Biofilms—communities of bacteria encased in a polymer-rich matrix—confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli, has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies.

Here, we report such a symbiotic role for filamentous phage in bacterial biofilm formation. Production of Pf phage by P. aeruginosa biofilms resulted in the spontaneous assembly of a highly ordered liquid crystalline matrix that enhanced biofilm function and, thereby, bacterial fitness.

Pf phage are involved in the progression of the P. aeruginosa biofilm life cycle by inducing cell death and the subsequent release of bacterial DNA (), a major component of the biofilm matrix (). However, the production of large amounts of Pf phage by P. aeruginosa biofilms—up to 10plaque-forming units (PFU)/ml—does not result in biofilm eradication (). Moreover, Pf phage production can be regulated by bacterial factors (). We hypothesized that Pf phage could serve a more symbiotic role within P. aeruginosa biofilms.

As P. aeruginosa biofilms develop, genes belonging to filamentous Pf1-like bacteriophage (Pf phage) are among the most highly transcribed (). Pf phage belong to the genus Inovirus which are long, negatively charged filaments about 2 μm in length and 6 to 7 nm in diameter. Their production by P. aeruginosa is stimulated in viscous environments () and under anaerobic growth conditions (), such as would be encountered within infected regions of CF lungs. Indeed, many CF clinical isolates carry Pf prophage (), including epidemic strains (). Laboratory strains of P. aeruginosa do as well, including PAO1 and PA14 (), which contain Pf4 and Pf5 prophage, respectively. Pf phage can also infect P. aeruginosa without integrating into the genome, as Pf1 does, whose host is P. aeruginosa strain K (PAK).

Pseudomonas aeruginosa is a major bacterial pathogen that causes about 10% of nosocomial infections and is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF) airway infections (). In CF, viscous secretions accumulate in the airways (), promoting chronic infection. The capacity of P. aeruginosa to establish chronic infections is dependent, in part, upon its ability to form biofilms—communities of bacteria encased in a polymer-rich matrix. Bacteria within biofilms display increased tolerance to antibiotics and desiccation, allowing them to persist in host tissues and on medical device surfaces ().

To differentiate the contributions of polymer concentration and liquid crystal assembly toward antibiotic killing, we compared mixtures of Pf4 and HMW-DNA (which form liquid crystals) against mixtures of LMW-DNA and Pf4 (which do not), as described for the desiccation experiments above. Mixtures containing liquid crystals inhibited the activity of tobramycin more so than isotropic mixtures of Pf4 and DNA ( Figure 7 A). Differential binding of tobramycin to HMW-DNA and LMW-DNA cannot explain these results ( Figure 7 A), suggesting that liquid crystal assembly further enhances tobramycin binding. Further, when mixtures of Pf4 and DNA were separated from buffer by a membrane, samples containing liquid crystals sequestered more tobramycin compared to isotropic samples ( Figure 7 B). To investigate sequestration further, we used a fluorescent Cy5-conjugated form of tobramycin to assess the binding of tobramycin to liquid crystals. Fluorescent imaging revealed that tobramycin was sequestered within liquid crystals ( Figure 7 C). The binding of microgram quantities of tobramycin did not affect the morphology of the liquid crystal, suggesting that the underlying molecular order was not severely disrupted. These results are consistent with the idea that liquid crystal assembly enhances the binding of aminoglycoside antibiotics, thereby mitigating bacterial killing.

To test this, increasing concentrations of tobramycin or ciprofloxacin were added to isotropic or liquid crystalline mixtures of Pf4 and DNA. After a 4-hr incubation to allow binding of antibiotics, E. coli was added and the minimum inhibitory concentration (MIC) was determined ( Figure 7 A). E. coli was used because it cannot be infected by Pf4 and to highlight the generality of the mechanism. Pf4 and DNA did not offer bacteria protection against ciprofloxacin, even in mixtures containing liquid crystals. In contrast, Pf4 and DNA did provide bacteria protection against tobramycin, consistent with the binding of tobramycin to polyanions.

(C) Binding of fluorescent tobramycin (Cy5-tobramycin, 40 μg/ml) to isotropic and liquid crystalline phases of Pf4 (10 10 PFU/ml) and HMW-DNA (2.5 mg/ml) was visualized by fluorescent microscopy. Scale bars, 20 μm.

(B) Tobramycin (200 μg/ml) was mixed with the indicated amounts of Pf4 and DNA (2.5 mg/ml) and placed into a dialysis cassette. The diffusion of unbound tobramycin across the membrane was monitored by HPLC-MS. Results were normalized to controls containing no Pf4 or DNA. Results are mean ± SD of duplicate experiments.

(A) Antibiotic killing in the presence of DNA and Pf4 was investigated. Increasing concentrations of tobramycin or ciprofloxacin were added to isotropic or liquid-crystal-containing mixtures of Pf4 (10 10 PFU/ml) and DNA (2.5 mg/ml). The arrow indicates the only sample with liquid crystals. E. coli was then added and samples were incubated overnight. The MIC for each antibiotic was plotted. Results are mean ± SD of three experiments.

Aminoglycosides are cationic and, hence, are bound by polyanions like DNA in the biofilm matrix () and at sites of infection (), reducing their efficacy. In contrast, ciprofloxacin does not interact electrostatically with polyanions and readily penetrates P. aeruginosa biofilms (). Given that biofilms with liquid crystalline matrices were more tolerant to aminoglycosides, we hypothesized that liquid crystal assembly might promote binding and sequestration of aminoglycosides in ways that prevent bacterial killing.

To address the possibility that tolerance to aminoglycosides was due to physiological changes induced by Pf4 or undefined genetic mutations in the SCV strain, we again used the Pf phage-null strain ΔPA0728/pilA. Planktonic ΔPA0728/pilA were added to isotropic or liquid crystalline mixtures of Pf4 and DNA containing tobramycin. Mixtures containing liquid crystals offered P. aeruginosa the most protection ( Figure 6 C) even though equivalent amounts of DNA and phage from the same stocks were present in both isotropic and liquid crystal-containing samples. Strains containing deletions of pilA, PA0728, or the double mutant ΔPA0728/pilA were equally sensitive to tobramycin, showing that these genes do not influence antibiotic tolerance ( Figure S7 B). Further, when Pf4 was replaced with the filamentous phage fd, similar trends were observed ( Figures S7 C and S7D). Together, these results indicate that liquid crystal formation by filamentous phage promote tolerance to aminoglycosides such as tobramycin.

Antibiotic tolerance is a hallmark of bacterial biofilms (). Hence, we investigated if the liquid crystalline organization of biofilm matrix might further increase antibiotic tolerance in P. aeruginosa biofilms. Indeed, relative to biofilms not organized into a liquid crystal, bacteria within liquid crystalline biofilms were more tolerant to aminoglycoside antibiotics but were equally sensitive to the fluoroquinolone ciprofloxacin ( Figures 6 A, 6B, and S7 A).

(C) To investigate whether or not Pf phage mediate tolerance to tobramycin through an extracellular mechanism, planktonic ΔPA0728/pilA, which cannot produce nor be infected by Pf4, was suspended in isotropic or liquid crystalline phases of Pf4 (10 10 PFU/ml) and DNA (2.5 mg/ml). Killing is represented as the log 10 reduction of viable cells recovered from cultures treated with tobramycin (10 μg/ml, 90 min) compared to untreated controls. Results are mean ± SD of three experiments.

(A and B) CFUs recovered from colony biofilms after treatment with increasing concentrations of (A) tobramycin or (B) ciprofloxacin are plotted. Results are mean ± SD of three experiments.

To investigate the impact of liquid crystal assembly on evaporation independent of polymer concentration, the HMW-DNA in liquid crystalline mixtures was replaced with LMW-DNA. The resulting mixtures contained the same concentrations of Pf4 and DNA, but since LMW-DNA did not promote liquid crystal assembly at the concentrations tested ( Figure 1 D, inset), the mixtures remained isotropic. These isotropic mixtures evaporated faster than liquid crystalline mixtures containing the same concentrations of DNA and Pf4, indicating that liquid crystal assembly, rather than high polymer concentration, promotes water retention ( Figure 5 C). Consistent with this conclusion, when biofilms with liquid crystalline matrices were desiccated, they retained more water (wet weight) and displayed increased desiccation tolerance compared to biofilms lacking liquid crystalline order ( Figures 5 D and 5E). Together, these results suggest that the assembly of the liquid crystalline biofilm matrix enhances water retention, increasing tolerance to desiccation.

To study water retention in mixtures of polymers and phage in the isotropic (unordered) or liquid crystalline phases, we measured evaporation rates using a plate reader. Samples were placed into a 96-well plate and incubated uncovered in a plate reader at 37C. Liquid crystals assembled from Pf4 and DNA evaporated at a slower rate compared to Pf4 or DNA alone ( Figure 5 C), indicating that the liquid crystalline phase retains water more efficiently than polymers in the isotropic phase.

The biofilm matrix protects bacteria against desiccation, promoting their survival (). Given that liquid crystals are inherently viscous () and viscous solutions generally display reduced rates of evaporation, we hypothesized that the liquid crystalline biofilm matrix might increase water retention and, in turn, augment desiccation tolerance.

We then asked how Pf4 and the liquid crystalline matrix contribute to biofilm functionality. One canonical feature of biofilms is their ability to adhere to surfaces. We measured the adhesiveness of biofilms with a liquid crystalline matrix and found that the addition of Pf4 promoted biofilm adhesion in PAO1 and ΔPA0728 ( Figure 5 A). Adhesion is also increased when Pf4 is supplied to PAK ( Figure 5 A), which cannot be infected by Pf4 ( Figure S5 E). To rule out infection-mediated effects on adhesion, we repeated these studies using a strain of P. aeruginosa (ΔPA0728/pilA) that can neither produce nor be infected by Pf4 ( Figures S5 E–S5G). This strain lacks PA0728 and type IV pili (pilA), the receptor that Pf4 use to infect P. aeruginosa (). To further exclude any possibility of infection, fd phage from E. coli were used instead of Pf phage. Using this system, we again find that phage addition promotes adhesion, as measured by an increase in adherent biomass ( Figure 5 B). Together with previous work implicating Pf phage in P. aeruginosa adhesion (), these data indicate that filamentous phage make structural contributions to biofilms that increase adhesion.

(E) Killing by desiccation is represented as the log 10 reduction of viable cells recovered from control biofilms compared to desiccated biofilms. Results are mean ± SD of three experiments.

(D) Colony biofilms formed from the indicated strains were desiccated for 18 hr at 37 ° C in an ambient incubator. The percent water loss was measured as the wet weight post-desiccation/wet weight pre-desiccation. Results are mean ± SD of six experiments for rough and SCV biofilms and three experiments for ΔPA0728 ± Pf4 biofilms.

(C) Evaporation of isotropic and liquid crystalline phases of Pf4 and DNA was monitored by absorbance (Abs, 600 nm). Absorbance was normalized to initial readings for each sample. As samples dried, the absorbance increased and stabilized once dried to completeness. In this way, the retention of water was monitored over time. Results are mean of three experiments; error bars are omitted for clarity.

(B) Adhesion of biofilms formed by strain ΔPA0728/pilA in the setting of increasing concentrations of fd phage. Results are mean ± SD of three experiments.

(A) Biofilm adhesion after 24 hr of growth as measured by the crystal violet adhesion assay. Results are mean ± SD of three experiments.

Taken together, these data are consistent with the model that, in the absence of sufficient amounts of multivalent cations, Pf phage assemble the P. aeruginosa biofilm matrix into a highly ordered liquid crystalline structure.

Given that growth in a biofilm can produce genetic diversity (), the SCV strain used in our studies could contain mutations that promote other mechanisms of liquid crystal formation in the biofilm matrix. As a control, birefringence was measured in colony biofilms formed by P. aeruginosa ΔPA0728, which cannot produce Pf4 (). Birefringence in this strain was low, similar to that of wild-type “rough” P. aeruginosa, indicating that the low amounts of Pf4 produced by rough wild-type P. aeruginosa are insufficient to promote liquid crystal assembly. Supplementation of ΔPA0728 with Pf4 produced colony biofilms with a smooth morphology similar to SCVs and increased birefringence ( Figure 4 C). Similar results were obtained with PAK and its Pf phage, Pf1 ( Figure S6 A). We also observed increased birefringence in flowcell biofilms that correlated with Pf4 production ( Figures S6 B–S6D), indicating that Pf-dependent matrix organization can occur under diverse growth conditions. Conversely, when grown on agar supplemented with Ca, the birefringence of SCV colony biofilms was reduced ( Figure S6 E), showing that divalent cations disrupt the liquid crystalline order of the biofilm matrix, likely through crosslinking interactions.

Many biological structures, due to their ordered nature, are birefringent. For example, lipid membranes, cell walls, and polymers can be birefringent, so long as they display sufficient order (). This could explain the basal levels of birefringence in biofilms producing little or no phage ( Figure 4 C). Removal of the matrix by washing of the bacterial cells resulted in a substantial decrease in birefringence ( Figure 4 C), illustrating that the liquid crystalline nature of the biofilm was manifested primarily from the extracellular matrix and not the bacterial cells.

In light of these findings, we hypothesized that Pf phage might promote similar liquid crystalline organization of the P. aeruginosa biofilm matrix. To test this, we analyzed colony biofilms formed from P. aeruginosa PAO1 small colony variants (SCVs). SCVs arise spontaneously within P. aeruginosa biofilm communities and produce abundant amounts of Pf4 (). SCVs display enhanced adherence and antibiotic tolerance, properties that are often implicated in intractable CF airway infections (). Compared to the larger wild-type colony biofilms with rough edges ( Figure S5 A), SCVs produced ∼5 × 10-fold more endogenous Pf4 ( Figure 4 A). When subcultured, SCVs stopped producing Pf4 and reverted back to the rough morphology ( Figure S5 B), consistent with a role for Pf4 in producing the SCV phenotype (). SCVs were intensely birefringent while rough colonies were minimally so ( Figures 4 B and 4C). The extended birefringence of SCV biofilms could be visualized by rotating the biofilm between crossed polarizing lenses ( Figure 4 D; Movies S2 and S3 ). These data are consistent with a role for Pf phage in the organization of bacterial colonies into liquid crystals.

(D) Representative images for SVC and “rough” colony biofilms (placed between glass plates) visualized through crossed polarizing lenses. Birefringence is visualized as bright areas where light passes through both polarizing lenses. The birefringence patterns change when the sample is rotated with respect to the polarizing lenses, revealing extended areas of birefringence.

(C) Birefringence (|sin(δ)|) of the indicated colony biofilms was quantified after normalizing for sample thickness. Birefringence was again measured after washing of the bacteria to remove the extracellular matrix. Results are mean ± SD of four experiments.

(A) Pf4 production by colony biofilms formed from the indicated strains were enumerated as PFU/ml. Adjusted total Pf phage content are also plotted on the right axis, see text and Figure S4 A. Results are mean ± SD of three experiments.

We also investigated the functional significance of liquid crystal assembly by Pf phage in sputum polymers. Given that liquid crystals are inherently viscous () and impaired clearance of viscous airway secretions contributes to airway obstruction in CF (), we hypothesized that the addition of Pf4 would increase the viscosity of mucin + DNA mixtures. To test this, viscosity was measured with a capillary viscometer. Indeed, the viscosity of mucin + DNA mixtures increased with the addition of Pf4 ( Figure 3 E), demonstrating that filamentous phage enhance the viscosity of disease relevant polymers. While crosslinking would also increase viscosity, the associated increase in birefringence implicates liquid crystal formation in the increased viscosity of these disease relevant polymer mixtures.

We then asked if Pf phage conferred liquid crystalline properties to CF sputum. Sputum from Pf-positive patients was more birefringent than sputum from Pf-negative patients ( Figure 3 D). Supplementation of Pf-negative sputum with Pf4 made these samples more birefringent. It is notable that sputum samples containing Pf phage displayed increased birefringence, despite the fact that the average Pf phage content in these samples (∼10Pf phage/ml, Figure 3 C) was lower than what was required in vitro to assemble liquid crystals in DNA (10Pf phage/ml; Figure 2 C) or mixtures of mucin and DNA (10Pf phage/ml; Figure 3 B). We speculate that in addition to DNA and mucin, multiple polymers present in CF sputum (e.g., F-actin, alginate, and HA) might provide a sufficiently crowded environment at high enough ionic strength to allow the assembly of liquid crystals from lower concentrations of Pf phage. Moreover, there may be phage variants within CF sputum not detected by our qPCR assay, as the mutation rate for Pf phage is quite high ().

To determine if Pf phage might play a similar role in clinical samples, we quantified Pf phage in CF sputum. Because plaques could be generated by other bacteriophage in sputum, we developed a specific qPCR method to detect intact Pf phage in CF sputum, as detailed in the Experimental Procedures section. Quantitation of purified Pf4 by qPCR produced similar results to enumeration by PFU ( Figure S4 B). All CF patients colonized with P. aeruginosa were positive for Pf phage ( Figure 3 C). Conversely, Pf phage were not detected in sputum collected from patients not infected with P. aeruginosa.

To assess if Pf phage promote liquid crystal formation under physiologically relevant conditions, we added Pf4 to disease-relevant concentrations of mucin (8% solids) mixed with DNA (HMW, 4 mg/ml) (). Supplementation of this host polymer mixture with Pf4 resulted in a dose-dependent increase in birefringence at phage concentrations ≥10PFU/ml, with filamentous structures forming at the highest Pf4 concentrations ( Figures 3 A and 3B ). Of note, this was lower than the concentration of phage required to form liquid crystals in 4 mg/ml of DNA alone (10PFU/ml; Figure 2 C), suggesting that the presence of multiple polymers amplified the depletion force, allowing lower concentrations of phage to assemble into liquid crystals.

(E) Changes in the viscosity (mPa⋅s) of mucin + DNA samples described in (A) were measured after supplementation with Pf4. Results are mean ± SD of four experiments.

(D) The birefringence (|sin(δ)|) of sputum samples described in (C) was quantified. The addition of 10 8 PFU/ml Pf4 to P. a. (−) sputum augmented birefringence. Results are mean ± SD.

(C) Pf phage were quantified by qPCR in sputum collected from CF patients infected by P. aeruginosa (P. a. (+), n = 10) or patients not infected by P. aeruginosa (P. a. (−), n = 5). Results are plotted as the geometric mean; nd, not detected.

(B) Birefringence was quantified as |sin(δ)| in mixtures of mucin and DNA described in (A) supplemented with increasing amounts of Pf4. Results are mean ± SD of three experiments.

(A) Representative images of mixtures of mucin (8% solids) and DNA (HMW, 4 mg/ml) in the presence or absence of Pf4. Transmitted light is displayed as I/I o where I represents intensity of light emerging from the sample and I o represents intensity of incident light. Birefringence is displayed as |sin(δ)|. Arrows indicate filament assembly. Scale bars, 200 μm.

To understand how Pf phage numbers relate to liquid crystal formation, we first needed accurate methods to quantify phage. Phage are typically enumerated by PFU. However, PFU enumeration might be expected to underestimate phage numbers, either because multiple phage could produce a single PFU or because non-infectious phage particles cannot be detected by PFU enumeration. For this reason, we also measured total phage content using spectroscopy (). This revealed that PFU enumeration did indeed underestimate total Pf phage content ( Figure S4 A), suggesting a large amount of non-infectious phage particles were present in our samples. Therefore, results involving Pf4 quantitation are presented in both PFU/ml (to facilitate comparison to the existing microbiology literature) and adjusted total phage content in mg/ml (calculated from the standard curve in Figure S4 A).

We also considered an alternative model where, rather than liquid crystals, Pf phage and polymers crosslink into a birefringent gel. However, if this were the case, we would expect a positive relationship between phage concentration, polymer concentration, and structure formation, as more polymer would be required to crosslink increasing amounts of phage. Instead, we find a negative relationship between these variables and liquid crystal formation (dashed lines in Figures 2 B–2D), consistent with depletion attraction. To test the impact of crosslinking on birefringence, we added calcium (Ca), which can crosslink polyanions like Pf phage (), to mixtures of Pf4 and HA. We found that the tactoidal morphology was disrupted by Caand replaced by large aggregates ( Figures S2 D and S2E). These aggregates remained intact after dilution with water ( Figure S2 F), consistent with irreversible crosslinking (). Crosslinking also decreased the birefringence of these structures ( Figure S3 ), indicating that the ordered, liquid crystalline structure was disrupted. These data indicate that divalent cations can bind and crosslink Pf phage and polymers, but depletion attraction was likely responsible for the birefringent structures we observed. However, crosslinking of polymers and Pf phage may be relevant to biofilm formation in particular contexts, such as settings with high concentrations of multivalent cations. While the physics of the interactions between filamentous phage and polymers are well established (), their relevance to microbiology, biofilms, and disease is unknown.

Using this analytical tool, we found that, in monovalent buffer, neither Pf4 nor polymer solutions (alginate, DNA, HA, and PEG, used for phage purification) alone were birefringent. A mixture of Pf4 and these polymers, however, resulted in the assembly of intensely birefringent structures ( Figure 2 A), indicating that Pf phage are necessary for the observed birefringence. Increasing the concentration of either polymer or Pf4 promotes the assembly of these structures, as indicated in the phase diagrams in Figures 2 B–2D. This is consistent with organization via depletion attraction; as polymer concentration increases, the system becomes increasingly crowded, allowing lower concentrations of phage to assemble into liquid crystals ().

(B–D) Phase diagrams showing the relationship between polymer concentration, Pf4 concentration, and liquid crystal assembly. Note the negative slope of the phase boundary (dotted lines), indicative of depletion attraction. See text for details.

(A) The birefringence of the indicated polymer (all 10 mg/ml) were quantified (|sin(δ)|) alone or in the presence of Pf4 (mixed 1:1 with 1 × 10 10 PFU/ml). Scale bars, 10 μm.

To quantify birefringence, we used an optical imaging system developed for birefringent media called Rotopol (). This device quantitates the phase difference of the polarized light beams emerging from the liquid crystal (i.e., the optical anisotropy) as |sin(δ)| where δ = 2πΔnL/λ, Δn represents birefringence, L represents optical path, and λ represents wavelength (550 nm). A computer-driven rotatable polarizer probes the light intensities of incoming circularly polarized light as it is changed by the birefringence of the sample.

One prediction of this model is that the higher order structures we observed in mixtures of filamentous phage and polymers in monovalent solutions are liquid crystals. To test this, we assessed their optical properties. Liquid crystals are birefringent; they split light into two beams with perpendicular polarization, a consequence of their highly ordered state. Therefore, birefringence is a direct measurement of the molecular alignment of the sample.

Taken together, these observations are consistent with the spontaneous self-assembly of filamentous phage and microbial or host polymers into higher order structures by depletion attraction. Depletion attraction is a cohesive force that operates between crowded, like-charged elements in environments where sufficient ionic strength exists to screen their repulsive forces, thus allowing tightly packed structures to assemble (). When filamentous particles, like phage, are confined to such structures, they spontaneously align and form liquid crystals with tactoidal morphology (). Polymer size and concentration are proportional to the range and magnitude of depletion attraction, respectively (), with larger and/or more concentrated polymers favoring the assembly of liquid crystals from lower concentrations of filamentous phage (). The disassembly of these structures upon dilution is also characteristic of depletion attraction ().

The formation of these structures was also dependent upon polymer size and concentration. High molecular weight (HMW) (∼2 kb) DNA of similar length to those found in CF sputum () form structures with Pf4 ( Figure 1 D) while low molecular weight (LMW) (<0.3 kb) DNA did not ( Figure 1 D, inset). Similar results were seen with HA, where digested HA fragments did not support structure formation ( Figures S1 H and S1I).

We next asked what environmental factors influenced the formation of these phage-mediated structures. We found that structure formation was dependent upon ionic strength; they did not efficiently assemble in deionized water and were more condensed and extended in monovalent buffers of increasing tonicity ( Figures 1 G–1I). Raman spectroscopy revealed that HA tightly interacts with Pf4, transforming its secondary structure by suppressing the vibrational freedom of amide groups on Pf4 coat proteins ( Figure S2 A). This suppression was lost when monovalent salts were washed out, indicating HA was not as tightly associated with Pf4 at low ionic strength ( Figure S2 B). We also found that the tactoidal structures formed in monovalent solutions dissociated when diluted with water ( Figure S2 C), indicating the assembly of these structures is reversible.

Proteomic analysis demonstrated that CoaB, the major coat protein of the filamentous bacteriophage Pf4, was abundant in P. aeruginosa supernatants. Thus, we assessed if Pf phage were the factor responsible for the assembly of these structures. Addition of HA to a biofilm formed by a strain of P. aeruginosa lacking the Pf phage integrase (ΔPA0728), a gene essential for Pf4 production () ( Figures S5 C and S5D), did not promote structure formation ( Figures S1 F and S1G). Conversely, purified Pf4 mixed with HA rapidly (∼1–5 min) formed similar structures ( Movie S1 ). These structures could be destroyed with HA’ase treatment ( Figures S1 H and S1I). Of note, similar structures were observed whether Pf4 phage were purified using PEG precipitation or a cesium chloride gradient method ( Figure S1 J). Similar structures were also seen upon the mixing of Pf4 with DNA, alginate, HA, or a broad, chemically diverse range of microbial and host polymers, including human serum ( Figures 1 D–1F and S1 K–S1Q). Additionally, the filamentous phage fd, which infects Escherichia coli, formed similar structures when mixed with HA or DNA ( Figures S1 R and S1S). These observations show that filamentous phage derived from different bacterial species interact with diverse polymers to rapidly assemble higher order structures.

While performing unrelated experiments that involved supplementing cultures of P. aeruginosa with hyaluronan (HA), a host glycosaminoglycan present in CF airways and abundant in inflamed tissues (), we observed the formation of morphologically complex biofilms composed of interlaced structures ( Figures 1 A and 1B ). The addition of hyaluronidase (HA’ase) dissolved these structures ( Figures S1 A and S1B), indicating that HA was essential for their formation. When filtered biofilm supernatants were mixed with HA, DNA, or alginate (a microbial polymer important in CF), similar interwoven structures assembled ( Figures 1 C and S1 C–S1E), suggesting that P. aeruginosa biofilms produce an extracellular factor that interacts with polymers to assemble well-organized structures.

(C) The addition of P. aeruginosa biofilm supernatant to HA (5 mg/ml) results in the spontaneous formation of higher order structures.

Discussion

The production of filamentous Pf phage by P. aeruginosa biofilms is associated with the organization of the matrix into a liquid crystalline structure that promotes the fundamental, pathogenic features of biofilms, including adhesion and tolerance to desiccation and antibiotics.

These findings are particularly relevant to the pathophysiology of CF, where the high viscosity and polymer content of airway secretions are thought to contribute potently to disease symptoms. Here, we show that Pf phage enhance the viscosity of polymers abundant in CF airway secretions. Although an increase in birefringence was correlated with an increase in viscosity, crosslinking interactions could also be operating between Pf phage and components of these complex polymer mixtures, contributing to viscosity.

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et al. Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa. Pf phage may also promote the transmissibility of P. aeruginosa. The transmission of P. aeruginosa from one CF patient to another can occur through aerosols or contaminated surfaces, and desiccation tolerance is thought to be critical to transmission (). Our results suggest that the liquid crystal organization of the matrix slows the evaporation of water, allowing biofilms to better tolerate desiccation. This property alone could have implications on the transmissibility of P. aeruginosa between CF patients, particularly in epidemic strains that contain Pf prophage, such as the Liverpool epidemic strain ().

Wnorowska et al., 2015 Wnorowska U.

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Kjelleberg S. The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage. The liquid crystalline biofilm matrix also provides P. aeruginosa protection against aminoglycoside antibiotics. Given that tobramycin accumulates within liquid crystals composed of Pf phage, the increased tolerance of P. aeruginosa biofilms to these antibiotics may be mediated by enhanced binding to the liquid crystalline matrix. Binding is perhaps facilitated by the highly ordered nature of these liquid crystals; they are densely packed with filamentous phage, so much so that they change the polarization of light. Thus, the negative charge density within these liquid crystals would be high, attracting cationic antibiotics. In addition to aminoglycosides, liquid crystals composed of Pf phage might be expected to efficiently bind other cationic antimicrobials important in airway defense. For example, cationic antimicrobial peptides bind to Pf phage through ionic interactions (). Together, our data on adhesion, sputum polymer viscosity, and antibiotic tolerance suggest that Pf phage contribute to the persistence of P. aeruginosa biofilm infections and may help explain how Pf phage influence P. aeruginosa virulence in vivo ().

Rice et al., 2009 Rice S.A.

Tan C.H.

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Kung V.

Woo J.

Tay M.

Hauser A.

McDougald D.

Webb J.S.

Kjelleberg S. The biofilm life cycle and virulence of Pseudomonas aeruginosa are dependent on a filamentous prophage. In addition to influencing persistence phenotypes, Pf phage are important to the progression of the biofilm lifecycle by inducing cell lysis (at least in a subset of the bacterial population), resulting in the accumulation of extracellular DNA in the biofilm matrix (). The concurrent increase in both DNA and phage concentrations would favor the organization of the biofilm matrix into a liquid crystalline structure. In addition to P. aeruginosa, filamentous phage of the genus Inovirus are produced by many Gram-negative bacterial species, including pathogens such as E. coli, Stenotrophomonas maltophilia, Vibrio cholerae, and others. Given that the E. coli phage fd likewise assembled liquid crystals, we postulate that filamentous phage may contribute to other chronic infections by affecting biofilm structure and function.

Finally, our data point toward potential strategies to prevent or treat biofilm infections by targeting filamentous phage and the liquid crystal organization of the biofilm matrix. Perturbing the soft matter physics of the biofilm matrix might increase the susceptibility of bacterial biofilms to host defenses or existing therapeutics.