Figure 5.

GacS autophosphorylates in trans.A, an autophosphorylation assay followed by Zn²⁺-Phos-tag SDS-PAGE was used to examine the autophosphorylation of GacS_cyt and GacS_cyt variants in the absence and presence of ATP. GacS_cyt wild-type, GacS_cyt H293A (a variant that cannot undergo autophosphorylation), GacS_cyt G472A G747A (a variant that cannot bind ATP), and an equimolar ratio of GacS_cyt H293A and GacS_cyt G472A G474A were used to assess the ability of GacS to autophosphorylate in trans or in cis. GacS_cyt and the GacS_cyt variants are 77 kDa. Each lane contains 7.74 μg protein. GacS_cyt has three potential phosphorylation sites (the catalytic histidine in the DHp domain, the conserved aspartate in the receiver domain, and the conserved histidine in the Hpt domain). B, autophosphorylation assay. Individual variant constructs (GacS_cyt H293A and GacS_cyt G472A G474A) are unable to undergo cis autophosphorylation, but when both variant constructs are introduced into the autophosphorylation assay, they can autophosphorylate in trans. The histidine kinase region is shown for clarity even though the assay was performed with the cytosolic region of GacS.