Fig 2. Role of distinct domains in DgcE dimerization.

A: The isolated region containing all three PAS domains (PAS₃), as well as the isolated GGDEF and EAL domains of DgcE were tested for dimerization in adenylate cyclase-based two-hybrid assays. B: DgcE variants lacking specific domains as indicated were tested by two-hybrid assays. Spotted co-transformants were grown at 28°C for 1 day. As a positive control, the leucin zipper part ('zip') of the yeast GCN4 protein was used. Using pKNT25 and pUT18, the T25 and T18 fragments of adenylate cyclase are attached at the C-termini of the indicated isolated domains and the different DgcE variants. Two-hybrid plasmid vectors used in each combination are listed at the left side of the panels, with 'insert' standing for the indicated proteins cloned into these vectors, vector names alone stand for the empty vectors in control combinations. C: The indicated parts of DgcE were tested for dimerisation using an alternative two-hybrid assay (Bacteriomatch; for details, see Materials and Methods). As a positive control, the strongly interacting RpoS and RssB proteins were used.