FIG. 7.

Western blot analysis of the HPr and its phosphorylated forms (HPr-His15-P or HPr-Ser46-P) to determine the amount of HPr-Ser46-P in L. monocytogenes strains. Equal amounts of cell extracts untreated (−) or incubated at 70°C for 10 min (+) to hydrolyze the heat-labile HPr-His15-P were separated on a 15% nondenaturing polyacrylamide gel and immunoblotted using specific rabbit polyclonal antibodies against HPr. The positions of HPr, HPr-Ser46-P, and HPr-His15-P are indicated. Equivalent loading of the gels was controlled by Coomassie-staining (data not shown). (A) Detection of HPr and its phosphorylated forms (HPr-His15-P or HPr-Ser46-P) in WT EGD-e and the ccpA mutant grown in MM supplemented with 50 mM glucose to OD₆₀₀s of 0.6 and 1.0. The hprK mutant (control; only able to phosphorylate HPr at His15; grown in MM supplemented with 50 mM glucose to an OD₆₀₀ of 1.0) and purified HPr and HPr-Ser46-P with and without incubation were separated on the nondenaturing polyacrylamide gel to show that the incubation at 70°C for 10 min has no effect on HPr or HPr-Ser46-P. (B) Determination of HPr and its phosphorylated forms (HPr-His15-P or HPr-Ser46-P) in WT EGD-e grown in MM supplemented with 50 mM glucose (G), 50 mM cellobiose (C), or 50 mM mannose (M) to OD₆₀₀s of 0.6 and 1.0. The hprK mutant (control; see panel A) and purified HPr and HPr-Ser46-P were separated on the gel to indicate the different HPr forms. ::ccpA and ::hprK, insertion mutants of ccpA and hprK, respectively.