Figure 1.
Detection of proteins phosphorylated in a stage-specific manner during biofilm development.
(A) Protein phosphorylation and (B) dephosphorylation events. (A, B) Protein extracts obtained from planktonic and biofilm cells following 8, 24, 72, 144, and 216 hr of growth under flowing conditions. Proteins were first separated by 2D/PAGE and subsequently subjected to immunoblot analysis. Phosphorylation events are subcategorized according to their appearance over the course of biofilm development as being stage-specific (phosphorylation events that were only detected at one growth stage, e.g. planktonic cells or 8 h old biofilms), biofilm-specific (protein phosphorylation only detected in biofilm cells regardless of their age), and constitutive (present independent of growth condition or biofilm age). Furthermore, protein phosphorylation events that occurred at different stages over the course of biofilm formation are subcategorized as reversible and irreversible attachment, biofilm formation and maturation depending on when and for how long protein phosphorylation was detected. The values on each bar indicate the number of protein phosphorylation events detected per (sub)category. (C) Protein phosphorylation patterns over the course of biofilm development. Phosphoproteomes of P. aeruginosa PAO1 biofilms at different stages of development were analyzed using [i] immunoblotting of 2D gels and [ii] cleavable isotope-coded affinity tag (cICAT) mass spectrometric analysis of metaloxide affinity-enriched phosphoproteins. Biofilm protein phosphorylation patterns are shown as percent difference relative to the PAO1 planktonic phosphorylation patterns. Error bars indicate one standard deviation based on triplicate experiments.
Table 1.
Identification of regulatory proteins that are differentially phosphorylated over the course of P. aeruginosa biofilm formation.
Figure 2.
TCS mutants are arrested in biofilm development.
Biofilms of strains (B) inactivated in or (C, D) overexpressing bfiS (PA4197), bfmR (PA4101), and mifR (PA5511), grown for 144 hours, were visualized by CSLM and compared to (A) wild type PAO1 biofilms at various stages of development. Biofilms were stained with the LIVE/DEAD BacLight viability stain (Invitrogen Corp.). White bar = 100 µm.
Table 2.
COMSTAT analysis of P. aeruginosa wild type and mutant biofilm structure.
Figure 3.
Biofilm formation by P. aeruginosa ΔgacS.
Confocal images were acquired following 24, 72, 120, 144, and 192 hours of biofilm growth under flowing conditions. Arrows indicate non-adherent, sloughing particles. White bar = 100 µm.
Figure 4.
Comparison of protein phosphorylation patterns (A) and protein production patterns (B) of wild type and mutant biofilms impaired in the developmental progression.
(A) The phosphoproteomes of 144-hr-old biofilms of ΔbfiS (light grey bar), ΔbfmR (dark grey bar), and ΔmifR (black bar) were compared to those of PAO1 biofilms following 8, 24, 72, and 144 hr of growth (black diamond, dashed line). Biofilm protein phosphorylation patterns were analyzed using a combination of immunoblotting of 2D gels and cleavable isotope-coded affinity tag (cICAT) mass spectrometric analysis of metaloxide affinity-enriched phosphoproteins and are shown as percent difference (%) relative to the planktonic phosphorylation patterns. (B) Similarity of 2D-protein production patterns as determined by Heuristic clustering. Experiments were carried out in triplicate for each strain and/or biofilm age.
Figure 5.
Inactivation of bfiS (PA4197), bfmR (PA4101), and mifR (PA5511) expression in mature biofilms results in biofilm architectural collapse and biomass loss.
P. aeruginosa mutants complemented with plasmid-borne copies of the respective genes placed under the regulation of the arabinose-inducible PBAD were grown under continuous flow conditions in glutamate minimal medium [17] in the presence of 0.1% arabinose for 144 hr after which time the biofilms were visualized by confocal microscopy (0 hr). Then, arabinose was eliminated from the growth medium and the biofilm architecture monitored post arabinose removal at the times indicated. PAO1 strain harboring the empty pJN105 vector was used as control. Biofilms were stained with the LIVE/DEAD BacLight viability stain (Invitrogen Corp.). White bars = 100 µm.
Table 3.
COMSTAT analysis of P. aeruginosa wild type and complemented mutant biofilm structure following removal of arabinose and thus, lack of expression of PA4101, PA4197 and PA5511, respectively.
Figure 6.
Model for the Role of novel two-component systems BfiRS, BfmRS, and MifRS in biofilm development and potential link to the multi-component signaling network LadS/RetS/GacAS/RsmA.
The three novel P. aeruginosa two-component systems (TCS) are essential in regulating the transition to irreversible attachment (BfiRS, stage 1–2), maturation-1 (BfmRS, stage 2–3), and maturation-2 (MifRS, stage 3–4) during biofilm development in response to as of yet unknown intra- and/or extracellular signals. Phosphorylation and thus, activation occurs in a sequential manner (BfiS<GacS<BfmR/BfmS<MifR), suggesting the presence of a TCS signal transduction network during the progression of biofilm development. Furthermore, this regulatory cascade involved in stage-specific biofilm development appears to be linked, via the BfiRS system, to the LadS/RetS/GacAS/RsmA network that reciprocally regulates virulence and surface attachment. Similarly to LadS, BfiS plays a role in GacS phosphorylation. Here, GacS has been shown to play a dual role in regulating biofilm developmental steps depending on the phosphorylation status. Moreover, RsmA represses the expression of the BfiS cognate response regulator, BfiR. Adapted from [9],[77].
Table 4.
Bacterial strains and plasmids.