. a.a, amino acid. (B) Total (T) and secreted (S) proteins isolated from ΔimpL_M strains harboring plasmids expressing either the wild-type (pImpL_M) or mutated ImpL_M (pImpL_M^K145A, pImpL_M^{G144A K145A}, or pImpL_M^{ΔWalker A}) protein. Cells grown in AB-MES (pH 5.5) for 6 h at 25°C were resolved by 12% glycine-SDS-PAGE and subjected to immunoblot analysis with C-ImpL_M, Hcp, and ActC antibodies for expression and secretion analysis. The positions of molecular mass markers (in kDa) are indicated on the left.