FIG. 6.

In vitro phosphorylation assay to detect HPrK/P (MPN223) in M. pneumoniae crude extracts. M. pneumoniae His₆-tagged HPr (20 μM) was incubated with 5 μg of crude extract and 0.5 mM ATP in assay buffer in a final volume of 20 μl at 37°C for 120 min. Subsequently, the HPrK/P was heat inactivated by boiling for 10 min. The proteins were analyzed by using 10% native PAGE. M. pneumoniae crude extracts were from cells that had been cultivated in the presence of different sugars as indicated. The first lanes are positive controls with M. pneumoniae His₆-tagged HPr (first lane) and His₆-tagged HPr that had been phosphorylated at Ser-46 in vitro (second lane).