BphP and AlgB are a two-component HK–RR pair.

Abstract

(A) Shown is a representative isolation of a suppressor mutation of the ΔkinB smooth colony biofilm phenotype. The white arrow in the left panel indicates a region of rugose sectoring in the ΔkinB smooth colony biofilm that is diagnostic of the emergence of a suppressor mutation. The right panel shows the colony biofilm phenotype of a mutant following isolation. (B) Chromosomal arrangements of the algB (red), kinB (blue), bphO (yellow), and bphP (green) genes. Large white arrows represent open reading frames (lengths not to scale), black bent arrows indicate promoters, and black circles indicate the locations of suppressor mutations. (C) Colony biofilm phenotypes of WT PA14 and the designated mutants on Congo red agar medium after 72 h of growth. palgB refers to algB under the Plac promoter on the pUCP18 plasmid. Scale bar is 2 mm for all images. (D) Pyocyanin production (OD695) was measured in WT PA14 and the designated mutants. pbphP refers to bphP under the Plac promoter on the pBBR-MCS5 plasmid. Error bars represent SEM for 3 biological replicates. (E) Autophosphorylation of BphP–BV and phosphotransfer to AlgB. (Left) Autophosphorylation of BphP–BV was carried out for 30 min and samples were removed at the indicated times for electrophoresis. (Right) An equimolar amount of AlgB was added to phospho-BphP-BV for 30 min and samples were removed at the indicated times for electrophoresis. (F) Dephosphorylation of AlgB-P by KinB or KinBP390S. Phosphotransfer to AlgB from phospho-BphP-BV was carried out for 30 min. ATP was removed from the reaction, and either KinB or KinBP390S was added. Samples were removed at the indicated times for electrophoresis. The top panel shows representative images of gels. The bottom graph shows percent AlgB-P levels at each time point with SEM for 3 independent replicates. Band intensities for AlgB-P when KinB was added (circles) and when KinBP390S was added (squares) were normalized to the levels at time 0. To assess the quality of protein preparations used in panels E and F, see S4B Fig. Data for the graphs in panels D and F can be found in supplemental file S1 Data. The original autoradiographs with the data for panels E and F are available in supplemental file S2 Data. BV, biliverdin; OD, optical density; SEM, standard error of the mean; WT, wild type.</p