Inactivation of two-component regulatory systems that are phosphorylated in a stage-specific manner leads to altered biofilm formation 
As differential and sequential phosphorylation of regulatory proteins was detected over the course of P. aeruginosa biofilm development, we asked whether inactivation of these regulatory proteins would alter or affect the stage-specific progression of biofilm formation.
We therefore focused on biofilm-specific regulatory proteins.
Since the proteins PA4101, PA4197, and PA5511 were found to be phosphorylated following 8, 24, and 72 hr of biofilm growth, respectively (Table 1), corresponding to three biofilm developmental stages [9],[12], mutants in these three genes were chosen and allowed to form biofilms for 144 hr in flow cells to test for biofilm formation defects.
Under the conditions tested, wild type P. aeruginosa biofilms reached maturity following 144 hr of growth as characterized by biofilms being composed of large microcolonies exceeding 100 microm in diameter (Fig. 2A).
In contrast, PA4197 and PA4101 mutant biofilms lacked microcolonies after 144 hr of growth (Fig. 2B) and were only composed of a thin layer of cells at the substratum with an average height of 0.5 and 1.4 microm, respectively (Table 2).
However, in contrast to PA4197 mutant biofilms, PA4101 mutant biofilms demonstrated the formation of some cellular aggregates which were less than 10 microm in height (Fig. 2B).
Furthermore, the mutant biofilms differed significantly from wild type biofilms with respect to biomass, surface coverage, and roughness coefficient.
Complementation of both PA4101 and PA4197 mutants restored biofilm formation to wild type levels (Fig. 2C, Table 2).
These results allowed us to firmly conclude that the mutant biofilm phenotypes are caused by a defect in the PA4197 and PA4101 ORF.
Based on the role of PA4197 in the initiation of biofilm formation, we named the PA4197 ORF Biofilm initiation Sensor (BfiS).
BfiS is an unusual sensor that harbors a His kinase A domain typically found in two-component system (TCS) sensor proteins, a Histidine kinase-like ATPase domain involved in autophosphorylation but also in protein dephosphorylation events, and a PAS signal receiver domain [53].
The cognate response regulator BfiR (PA4196) harbors a CheY-like signal receiver domain and a LuxR-like DNA binding domain, which is also present in the quorum-sensing regulatory proteins LasR, RhlR, and QscR and in response regulators with established roles in biofilm formation (GacA, RocA1/SadA) [53].
BfiR also harbors region 4 of Sigma-70 (RpoD)-like sigma factors, a domain involved in binding to -35 promoter elements [53].
Due to its role in biofilm maturation, we named the PA4101 ORF Biofilm maturation Regulator (BfmR).
The protein harbors an OmpR-like transcriptional regulator domain encompassing the common signal receiver and DNA-binding effector domains [53].
The cognate sensor BfmS (PA4102) is unusual in that it lacks an autophosphorylation site typically found in sensor kinases [53].
As shown in Table 1, the probable TCS regulatory protein PA5511 was phosphorylated following 72 hr of surface-associated growth.
PA5511 mutant biofilms grown for 144 hr lacked clusters and microcolonies typically found in wild type biofilms following 72-144 hr of growth (Fig. 2A-B).
Complementation restored biofilm formation to wild type levels (Fig. 2C, Table 2).
However, when placed in a PAO1 background (PAO1/pJN5511), overexpression of PA5511 resulted in biofilms composed of large microcolonies exceeding 250 microm in diameter (compared to an average cluster diameter of 150 microm in P. aeruginosa PAO1, Fig. 2A, D).
Since cluster formation correlated with PA5511 expression levels, we named PA5511 Microcolony formation Regulator (MifR).
MifR harbors a CheY-like receiver and a sigma-54 interaction domain [53].
The protein is on average 30-50% identical to known P. aeruginosa NtrC-like enhancer binding proteins including PilR, FleQ, FleR, AlgB, CbrB, and NtrC [53],[54].
The cognate sensor (MifS, PA5512) is a typical sensor kinase harboring both a His kinase A and a His kinase-like ATPase domain [53].
Since individual carbon and nitrogen sources have been demonstrated to modulate P. aeruginosa in vitro biofilm development and architecture [16], [55]-[58], surface motility [59] and P. aeruginosa cell-cell signaling (quorum sensing) [60]-[63], the biofilm architecture of all four mutant biofilms was tested using three different media including LB medium and two minimal media containing glutamate [17] or citrate [64] as sole carbon source.
Under the conditions tested, the biofilm architecture of all three mutants was similar to the biofilm architecture shown in Fig. 2 independent of the media used.
