Figure 3: hrpL promoter functional UAS site mapping and promoter binding.

(a) Overview of upstream promoter truncation constructs of the hrpL promoter DNA::lacZ repoter gene (A: −600 to +58; B: −147 to +58; C: −101 to +58 and D: −36 to +58; numbering with respect to the transcription start-site, +1) used to map functional UAS sequences and showing candidate UAS upstream activator sequences (black boxes), IHF (integration host factor) and σ⁵⁴-RNAP-binding sites. (b) In vivo transcription from reporter constructs shown in a, in either wild-type E.coli (MC4100, black bars) or an isogenic strain deleted for IHF coding sequence (grey bar). (c) Electrophoretic mobility shift assay using the hrpL promoter probe with increasing HrpR, HrpS, pre-mixed HrpR and HrpS (HrpR+HrpS) concentrations (as indicated). (d) As in c with co-expressed, co-purified HrpRS (HrpRS). (e) Electrophoretic mobility shift assay of the hrpL promoter probe with (10 nM) IHF. In b, all assays were minimally performed in triplicate and standard errors of the mean are shown.