Introduction 
Biofilms are composed of microorganisms attached to a solid surface and encased in a hydrated polymeric matrix of their own synthesis. Biofilms form when bacteria adhere to surfaces in moist environments. Biofilm-associated microorganisms have been shown to colonize a wide variety of medical devices and have been implicated in over 80% of chronic inflammatory and infectious diseases including chronic otitis media, native valve endocarditis, gastrointestinal ulcers, urinary tract and middle ear infections, and chronic lung infections in cystic fibrosis (CF) patients [1],[2]. The human pathogen Pseudomonas aeruginosa is considered one of the primary causes of mortality in patients with CF, the most common life-threatening hereditary disease in Caucasians [3],[4]. In addition, P. aeruginosa causes a variety of diseases in individuals predisposed to infections as the result of severe burns, wounds, urinary tract or corneal injury, or immunocompromised status [5]-[8]. Biofilm cells differ from their planktonic counterparts in the genes and proteins that they express, resulting in distinct phenotypes including altered resistance to antibiotics and the human immune system [2],[9],[10]. Thus, it is not surprising that biofilms are considered to be differentiated communities compared to their planktonic counterparts [9],[11]. This is supported by the finding that various microorganisms, including P. aeruginosa have been shown to form biofilms in a stage-specific and coordinated manner. Biofilm formation is initiated with surface attachment by planktonic bacteria, followed by formation of clusters and microcolonies and subsequent development of differentiated structures in which individual bacteria as well as the entire community are surrounded by exopolysaccharides. The biofilm developmental cycle comes full circle when biofilms disperse [12],[13]. This process has been shown to be governed by the activities of regulatory networks that coordinate the temporal expression of various motility, adhesion, and exopolysaccharide genes in response to inter- and intracellular signaling molecules and environmental cues. Vallet et al. [14] described a transcriptional regulator MvaT in P. aeruginosa that represses the expression of cup genes involved in the chaperone-usher fimbrial assembly pathway. MvaT deletion mutants exhibited enhanced attachment. In contrast, type IV pili and flagella deletion mutants exhibited reduced attachment indicating that attachment and biofilm formation are mediated by extracellular appendages [12], [15]-[17]. Furthermore, the intracellular signaling molecule bis-(3'-5')-cyclic diguanylic guanosine monophosphate (cyclic-di-GMP), first described to control extracellular cellulose biosynthesis in Acetobacter xylinum [18],[19], has been demonstrated in several microorganisms to modulate biofilm formation via the production of exopolysaccharides or matrix components, control auto-aggregation of planktonic cells, and regulate swarming motility [20]-[32]. In P. aeruginosa, at least two pathways have been identified to modulate cyclic-di-GMP and thus, biofilm formation. These are the wsp chemosensory signal transduction pathway [25] and a genetic pathway composed of the phosphodiesterase BifA, the inner membrane-localized diguanylate cyclase SadC and the cytoplasmic protein SadB [20],[21],[33]. Both are involved in the reciprocal cyclic-di-GMP-dependent regulation of Pel and Psl exopolysaccharide production as P. aeruginosa transitions from a planktonic to a surface associated lifestyle. Both Pel and Psl exopolysaccharides contribute to the overall architecture of biofilms and are essential for surface interaction and biofilm initiation [34],[35]. Expression of the pel and psl genes is coordinated by the global regulator RetS, a hybrid sensor kinase-response regulator protein, that plays a key role in the reciprocal regulation of virulence factors and biofilm formation required for acute versus chronic infection [36]. RetS belongs to the family of two-component regulatory systems (TCS) which translate external signals into adaptive responses by a variety of mechanisms, including control of gene expression and methylation of target proteins. RetS is postulated to act in concert with two other TCS sensor kinase-response regulator hybrids, GacS and LadS, to coordinate the expression of determinants involved in biofilm formation and the production of determinants required for cytotoxicity in P. aeruginosa via the small regulatory RNA rsmZ [36],[37]. Inactivation of RetS results in reduced cytotoxicity but increased attachment and biofilm formation, while inactivation of both LadS and GacS results in increased virulence but decreased biofilm formation capacity [36],[37]. This multi-component switch thus orchestrates the transition from the planktonic to the biofilm mode of growth by P. aeruginosa via phosphorylation events of the two-component regulatory system GacA/GacS [36]-[38]. Overall, the findings suggest that the transition to a surface associated lifestyle proceeds via several pathways, probably in response to environmental cues or signals present during attachment, and involves the coordinated transduction of phosphorylation events via two-component regulatory systems (TCS). This raises the question of whether the transition to later stages of biofilm formation, which coincide with distinct phenotypes compared to planktonic and initial attached bacterial cells, also involves sensing of environmental signal(s) and requires the coordinated transduction of phosphorylation events (phosphorelays). Here we demonstrate that P. aeruginosa exhibits distinct protein phosphorylation patterns at various stages of biofilm development. Furthermore, we report the identification of three novel two-component regulatory systems named BfiRS (PA4196-4197), BfmRS (PA4101-4102), and MifRS (PA5511-5512) that coordinate phosphorylation events required for the progression of P. aeruginosa biofilm development in a stage-specific manner. These systems together form a coordinated signaling network that regulates three committed steps of the P. aeruginosa biofilm life cycle, in particular the transition to three later biofilm developmental stages following initial attachment, namely initiation of biofilm formation (BfiRS), biofilm maturation (BfmRS), and microcolony formation (MifRS).
