WalH and WalI are membrane-anchored extracellular proteins.

(A) E. coli DH5α cells producing ‘PhoA’-‘LacZ’ fusion proteins (WalH_1-40-PhoA_21-471-LacZ_4-60, WalI_1-40-PhoA_21-471-LacZ_4-60, or WalJ_1-40-PhoA_21-471-LacZ_4-60) were plated on indicator medium with two chromogenic substrates, Red-Gal (for β-galactosidase activity) and X-Pho (for phosphatase activity). Blue coloring of the colonies (high phosphatase activity) indicates a membrane or extracellular localization of the fusion point. Red coloring of the colonies (high β-galactosidase activity) indicates cytosolic localization of the fusion point. (B) Schematic representation of Wal protein localization and topology with respect to the cell membrane. In the case of WalK and WalR, topology and localization were deduced from primary sequence analysis using the Phobius Hidden Markov Model (indicated by stars).