The BDSF quorum sensing receptor RpfR regulates Bep exopolysaccharide synthesis in Burkholderia cenocepacia via interaction with the transcriptional regulator BerB

Abstract The polysaccharide Bep is essential for in vitro biofilm formation of the opportunistic pathogen Burkholderia cenocepacia. We found that the BDSF quorum sensing receptor RpfR is a negative regulator of the bep gene cluster in B. cenocepacia. An rpfR mutant formed wrinkled colonies, whereas additional mutations in the bep genes or known bep regulators such as berA and berB restored the smooth colony morphology of the wild-type. In accordance with previous work, we found that there is a good correlation between intracellular c-di-GMP levels and bep expression when the c-di-GMP level is increased or decreased through ectopic expression of the diguanylate cyclase YedQ or the c-di-GMP phosphodiesterase PA5295. However, when the intracellular c-di-GMP level is changed by site directed mutagenesis of either the EAL or GGDEF domain of RpfR there is no correlation between intracellular c-di-GMP levels and bep expression. Apart from rpfR each of the 25 c-di-GMP phosphodiesterase and diguanylate cyclase genes encoded by B. cenocepacia can be knocked out without an effect on berA and bep gene expression. Moreover, bacterial two hybrid assays provided evidence that RpfR and BerB physically interact. Our results suggest that the specificity of RpfR-mediated regulation of the bep genes is governed by RpfR-BerB protein interaction. We suggest a model where RpfR binds BerB at low c-di-GMP levels to sequester this RpoN-dependent activator to an RpfR/RpfF complex such that BerB cannot activate expression of the transcriptional activator BerA and thus transcription of the bep genes is not activated. If the c-di-GMP levels rise, possibly by the enzymatic action of RpfR, BerB binds c-di-GMP and is released from the RpfR/RpfF complex and associates with RpoN to activate transcription of berA, and the BerA protein subsequently activates transcription of the bep genes.