Fig. 4.

The conserved His208 residue in BglG is essential for its phosphorylation by HPr or FruB in vitro. Shown are PEP-dependent phosphorylation assays containing EI and HPr (lanes 1–4) or EI and FruB (lanes 5–8). In addition, the following BglG variants were present: Strep–BglG (lanes 1 and 5), Strep–BglG–H208A (lanes 2 and 6), Strep–S_tag–BglG (lanes 3 and 7), or Strep–S_tag–BglG–H208A (lanes 4 and 8). Assays contained either [³²P]-PEP (Upper) or 1 μM cold PEP (Lower). Proteins were subsequently separated on 15% SDS-polyacrylamide gels and gels were analyzed by phosphoimaging (Upper) or Coomassie brilliant blue staining (Lower).