Fig 4. A role for RdcA and RdcB in DgcE-controlled biofilm-associated functions.

A: Macrocolony morphology of strain AR3110 and its derivatives carrying the indicated single or double mutations. B: CsgD levels determined by immunoblot analysis in strain AR3110 carrying the indicated mutations. Samples were obtained at an OD₅₇₈ of 3.6–3.8, with 6 μg total protein loaded per lane. C: csgB::lacZ expression measured after growth of strain W3110 Δlac(I-A) carrying the indicated mutations in LB at 28°C for 24 h. D: Cellular c-di-GMP concentration determined in strain W3110, sampled at an OD₅₇₈ of 3. E: Effects of mutations in rdcA and/or rdcB on macrocolony morphology of AR3110 derivatives carrying the C-terminally truncated dgcE^ΔEAL allele. F: Expression of RdcA and DgcE during the growth cycle. Both proteins carried a chromosomally encoded C-terminal 3xFLAG tag for immunoblot detection, which was performed with samples harvested at the OD₅₇₈ of 1 and 2.5, after 11 h of growth (where cells have reached an OD₅₇₈ of approximately 4.5) and after overnight growth (on). The 3xFLAG-free, but otherwise isogenic strain W3110 (wt) was used as a control.