Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture

Aamir Ghafoor; Iain D Hay; Bernd H A Rehm

doi:10.1128/AEM.00637-11

. 2011 Aug;77(15):5238–5246. doi: 10.1128/AEM.00637-11

Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture ^{^▿}^†

Aamir Ghafoor ¹, Iain D Hay ¹, Bernd H A Rehm ^1,^*

PMCID: PMC3147449 PMID: 21666010

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.

INTRODUCTION

Pseudomonas aeruginosa has been widely studied as a model organism for biofilm formation. P. aeruginosa is an opportunistic human pathogen which causes infection in burn wounds and in the lungs of patients suffering from the genetic disease called cystic fibrosis (CF). The ability to form biofilms is critical for its survival in the CF lung environment and enhances its resistance to antimicrobial treatment and host defense mechanisms (9). P. aeruginosa biofilms are also common on medical devices, such as contact lenses and catheters (20). At least three exopolysaccharides have been shown to be produced by P. aeruginosa: alginate, Psl, and Pel (27). Each of these exopolysaccharides has been found to be involved in biofilm formation (27).

Alginates, which are overproduced by P. aeruginosa after infection of CF patients, are linear polyanionic exopolysaccharides composed of uronic acids (25). Alginate-overproducing mutants form large finger-like microcolonies compared to wild-type strains (7). Alginate has been shown to contribute to decreased susceptibility of biofilms to antibiotic treatment and human antibacterial defense mechanisms (19, 23).

The Psl polysaccharide is rich in mannose and galactose and is involved in initial attachment and mature biofilm formation (12). Psl is produced during planktonic growth, mediating attachment to surfaces and contributing to microcolony formation. In mature biofilms, Psl is associated with the caps of mushroom-like microcolonies, forming a peripheral meshwork covering the cap region (12, 13).

Pel is a glucose-rich, cellulose-like polymer essential for the formation of a pellicle at the air-liquid interface (5). Increased Pel production has also been associated with the wrinkled colony phenotype (6). It has recently been shown that Pel plays a role in cell-to-cell interactions in P. aeruginosa PA14 biofilms, providing a structural scaffold for the community at early stages of biofilm formation (4).

Besides the important role of the exopolysaccharides in biofilm formation, extracellular DNA (eDNA) has been shown to be an important component of the biofilm matrix (1, 15). eDNA was found to mediate cell-cell interactions in biofilms (1). Moreover, it was shown that removal of eDNA by DNase treatment at initial stages of biofilm formation interfered with maturation of the biofilm. However, in mature biofilms, such DNase treatment showed little impact on biofilm architecture (34). eDNA was found to be present mostly in the stalk region of microcolonies (1). A recent study has suggested that Psl and eDNA are spatially separated from each other, with Psl being present at the periphery of the biofilm and eDNA mostly being in the Psl-free biofilm matrix (12).

This study aims to shed light on how these exopolysaccharides and eDNA contribute to biofilm formation and architecture, particularly in view of the synergistic effects between the different exopolysaccharides.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The bacterial strains, plasmids, and oligonucleotides used in this study are listed in Table S1 in the supplemental material. LB medium was used to grow all Escherichia coli strains at 37°C. When needed, the following antibiotics were added to the medium at the indicated concentrations: ampicillin, 75 μg/ml, and gentamicin, 10 μg/ml. P. aeruginosa PAO1 and PDO300 (14) and their isogenic mutants were grown in LB medium, Pseudomonas isolation (PI) medium (20 g of peptone, 10 g of K₂SO₄, 1.4 g MgCl₂·6H₂O, 25 mg of triclosan, and 20 ml of glycerol per liter), or PI agar (PIA) medium at 37°C; and if required, gentamicin and carbenicillin were added at concentrations of 100 to 300 μg/ml and 300 μg/ml, respectively.

Isolation, analysis, and manipulation of DNA.

General cloning procedures were performed as described previously (28). DNA primers, deoxynucleoside triphosphates, and Taq and Platinum Pfx polymerases were purchased from Life Technologies (Auckland, New Zealand). DNA sequences of new plasmid constructs were confirmed by DNA sequencing, according to the chain termination method, using an ABI 310 automatic sequencer.

Construction of single-, double-, and triple-deletion mutants.

Two regions of the pelF gene were amplified by using Pfx polymerase with primers PelF-N1, PelF-N2, PelF-C1, and PelF-C2. Both PCR products were hydrolyzed with BamHI, ligated together, and inserted into vector pGEM-T Easy from Promega (Sydney, Australia), resulting in pGEM-T Easy::PelF-NC. A 1,100-bp fragment containing the aacC1 gene (encoding gentamicin acetyltransferase) flanked by two Flp recombinase target sites was released when vector pPS856 (8) was hydrolyzed with BamHI. The 1,100-bp BamHI fragment (aacC1 gene) was inserted into the BamHI site of plasmid pGEM-T Easy::PelF-NC, resulting in plasmid pGEM-TEasy::ΔpelF ΩGm (where Gm is the gentamicin resistance cassette). A 1,949-bp ΔpelF ΩGm DNA fragment was amplified by Pfx polymerase using primers PelF-N1and PelF-C2, and the corresponding PCR product was inserted into the SmaI site of vector pEX100T (8, 30), resulting in plasmid pEX100T::ΔpelF ΩGm. The gene-knockout plasmid was transferred into PAO1 via electroporation as previously described (3). Transformants were selected on LB medium containing 100 μg of gentamicin/ml and subsequently plated on mineral salt medium containing 300 μg of gentamicin/ml and 5% (wt/vol) sucrose. Those cells able to grow on this selective medium will have emerged from double-crossover events. Gene replacement was confirmed via PCR with primers PelF XUP and PelF XDN. E. coli S17-1 was used to transfer the Flp recombinase-encoding vector pFLP2 (8) into P. aeruginosa PAO1 ΔpelF ΩGm. Transfer of plasmid pFLP2 into recipient cells was confirmed by selecting carbenicillin-resistant cells on PIA medium containing carbenicillin (300 mg/ml). These carbenicillin-resistant bacterial cells were cultivated on PIA medium containing 5% (wt/vol) sucrose, and cells grown on this medium were analyzed by observing their sensitivity to gentamicin and carbenicillin. PCR with primers PelF XUP and PelF XDN was done to confirm the loss of the gentamicin resistance cassette. Consequently, P. aeruginosa PAO1 ΔpelF and PDO300 ΔpelF were generated. Accordingly, plasmids pEX100::Δalg8 ΩGm (26) and pEX100T::ΔpslA ΩGm (21) were used for the disruption of the alg8 and/or pslA gene in P. aeruginosa PAO1. To generate Psl- and alginate-negative double mutants, single mutants PAO1 ΔpslA and PDO300 Δalg8 were transformed with pEX100T::ΔpslA ΩGm and pEX100T::Δalg8 ΩGm, respectively, and the resultant markerless PAO1 ΔpslA alg8 and PDO300 ΔpslA alg8 strains were generated as described above. To generate the respective pel-deficient double and triple mutants, pEX100T::ΔpelF ΩGm was transferred into markerless single and double mutants PAO1 Δalg8, PAO1 ΔpslA (21), PDO300 Δalg8 (26), PDO300 ΔpslA, PAO1 ΔpslA alg8, and PDO300 ΔpslA alg8. The gentamicin resistance cassette was removed to obtain PAO1 Δalg8 ΔpelF, PAO1 ΔpslA ΔpelF, PDO300 Δalg8 ΔpelF, PDO300 ΔpslA ΔpelF, PAO1 ΔpslA alg8 ΔpelF, and PDO300 ΔpslA alg8 ΔpelF. All mutants were stored at −80°C for future use.

Complementation of isogenic pelF deletion mutant.

For complementation of the isogenic knockout mutant, P. aeruginosa PAO1 ΔpslA ΔpelF/pBBR1-MCS5::pelF was constructed. The pelF gene was amplified by PCR using the primers PelFEcRfor and PelFClaRev, together with chromosomal DNA from P. aeruginosa PAO1. The PCR product was inserted into the vector pGEM-T Easy. SalI, one of the multiple cloning sites provided in vector pGEM-T Easy, was available upstream of the gene cloned into the vector pGEM-T Easy. Thus, the pelF gene was hydrolyzed with SalI and ClaI (for which a restriction site was available in the downstream primer) and inserted into the broad-host-range vector pBBR1-MCS5 hydrolyzed by SalI and ClaI. In the resulting plasmid, pBBR1-MCS5::pelF, the pelF gene was arranged linear to and downstream of the lac promoter. The donor strain, E. coli S17-1, harboring pBBR1-MCS5::pelF was allowed to conjugate with PAO1 ΔpslA ΔpelF. Recipient PAO1 ΔpslA ΔpelF cells carrying pBBR1-MCS5::pelF were isolated by selection on PIA medium containing gentamicin (300 μg/ml). To confirm the successful transfer of plasmid from donor to recipient strain, plasmids were isolated and digested with the respective restriction enzymes.

Construction of broad-host-range promoter-probe vector pTZ110::P_pel.

The broad-host-range promoter-probe vector pTZ110::P_pel was constructed as follows. The primers PpelEcRfor and PpelBaRev and genomic DNA from P. aeruginosa PAO1 were used to amplify a 1,000-bp upstream region of pelA comprising the putative promoter region of the pel gene operon. The PCR product was inserted into the vector pGEM-T Easy and transformed into E. coli TOP10 cells. Hydrolysis with EcoRI (for which a site was present in the upstream primer) and BamHI (for which a site was present in the downstream primer) yielded a 1,000-bp putative promoter region, P_pel, which was subsequently cloned into the corresponding sites of pTZ110. In this plasmid, the promoter region was upstream of lacZ. This plasmid was transferred to P. aeruginosa strains through conjugation via E. coli S17-1, and transconjugants were selected for on LB agar containing 300 μg/ml carbenicillin.

β-Galactosidase assay.

β-Galactosidase activity was measured as described by Miller (17) and is expressed in Miller units (MU). The data presented below are the results obtained from three independent experiments. The variance is indicated by error bars in the figures.

Pellicle formation assay.

Pellicle formation was assessed as previously described (5). Briefly, borosilicate glass tubes (18 mm by 150 mm) containing 10 ml of PI medium (broth) were inoculated with each mutant. Tubes were incubated without shaking at 37°C. Pellicle formation at the air-liquid interface after 4 days was assayed by visual inspection.

Congo red binding assay.

The assay to assess the ability of mutants to produce Pel polysaccharide was adapted from Spiers et al. (31). Each mutant was incubated in 2 ml of PI medium for 48 h at 37°C without shaking. The bacterial content, along with polysaccharides produced by bacterial cells, was collected by centrifugation, and the supernatant was discarded. The pellet was washed with PI medium and transferred into 2-ml microcentrifuge tubes. The pellet was resuspended in 1 ml of 20 mg ml⁻¹ Congo red in PI medium and incubated for 90 min while it was shaken. Bacterial content and bound Congo red were sedimented by centrifugation at 15,870 × g for 5 min. The supernatant was collected and the optical density at 490 nm (OD₄₉₀) was noted. The total Congo red percentage left in supernatant was measured.

Solid surface attachment (SSA) assay.

Attachment to a solid surface was assessed as previously described (16) and with some modifications. In brief, pertinent strains were grown overnight in PI broth medium and the OD at a wavelength of 600 nm was measured. An appropriate amount of overnight culture was added into PI broth to obtain 1:100 dilutions. Eight wells of a 96-well plate was inoculated with 100 μl of diluted culture of a particular strain, and the plate was incubated at 37°C for 2 −96 h. In order to remove planktonic/nonadherent bacteria at the end of each incubation time, plates were washed using either a vigorous or a gentle washing procedure as previously described (7).

DNase treatment of biofilms.

P. aeruginosa PAO1 ΔpslA Δalg8 and P. aeruginosa PAO1 ΔpelF Δalg8 were grown in flow cells for 96 h. DNase I from Sigma (St. Louis, MO) was dissolved in medium at a concentration of 500 μg/ml and injected into the flow cell. This was incubated for 30 min without flow. Flow was restored to 0.3 ml/min for 15 min, after which the DNase-treated biofilms were stained as described below.

Continuous-culture flow cell biofilms.

For biofilm analysis, each mutant was grown in continuous-culture flow cells for 4 days at 37°C as previously described (2). The flow cells used in this study had dimensions of 4 mm by 40 mm by 1.5 mm. Each channel was filled with PI medium, inoculated with a total of 0.5 ml overnight culture of the respective mutant containing approximately 2 × 10⁹ cells per ml, and incubated without flow for 4 h at 37°C. PI medium was then allowed to flow at a mean flow of 0.3 ml min⁻¹, corresponding to a laminar flow with a Reynolds number of 5. The flow cells were then incubated at 37°C for 96 h. Biofilms were stained using a LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Inc., Eugene, OR) and visualized using confocal laser scanning microscopy (CLSM; Leica SP5 DM6000B microscope). Images of P. aeruginosa and all of its mutants were captured using a ×63 objective lens. Images were analyzed using IMARIS (Bitplane, Inc.) software.

Quantitative analysis of biofilms using IMARIS image analysis software (Bitplane, Inc.).

Biofilm appearance, biofilm volume, dead-to-live ratio, thickness, and compactness of biofilms were the parameters used to compare the architectural differences of the biofilms formed by the various mutants. To obtain volume per unit area (μm³/μm²), a ratio between total volume and total area covered by biofilm was calculated. The compactness of the biofilm was assessed as total fluorescence per volume of biofilm. To obtain the ratio between the number of dead cells to the number of living cells per biofilm volume, the ratio between red fluorescence and green fluorescence was calculated. In each biofilm, thickness of the base and microcolonies were measured separately, and standard deviations were calculated.

RESULTS

Generation of isogenic pelF deletion mutants deficient in Pel polysaccharide production.

Deletion of the pelF gene in PAO1 resulted in a Pel-deficient phenotype. Pellicle formation at the air-liquid interphase was absent in the ΔpelF mutants PAO1 ΔpelF, PAO1 ΔpelF Δalg8, PAO1 ΔpslA ΔpelF, and PAO1 ΔpslA ΔpelF Δalg8 (Fig. 1A). The Congo red binding assay also showed that Pel-deficient mutants bind less Congo red, leaving most of it in the cell-free supernatant, whereas Pel polysaccharide-producing mutants PAO1, PAO1 Δalg8, PAO1 ΔpslA, and PAO1 ΔpslA Δalg8 showed increased binding of Congo red, leaving less Congo red in the cell-free supernatant (Fig. 1B).

Fig. 1. — Assessment of Pel formation in various exopolysaccharide-deficient PAO1 mutants. ΔF, PAO1 Δ*pelF*; Δ8, PAO1 Δ*alg8*; ΔA, PAO1 Δ*pslA* ΔAΔF, PAO1 Δ*pslA* Δ*pelF*; ΔAΔ8, PAO1 Δ*pslA* Δ*alg8*; ΔFΔ8, PAO1 Δ*alg8* Δ*pelF*; ΔAΔFΔ8, PAO1 Δ*pslA* Δ*pelF* Δ*alg8*. (A) Pellicle formation at air-liquid interphase when each mutant was grown in 10 ml of PI medium for 4 days as a static biofilm. (B) Congo red binding assay. All strains grown as static biofilms for 4 days were mixed with 20 mg/ml Congo red and left for 90 min while it was shaken. The biomass was sedimented, and unbound Congo red was detected at 490 nm. The percentage of Congo red left in the supernatant is shown here.

Similar Congo red staining results were shown for Pel-deficient mutants PDO300 ΔpelF, PDO300 Δalg8 ΔpelF, PDO300 ΔpslA ΔpelF, and PDO300 ΔpslA Δalg8 ΔpelF (data not shown).

Complementation of ΔpelF deletion mutant.

To confirm that disruption of the pelF gene had no polar effect on genes downstream of pelF within the pel operon, plasmid pBBR1-MCS5::pelF was constructed and transformed into P. aeruginosa PAO1 ΔpslA ΔpelF. Since the lack of Pel production of the pelF mutant was particularly obvious in the Psl-negative background, the double mutant PAO1 ΔpslA ΔpelF was used for complementation experiments. The mutant PAO1 ΔpslA ΔpelF harboring plasmid pBBR1-MCS5::pelF was restored in its ability to produce Pel, which was shown by the formation of a pellicle at the air-liquid interphase and increased Congo red binding (Fig. 1B).

Generation of various isogenic deletion mutants deficient in production of various exopolysaccharides.

To study the synergistic or antagonistic effects of the three polysaccharides on biofilm formation, single, double, and triple mutants of strains PAO1 and PDO300 were generated. Previously, a PAO1 ΔpslA mutant was generated and characterized and was shown to be deficient in attachment to a solid surface at an early stage of biofilm formation (21). PAO1 ΔpslA was used to generate Psl/alginate-, Psl/Pel-, and Psl/alginate/Pel-deficient mutants. The resultant mutants, PAO1 ΔpslA Δalg8, PAO1 ΔpslA ΔpelF, and PAO1 ΔpslA Δalg8 ΔpelF, respectively, showed impaired attachment in the SSA assay (Fig. 2A). The PDO300 ΔpslA, PDO300 ΔpslA Δalg8, PDO300 ΔpslA ΔpelF, and PDO300 ΔpslA Δalg8 ΔpelF strains generated in this study also showed impaired solid surface attachment (see Fig. S1 in the supplemental material).

Fig. 2. — Attachment of various *P. aeruginosa* PAO1 strains to a solid surface. The SSA assay was used to assess the impacts of various exopolysaccharide deficiencies on attachment. ΔF, PAO1 Δ*pelF*; Δ8, PAO1 Δ*alg8*; ΔA, PAO1 Δ*pslA*; ΔAΔF, PAO1 Δ*pslA* Δ*pelF*; ΔAΔ8, PAO1 Δ*pslA* Δ*alg8*; ΔFΔ8, PAO1 Δ*alg8* Δ*pelF*; ΔAΔFΔ8, PAO1 Δ*pslA* Δ*pelF* Δ*alg8*; media, uninoculated *Pseudomonas* isolation medium control. (A) Differences during early attachment phase at 2-, 4-, and 6-h time points; (B) differences between loosely and tightly attached 4-day-old biofilms (adherent biofilms after soft and vigorous washing, respectively). Values and error bars represent the averages and standard deviations, respectively, for 24 independent replicates.

PDO300 Δalg8 has already been previously characterized to be deficient in alginate production (26). All alg8-knockout mutants of PDO300, PDO300 ΔpslA Δalg8, PDO300 Δalg8 Δpel, and PDO300 ΔpslA Δalg8 ΔpelF, were nonmucoid when they were grown on PIA plates and showed no alginate production when they were assessed for alginate quantification (see Table S2 in the supplemental material). Isogenic mutants PAO1 Δalg8, PAO1 ΔpslA Δalg8, PAO1 Δalg8 ΔpelF, and PAO1 ΔpslA Δalg8 ΔpelF were generated as outlined in Table S1 in the supplemental material. An overview of all strains used and generated in this study and their exopolysaccharide biosynthesis-relevant genotype and phenotype are presented in Table 1 .

Table 1.

Exopolysaccharide biosynthesis-relevant genotypes and phenotypes of strains used and/or generated in this study

Strain	Exopolysaccharide produced by strains^a
Strain	Alginate	Pel	Psl^b
PAO1	+	+	+
PAO1 ΔpelF	+	−	+
PAO1 Δalg8	−	++	+
PAO1 ΔpslA	+	+++	−
PAO1 ΔpslA ΔpelF	+	−	−
PAO1 ΔpslA Δalg8	−	+++	−
PAO1 ΔpelF Δalg8	−	−	+
PAO1 ΔpslA ΔpelF Δalg8	−	−	−
PDO300	+++	+	+
PDO300 ΔpelF	++++	−	+
PDO300 Δalg8	−	++	+
PDO300 ΔpslA	++	+++	−
PDO300 ΔpslA ΔpelF	++++	−	−
PDO300 ΔpslA Δalg8	−	+++	−
PDO300 ΔpelF Δalg8	−	−	+
PDO300 ΔpslA ΔpelF Δalg8	−	−	−

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−, polysaccharide production not detectable; +, polysaccharide production detectable, with increased numbers of of plus signs indicating increased relative production.

Psl quantification was not done due to unavailability of a specific and differentiating detection method. However, its production was indicated by determining the total exopolymers produced by the alginate/Pel-deficient mutant.

Effect of anti-sigma factor MucA on transcription of pel psl operon.

All the mutants in this study were derivatives of PAO1 and its isogenic mucA-negative mutant, PDO300. MucA is a membrane-anchored anti-sigma factor which sequesters AlgU, an alternative sigma factor required for transcription of the various genes, including those in the alginate operon (29). To assess the impact of this mucA mutation in alginate-overproducing strain PDO300 on the expression of the psl and pel biosynthesis genes, the levels of transcription from the psl and pel promoters were analyzed in the PAO1 strain and its mucA-negative derivative, PDO300. To analyze the activity of the psl and pel promoter, respectively, plasmids pTZ110::P_pel and pTZ110::P_psl (21) were introduced into PAO1 and PDO300, respectively. The measured β-galactosidase activity was used to deduce the respective promoter activities. The pel promoter in PAO1 and PDO300 mediated β-galactosidase activities of 683 ± 158 and 564 ± 68 Miller units, respectively. The psl promoter in PAO1 and PDO300 mediated β-galactosidase activities of 2,704 ± 477 and 1,923 ± 183 Miller units, respectively.

Analysis of roles of the various exopolysaccharides with respect to attachment to solid surfaces.

An SSA assay was conducted to observe the attachment of cells at initial and mature stages of biofilm development. After 2, 4, and 6 h of incubation, the strains producing Psl (PAO1, PAO1 ΔpelF, PAO1 Δalg8, PAO1 ΔpelF Δalg8) showed increased attachment compared to Psl-negative mutants. After 4 days of growth, psl-negative, pel-producing mutants PAO1 ΔpslA and PAO1 ΔpslA Δalg8 showed increased attachment compared to Pel-deficient mutants. Results are summarized in Fig. 2. Similar results were found for PDO300 and derived isogenic mutants (see Fig. S1 in the supplemental material).

Analysis of biofilms formed by mutants deficient in production of one or more exopolysaccharides.

Representative biofilm images of respective exopolysaccharide-negative mutants of P. aeruginosa were obtained using CLSM (Fig. 3; see Fig. S2 and S3 in the supplemental material). The CLSM stack images were reconstructed into three-dimensional (3D) images using IMARIS software. The results showed that PAO1 Δalg8 was not able to form mushroom-like structures. Mutants, including PAO1 ΔpelF and PAO1 ΔpelF Δalg8, capable of producing Psl and alginate or only Psl, respectively, formed mushroom-like structures. Interestingly, PAO1 ΔpelF, which produces Psl and alginate, formed mushroom-like structures with a higher density of living cells than PAO1 ΔpelF Δalg8 biofilms, which additionally lacked alginate and formed mushroom-like structures with caps almost devoid of living cells (Fig. 3C and E). PAO1 ΔpslA and PAO1 ΔpslA Δalg8 were still able to form biofilms, but these biofilms were flat and much more compact than the biofilms formed by all other studied mutants, and both live and dead cells were present in these biofilms (Fig. 3D and F). Interestingly, it was observed that PAO1 ΔpslA ΔpelF and PAO1 ΔpslA ΔpelF Δalg8 were not able to form any biofilm after 96 h, and only a layer of single cells was observed (see Fig. S2 in the supplemental material). Results similar to those obtained for the various PAO1 mutants were obtained when the biofilms of the respective PDO300 mutants were analyzed (see Fig. S3A to G in the supplemental material), except for PDO300ΔpelF, which could not be assessed due to the excessive formation of exopolymeric matrix material and the subsequent blocking of the flow cell. The levels of alginate produced by this strain were found to be about 10-fold increased compared with those produced by parent strain PDO300 (see Table S2 in the supplemental material). Although mutant PDO300 ΔpelF ΔpslA showed a similar overproduction of alginate, biofilms could still be grown in flow cells without blockage.

DNase treatment of mutants PAO1 ΔpslA Δalg8 and PAO1 ΔpelF Δalg8.

The biofilms of the PAO1 ΔpslA Δalg8 and PAO1 ΔpelF Δalg8 strains showed increased levels of red fluorescence, indicating dead cells and eDNA. To assess the role of eDNA in biofilms, DNase treatment was conducted. This resulted in almost complete removal of red fluorescent structures (Fig. 4A to D), as was indicated by decreases of the red fluorescence-to-green fluorescence ratio from 1.08 to 0.45 in the case of PAO1 ΔpslA Δalg8 and 0.8 to 0.2 in the case of PAO1 ΔpelF Δalg8.

Fig. 4. — DNase treatment of large amounts of extracellular DNA-producing mutants biofilms. (A) Images of PAO1 Δ*pslA* Δ*alg8* without DNase treatment; (B) images of PAO1 Δ*pslA* Δ*alg8* after DNase treatment; (C) images of PAO1 Δ*pelF* Δ*alg8* without DNase treatment; (D) images of PAO1 Δ*pelF* Δ*alg8* after DNase treatment.

Quantitative analysis of biofilms.

The biofilm volume, dead-to-live ratio, thickness, and compactness of biofilms were assessed using IMARIS software. To obtain the volume per area ratio, the total volume (x × y × z = μm³) and total area (x × y = μm²) covered by each biofilm were measured. The highest volume-to-area ratio was found for PAO1 and PAO1 ΔpslA Δalg8 biofilms. PAO1 Δalg8 showed the lowest volume-to-area ratio (Table 2).

Table 2.

Characteristics of biofilms formed by mutants

Biofilm-forming strain	Vol (μm³/μm²)	Dead/live ratio^a
PAO1	47.21	0.33
PAO1 ΔpelF	35.63	0.53
PAO1 Δalg8	28.91	0.41
PAO1 ΔpslA	25.09	0.65
PAO1 ΔpslA Δalg8	49.37	1.08
PAO1 ΔpelF Δalg8	42.57	0.81

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Ratio between red and green fluorescence shown by each biofilm-forming mutant.

The biofilms formed by PAO1ΔpslA, PAO1 ΔpslA Δalg8, and PAO1 Δalg8 were found to be more compact than the other biofilms. Intensity of fluorescence per unit biofilm volume was used to compare the compactness of biofilms. All biofilms formed by Pel-producing PAO1, PAO1 Δalg8, PAO1 ΔpslA, and PAO1 ΔpslA Δalg8 showed 1.83 × 10³, 1.43 × 10³, 3.75 ×10³, and 1.79 × 10³ relative light intensity units per μm³, respectively, which is greater than that of biofilms formed by Pel-deficient mutants PAO1 ΔpelF and PAO1 ΔpelF Δalg8, showing 1.07 × 10³ and 8.82 × 10² relative light intensity units per μm³, respectively. The ratio of dead cells to live cells in the various biofilms showed that in the absence of alginate, biofilms contain more dead cells and extracellular DNA. Dead cell-to-live cell ratios in biofilms produced by alginate-negative mutants PAO1 Δalg8, PAO1 ΔpslA Δalg8, and PAO1 ΔpelF Δalg8 were higher than those in alginate-producing PAO1, PAO1 Δpsl, and PAO1 ΔpelF biofilms (Table 2).

Biofilms formed by all strains contained a base layer of various heights. Psl-producing PAO1, PAO1 ΔpelF, and PAO1 ΔpelF Δalg8 formed structured biofilms having a base layer one-fourth to one-fifth the total height of the microcolony, and the microcolony could be clearly differentiated into stalk and cap structures. On the other hand, Psl-deficient mutants PAO1 ΔpslA and PAO1 ΔpslA Δalg8 formed unstructured flat biofilms, where the only discernible features were short dome-like structures. Interestingly, PAO1 Δalg8, which produces Psl and Pel, showed an unstructured flat biofilm similar to that of Pel-overproducing, Psl-deficient mutants (Fig. 5).

Fig. 5. — Height of microcolonies and base layer of each biofilm-forming mutant measured from 3D pictures of biofilms grown for 96 h. White, height of base of the biofilm. ΔF, PAO1 Δ*pelF*; Δ8, PAO1 Δ*alg8*; ΔA, PAO1 Δ*pslA*; ΔAΔ8, PAO1 Δ*pslA* Δ*alg8*; ΔF Δ8, PAO1 Δ*alg8*Δ*pelF*.

DISCUSSION

In this study the role of single exopolysaccharides and various combinations of exopolysaccharides in biofilm formation by P. aeruginosa was investigated. Three exopolysaccharides, Psl, Pel, and alginate, have been described to be produced by P. aeruginosa as well as have been found to play a role in biofilm formation and biofilm architecture (24). To elucidate the role of each exopolysaccharide, key biosynthesis genes required for the production of the respective exopolysaccharide were disrupted independently and in various combinations. Hence, isogenic deletion mutants of strain PAO1 and its mucA-negative derivative PDO300 deficient in the production of one or more exopolysaccharides were generated for comparative analysis.

Previous work had established that knocking out alg8 from P. aeruginosa results in the complete loss of alginate production, as shown by the lack of alginate and uronic acids in culture supernatants (26). Similarly, deletion of pslA had been shown to abolish Psl production, which impaired surface attachment (21). Hence, mutants deficient in alginate and Psl were generated in this study by knocking out alg8 and pslA, respectively. Previously, it was shown that a third exopolysaccharide, Pel, is produced by P. aeruginosa and was found to be important for the formation of static biofilms by various strains (5). Seven genes contained in a single operon (pelA to pelG) are required for production of the Pel exopolysaccharide (5, 33). The pelF sequence analysis suggested that it encodes a glycosyltransferase, presumably involved in the polymerization of the Pel exopolysaccharide (5). Deletion of pelF from the genome of P. aeruginosa PAK significantly reduced biofilm formation compared to that by the parent strain (33). The inability of mutants to produce Pel had been characterized by a lack of pellicle formation at the air-liquid interphase and reduced Congo red binding (5, 11). Here, pelF was deleted from PAO1 and PDO300 in order to generate isogenic mutants deficient in Pel exopolysaccharide biosynthesis. All pelF mutants showed reduced Congo red binding and the absence of pellicle formation. Although the specificity of this Congo red binding assay has not been established, it has been widely used as an indicator of Pel production (5, 32, 33). Our results showed that all mutants deficient in pellicle formation at the air liquid-interphase also showed reduced Congo red binding (Fig. 1).

Previous studies showed that Psl is involved in initial attachment of cells (2, 6, 10, 21), whereas Pel plays a role in the later stages of biofilm maturity (6). Our results were consistent with these findings. Mutants deficient in Psl showed reduced crystal violet staining of static biofilms at an early stage of biofilm development. Furthermore, neither alginate nor Pel appeared to compensate for this deficiency (Fig. 2A and B). However, in mature biofilms, increased crystal violet staining of the Pel-overproducing mutant PAO1 ΔpslA Δalg8 suggested that Pel at later stages of biofilm development mediates entrapment of more cells (Fig. 2). Since Pel has been shown to contribute to cell-to-cell interactions in P. aeruginosa PA14 (4), increased Pel production could have further increased cell-cell interactions. PA14 is a naturally Psl-deficient strain, and the Psl-deficient mutants generated here are similar to strain PA14 in this respect. Therefore, it is conceivable that increased crystal violet staining resulted from an increased number of bacterial cells in the biofilm held together by the excessive Pel produced by these mutants.

Here it was shown that mutants lacking alginate production showed an increased ratio of dead to live cells (Table 2; Fig. 3). However, propidium iodide also stains eDNA (18). PAO1 ΔpslA Δalg8 and PAO1 ΔpelF Δalg8 biofilms showed extensive surface structures predominantly composed of eDNA, as was suggested by DNase treatment (Fig. 4). Previous studies suggested that eDNA plays an important role in biofilms as a cation-chelating and antibiotic resistance-inducing matrix component (18, 34). Since large amounts of eDNA were observed only in mutants lacking alginate production, increased cell death might have led to more eDNA being released, functionally and structurally replacing the polyanionic alginate.

The role of alginate for entrapping live cells in the cap of the mushroom-like structure was underpinned by the observation that the caps in biofilms formed by PAO1 ΔpelF Δalg8 (which produces only Psl) were almost devoid of live cells, whereas those formed by Psl- and alginate-producing strains PAO1, PAO1 ΔpelF (Fig. 3), and PDO300 (see Fig. S3A in the supplemental material) showed a high density of live cells in the cap region. This suggested that the caps of the mushroom-like structures are not made up of only Psl but that alginate plays an important role in retaining live cells and/or supporting the viability of cells in these caps.

Interestingly, analysis of biofilms formed by PAO1 ΔpslA Δalg8 suggested that Pel and eDNA together with live bacterial cells constituted a connected meshwork showing increased cell-to-cell interactions and, hence, an increased compactness of the biofilms (Fig. 3 and 4). This is consistent with the findings of a study that suggested that increased Pel production enhanced cell-to-cell interactions as well as increased biomass of biofilms (4). This compactness might be the reason why that even when Psl is produced (PAO1Δalg8) elevated mushroom-like structures were not formed (Fig. 5). The increase in volume and height (Fig. 5) and the decrease in compactness (Fig. 3C and E) of biofilms produced by Pel-deficient mutants PAO1 ΔpelF and PAO1 ΔpelF Δalg8 compared to those produced by the parent strain provided further evidence that Pel contributes to the compactness of the biofilm. Unlike Pel, biofilms of PAO1 ΔpelF Δalg8 with Psl along with eDNA did not show such cell-to-cell interactions. This is in accordance with the findings of previous studies, which suggested that Psl and eDNA in a biofilm are not located in close vicinity (12).

Interestingly, the Psl-negative mutant showed more Pel production in static biofilms (Fig. 1) and the Pel-deficient mutant showed increased alginate production when it was grown on solid media (see Table S2 in the supplemental material) compared with the respective wild-type strains. This could be due to competition of the biosynthesis pathways of the Pel, Psl, and alginate with respect to metabolic precursors. Polymer biosynthesis pathways in P. aeruginosa had been suggested to be competitive with respect to common precursors (22, 24). However, the possibility that the absence of one or two exopolysaccharides impacts the regulation level of biosynthesis of the still produced polysaccharide or polysaccharides cannot be excluded.

To assess how the anti-sigma factor MucA impacts the ability to produce Pel and Psl at the transcriptional level, the activation of the pel and psl promoters was assessed in the PAO1 and PDO300 strains, respectively. This showed that the lack of MucA did not significantly impact the pel promoter activity but decreased the psl promoter activity by about 30%. It is known that a deficiency of MucA results in increased alginate production and transcriptional activation of the alginate operon (29). Here it seemed that this increase in alginate production and/or the unleashed AlgU had a negative effect on the transcriptional activity of the psl operon. This suggested some regulatory cross talk between the regulation of the biosynthesis of various exopolysaccharides. Further investigations are required to shed further light on regulation of polysaccharide biosynthesis.

Overall, this study showed that thick-structured biofilms are still formed by mutants producing one or two polysaccharides, except when only alginate is produced. Only when neither psl nor pel (PAO1 ΔpslA ΔpelF) was produced, cells were not able to form biofilms (Fig. 4G and H). Experimental evidence that the deficiency in one or two polysaccharides enhanced production of the remaining polysaccharides or polysaccharide, respectively, was provided (Fig. 1; see Table S2 in the supplemental material). These data shed new light on how the three polysaccharides and eDNA interactively contribute to P. aeruginosa biofilm formation and architecture.

Supplementary Material

[Supplemental material]

supp_77_15_5238__index.html^{(1.3KB, html)}

ACKNOWLEDGMENT

This study was supported by research grants to B.H.A.R. from Massey University. A.G. was supported by the Higher Education Commission of Pakistan.

We are grateful to Adrian Turner (School of Biological Sciences, University of Auckland) for his permission to use the IMARIS software.

Footnotes

^†

Supplemental material for this article may be found at http://aem.asm.org/.

^▿

Published ahead of print on 10 June 2011.

REFERENCES

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Supplemental material]

supp_77_15_5238__index.html^{(1.3KB, html)}

supp_77_15_5238__Supplemental_Material_revised.doc^{(5.8MB, doc)}

[B1] 1. Allesen-Holm M., et al. 2006. A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol. Microbiol. 59:1114–1128 [DOI] [PubMed] [Google Scholar]

[B2] 2. Campisano A., Schroeder C., Schemionek M., Overhage J., Rehm B. H. 2006. PslD is a secreted protein required for biofilm formation by Pseudomonas aeruginosa. Appl. Environ. Microbiol. 72:3066–3068 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Choi K. H., Kumar A., Schweizer H. P. 2006. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methods 64:391–397 [DOI] [PubMed] [Google Scholar]

[B4] 4. Colvin K. M., et al. 2011. The Pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa. PLoS Pathog. 7:e1001264. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5. Friedman L., Kolter R. 2004. Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms. Mol. Microbiol. 51:675–690 [DOI] [PubMed] [Google Scholar]

[B6] 6. Friedman L., Kolter R. 2004. Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix. J. Bacteriol. 186:4457–4465 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Hay I. D., Gatland K., Campisano A., Jordens J. Z., Rehm B. H. 2009. Impact of alginate overproduction on attachment and biofilm architecture of a supermucoid Pseudomonas aeruginosa strain. Appl. Environ. Microbiol. 75:6022–6025 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8. Hoang T. T., Karkhoff-Schweizer R. R., Kutchma A. J., Schweizer H. P. 1998. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86 [DOI] [PubMed] [Google Scholar]

[B9] 9. Hoiby N., Ciofu O., Bjarnsholt T. 2010. Pseudomonas aeruginosa biofilms in cystic fibrosis. Future Microbiol. 5:1663–1674 [DOI] [PubMed] [Google Scholar]

[B10] 10. Jackson K. D., Starkey M., Kremer S., Parsek M. R., Wozniak D. J. 2004. Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation. J. Bacteriol. 186:4466–4475 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Lee V. T., et al. 2007. A cyclic-di-GMP receptor required for bacterial exopolysaccharide production. Mol. Microbiol. 65:1474–1484 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Ma L., et al. 2009. Assembly and development of the Pseudomonas aeruginosa biofilm matrix. PLoS Pathog. 5:e1000354. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Ma L., Jackson K. D., Landry R. M., Parsek M. R., Wozniak D. J. 2006. Analysis of Pseudomonas aeruginosa conditional Psl variants reveals roles for the Psl polysaccharide in adhesion and maintaining biofilm structure postattachment. J. Bacteriol. 188:8213–8221 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14. Mathee K., et al. 1999. Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology 145(Pt 6):1349–1357 [DOI] [PubMed] [Google Scholar]

[B15] 15. Matsukawa M., Greenberg E. P. 2004. Putative exopolysaccharide synthesis genes influence Pseudomonas aeruginosa biofilm development. J. Bacteriol. 186:4449–4456 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Merritt J. H., Kadouri D. E., O'Toole G. A. 2005. Growing and analyzing static biofilms, p. 1B.1.1–1B1.7. In Coico R., Kowalik T., Quarles J., Stevenson B., Taylor R. (ed.), Current protocols in microbiology , vol. 1 John Wiley & Sons, Hoboken, NJ: [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17. Miller J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [Google Scholar]

[B18] 18. Mulcahy H., Charron-Mazenod L., Lewenza S. 2008. Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS Pathog. 4:e1000213. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Nivens D. E., Ohman D. E., Williams J., Franklin M. J. 2001. Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J. Bacteriol. 183:1047–1057 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20. O'Toole G., Kaplan H. B., Kolter R. 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54:49–79 [DOI] [PubMed] [Google Scholar]

[B21] 21. Overhage J., Schemionek M., Webb J. S., Rehm B. H. 2005. Expression of the psl operon in Pseudomonas aeruginosa PAO1 biofilms: PslA performs an essential function in biofilm formation. Appl. Environ. Microbiol. 71:4407–4413 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Pham T. H., Webb J. S., Rehm B. H. 2004. The role of polyhydroxyalkanoate biosynthesis by Pseudomonas aeruginosa in rhamnolipid and alginate production as well as stress tolerance and biofilm formation. Microbiology 150:3405–3413 [DOI] [PubMed] [Google Scholar]

[B23] 23. Pier G. B., Coleman F., Grout M., Franklin M., Ohman D. E. 2001. Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis. Infect. Immun. 69:1895–1901 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Rehm B. H. 2010. Bacterial polymers: biosynthesis, modifications and applications. Nat. Rev. Microbiol. 8:578–592 [DOI] [PubMed] [Google Scholar]

[B25] 25. Remminghorst U., Hay I. D., Rehm B. H. 2009. Molecular characterization of Alg8, a putative glycosyltransferase, involved in alginate polymerisation. J. Biotechnol. 140:176–183 [DOI] [PubMed] [Google Scholar]

[B26] 26. Remminghorst U., Rehm B. H. 2006. In vitro alginate polymerization and the functional role of Alg8 in alginate production by Pseudomonas aeruginosa. Appl. Environ. Microbiol. 72:298–305 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Ryder C., Byrd M., Wozniak D. J. 2007. Role of polysaccharides in Pseudomonas aeruginosa biofilm development. Curr. Opin. Microbiol. 10:644–648 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Sambrook J., Fritsch E. F., Maniatis T. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [Google Scholar]

[B29] 29. Schurr M. J., Yu H., Martinez-Salazar J. M., Boucher J. C., Deretic V. 1996. Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis. J. Bacteriol. 178:4997–5004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Schweizer H. P., Hoang T. T. 1995. An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15–22 [DOI] [PubMed] [Google Scholar]

[B31] 31. Spiers A. J., Bohannon J., Gehrig S. M., Rainey P. B. 2003. Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Mol. Microbiol. 50:15–27 [DOI] [PubMed] [Google Scholar]

[B32] 32. Vasseur P., Soscia C., Voulhoux R., Filloux A. 2007. PelC is a Pseudomonas aeruginosa outer membrane lipoprotein of the OMA family of proteins involved in exopolysaccharide transport. Biochimie 89:903–915 [DOI] [PubMed] [Google Scholar]

[B33] 33. Vasseur P., Vallet-Gely I., Soscia C., Genin S., Filloux A. 2005. The pel genes of the Pseudomonas aeruginosa PAK strain are involved at early and late stages of biofilm formation. Microbiology 151:985–997 [DOI] [PubMed] [Google Scholar]

[B34] 34. Whitchurch C. B., Tolker-Nielsen T., Ragas P. C., Mattick J. S. 2002. Extracellular DNA required for bacterial biofilm formation. Science 295:1487. [DOI] [PubMed] [Google Scholar]

PERMALINK

Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture ▿ †

Aamir Ghafoor

Iain D Hay

Bernd H A Rehm