The important human pathogen Pseudomonas aeruginosa has been linked to numerous biofilm-related chronic infections. Here, we demonstrate that biofilm formation following the transition to the surface attached lifestyle is regulated by three previously undescribed two-component systems: BfiSR (PA4196-4197) harboring an RpoD-like domain, an OmpR-like BfmSR (PA4101-4102), and MifSR (PA5511-5512) belonging to the family of NtrC-like transcriptional regulators. These two-component systems become sequentially phosphorylated during biofilm formation. Inactivation of bfiS, bfmR, and mifR arrested biofilm formation at the transition to the irreversible attachment, maturation-1 and -2 stages, respectively, as indicated by analyses of biofilm architecture, and protein and phosphoprotein patterns. Moreover, discontinuation of bfiS, bfmR, and mifR expression in established biofilms resulted in the collapse of biofilms to an earlier developmental stage, indicating a requirement for these regulatory systems for the development and maintenance of normal biofilm architecture. Interestingly, inactivation did not affect planktonic growth, motility, polysaccharide production, or initial attachment. Further, we demonstrate the interdependency of this two-component systems network with GacS (PA0928), which was found to play a dual role in biofilm formation. This work describes a novel signal transduction network regulating committed biofilm developmental steps following attachment, in which phosphorelays and two sigma factor-dependent response regulators appear to be key components of the regulatory machinery that coordinates gene expression during P. aeruginosa biofilm development in response to environmental cues.
Biofilms are complex communities of microorganisms encased in a matrix and attached to surfaces. It is well recognized that biofilm cells differ from their free swimming counterparts with respect to gene expression, protein production, and resistance to antibiotics and the human immune system. However, little is known about the underlying regulatory events that lead to the formation of biofilms, the primary cause of many chronic and persistent human infections. By mapping the phosphoproteome over the course of P. aeruginosa biofilm development, we identified three novel two-component regulatory systems that were required for the development and maturation of P. aeruginosa biofilms. Activation (phosphorylation) of these three regulatory systems occurred in a sequential manner and inactivation arrested biofilm formation at three distinct developmental stages. Discontinuation of bfiS, bfmR, or mifR expression after biofilms had already matured resulted in disaggregation/collapse of biofilms. Furthermore, this regulatory cascade appears to be linked via BfiS-dependent GacS-phosphorylation to the previously identified LadS/RetS/GacAS/RsmA network that reciprocally regulates virulence and surface attachment. Our data thus indicate the existence of a previously unidentified regulatory program of biofilm development once P. aeruginosa cells have committed to a surface associated lifestyle, and may provide new targets for controlling the programmed differentiation process of biofilm formation.