RhlR represses biofilm formation via KinB.

Abstract

(A) Colony biofilm phenotypes of WT PA14 and the designated mutants on Congo red agar medium after 72 h of growth. kinB::Tn refers to a mutant identified in a genetic screen harboring a transposon insertion in kinB. pkinB, prhlR, and palgB refer to kinB, rhlR, and algB, respectively, under the Plac promoter on pUCP18. Scale bar for all images is 2 mm. (B) Relative expression levels of rpoD and pelA measured by qRT-PCR in WT and ΔkinB mutant colony biofilms grown as in (A). The pelABCDEFG operon encodes proteins required for the production of the Pel polysaccharide, which is essential for the formation of pellicles, SSA biofilms, and colony biofilms [29]. (C) Phos-tag western blot analysis of the indicated strains probed for 3xFLAG-AlgB. (D) Relative algB transcript levels measured by qRT-PCR in WT PA14 and the ΔrhlR mutant grown planktonically to OD600 = 1.0. (E) Relative rhlR transcript levels measured by qRT-PCR in WT PA14 and the ΔkinB mutant grown planktonically to OD600 = 1.0. (F) Pyocyanin production (OD695) was measured in WT PA14 and the designated mutants. Production from the WT was set to 100%. (G) Relative expression of rpoD, hsiC2, hcnA, lecA, and lecB measured by qRT-PCR in WT PA14 and the designated mutants grown planktonically to OD600 = 1.0. rpoD is used as the control for comparison. For panels B, D, E, and G, data were normalized to 16S RNA levels, and the WT levels were set to 1.0. For data in panels B, D, E, F, and G, error bars represent SEM for 3 biological replicates. Data for panels B, D, E, F, and G can be found in supplemental file S1 Data. The original western blot showing the data for panel C is available in supplemental file S2 Data. AU, arbitrary unit; OD, optical density; qRT-PCR, quantitative Reverse Transcriptase-Polymerase Chain Reaction; SEM, standard error of the mean; SSA, solid-surface–associated; WT, wild type.</p