Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix

Computational analyses predict that PA4625 encodes a filamentous β-helical protein secreted by its transporter, encoded by PA4624

The Pseudomonas genome website has annotated PA4625 and PA4624 as hypothetical proteins, putatively encoded by a two gene operon. To gain insight into potential functions for PA4625 and PA4624, we performed a bioinformatics analysis. A Phyre analysis was conducted to identify remote homology to known structures (Bennett-Lovsey et al., 2008). Phyre revealed that PA4625 and PA4624 are most likely members of a TPS. The PA4625 gene is predicted to encode a 220 kDa protein with a type I export signal (shaded in blue, Fig. 1C). The N-terminal region was most similar to the secretion domains of the large adhesins, FHA and HMW1 of B. pertussis and Haemophilus influenzae respectively. PA4624 is most similar to a membrane protein, FhaC, which is a member of omp85/tpsB transporter family. Based upon these homologies, and the fact that their expression was controlled by c-di-GMP, we renamed PA4625 and PA4624 as cdrA (cyclicdiguanylate-regulated TPS partnerA) and cdrB (cyclicdiguanylate-regulated TPS partnerB).

Fig. 1

A diagram of a tertiary structure model of CdrA. The model was constructed by the de novo modelling of six separate segments and reattaching these segments by overlapping regions of structures. A. The global model of CdrA predicts a β-helix dominated structure with several exposed loops containing α-helices. The arrow indicates a predicted integrin binding motif. B. The same model was colour-coded to represent a trinary confidence measure based on secondary structure. Green indicates high confidence, yellow indicates intermediate confidence and red indicates low confidence. C. The N-terminal domain of CdrA contains a predicted signal peptide (blue), a haemagglutination site (orange) and a putative sugar-binding domain (purple). D. An exposed loop of the CdrA model contains a predicted integrin binding motif (red). Amino acids of the integrin binding motif are highlighted in red.

The secondary structure of CdrA was predicted by PsiPred (Jones, 1999; Bryson et al., 2005) to be dominated almost entirely by β-strands (see Fig. S1). Given this result and the size of the protein, we predict that CdrA has a beta helix tertiary structure (Fig. 1A). The tertiary structure was modelled by an iterative approach in which segments of the protein were modelled de novo and reattached via structural alignment of overlapping regions (presented in Fig. 1). The secondary structure and function of CdrB was predicted by I-TASSER (Zhang, 2008) to be most similar to FhaC which forms a β-barrel channel in the outer membrane for the outward translocation of FHA. The highest-ranking tertiary structure of CdrB as predicted by I-TASSER is presented in Fig. S2.

A search of the Conserved Domains Database (Marchler-Bauer et al., 2007) revealed that amino acids 45–153 of CdrA contain a carbohydrate-dependent haemagglutination activity domain (orange shading, Fig. 1A and C). These domains are found in secreted proteins which include haemagglutinins, haemolysins and members of the TPS secretion family. In addition, a sugar-binding domain harbouring a glycine-rich motif (PS50315) spanning amino acids 332–477 was predicted by PROSITE and PDBeMotif 1.0 (purple shading, Fig. 1A and C). A 42 amino acid signal peptide sequence (shaded blue, Fig. 1A and C) was predicted by SignalP 3.0 (Bendtsen et al., 2004) to be cleaved between residues A42 and A43 of CdrA (P = 0.993). In other TPS secretion systems, similar signal peptides have been shown to be important for Sec-dependent secretion across the inner membrane (Chevalier et al., 2004). Analysis of the primary amino acid sequence also identified an Arg-Gly-Asp (RGD) sequence motif at amino acids 1019–102, which may function as an integrin recognition site similar to eukaryotic extracellular matrix proteins (arrow in Fig. 1A and red shading, Fig. 1D).

Verification that cdrA transcription is c-di-GMP-dependent and that cdrAB constitutes an operon

Previous transcriptional profiling studies suggested that cdrAB expression is elevated under conditions of high intracellular c-di-GMP (Hickman et al., 2005; Hickman and Harwood, 2008; Starkey et al., 2009). To verify these results, we measured cdrA transcript levels in the wild-type and a wspF mutant strain (high c-di-GMP). The levels of relative cdrA transcript were 27-fold higher in the wspF mutant strain (Fig. 2).

Fig. 2

RT-PCR data of cdrA expression under conditions of high and low c-di-GMP. cdrA is highly expressed under conditions where c-di-GMP is elevated. Relative transcript levels for cdrA are shown for wild-type P. aeruginosa (PAO1), a wspF mutant (elevated c-di-GMP), PAO1 expressing a diguanylate cyclase, PA1120 (elevated c-di-GMP), a wspF mutant expressing a c-di-GMP degrading phosphodiesterase PA2133 (low c-di-GMP), a wspFpelApslBCD mutant harbouring a vector control (elevated c-di-GMP) and a wspFpelApslBCD mutant expressing a c-di-GMP degrading phosphodiesterase (low c-di-GMP). Data are shown for four biological replicates.

To verify that cdrAB expression is controlled by c-di-GMP and not a side-effect of auto-aggregation seen in the wspF background, we engineered a wspFpelApslBCD triple mutant strain that retains elevated c-di-GMP, but does not auto-aggregate (Starkey et al., 2009). First we verified that wspFpelApslBCD displayed elevated levels of cdrA expression compared with the parental PAO1 strain (Fig. 2). Next we examined the effect of c-di-GMP depletion on cdrA expression. Depletion of c-di-GMP was achieved through expression of a c-di-GMP phosphodiesterase encoded by the PA2133 gene (Hickman et al., 2005). Under conditions where c-di-GMP has been depleted in the wspFpelApslBCD mutant, cdrA transcripts were 7-fold lower than the wspFpelApslBCD strain harbouring the vector control (Fig. 2).

The Operon Finding Software v2.1 of the Pseudomonas genome website predicts that cdrA and cdrB are part of a two gene operon. To test this, we designed primers to be used in reverse transcription polymerase chain reaction (RT-PCR) that are internal to the cdrA and cdrB open reading frames. Only a dicistronic mRNA containing coding sequence of both ORFs would produce an amplicon. We tested mRNA harvested from both a PAO1 and a PAO1wspF mutant background. Both PAO1 and PAO1wspF strains produced a DNA band of the correct size (see Fig. S3), indicating that cdrA and cdrB are transcribed as part of the same transcript.

CdrA appears to be processed, and is found associated with cells as well as in culture supernatants

CdrA sequence analysis predicts that it is secreted or cell-surface associated. To determine CdrA localization and whether it is post-translationally processed like other TPS adhesins (e.g. FHA), we generated polyclonal antisera to a fragment of CdrA. Western analysis indicates that two primary forms of CdrA are produced, an unprocessed version of the protein (∼220 kDa) and a smaller, processed version (∼150 kDa) (Fig. 3). Primarily full-length CdrA appears to be cell-associated, while only the processed version is present in culture supernatants (Fig. 3A). This is consistent with previous reports of FHA, which is also both cell surface-associated and secreted into the extracellular environment (Mazar and Cotter, 2007). No CdrA was seen in the supernatant of the strain overexpressing only cdrA, presumably because of the absence of CdrB to transport CdrA across the outer membrane. As expected, levels of CdrA were elevated in a wspF background compared with PAO1 only (Fig. 3).

Fig. 3

Western blot analysis of CdrA. Polyclonal antisera was used to monitor CdrA expression. In the top panel, CdrA was detected in the cellular fractions and culture supernatants in strains engineered to overexpress CdrA. The detection of CdrA in supernatants was dependent upon CdrB. In the lower two panels, CdrA expression was only detected in planktonic cultures of a ΔwspF background, not PAO1.

N-terminal sequencing was performed to gain insight into how CdrA is processed. The N-terminal sequence of the processed 150 kDa protein begins at amino acid 438. Were processing only to occur at the N-terminus, this would produce a 175 kDa protein, indicating that processing occurs at the C-terminus as well.

CdrAB expression increases biofilm formation in wild-type and RSCV strains

The finding that CdrAB is co-regulated with other key matrix components, Pel and Psl, led us to hypothesize that it may contribute to the biofilm matrix. Therefore, we examined the effects of CdrAB on biofilm development. Initially, we evaluated the effects of cdrAB overexpression on biofilm formation. We constructed a plasmid, pBADcdrAB, for arabinose-inducible expression of cdrAB. Biofilm formation was increased 6.4-fold when cdrAB was overexpressed as compared with the vector control (pMJT-1) (Fig. 4A).

Fig. 4

The effect of cdrAB expression on biofilm formation in a static microtitre dish assay. A. Biofilm formation as measured by crystal violet staining by a cdrAB overexpression strain. Open bars indicate control treatments without arabinose induction and grey bars indicate 0.2% arabinose-induced treatment. B. cdrA contributes to biofilm formation. Biofilm formation by wspF, wspFcdrA, wspFpelApslBCD and wspFpelApslBCDcdrA mutant strains were quantified by measuring crystal violet staining after 20 h of static growth in a microtitre dish assay.

To evaluate the impact of loss of CdrA expression on biofilm formation, we constructed in-frame deletions of cdrA in wild-type PAO1 and wspF backgrounds. This mutation had no effect on the RSCV colony morphology on solid medium (data not shown). In static microtitre-dish assays, a ΔcdrA mutation had minimal effect on accumulation of biofilm biomass in a PAO1 and a wspF background (Fig. 4B).

Exopolysaccharides have been shown to mask the effects of potential surface adhesins (Schembri et al., 2004), so to eliminate potential interference from either the Pel or Psl polysaccharides we tested the effects of a cdrA mutation in a strain lacking Pel and Psl. Even though strains lacking Pel and Psl were substantially reduced in their ability to form biofilms (because Psl is important for surface attachment), the wspFpelApslBCD mutant produced 18-fold more biofilm biomass as compared with a wspFpelApslBCDcdrA strain (Fig. 4B).

Next, we examined biofilm formation under conditions of continuous flow. Analyses of the cdrAB overexpression strain supported the microtitre-dish binding assay data. PAO1 pMJT-1 formed typical mushroom-shaped structures that are commonly observed for these conditions. In contrast, the cdrAB overexpression strain formed larger structures that were thicker and encompassed a greater volume of biofilm biomass (data not shown). However, unlike the wspF background which produced large compact cell aggregates (Fig. 5B), the biofilm formed by the cdrAB overexpression strain was characterized by more, but less tightly packed small cell aggregates.

Fig. 5

CdrA contributes to biofilm development under conditions of liquid flow. A. Top view and side view of PAO1 and PAO1ΔcdrA biofilms at 20× magnification. B. Top view and side view of PAO1 ΔwspF and PAO1 ΔwspFcdrA biofilms at 40× magnification. C. Relative transcript levels of cdrA for PAO1 cells grown planktonically for 24 h as compared with cells grown in a biofilm. D. Western blot analysis using CdrA antisera of PAO1 grown planktonically and as a biofilm for 24 h.

The PAO1cdrA strain produced biofilms that were thinner and less structured than the wild-type (Fig. 5A). A wspF mutant produced biofilms typical of RSCV strains, characterized by a large amount of biomass and several large cell aggregates. A cdrA mutation in the wspF background had a dramatic phenotype. The biofilm cells were loosely packed, lacking the compact cell clusters of the wspF strain (Fig. 5B). Cells in the wspF strain were tightly associated with the biofilm, while in the wspFcdrA mutant strain, they were loosely associated and easily sloughed off. An illustration of this property is that wspFcdrA biofilms can be easily perturbed by changes in physical parameters such as flow. Simply interrupting flow through the flow cell reactor by periodically pinching the inlet tubing displaced the wspFcdrA biofilm (Movie S2), while the wspF biofilm (Movie S1) was not affected.

The flow cell data suggest that CdrA may play an important role in biofilms formed by both wild-type and wspF strains under conditions of flow. Previous expression analyses (Fig. 2) indicated that cdrA transcript levels were very low in wild-type planktonic culture. To determine if cdrA expression is elevated when PAO1 is grown as a biofilm, transcript levels were measured in cells grown under continuous flow in a tube biofilm reactor and compared with logarithmic and stationary phase planktonic cells. Transcript expression levels of cdrA in a biofilm were 15.4-fold higher than planktonic cells (Fig. 5C). These data indicate that cdrA is expressed during biofilm growth. Elevated CdrA expression in biofilm cultures of PAO1 was also observed in CdrA Western blots (Fig. 5D).

CdrAB expression promotes auto-aggregation in liquid culture and small, congo-red binding colonies on solid medium

To gain further insight into CdrA function, we examined the effects of overexpression on P. aeruginosa phenotypes. Induction of cdrAB expression resulted in liquid culture auto-aggregation visible at the microscopic (Fig. 6A) and macroscopic level (Fig. 6B). Overexpression of cdrA or cdrB independently does not result in auto-aggregation. This is consistent with the data presented in Fig. 3, suggesting that CdrB is required for CdrA secretion.

Fig. 6

Overexpression of cdrAB results in aggregation in liquid culture and small colonies on solid medium. A. Phase contrast micrographs of P. aeruginosa PAO1 harbouring the expression vector pBADcdrAB and pMJT-1. Bacteria were cultured in LB medium and 0.5% arabinose. B. Liquid cultures of P. aeruginosa PAO1 harbouring the expression vectors pBADcdrA, pBADcdrB, pBADcdrAB, and pMJT-1 cultured in LB medium and 1.0% arabinose. C. Colonies of PAO1 harbouring the expression vector pBADcdrAB cultivated on VBMM congo red agar plates supplemented with increasing concentrations of arabinose.

Induction of cdrAB on solid medium results in a small colony phenotype that binds congo red. Increasing concentrations of arabinose result in a corresponding decrease in the size of the colony resulting in a small colony phenotype that is similar to an RSCV in size, but does not appear to be rugose (Fig. 6C). Overexpression was found to have no effect on growth, ruling out the possibility that cdrAB expression exerts a toxic effect on the cell (see Fig. S4). Another interpretation of a small colony phenotype is that CdrAB overexpression may reduce surface motility. Consistent with this observation, we demonstrated that CdrAB overexpression reduced P. aeruginosa swarming motility (see Fig. S5).

CdrAB-mediated auto-aggregation requires the Psl polysaccharide

Although our data above indicate an important role for CdrA in biofilms, we had no mechanistic understanding. The liquid culture auto-aggregation associated with cdrAB overexpression provided our first clue. The presence of an N-terminal carbohydrate binding domain, and the co-regulation of cdrAB with the pel and psl gene clusters led us to propose that CdrA may bind to one of these polysaccharides. There is precedence for sugar binding in this particular family of adhesins (Menozzi et al., 1994b). Like CdrA, expression of FHA of B. pertussis induces liquid culture auto-aggregation. FHA auto-aggregation can be disrupted by the exogenous addition of sugars to which it binds (Menozzi et al., 1994a).

We used the cdrA-mediated auto-aggregation phenotype (Fig. 6B) as a means to identify potential binding targets. As seen with FHA, we predicted that CdrA-binding targets would disrupt CdrA-dependent auto-aggregation. To test whether Pel and/or Psl could be binding targets, we examined the effect of cdrAB overexpression in pel and psl single and double mutant strains. Strains deficient in the production of Psl did not aggregate when cdrAB was overexpressed, indicating that CdrA may interact with the Psl polysaccharide under these conditions (Fig. 7A). PAO1 harbouring the vector control and strains containing the uninduced cdrAB overexpression construct did not auto-aggregate (data not shown).

Fig. 7

CdrA-mediated aggregation is dependent on Psl and inhibited by the addition of mannose. A. Aggregation of wild-type PAO1 and mutant strains that no longer produce the Pel and/or Psl exopolysaccharides were evaluated after induction of cdrAB with 1% arabinose and 3 h of growth. B. Structure of the Psl polysaccharide. C. Inhibition of aggregation by the addition of sugars. Analysis of the sugar-binding specificity of cdrA was monitored indirectly by measuring the % relative aggregation of PAO1 pBADcdrAB strain in which cdrAB expression has been induced by addition of 1% arabinose.

One interpretation of these data is that a strain unable to make Psl is also impaired in production of CdrA. Using Western analysis, we demonstrated that CdrA levels in the supernatant were not affected by the pslBCD mutation (Fig. 3). However, cell-associated CdrA levels appeared to be lower. Conversely, using antisera specific to Psl, we also showed that a cdrA mutation did not significantly affect Psl production (see Fig. S6). We also evaluated aggregation in P. aeruginosa strain PA14, which lacks some of the genes required for Psl production (Friedman and Kolter, 2004a). Overexpression of cdrAB in this strain also failed to induce auto-aggregation (Fig. 7A).

Mannose inhibits CdrA-mediated auto-aggregation

Previous studies have determined that Psl is a mannose-rich exopolysaccharide (Friedman and Kolter, 2004b; Ma et al., 2007). Because Psl is a repeating pentasaccharide composed of mannose, rhamnose and glucose monosaccharides (in a 3:1:1 ratio), we surveyed these sugars and other selected sugars for the ability to disrupt CdrA-mediated auto-aggregation (Fig. 7B and C). Addition of D-mannose and methyl α-D-mannopyranoside reduced the relative auto-aggregation 61% and 66% respectively, as observed visually and quantified by measuring absorbance (Fig. 7C and Fig. S7). Inhibition of aggregation by mannose provides additional support for an interaction with the Psl exopolysaccharide. The other sugars found in Psl, L-rhamnose and D-glucose, had little effect on auto-aggregation.

Auto-aggregation was also diminished in the presence of other sugars. The addition of D- and L-Fucose, D-Fructose and GlcNAc resulted in a 40–46% decrease in relative aggregation. These results indicate that CdrA may be a multivalent adhesin that has the ability to recognize multiple carbohydrates. This is the case for FHA as well, which is capable of binding multiple, structurally distinct sugars. In every case tested, addition of sugars to P. aeruginosa not overexpressing cdrAB did not impact the aggregation state or growth of the culture (data not shown).

Extracellular CdrA directly binds to Psl

Data presented in Figs 6 and 7 indirectly suggest CdrA is capable of binding Psl. To directly test this hypothesis, we performed co-immunoprecipitation (Co-IP) experiments. We coated magnetic beads with anti-Psl antibodies. We then incubated these beads with cell free supernatant of cdrAB overexpressing strains in wild-type and psl mutant backgrounds. The rationale is that if CdrA is binding to extracellular Psl, when this Psl interacts with the Psl antibodies on the beads, CdrA will be co-precipitated. Protein eluted off the washed beads are then run on an SDS-PAGE and probed for CdrA using CdrA antisera. Figure 8 shows that CdrA failed to associate with the beads when P. aeruginosa was not producing Psl (PAO1 pslBCD/pBADcdrAB), while it did in a strain producing Psl (PAO1/pBADcdrAB). As a negative control, the same experiment was performed with a secreted, overexpressed protein, Hcp-1. This protein is a substrate of type VI secretion and is highly abundant in culture supernatant when overexpressed. This protein failed to associate with Psl in our assay (data not shown).

Fig. 8

Psl and CdrA Co-immunoprecipitation analysis. This figure depicts a western analysis using CdrA antisera. The Co-IP eluants show what eluted off of Psl antisera coated beads after incubation with supernatants of selected strains. The input pool shows the amount of CdrA present in the supernatants that were incubated with the beads.

CdrA is required for Psl localization in biofilms, contributing to their integrity

The Psl exopolysaccharide has been shown to be important for attachment to surfaces and maintenance of biofilm structure (Friedman and Kolter, 2004b; Jackson et al., 2004; Matsukawa and Greenberg, 2004; Ma et al., 2006). Because CdrA appears to bind Psl, we hypothesized that its function in a biofilm community might be to interact with Psl as part of the extracellular matrix, perhaps tethering cells to each other, Psl, or both.

To test this hypothesis we examined the effects of a cdrA mutation on Psl distribution in a biofilm. Lectin staining techniques have been developed for the purposes of visualizing Psl. These lectins show specificity for the Manα1,3Man or Manα1,6Man structural components of the Psl EPS (Ma et al., 2007). Consistent with previous results, a wspF strain produces a biofilm characterized by large cell aggregates in which the Psl forms a tightly associated shell around the aggregate (Fig. 9A and C) (Ma et al., 2009). In sharp contrast, a cdrA mutation in this background produces a loosely structured biofilm in which the Psl is not tightly associated with the cell aggregates, and appears to be released into the flow stream (Fig. 9B and D). These results suggest a role for CdrA in the association and stabilization of Psl with bacterial cells and the biofilm matrix.

Fig. 9

Lectin staining reveals CdrA is required for Psl association with a developing biofilm. Biofilms produced by Pseudomonas aeruginosa PAO1 ΔwspF (A, side view; C, bottom-up view) and ΔwspFcdrA (B, side view; D, bottom-up view) were stained with lectin. Bacterial cells were stained with Syto9 (green) and Psl stained with HHA-Tritc labelled lectin (red).

Clinical isolates have the cdrAB genes and the capacity to functionally express CdrA

We also asked whether P. aeruginosa clinical isolates have the genetic capacity to produce CdrA. We isolated chromosomal DNA from 11 independent CF clinical isolates and assayed for the presence of the cdrA gene using PCR (Fig. S8). We found that all 11 isolates produced a band of the expected size. Wolfgang et al. also reported the presence of cdrAB in all 16 of their clinical and environmental isolates (Wolfgang et al., 2003). We next tested whether cultures from the isolates were producing CdrA by western analysis. In this case six of the isolates produced a protein of about the right size that was immunoreactive with the CdrA specific antisera (Fig. S8). These data collectively suggest that cdrA is not specific to the PAO1 strain and could play a role in biofilm formation for some clinical strains.

Bacterial strains and growth conditions

Strains and plasmids used for this study are listed in Table 1. Bacteria were grown overnight at 37°C with shaking at 250 r.p.m. in Luria–Bertani (LB) broth unless otherwise stated. L-arabinose at the reported concentrations or 0.5 mM IPTG was supplemented into the medium for inducible-expression experiments. Motility plate assays were used to determine differences in swarming motility. LB agar medium was solidified with 0.45% Noble agar. Plates were inoculated using a sterilized platinum wire with log-phase cells and swarming diameter was measured 40 h after incubation at 30°C.

Strain and plasmid construction

DNA manipulations were performed using standard methods. Genomic DNA was prepared using the DNeasy Blood & Tissue Kit (QIAGEN). PCR amplicons were excised and purified from agarose gels by gel extraction with the Qiaex II kit (QIAGEN). Deletion mutants of pelA, pslBCD, wspF and cdrA were constructed by allelic exchange using standard procedures for P. aeruginosa. Plasmids engineered with the deletion constructs were mated into P. aeruginosa and selected on VBMM agar (Vogel and Bonner, 1956) containing 100 µg ml⁻¹ gentamicin. Strains were further selected on LB agar plates containing 5% sucrose (w/v) to isolate double recombinants. Deletion mutants were confirmed by PCR with primer sets internal and external to the deletion. Arabinose inducible cdrA, cdrB and cdrAB plasmids were constructed by PCR amplification and cloned into the multicloning site of the arabinose-inducible expression vector, pMJT-1. XbaI and EcoRI restriction sites were engineered into the PCR primers to directionally clone each amplicon.

Isolation of total RNA, RT-PCR and evidence that cdrAB is an operon

Cells were grown in LB broth at 37°C overnight for RT-PCR. Tube biofilm experiments were conducted as previously described (Schaefer et al., 2001; Kirisits et al., 2005). P. aeruginosa PAO1 was injected into 6.35 mm inner-diameter tube and incubated for 30 min. Control cells were incubated staticly. Biofilms were grown at room temperature in LB medium containing 100 mM MOPS at 50 ml h⁻¹ flow rate for 48 h.

Total RNA was isolated by using the RNAprotect Bacteria Reagent and RNeasy kit (QIAGEN). Genomic DNA was eliminated by RQ1 RNase-Free DNase (Promega) treatment during the isolation procedure. Reverse transcription was performed on 1 µg of RNA by using the SuperScriptIII First-Strand Synthesis System (Invitrogen) according to the manufacturer's recommendations. Real-Time PCR was performed with the 7500 Real-Time PCR System with the SYBR Green PCR Master Mix (Applied Biosystems). Cycling parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Standard curves for quantification of transcripts were performed using dilutions of P. aeruginosa chromosomal DNA from 1 X 10⁻⁴ ng to 10 ng (Lequette et al., 2006). Transcript levels of all genes tested were normalized to transcript levels of the ampR gene (Lequette et al., 2006). PCR reactions were performed in quadruplicate for each gene. Data represent the average and the standard deviation between samples.

Transcription of cdrAB as an operon was verified by RNA extraction and cDNA synthesis as described above. PCR from cDNA was utilized to amplify an 854 bp region spanning the N-terminus of cdrA and the C-terminus of cdrB. PCR experiments included reactions consisting of cDNA, genomic DNA, RNA and cDNA no reverse transcriptase controls.

Modelling experiments

CdrA is a 2154 amino acid protein. De novo modelling programmes or servers are not reliable at such sizes. To overcome this limitation, CdrA was modelled as six separate segments and reattached by structural alignments of overlapping domains. Three different sets of segment structures were used to assemble CdrA structures. Three whole protein models were made from each set of segment models, each with a different set of overlapping regions. Thus, nine total models were made with this protocol. The top models out of these nine were chosen by a recent version of the residue-specific all-atom probability discriminatory function (RAPDF) (Samudrala and Moult, 1998).

Domain boundaries were predicted by the DomPred algorithm on the 3D Jury server (Marsden et al., 2002). Segment structures were modelled using the I-TASSER server. The I-TASSER algorithm is an enhanced threading algorithm and is the most reliable de novo modelling technique as of the CASP8 competition (http://www.predictioncenter.org/casp8) (Wu et al., 2007; Zhang, 2008). After each segment was attached, the resulting structure was decreased in potential energy by a combination of the side-chains with a Rotamer Library (SCWRL3) algorithm (Canutescu et al., 2003) and the Energy Calculations and Dynamics Program (ENCAD) (Levitt et al., 1995). A trinary confidence measure was implemented by comparing the results of sequence based secondary structure prediction to secondary structure elements assigned to the model. The sequence based secondary structure predictions were performed by PsiPred (Bryson et al., 2005). The model based secondary structure assignments were performed by the Definition of Secondary Structure of Proteins (DSSP) algorithm (Kabsch and Sander, 1983). Amino acids that had matching secondary structure assignments were denoted with a high confidence. Amino acids in which a secondary structure prediction was made by one method but not the other was denoted with an intermediate confidence. Amino acids in which opposite secondary structures assigned by the two methods were denoted with a low confidence.

Three separate sets of segments were modelled using the I-TASSER server. For each set of segments, three different arrangements of overlapping regions were used to build the whole protein. Nine different models were produced by repeating this method with altered parameters. The best of these nine final models was chosen by an all-atom scoring function (Samudrala and Moult, 1998) The RAPDF (Samudrala and Moult, 1998) was used to rank these models by likelihood of accuracy. Our two highest scoring models had a similar overall tertiary structure. We prefer the second highest scoring model because of a structurally independent putative signal peptide and an exposed putative integrin binding domain. Confidence of the model was determined by comparing the secondary structure that was predicted from the model with the secondary structure inferred from the sequence (Fig. 1B) (Kabsch and Sander, 1983). Secondary structure predictions were identical for 57% of the model.

Generation of CdrA antibody and Western blot detection

A unique domain of the cdrA gene (bp 2308–2808) from P. aeruginosa PAO1 was amplified by PCR and cloned into pET-20b(+) vector (Novagen) to construct plasmid pBB022 for the expression of His-tagged CdrA protein (aa 770–936). Protein expression was induced at 16°C by addition of 0.5 mM IPTG at 0.5 OD₆₀₀ and subsequent overnight incubation. The recombinant CdrA protein was purified under denaturing conditions by affinity chromatography on Ni-NTA resin as recommended by the manufacturer (Qiagen). Rabbit polyclonal antibodies against CdrA (αCdrA) were raised in New Zealand white rabbits by immunization (Express Line Plus Protocol) of each rabbit with 2 mg of recombinant CdrA protein (Lampire). Polyclonal antibody against CdrA was obtained.

For immunoblotting experiments, overnight cultures of P. aeruginosa were inoculated into medium as indicated and grown to mid-log phase. Whole cell lysates and proteins concentrated from supernatants were prepared as described by Mougous et al. with modification (Mougous et al., 2006). Bacterial cells were harvested by centrifugation, resuspended in 50 µl of sample buffer mixed with 100 µl of Laemmli buffer and heated to 99°C for 5 min. The protein concentration of each whole cell lysate sample was measured with the Pierce® 660nm protein assay (Thermo Scientific) and 10 µg of protein of the respective sample was used for electrophoresis, blotted and incubated with αCdrA (1:2000). Proteins residing in the supernatant were concentrated from 1.6 ml of supernatant by TCA precipitation (Mougous et al., 2006). Normalization and sample loading were based on the corresponding protein concentration of whole cell lysates from which the supernatants were derived. Detection was performed with the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Western immunoblotting of polysaccharide extracts from colony biofilms

Colony biofilms were grown on sterile 0.22 Micron polycarbonate filters (GE Water & Process Technologies) on LB agar medium for 48 h (Rani et al., 2007). Filters were transferred to fresh medium daily. Bacterial samples from two filters were resuspended in 100 µl of 0.5 M EDTA and boiled for 5 min and processed. Supernatants were collected and the protein levels were quantified by Bradford Assay. Samples were additionally treated with proteinase K (final concentration 0.5 mg ml⁻¹⁾ for 60 min at 60°C and 30 min at 80°C. Polysaccharide extracts were normalized based on protein concentration of samples prior to treatment with proteinase K and 10 µg of samples were spotted on a nitrocellulose membrane and allowed to air dry for 30 min. Immunoblots were probed with α-PSL (Byrd et al., 2009).

Co-IP of CdrA bound to Psl polysaccharide

Supernatants were buffered to a final concentration of 1× PBS and 1× Roche protease inhibitor EDTA-free cocktail. Protein was concentrated from buffered supernatants by centrifugation filtration (Amicon Ultracel-10K). Dynabeads Protein A magnetic beads (Invitrogen) were incubated with 5 µl of α-PSL (Byrd et al., 2009) according to the manufacturer's recommendations with the following modifications. Beads were washed twice after antibody binding with PBS Tween 20 and the target antigen was incubated 20 min with the Dynabeads-antibody complex. Target antigen was eluted with 20 µl of 2× Laemmli buffer and separated by SDS PAGE. Western blot analysis was performed with α-CdrA. To verify that equal amounts of CdrA were present in the input pools of strains overexpressing CdrA, supernatants were TCA precipitated from the various treatments and included as an additional control. Additionally, the levels of the secreted protein Hcp1 were evaluated to determine the specificity of Co-IP with a non-target antigen.

Static microtitre biofilm formation assays

Static biofilm formation was evaluated by monitoring biofilm biomass accumulation using the method of O'Toole and Kolter (1998a,b); with minor modifications. Overnight cultures were subcultured and grown to mid-log growth phase. Nunc Bacti 96 well microtitre plates containing 95 µl of VBMM medium were inoculated with and 5 µl of culture and incubated for 20 h at 37°C under static conditions. Plates were washed to remove nonadherent cells by submerging in deionized water and decanting. Biofilm biomass was stained by the addition of 150 µl of 0.1% (w/v) crystal violet added to each well, incubated for 15 min, rinsed with water, and solubilized by the addition of 200 µl of 95% ethanol. Absorbance was measured at 595 nm. Data represent the mean of six replicates.

Biofilm flow cell experiments

Biofilms were cultivated at 37°C for 48 h in polycarbonate flow cells with individual channel dimensions of 1 by 4 by 40 mm in 1% LB (v/v) medium. Flow cells were inoculated with 100 µl of bacterial culture at an OD600 of 0.05 in 1% LB medium diluted from exponentially growing cultures. Bacterial cells were allowed to attach to the inverted glass coverslip for 1 h before the initiation of medium flow at a constant rate of ∼3.75 ml h⁻¹ using a Watson Marlow 205S peristaltic pump (Watson Marlow, Falmouth, UK).

Aggregation and sugar analysis

Stationary-phase cultures were diluted 30-fold into medium supplemented with 1% (w/v) arabinose, 300 µg ml⁻¹ carbenicillin and various sugars at a final concentration of 5 mg ml⁻¹. Sugars were obtained from commercial sources. Aggregation was evaluated by visual assessment and/or measurement of the absorbance at 600 nm after 3 h of growth. The % relative aggregation was calculated by dividing the treatment by the average of the control strain and multiplying by 100.

Lectin staining

Biofilms were stained with TRITC-labelled HHA lectin (EY laboratories), and SYTO 9 (Invitrogen) and visualized at 63× magnification by CLSM after 48 h of growth at 37°C. The specificity of the HHA lectin for Psl has been previously described (Ma et al., 2009). Flow was suspended and 0.3 ml of 0.02 mM SYTO9 and 100 µg ml⁻¹ TRITC-HHA was injected upstream of the inlet flow. Biofilms were stained for 15 min in the dark and examined by confocal laser scanning microscopy (CLSM) 15 min after flow was resumed (Hentzer et al., 2001).