Results 
The formation of biofilms has been proposed to be controlled in response to environmental signals [39].
Given that protein phosphorylation is a common modification system used in signal transduction that changes the function of proteins in response to environmental stimuli [38], we chose a phosphoproteomic approach for the detection and identification of regulatory pathways active following the transition to the surface attached mode of growth.
Detection of differentially phosphorylated proteins over the course of biofilm formation While phosphoproteomic analyses have become widespread in studies of regulation, signaling, development, the characterization of bacterial species and host responses during pathogenesis [40]-[49], only a limited number of studies have demonstrated that bacterial phosphoproteomes are dynamic [44],[46],[48].
We therefore used a combination of 2D/PAGE and immunoblot analysis using commercially available anti-phospho Ser/Thr antibodies (see Suppl. Fig. S1A-B for an example) to probe for the presence of signal transduction events that occur over the course of biofilm formation.
Immunoblots of whole cell extracts obtained from planktonic cells and biofilm cells representing five developmental stages (reversibly and irreversibly attached cells, maturation-1 and -2 and dispersion stage; following 8, 24, 72, 144, and 216 hr of growth, respectively, see [12],[50] for timing of biofilm stages) were thus analyzed for the presence of planktonic- and biofilm-specific phosphorylation events.
The planktonic mode of growth coincided with 24 phosphorylated proteins that were not phosphorylated following the transition of P. aeruginosa to surface-associated mode of growth (Fig. 1A, stage-specific events).
Additional stage-specific events were detected for biofilms differing in age.
For instance, 8 hr and 24 hr old biofilms displayed 23 and 21 phosphorylation events, respectively, not detected at any other stage.
Regardless of the biofilm developmental stage, 7 phosphorylation events were detected that were absent in planktonic cells (Fig. 1A, biofilm-specific events).
In both modes of growth, 26 proteins were constitutively phosphorylated.
In addition to biofilm stage-specific phosphorylation of proteins, protein phosphorylation events were detectable at more than one biofilm growth stage indicating that the transition to surface-associated growth coincides with distinct protein phosphorylation and dephosphorylation events.
As shown in Fig. 1A, these phosphorylation events are subcategorized as occurring during the reversible and irreversible attachment, biofilm formation and maturation stage depending on when and for how long protein phosphorylation was detected.
For instance, four proteins were phosphorylated both in planktonic and reversible attached cells (8 hr biofilms) but not at any other biofilm stage (Fig. 1A, reversible attachment) while 4 different proteins were phosphorylated only in planktonic cells and biofilm cells after 8 hr and 24 hr of growth under flowing conditions (Fig. 1A, irreversible attachment).
Furthermore, evidence of proteins being dephosphorylated over the course of biofilm formation was detected.
Multiple proteins were found to be dephosphorylated at either a single or at multiple stages over the course of biofilm formation and maturation (Fig. 1B).
Moreover, the similarity of the biofilm phosphoproteome to the planktonic phosphorylation patterns decreased from 59% in 8-hr-old biofilms to 35% in 144-hr-old, mature biofilms.
The reduced similarity in phosphorylation events between biofilms and planktonic cells was mainly due to biofilm specific phosphorylation events detected at one or more stages of development.
Dispersion-stage biofilms (216-hr-old) shared 43% similarity with the phosphorylation patterns of planktonically-grown P. aeruginosa cells (not shown).
The increase in similarity between the planktonic and the 216-hr-old biofilm phosphoproteomes is consistent with previous reports indicating that cells within dispersion-stage biofilms are returning to the planktonic mode of growth [12],[50].
Protein phosphorylation in bacteria is not restricted to serine and threonine amino acid residues; however, the analysis of phosphorylation events by immunoblotting is limited to the availability of anti-phospho Ser/Thr (and tyrosine) antibodies.
We therefore also purified phosphorylated proteins using metal oxide affinity chromatography (MOAC, see Fig. S1), a gel-independent approach allowing for the enrichment of phosphoproteins independent of the phosphorylation site with an up to 100% specificity [51],[52], followed by cleavable isotope coded affinity tag (cICAT) labeling and analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS).
This quantitative mass spectrometric approach was used to analyze protein phosphorylation patterns of biofilm cells grown to the reversible, irreversible, maturation-1 and maturation-2 biofilm stages (8-, 24-, 7-2, and 144-hr-old biofilms, respectively [12]) in comparison to those of planktonic cells.
Similarly to the results obtained via immunoblot analysis, the changes in phosphorylation events over the course of biofilm development detected using LC-MS-MS analysis appeared to be stage-specific (two examples are shown in Suppl. Fig. S2), with the similarity to the planktonic patterns decreasing from 72% in 8 hr biofilms to 38% in 144 hr biofilms (Fig. 1C).
The overall stage-specific (de)phosphorylation events as well as the differences in the phosphoproteome were similar to those detected by immunoblot analysis using anti-Ser/Thr antibodies.
This is the first description of the dynamic changes of the phosphoproteome occurring during biofilm development.
The combination of approaches used here has not been previously used to identify phosphorylated proteins in biofilms.
