FIG. 7.

In vitro phosphorylation assay to detect enzyme I (MPN627) in M. pneumoniae crude extracts. M. pneumoniae His₆-tagged HPr (20 μM) was incubated with 1 μg of crude extract and 0.5 mM PEP in assay buffer in a final volume of 20 μl at 37°C for 30 min. Assay mixtures that had been incubated for an additional 10 min at 70°C to hydrolyze the heat-labile HPr(His∼P) and samples where PEP had been omitted served as negative controls. The proteins were analyzed by using 10% native PAGE. The crude extracts were prepared from cells that had been cultivated in the presence of different sugars as indicated. The first three lanes are positive controls with M. pneumoniae His₆-tagged HPr (first lane), in vitro phosphorylated His₆-tagged HPr(His∼P) using B. subtilis enzyme I (second lane), and the same assay mixture after 10 min at 70°C (third lane).