WO2016014575A1 - Organization of polymers by inovirus bacteriophage - Google Patents

Abstract

Described herein is a method for organizing polymers, comprising mixing a first composition comprising purified Inovirus bacteriophage (e.g., Pf-family bacteriophage) with a second composition comprising one or more polymers. Also described is a liquid crystal comprising Inovirus bacteriophage (e.g., Pf-family bacteriophage) and one or more polymers, wherein the liquid crystal is substantially free of any bacterial strain (e.g., Pseudomonas aeruginosa strain) capable of producing Inovirus bacteriophage.

Description

ORGANIZATION OF POLYMERS BY INOVIRUS BACTERIOPHAGE CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/027,698 filed July 22, 2014, the content of which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with Government support under contract HL007287 awarded by the National Institutes of Health. The Government has certain rights in this invention. BACKGROUND [0003] Liquid crystals have properties between a liquid and a solid crystal, and often comprise molecules that are organized in a highly ordered, crystalline way but flow like a liquid. A need exists for an effective and efficient method for forming liquid crystals from a variety of polymer precursors. SUMMARY [0004] Many embodiments described herein relate to a method for organizing polymers, comprising mixing a first composition containing bacteriophage belonging to the Inovirus family of viruses with a second composition comprising one or more polymers. [0005] In some embodiments, the Inovirus bacteriophage are Enterobacteria phage. In some embodiments, the Inovirus bacteriophage are Pseudomonas phage. In some embodiments, the Inovirus bacteriophage are Ralstonia phage. In some embodiments, the Inovirus bacteriophage are Vibrio phage. In some embodiments, the Inovirus bacteriophage are Propionibacterium phage. In some embodiments, the Inovirus bacteriophage are Stenotrophomonas phage. In some embodiments, the Inovirus bacteriophage are

Xanthomonas phage. [0006] In some embodiments, the Inovirus bacteriophage are selected from Enterobacteria phage I2-2, Enterobacteria phage If1, Enterobacteria phage Ike, Enterobacteria phage M13, Enterobacteria phage fd, Enterobacteria phage f1, Propionibacterium phage B5, Pseudomonas phage Pf1, Pseudomonas phage Pf3, Pseudomonas phage Pf4, Pseudomonas phage Pf5, Ralstonia phage p12J, Ralstonia phage PE226, Ralstonia phage RSM1, Ralstonia phage RSM3, Ralstonia phage RSS0, Ralstonia phage RSS1, Stenotrophomonas phage phiSMA9, Vibrio cholerae phage KSF-1phi, Vibrio cholerae phage VGJphi, Vibrio parahaemolyticus phage VfO4K68, Vibrio parahaemolyticus Bacteriophage VfO4K68, Vibrio

parahaemolyticus phage VfO3K6, Vibrio phage CTX, Vibrio phage fs1, Vibrio phage fs2, Vibrio phage VCY-phi, Vibrio phage VEJphi, Vibrio phage Vf12, Vibrio phage Vf33, Vibrio phage VSK, Xanthomonas phage Cf1c, variants or modified bacteriophages thereof, and related bacteriophages. [0007] In some embodiments, the Inovirus bacteriophage are Pf-family bacteriophage. In some embodiments, the Pf-family bacteriophage are Pf4 bacteriophage, Pf5 bacteriophage, or Pf1 bacteriophage. [0008] In some embodiments, the Pf-family bacteriophage are Pf4 bacteriophage produced by P. aeruginosa strain PAO1, Pf5 bacteriophage produced by P. aeruginosa strain PA14, or Pf1 bacteriophage produced by P. aeruginosa strain PAK. [0009] In some embodiments, the polymer is a biological polymer or synthetic polymer. [0010] In some embodiments, the polymer is a biological polymer selected from the group consisting of DNA, alginate, hyaluronan, collagen, heparin, heparin sulfate, fibronectin, chondroitin sulfate, perlican, and other polymers present in human serum or sputum. In some embodiments, the polymer is a biological polymer selected from the group consisting of cellulose, chitosan, chitin, lignins, polycaprolactones, polyesteramides, polylactids, elastin, keratin, myosin, actin, polyhydroxyalkanoates, wool, albumin, rubber, and other protein- based polymers and polysaccharides. [0011] In some embodiments, the polymer is a synthetic polymer selected from the group consisting of chitosan, dextran, dextran sulfate, polyacrylic acid, polyethylene glycol, polystyrene sulfonate, and polyethylene glycol diacrylate. In some embodiments, the polymer is a synthetic polymer different from, or substantially free of, dextran. In some embodiments, the polymer is a synthetic polymer selected from the group consisting of low- density polyethylene (LDPE), high-density polyethylene (HDPE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), nylon, nylon 6, nylon 6,6, thermoplastic polyurethanes (TPU), polytetrafluoroethylene, polyvinylchloride, polyesters, polyanhidrides, polychlorotrifluoroethylene, Kevlar, Neoprene, Teflon, Zylon, Viton, Ultem, Orlon, Mylar, silk, polyacetylene, polyphenylene vinylene, polypyrrole, polythiophene, polyaniline, and polyphenylene sulfide. [0012] In some embodiments, the weight ratio between the Inovirus bacteriophage (e.g., Pf- family bacteriophage) to the polymer is about 1:1,000,000 to about 100:1, or about 1:100,000 to about 100:1, or about 1:10,000 to about 100:1, or about 1:1,000 to about 100:1, about 1:500 to about 50:1, or about 1:200 to about 20:1, or about 1:100 to about 10:1, or about 1:50 to about 5:1, or about 1:20 to about 2:1, or about 1:10 to about 1:1, or about 1:5 to about 1:2. [0013] In some embodiments, the Inovirus bacteriophage have an average length of, for example, at least about 700 nm, or at least about 1,000 nm, or at least about 1,500 nm, or at least about 2,000 nm, or at least about 2,500 nm, or at least about 3,000 nm, or at least about 3,400 nm. In some embodiments, the Inovirus bacteriophage (e.g., Pf-family bacteriophage) comprises Pf4 having an average length of, for example, at least about 3,400 nm (about 12,437 bp genome size). In some embodiments, the Inovirus bacteriophage (e.g., Pf-family bacteriophage) comprises Pf1 having an average length of, for example, at least about 2,000 nm (about 7,349 bp genome size). [0014] In some embodiments, the Inovirus bacteriophage (e.g., Pf-family bacteriophage) has an average linear charge density of, for example, at least about 5 e/nm, or at least about 8 e/nm, or at least about 10 e/nm, or at least about 12 e/nm. [0015] In some embodiments, the method further comprises obtaining a liquid crystal comprising the Inovirus bacteriophage (e.g., Pf-family bacteriophage) and the polymer. In some embodiments, the liquid crystal has a birefringence |sin(δ)| of at least about 0.3, or at least about 0.4, or at least about 0.5, or at least about 0.6. [0016] Further embodiments described herein relate to a liquid crystal formed by the method described herein. These liquid crystals can take the form of coatings, films, nano- wires, and filaments. [0017] Additional embodiments described herein relate to a liquid crystal comprising Inovirus bacteriophage (e.g., Pf-family bacteriophage) and one or more polymers, wherein the liquid crystal is substantially free of any bacterial strain (e.g., P. aeruginosa strain) capable of producing the Inovirus bacteriophage. In some embodiments, the liquid crystal is free of any living bacteria. In some embodiments, the liquid crystal comprises less than about 5 wt.% or less than about 5 vol.% of bacterial contaminants. [0018] In some embodiments, the liquid crystal is formed by Enterobacteria phage. In some embodiments, the liquid crystal is formed by Pseudomonas phage. In some

embodiments, the liquid crystal is formed by Ralstonia phage. In some embodiments, the liquid crystal is formed by Vibrio phage. In some embodiments, the liquid crystal is formed by Propionibacterium phage. In some embodiments, the liquid crystal is formed by

Stenotrophomonas phage. In some embodiments, the liquid crystal is formed by

Xanthomonas phage. [0019] In some embodiments, the Inovirus bacteriophage are selected from Enterobacteria phage I2-2, Enterobacteria phage If1, Enterobacteria phage Ike, Enterobacteria phage M13, Enterobacteria phage fd, Enterobacteria phage f1, Propionibacterium phage B5, Pseudomonas phage Pf1, Pseudomonas phage Pf3, Pseudomonas phage Pf4, Pseudomonas phage Pf5, Ralstonia phage p12J, Ralstonia phage PE226, Ralstonia phage RSM1, Ralstonia phage RSM3, Ralstonia phage RSS0, Ralstonia phage RSS1, Stenotrophomonas phage phiSMA9, Vibrio cholerae phage KSF-1phi, Vibrio cholerae phage VGJphi, Vibrio parahaemolyticus phage VfO4K68, Vibrio parahaemolyticus Bacteriophage VfO4K68, Vibrio

parahaemolyticus phage VfO3K6, Vibrio phage CTX, Vibrio phage fs1, Vibrio phage fs2, Vibrio phage VCY-phi, Vibrio phage VEJphi, Vibrio phage Vf12, Vibrio phage Vf33, Vibrio phage VSK, Xanthomonas phage Cf1c, variants or modified bacteriophages thereof, and related bacteriophages. [0020] In some embodiments, the liquid crystal is formed by Pf-family bacteriophage. In some embodiments, the liquid crystal is formed by Pf4 bacteriophage, Pf5 bacteriophage, or Pf1 bacteriophage. [0021] In some embodiments, the liquid crystal is formed by a biological polymer or synthetic polymer. In some embodiments, the weight ratio between the Inovirus

bacteriophage (e.g., Pf-family bacteriophage) to the polymer is about 1:1,000,000 to about 100:1, or about 1:100,000 to about 100:1, or about 1:10,000 to about 100:1, or about 1:1,000 to about 100:1, or about 1:500 to about 50:1, or about 1:200 to about 20:1, or about 1:100 to about 10:1, or about 1:50 to about 5:1, or about 1:20 to about 2:1, or about 1:10 to about 1:1, or about 1:5 to about 1:2. [0022] In some embodiments, the liquid crystal is formed by Inovirus bacteriophage having an average length of, for example, at least about 700 nm, or at least about 1,000 nm, or at least about 1,500 nm, or at least about 2,000 nm. In some embodiments, the liquid crystal is formed by Inovirus bacteriophage having an average length of, for example, at least about 2,000 nm, or at least about 2,500 nm, or at least about 3,000 nm, or at least about 3,400 nm. In some embodiments, the liquid crystal is formed by Inovirus bacteriophage (e.g., Pf-family bacteriophage) having an average linear charge density of at least about 5 e/nm, or at least about 8 e/nm, or at least about 10 e/nm, or at least about 12 e/nm. [0023] In some embodiments, the liquid crystal has a birefringence |sin(δ)| of at least about 0.3, or at least about 0.4, or at least about 0.5, or at least about 0.6. [0024] These and other features, together with the organization and manner of operation thereof, will become apparent from the following detailed description when taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0025] Figure 1 includes (A) image of P. aeruginosa PAO1 colonies isolated from P. aeruginosa biofilms under transmitted light and a specialized polarizing microscope able to measure birefringence; (B) chart showing P. aeruginosa biofilm isolates to be more birefringent (a direct measure of molecular order) than washed cells from biofilms or cell pellets from planktonically grown P. aeruginosa; (C) chart showing that compared to isolates from biofilms, various polymers exhibit low birefringence, even at high

concentrations (10 mg/ml); and (D) chart showing that compared to PAO1 colonies, birefringence is minimally impacted in P. aeruginosa colonies not capable of producing the major polysaccharides Pel, Psl, and alginate, even when grown in the presence of DNase. [0026] Figure 2 includes (A-B) charts showing smooth colonies that are intensely birefringent are associated with the presence of Pf4; and (C-D) charts showing birefringence can be enhanced in P. aeruginosa by addition of purified Pf4 and rescued in strain ΔPA0728 which is not capable of producing Pf4. [0027] Figure 3 includes (A) image showing formation of birefringent droplets with tactoidal (crystalline) morphology when non-birefringent supernatants containing phage are spiked into solutions of alginate or DNA; (B) phase diagram showing purified Pf4 forming birefringent droplets with tactoidal morphology in a concentration dependent manner. [0028] Figure 4 includes (A-B) charts showing increased antibiotic tolerance of biofilms associated with the quantity of pf4 phage; (C) chart showing Pf4 increases the sensitivity of PAO1 planktonic cultures to antibiotics but does not impact the antibiotic sensitivity or growth of planktonic cultures of a Pf4 resistant strain (ΔpilA); (D) chart showing Pf4 induces a growth lag in PAO1 planktonic cultures; (E-F) charts showing the addition of DNA to planktonic cultures rescues Pf4 induced sensitivity to antibiotics and reduces the Pf4 induced growth lag. [0029] Figure 5 shows antibiotic tolerance in ΔPA0728/pilA biofilms, a strain that can not be infected by Pf4 bacteriophage, increased with the amount of Pf4 phage added to the biofilm matrix. [0030] Figure 6 shows the number of Pf phage particles per ml present in various CF sputum samples. [0031] Figure 7 use fluorescently labeled Pf phage or fluorescently labeled polymer (Hyaluronan) to show the organization of Pf phage and polymers into tactoidal structures and cables corresponding to the crystalline structures observed in Figure 3. The images demonstrate that bacteriophage (e.g., Pf4) can organize fluorescent-labeled polymers (e.g., HA) into filamentous or cable-like structures. [0032] Figure 8 use fluorescently labeled Pf phage or fluorescently labeled polymer (Hyaluronan) to show the organization of Pf phage and polymers into tactoidal structures and cables corresponding to the crystalline structures observed in Figure 3. The images demonstrate that fluorescently labeled bacteriophage (e.g., Pf4) are themselves organized by the presence of unlabeled polymers (e.g., HA) into structures. [0033] Figure 9 shows optical and Atomic force microscopy (AFM) images demonstrating the topography of the individual cables and tactoids formed by complexes of Pf

bacteriophage and polymer (e.g., HA). The thickness of an individual filament is about 40 nm, which is consistent with the model of a single phage with a random coil polymer shell in Figure 10. [0034] Figure 10 shows a model for the organization of a single Pf phage with a random coil polymer shell. It is proposed that repeating networks of Pf phage and polymers self- assemble through charge-based interactions into liquid crystals. These take the form of cables and tactoids but, in large numbers, can come together to form synthetic biofilm structures. [0035] Figure 11 shows (A) optical and (B-C) AFM images demonstrating that phage and polymer structures form synthetic biofilms that exhibit high levels of stability and tensile strength. These can coat surfaces (e.g., glass coverslip), can be torn (e.g., by a pipette tip), and can be lifted off as sheets. [0036] Figure 12 shows that the filamentous phage Pf4 interacts with host and microbial polymers to spontaneously assemble structural complexes. (A) P. aeruginosa forms a flat confluent biofilm in culture. (B) P. aeruginosa supplemented with 5 mg/ml HA forms dense, morphologically complex biofilms in culture. (C) The addition of P. aeruginosa biofilm supernatant to HA (5 mg/ml) results in the spontaneous formation of adherent structures. (D) Purified, fluorescently labeled Pf4 (8.8 x 10⁹ PFU/ml) mixed with 5 mg/ml DNA 2 kbp (HMW) in size forms large, interwoven structures while DNA <0.3 kbp (LMW) in size does not (see inset). (E) Purified, fluorescently labeled Pf4 (8.8 x 10⁹ PFU/ml) mixed with 5 mg/ml alginate forms large, interwoven structures. (F) Visualization of structures formed from Pf4 and HA by AFM semi-contact topography. The scale indicates height. (G)

Increasing the concentration of HA as indicated increases the size of the structures assembled upon mixing with Pf4 (8.8 x 10⁹ PFU/ml). (H) Pf4 (8.8 x 10⁹ PFU/ml) and HA (5 mg/ml) were suspended in: DI water, 1x PBS, or 10x PBS. [0037] Figure 13 shows mixtures of Pf4 and a diverse range of host and microbial polymers assemble birefringent liquid crystals. The birefringence of DNA, alginate, HA (all at 10 mg/ml) and Pf4 (1 x 10¹¹ PFU/ml) were quantified as | sin(δ) |. Polymers and Pf4 by themselves were not birefringent. However, 1:1 mixtures of polymer and Pf4 were intensely birefringent. Scale bars, 10 µm. [0038] Figure 14 shows that Pf4 assembles liquid crystalline structures in the presence of disease relevant polymers, increasing viscosity. (A) Birefringence was quantified as | sin(δ) | in mixtures of mucin (8% solids) and DNA (~2kbp in size, 4 mg/ml) supplemented with increasing amounts of the filamentous bacteriophage Pf4. Results are mean ± SEM of 3 experiments. (B) Representative images of mucin + DNA mixtures showing transmitted light (displayed as I/I_o where I = the intensity of light emerging from the sample and I_o = the intensity of incident light) and changes in birefringence (| sin(δ) |) upon the addition of Pf4. Arrows indicate filament assembly. Scale bars, 200 µm. (C) Pf phage were quantified by qPCR in sputum collected from patients infected by P. aeruginosa (P. a. (+), n = 10) or patients not infected by P. aeruginosa (P. a. (-), n = 5). Results are mean ± SEM, nd; not detected. (D) The birefringence (| sin(δ) |) of sputum samples described in (C) was quantified. The addition of 10⁸ PFU/ml Pf4 to P. a. (-) sputum augmented birefringence. Results are mean ± SEM of 3 experiments. (E) Changes in the viscosity (mPa*sec) of mucin + DNA samples described in (A) were monitored in response to supplementation with Pf4. Results are mean ± SEM of 4 experiments. [0039] Figure 15 shows that Pf4 organizes the P. aeruginosa biofilm matrix into a liquid crystalline structure. (A) Pf4 production by rough and SCV colony types was enumerated as PFUs/ml and normalized to bacterial CFUs/ml. Results are mean ± SEM of 3 experiments. (B and C) Representative images of rough and SCV colonies showing transmitted light (displayed as I/I_o where I = the intensity of light emerging from the sample and I_o = the intensity of the incident light) and birefringence (| sin(δ) |). Scale bars, 250 µm. (D)

Birefringence (| sin(δ) |) was quantified in rough and SCV colonies after normalizing for sample thickness. Birefringence was again measured after washing of the bacteria to remove the extracellular matrix. Results are mean ± SEM of 4 experiments. (E) Pf4 produced by ∆PA0728 or∆PA0728 supplemented with Pf4 (ΔPA0728 + Pf4) were enumerated as

PFUs/ml and normalized to bacterial CFUs/ml. Results are mean ± SEM of 3 experiments, nd; not detected. (F) Birefringence (| sin(δ) |) was quantified in∆PA0728 and∆PA0728 + Pf4. Results are mean ± SEM of 4 experiments. [0040] Figure 16 shows P. aeruginosa biofilms with liquid crystalline matrices display increased adhesion and tolerance to desiccation. (A) Biofilm adhesion after 24 hours of growth as measured by the crystal violet adhesion assay. Results are mean ± SEM of 3 experiments. (B) Evaporation of isotropic and liquid crystalline phases of Pf4 and DNA was monitored by absorbance (Abs, 600 nm). Absorbance was normalized to initial absorbance 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 3 experiments; error bars are omitted for clarity. (C) Colony biofilms formed from rough or SCV isolates were incubated for 18-h at 37°C in an ambient incubator. The percent water loss was measured as the wet weight pre desiccation / wet weight post desiccation. Results are mean ± SEM of 6 experiments. (D) Killing by desiccation is represented as the log₁₀ reduction of viable cells recovered from control biofilms compared to desiccated biofilms. Results are mean ± SEM of 3 experiments. [0041] Figure 17 shows that the liquid crystalline biofilm matrix increases antibiotic tolerance to antibiotics by enhancing aminoglycoside binding. (A-C) Killing by tobramycin (10 µg/ml), gentamicin (10 µg/ml), or ciprofloxacin (1 µg/ml) is represented as the log₁₀ reduction of viable cells recovered from biofilms treated with antibiotics (18-h) compared to untreated controls. Results are mean ± SEM of 3 experiments. (D) P. aeruginosa

ΔPA0728/pilA, which is not capable of producing or being infected by Pf4, was suspended in isotropic or liquid crystalline phases of Pf4 (10¹⁰ PFUs/ml) and DNA (2.5 mg/ml). The sample in the liquid crystalline phase is indicated; all other samples are isotropic. The cultures were then treated with 10 µg/ml tobramycin. Killing by tobramycin is represented as the log₁₀ reduction of viable cells recovered from cultures treated with antibiotics (90 minutes) compared to untreated controls. Results are mean ± SEM of 3 experiments. (E) Binding of antibiotics to DNA and Pf4 was investigated. Tobramycin (0-3 µg/ml) or ciprofloxacin (0-0.02 µg/ml) were added to isotropic or liquid crystalline phases of Pf4 (10¹⁰ PFUs/ml) and DNA (2.5 mg/ml). The arrow indicates the sample in the liquid crystalline phase. E. coli was then added and the samples were incubated overnight. The highest antibiotic concentration at which bacterial growth occurred was plotted. Results are mean ± SEM of 3 experiments. (F) Binding of tobramycin to isotropic and liquid crystalline phases of Pf4 (10¹⁰ PFUs/ml) and DNA (2.5 mg/ml) was investigated by adding fluorescently conjugated tobramycin (Cy5-tobramycin, 40 µg/ml) to the indicated samples. Scale bars, 20 µm. [0042] Figure 18 shows interactions between diverse polymers and filamentous Pf phage result in the assembly of large interwoven structures. (A-C) Pf4 (8.8 x 10⁹ PFU/ml) and HA of different molecular weight (5 mg/ml) interact to assemble morphologically distinct structures. Treatment of preformed structures with hyaluronidase (HA’ase) results in the destruction of the structures. Scale bars, 50 µm. (D and E) Biofilm supernatants form interwoven sheets when mixed with alginate or DNA. Scale bars, 50 µm. (F-L) Fluorescently labeled Pf4 (8.8 x 10⁹ PFU/ml) mixed with 5 mg/ml of the indicated polymer (or 1:1 with PBS in (M)) were imaged by fluorescent microscopy. Scale bars, 50 µm except in L where the scale bar represents 200 µm. (N and O) The filamentous phage fd, which is produced by E. coli, forms similar structures when mixed with HA or DNA. Scale bars, 20 µm. (P) Raman spectra of amide band regions indicate tight interactions of Pf4 and HA, resulting in suppressed β-sheet peaks of Pf4. (Q) The suppression of β-sheet peaks is weakened after removing salts by washing of the sample. [0043] Figure 19 shows small colony variant production, phage production, and phage sensitivity of various strains of P. aeruginosa. (A) P. aeruginosa PAO1 biofilms producing Pf4 generate both wild type colonies with rough edges and small colony variants (SCVs). Isolates were collected by dipping an inoculating loop into the biofilm followed by isolating for individual colonies by streaking on a Petri dish. (B) ΔPA0728 biofilm supernatants do not contain the major coat protein of Pf4, CoaB, as analyzed by SDS-PAGE and confirmed by mass spectrometry. (C) Pf4 DNA packaged within mature phage particles was detected by PCR primers that can amplify circularized Pf4 DNA (the replicative form, RF). (D) Purified Pf4 (8.8 x 10⁹ PFU/ml) was spotted onto lawns of the indicated strains of P.

aeruginosa. (E) Circular Pf4 DNA was not detected in ΔPA0728/pilA biofilm supernatants by PCR using the RF specific primers. (F) Supernatants collected from ΔPA0728/pilA biofilms did not produce any plaques on a lawn of ΔPA0728. [0044] Figure 20 shows birefringence analysis of P. aeruginosa flowcell biofilms. (A) The Rotopol imaging system was coupled with confocal microscopy to image PAO1 and PAO1 + Pf4 flowcell biofilms after three days of growth. Biofilms were stained for biomass using Syto9 and imaged using confocal microscopy. Birefringence was then measured in the same field of view using Rotopol, scale bar = 35 µm. (B) Phage production was quantified in the biofilm effluent over time by plating for PFUs on a lawn of ΔPA0728. (C) Birefringence was measured over time and normalized to cluster height. Cluster heights were measured in the Z- axis which were then used to normalize birefringence measurements to sample height using the formula where Δn=birefringence, L optical path (cluster height measured by confocal microscopy), and λ=wavelength (550 nm). Results are mean ± SEM of 3

experiments. [0045] Figure 21 shows that filamentous phage fd offers P. aeruginosa protection from tobramycin by interacting with polymers to form liquid crystals. Various isotropic mixtures of fd and HA or fd and DNA offer P. aeruginosa ΔPA0728 some protection against 10 µg/ml tobramycin (90 minute treatment time). Concentrations of fd and polymer where liquid crystals form offer the most protection (last bars). Replacing the polymer in the liquid crystal mixtures with low molecular weight oligomers (resulting in an isotropic rather than liquid crystalline mixture) results in a loss of the extra protection offered by liquid crystal assembly. Results are mean ± SEM of 3 experiments. [0046] Figure 22 shows liquid crystal formation mediated by Pf1 bacteriophage. (A) DNA, (B) hyaluronan, (C) alginate. Polymer and Pf1 were mixed together at given concentrations and viewed at 20x for biphasic separation (tactoid formation) after 30 minutes. If tactoids were observed, the solution was determined to be biphasic (nematic tactoids in an isotropic solvent). If no tactoids were observed, the solutions was determined to be isotropic.

Solutions will be observed again after overnight incubation to determine how time affects the phase line (dashed line). The phase line was placed between points were an isotropic to biphasic transition was observed. DETAILED DESCRIPTION [0047] It is discovered that Inovirus bacteriophage (e.g., Pf family bacteriophage) can organize bacterial biofilm matrix (e.g., P. aeruginosa biofilm matrix) into a structure with liquid crystal properties. For example, purified Pf4, Pf1, and fd bacteriophage, when disposed in solution with polymers, can spontaneously organize the polymers into a liquid crystal. The spontaneously organization of the polymers into liquid crystal structures by the Inovirus bacteriophage can be based on, for example, charge repulsion and depletion attraction. These phage-mediated structures are liquid crystals which can be remarkably tenacious and stable. Such synthetic structures can be used to create conductive surfaces, immune-modulatory materials, protective coatings, Magnetic Resonance Imaging (MRI) contrast agents, batteries and capacitors. [0048] The bacteriophage can also be genetically engineered and functionalized to endow the synthetic biofilms/structures with a diverse range of properties. In addition, the structures can be made within a magnetic field, conferring directionality. [0049] Bacteriophage-mediated polymer organization can be inexpensive, massively scalable, and efficient. The ability of purified Inovirus bacteriophage (e.g., Pf family bacteriophage) to organize polymers into adherent, stable, liquid crystalline structures with a diverse range of properties, which can be adjusted based on the polymers used, will have extensive commercial applications in various areas, including industrial engineering, bio- design, environmental engineering, batteries, and television screens or computer monitors. [0050] One aspect of the invention relates to a method for organizing polymers, comprising mixing a first composition comprising purified Inovirus bacteriophage (e.g., Pf-family bacteriophage) with a second composition comprising one or more polymers. Inoviruses are non-enveloped, filamentous, rod-shaped viruses typically about 6-7 nm in diameter and about 700-2000 nm in length, which often have single stranded DNA. [0051] The first composition can be, for example, a liquid composition. The second composition can be, for example, a liquid composition. The method can further comprise the step of, for example, coating the first composition, the second composition, or the mixture thereof onto a surface. [0052] The mixing of the first composition and the second composition can lead to the formation of, for example, a liquid crystal material comprising the Inovirus bacteriophage (e.g., Pf-family bacteriophage) and the polymer. The liquid crystal can have a birefringence of, for example |sin(δ)|=0.3 or more, or |sin(δ)|=0.4 or more, or |sin(δ)|=0.5 or more, or |sin(δ)|=0.6 or more. [0053] The mixture of the first composition and the second composition can comprise one or more salts such as, for example, monovalent salts such as sodium chloride, which are believed to facilitate the formation of the liquid crystal by masking long-range electrostatic repulsion of like-charged molecules. The mixture of the first composition and the second composition can have a pH level of, for example, between pH of about 6.5 and about 8.5, which is believed to facilitate the formation of the liquid crystal. The formation of the liquid crystal can be driven by, for example, repulsive forces between the bacteriophage and depletion attraction. [0054] The Inovirus bacteriophage can be, for example, Enterobacteria phage. The Inovirus bacteriophage can be, for example, Pseudomonas phage. The Inovirus

bacteriophage can be, for example, Ralstonia phage. The Inovirus bacteriophage can be, for example, Vibrio phage. The Inovirus bacteriophage can be, for example, Propionibacterium phage. The Inovirus bacteriophage can be, for example, Stenotrophomonas phage. The Inovirus bacteriophage can be, for example, Xanthomonas phage. [0055] The Inovirus bacteriophage can be, for example, selected from Enterobacteria phage I2-2, Enterobacteria phage If1, Enterobacteria phage Ike, Enterobacteria phage M13, Enterobacteria phage fd, Enterobacteria phage f1, Propionibacterium phage B5, Pseudomonas phage Pf1, Pseudomonas phage Pf3, Pseudomonas phage Pf4, Pseudomonas phage Pf5, Ralstonia phage p12J, Ralstonia phage PE226, Ralstonia phage RSM1, Ralstonia phage RSM3, Ralstonia phage RSS0, Ralstonia phage RSS1, Stenotrophomonas phage phiSMA9, Vibrio cholerae phage KSF-1phi, Vibrio cholerae phage VGJphi, Vibrio parahaemolyticus phage VfO4K68, Vibrio parahaemolyticus Bacteriophage VfO4K68, Vibrio

parahaemolyticus phage VfO3K6, Vibrio phage CTX, Vibrio phage fs1, Vibrio phage fs2, Vibrio phage VCY-phi, Vibrio phage VEJphi, Vibrio phage Vf12, Vibrio phage Vf33, Vibrio phage VSK, and Xanthomonas phage Cf1c. A mixture of two or more different Inovirus bacteriophages also can be used. [0056] The Inovirus bacteriophage can be, for example, Pf-family bacteriophage. The Pf- family bacteriophage can be, for example, Pf4 bacteriophage. The Pf-family bacteriophage can be, for example, Pf5 bacteriophage. The Pf-family bacteriophage can be, for example, Pf1 bacteriophage. The Pf-family bacteriophage can be, for example, Pf4 bacteriophage produced by P. aeruginosa strain PAO1. The Pf-family bacteriophage can be, for example, Pf5 bacteriophage produced by P. aeruginosa strain PA14. The Pf-family bacteriophage can be, for example, Pf1 bacteriophage produced by P. aeruginosa strain PAK. [0057] The Inovirus bacteriophage (e.g., Pf-family bacteriophage) can have an average length of, for example, at least about 700 nm, or at least about 1,000 nm, or at least about 1,500 nm, or at least about 2,000 nm, or at least about 2,500 nm, or at least about 3,000 nm, or at least about 3,400 nm, which is believed to be more effective in forming liquid crystals than shorter bacteriophage. [0058] The Inovirus bacteriophage (e.g., Pf-family bacteriophage) can have an average linear charge density of, for example, at least about 5 e/nm, or at least about 8 e/nm, or at least about 10 e/nm, or at least about 12 e/nm, which is believed to be suitable for organizing polymers into liquid crystals based on charge repulsion. The Inovirus bacteriophage can have, for example, a negative electric charge, which is believed to be more effective in forming liquid crystals than bacteriophage with no electric charge. For example, the CoaB protein of Pf-family bacteriophage can comprise at least 3, or at least 4, or at least 5 amino acids with negatively charged side chains. [0059] The polymer can be, for example, a biological polymer such as DNA, alginate, hyaluronan, collagen, heparin, heparin sulfate, fibronectin, chondroitin sulfate, perlican, or a polymer present in human serum. The polymer can be, for example, substantially free of dextran. [0060] The polymer can be, for example, a synthetic polymer such as chitosan, dextran, dextran sulfate, polyacrylic acid, polyethylene glycol, polystyrene sulfonate, and

polyethylene glycol diacrylate. A mixture of two or more different polymers also can be used. [0061] The weight ratio between the Inovirus bacteriophage (e.g., Pf-family bacteriophage) to the polymer can be, for example, about 1:500 to about 50:1, or about 1:200 to about 20:1, or about 1:100 to about 10:1, or about 1:50 to about 5:1, or about 1:20 to about 2:1, or about 1:10 to about 1:1, or about 1:5 to about 1:2. [0062] Another aspect of the present invention relates to a liquid crystal comprising

Inovirus bacteriophage (e.g., Pf-family bacteriophage) and one or more polymers. Said liquid crystal can be, for example, substantially free of any bacterial strain (e.g., P. aeruginosa strain) capable of producing the Inovirus bacteriophage. [0063] The liquid crystal can comprise, for example, Enterobacteria phage. The liquid crystal can comprise, for example, Pseudomonas phage. The liquid crystal can comprise, for example, Ralstonia phage. The liquid crystal can comprise, for example, Vibrio phage. The liquid crystal can comprise, for example, Propionibacterium phage. The liquid crystal can comprise, for example, Stenotrophomonas phage. The liquid crystal can comprise, for example, Xanthomonas phage. [0064] The liquid crystal can comprise, for example, a Inovirus bacteriophage selected from Enterobacteria phage I2-2, Enterobacteria phage If1, Enterobacteria phage Ike,

Enterobacteria phage M13, Enterobacteria phage fd, Enterobacteria phage f1 ,

Propionibacterium phage B5, Pseudomonas phage Pf1, Pseudomonas phage Pf3,

Pseudomonas phage Pf4, Pseudomonas phage Pf5, Ralstonia phage p12J, Ralstonia phage PE226, Ralstonia phage RSM1, Ralstonia phage RSM3, Ralstonia phage RSS0, Ralstonia phage RSS1, Stenotrophomonas phage phiSMA9, Vibrio cholerae phage KSF-1phi, Vibrio cholerae phage VGJphi, Vibrio parahaemolyticus phage VfO4K68, Vibrio parahaemolyticus Bacteriophage VfO4K68, Vibrio parahaemolyticus phage VfO3K6, Vibrio phage CTX, Vibrio phage fs1, Vibrio phage fs2, Vibrio phage VCY-phi, Vibrio phage VEJphi, Vibrio phage Vf12, Vibrio phage Vf33, Vibrio phage VSK, and Xanthomonas phage Cf1c. [0065] The liquid crystal can comprise, for example, Pf-family bacteriophage. The liquid crystal can comprise, for example, Pf4 bacteriophage, Pf5 bacteriophage, and/or Pf1 bacteriophage. In one specific embodiment, the liquid crystal comprises Pf4 bacteriophage. In another specific embodiment, the liquid crystal comprises Pf4 bacteriophage produced by P. aeruginosa strain PAO1. [0066] The liquid crystal can comprise, for example, Inovirus bacteriophage having an average length of, for example, at least about 700 nm, or at least about 1,000 nm, or at least about 1,500 nm, or at least about 2,000 nm, or at least about 2,500 nm, or at least about 3,000 nm, or at least about 3,400 nm. The liquid crystal can comprise, for example, Inovirus bacteriophage having an average charge density of, for example, at least about 5 e/nm, or at least about 8 e/nm, or at least about 10 e/nm, or at least about 12 e/nm. The liquid crystal can comprise a birefringence |sin(δ)| of, for example, at least about 0.3, or at least about 0.4, or least about 0.5, or at least about 0.6. [0067] Another aspect of the present invention relates to a liquid crystal described herein that is formed under a magnetic field wherein the bacteriophage and/or polymers are oriented by the applied magnetic field. [0068] A further aspect of the invention relates to the use of a liquid crystal described herein in applications such as liquid crystal displays, engineered phage catalytic, industrial coatings, energy conduction, and batteries. WORKING EXAMPLES EXAMPLE 1

Materials and Methods

[0069] Chemicals. All chemicals were purchased from Sigma unless indicated otherwise. [0070] Strains. P. aeruginosa strain PAO1,∆pilA, and∆pelf/pslD/algD were described previously.∆PA0728,∆PA0728/pilA were constructed by allelic exchange using previously described deletion constructs. [0071] Growth conditions. Cells were maintained in LB broth unless indicated otherwise. Biofilms were grown as described previously. [0072] Microscopy. Rotopol, light microscopy, fluorescent microscopy. [0073] Phage quantification. qPCR and absorbance based. [0074] Antibiotic tolerance assays. Colony biofilms were grown for 48 hours with a fresh plate provided every 24 hours. After this time, the colony biofilm was placed onto a plate containing antibiotics and incubated for the specified time at 37 C. Biofilms were then washed off of the membrane, vortexed thoroughly, and diluted in PBS to determine CFU/ml. [0075] Results. Pseudomonas aeruginosa is an opportunistic pathogen in multiple clinical settings, including devastating pulmonary infections in individuals with the genetic disorder cystic fibrosis (CF). P. aeruginosa forms biofilms, organized communities of bacteria encased in a polymer-rich extracellular matrix. Biofilms are ubiquitous in nature and are inherently resistant to antibiotics and the immune system. [0076] While examining colonies isolated from P. aeruginosa biofilms under a polarizing microscope, it was observed that some were intensely birefringent compared to freshly streaked colonies of PAO1 (Fig. 1A). Birefringence is a direct consequence of the

organization of the material and occurs when light passing through the material is split into two beams, each refracted at a different angle, and each polarized at a right angle to the other. It was hypothesized that birefringence was a property of the polymeric matrix encasing the colony. Consistent with this, it was found that P. aeruginosa biofilm isolates, but not washed cells from biofilms or cell pellets from planktonically grown P. aeruginosa, were birefringent (Fig. 1B). These data showed that the P. aeruginosa matrix can be highly organized resulting in birefringent optical properties. [0077] Biofilms are neither a liquid nor a solid. Other common birefringent materials that fall into this category are liquid crystals. It was unclear if the matrix of birefringent colony biofilms were organized like liquid crystals and what caused this liquid crystal-like organization. Since polymers abundant in biofilms such as alginate and DNA can, under certain conditions, form liquid crystals, one possibility is that matrix birefringence is manifested by polymers abundant in biofilms. However, compared to isolates from biofilms, these polymers exhibited low birefringence, even at high concentrations (10 mg/ml) (Fig. 1C). Moreover, compared to colonies of PAO1, birefringence was minimally impacted in P. aeruginosa colonies not capable of producing the major polysaccharides Pel, Psl, and alginate, even when grown in the presence of DNase (Fig.1D). These data showed that birefringence is unlikely to be solely dependent on individual or unorganized polymers common to the biofilm matrix. [0078] Birefringent colony biofilms were similar in other ways. For example, small, smooth morphologies were observed in many intensely birefringent PAO1 colony biofilms (Fig. 1A). The appearance of small colony variants from PAO1 biofilms was attributed to the activation of the filamentous prophage Pf4. It was found that the small, smooth colonies that were intensely birefringent also contained large amounts of Pf4 (Fig. 2A and 2B). Isolates containing less Pf4 were minimally birefringent. Moreover, birefringence could be enhanced in P. aeruginosa by addition of purified Pf4 and rescued in ΔPA0728, a strain not capable of producing Pf4 (Fig. 2C and 2D). The addition of phage to these cultures resulted in the appearance of small, smooth colonies. Based on these data, it was conclude that the filamentous phage Pf4 imparted liquid crystal-like anisotropic properties to the P. aeruginosa biofilm matrix. [0079] It was estimated that the majority of P. aeruginosa isolates harbor at least one prophage. Filamentous Pf prophage such as Pf4 are particularly abundant. Many CF clinical isolates carry Pf phage genes, including epidemic strains, as do laboratory strains of P.

aeruginosa, including PAO1 and PA14. Moreover, Pf phage particles present per ml of CF sputum (Fig. 6) are on par with P. aeruginosa cultures producing phage in vitro. Although specific contributions of Pf phage to P. aeruginosa biofilms are unclear, various in vitro biofilm models resulted in the up-regulation of these phage, as did hypoxic growth conditions that mimic the CF lung environment. This suggests that Pf filamentous phage are associated with the formation of P. aeruginosa biofilms. [0080] Filamentous phage possess fascinating physical properties. Given their long, rod- like shape and monodispersity, dense solutions of filamentous phage showed liquid crystalline behavior, but the potential contributions of this behavior to bacterial biofilms was recognized. [0081] Hence, it was investigated if Pf4 could also form liquid crystals. Phage

concentration is a factor in liquid crystal formation. Specifically, it was tested if planktonic culture supernatants containing concentrations of phage similar to the concentrations of phage present in birefringent colony biofilms could be birefringence. However, birefringence was actually low (sin(d) < 0.1) in these supernatants indicating the concentration of phage was too low for liquid crystal formation. It was reasoned that the polymer-rich environment of the matrix might influence liquid crystal formation. Indeed, polymers could influence liquid crystal formation by filamentous phage by providing a cohesive force that reduces the overall concentration of phage for liquid crystal formation. When non-birefringent supernatants containing phage were spiked into solutions of alginate or DNA, birefringent droplets with tactoidal morphology formed (Fig. 3A). These tactoidal droplets are similar in morphology to liquid crystal droplets formed from the filamentous phage Fd in the presence of polymer. These data showed that P. aeruginosa can produce concentrations of Pf4 in culture supernatants sufficient to form liquid crystals in a polymer-rich setting such as those found in microbial biofilms and disease settings such as human sputum. [0082] As with culture supernatants, purified Pf4 rapidly formed birefringent droplets with tactoidal morphology in a concentration dependent manner. As shown in the phase diagram (Fig. 3B), in some examples the threshold concentration of Pf4 necessary to spontaneously form liquid crystals with 2.5 mg/ml polymer was ~10⁸/ml. However, at higher concentrations of polymers, lower concentrations of Pf4 were sufficient to cause liquid crystal formation. [0083] Pf4 could form liquid crystals when mixed with a wide range of biological and synthetic polymers. The formation of liquid crystals from solutions of Pf4 and polymers is spontaneous and rapid. Video footage of such rapid Pf4 mediated liquid crystal formation was recorded. These results suggest that Pf4, together with extracellular polymers, drives the organization of the biofilm matrix of P. aeruginosa using the same mechanisms that govern the spontaneous formation of liquid crystals. [0084] The matrix is a hallmark of biofilm formation, while organizing it into a liquid crystal likely impacts P. aeruginosa biofilm physiology. The biofilm matrix is the first line of defense against the environment and directly influences the penetration of antibiotics, oxygen, and nutrients into the biofilm. Hence, it was then investigated how liquid crystal organization of the biofilm matrix might influence antibiotic tolerance in P. aeruginosa. [0085] Biofilms producing Pf4 at levels sufficient to increase birefringence resulted in increased tolerance to antibiotics (Fig. 4A and 4B). This increase in antibiotic tolerance might be due to physiological effects of Pf4 on P. aeruginosa (e.g., reduced growth rates, induced stress responses, etc.) rather than any exogenous impacts of the phage such as liquid crystal organization of the matrix. To test this possibility, planktonic cultures were infected with Pf4 and exposed to antibiotics. Pf4 infects P. aeruginosa through type IV pili. A strain lacking type IV pili (ΔpilA) was used as a control in these experiments. Pf4 dramatically increased the sensitivity of planktonic cultures of P. aeruginosa to antibiotics (Fig. 4C). However, Pf4 did not impact the antibiotic sensitivity or growth of ΔpilA planktonic cultures. Cell death due to Pf4 alone was moderate (Fig. 4C), which was expected given that filamentous phage of the genus Inovirus typically do not lyse their hosts, but are rather continuously extruded into the extracellular environment. Pf4 also induced a strong growth lag in planktonic cultures (Fig. 4D). These results show that an active infection leads to antibiotic sensitization of P. aeruginosa planktonic cultures. Therefore, physiological effects of the phage on P. aeruginosa may not solely explain the observed increase in antibiotic tolerance in biofilms. Rather, since Pf4 is induced in established biofilms, Pf4 may exogenously influences antibiotic tolerance in P. aeruginosa biofilms by organizing the matrix into a liquid crystal. [0086] To mimic the polymer rich environment of the matrix and induce liquid crystal formation by Pf4, planktonic cultures were maintained in increasing concentrations of exogenous polymer. The addition of DNA to these cultures rescued Pf4 induced sensitivity to antibiotics and reduced the Pf4 induced growth lag (Fig. 4E and 4F). [0087] To further investigate phage mediated liquid crystal modification of the biofilm matrix and antibiotic tolerance in P. aeruginosa biofilms, phage were added exogenously to a strain of P. aeruginosa not capable of producing Pf4 and lacking type IV pili

(ΔPA0728/pilA). Since phage cannot infect or replicate in this strain, the amount of exogenously added phage in the biofilm matrix can be controlled. Antibiotic tolerance in ΔPA0728/pilA biofilms increased with the amount of phage added to the biofilm matrix (Fig. 5), suggesting that phage influence antibiotic tolerance exogenously and not through known routes such as slowing growth or inducing stress responses. [0088] Conclusions. Given the liquid crystal properties of dense concentrations of filamentous phage, the ability of polymers to influence liquid crystals formation, and the widespread presence of Pf prophage in clinical P. aeruginosa isolates, it is concluded that Pf bacteriophage function to spontaneously organize and enhance the functionality of P.

aeruginosa biofilms. This strategy to organize and possibly retain polymeric materials in the biofilm matrix is well suited for the extracellular environment as it is entropically favorable. Since essentially any polymer can influence liquid crystal formation by Pf4, phage production would provide P. aeruginosa biofilms a unique mechanism to incorporate and/or retain potentially any polymer in the immediate environment (i.e., host polymers in the CF lung) into the matrix. This mechanism would provide P. aeruginosa (or any bacteria that produces filamentous phage) a large degree of plasticity when forming biofilms in various environments. [0089] In summary, filamentous Pf bacteriophage infect P. aeruginosa, a microbe responsible for severe, biofilm-associated infections in humans. In contrast to the view of phage as viral parasites, it was discovered these filamentous phage contribute as structural elements of the P. aeruginosa biofilm matrix. Pf phage spontaneously organize within the polymer-rich biofilm matrix into a structure with liquid crystal properties. This organizational strategy results in increased antibiotic tolerance of P. aeruginosa biofilms. Pf phage thereby contribute to microbial fitness, and the pathogenesis of P. aeruginosa mediated disease. In addition to Pf phage’s organization of the P. aeruginosa biofilm matrix into a liquid crystal, the same mechanism is also suitable for organizing other biological or synthetic polymers into liquid crystals. EXAMPLE 2

Introduction

[0090] Pseudomonas aeruginosa is a major bacterial pathogen. It causes ~10% of nosocomial infections and is responsible for much of the morbidity and mortality associated with cystic fibrosis (CF) airway infections. Viscous secretions accumulate in CF airways, trapping bacteria and 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 airways and on other surfaces. [0091] As P. aeruginosa biofilms develop, genes belonging to filamentous Pf1-like bacteriophage (Pf phage) are amongst the most highly transcribed. Pf phage (of the genus Inovirus) are long, negatively charged filaments ~2 µm in length and 6-7 nm in diameter. Production of Pf phage can be stimulated by anaerobic growth conditions, as may 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. [0092] Pf phage have been linked to the progression of the P. aeruginosa biofilm life cycle by inducing cell death and the subsequent release of DNA, a major component of the biofilm matrix. However, the production of large amounts of Pf phage by P. aeruginosa biofilms (up to 10¹¹ plaque forming units (PFUs) /ml) does not result in biofilm eradication. Experimental Procedures [0093] Chemicals and reagents. Salmon sperm DNA (~2 kbp, D1626), porcine gastric mucin, sodium alginate, collagen, heparin sulfate, perlecan, chondroitin sulfate, fibronectin, and human serum were purchased from Sigma-Aldrich Co., St. Louis, MO. Sodium hyaluronate was purchased from Glycosan Biosystems. Fragmented salmon sperm DNA (<0.3 kbp) was purchased from USB Corp., Cleveland, OH. Tobramycin was obtained from APP Pharmaceuticals, LLC, Schaumburg, IL. Gentamicin was obtained from Sigma-Aldrich Co., St. Louis, MO. Ciprofloxacin was obtained from Hospira, Inc., Lake Forest, IL. [0094] Bacterial strains, media, and culture conditions. Bacterial strains, plasmids, and PCR primers are listed in Table S1. Unless specified otherwise, bacteria were grown at 37°C with shaking in Luria-Bertani (LB) medium. [0095] Construction of strains∆PA0728 and∆PA0728/pilA. Plasmid pEX-∆PA0728 was introduced to PAO1 or∆pilA (Table S1) to create strains∆PA0728 and∆PA0728/pilA, respectively, by allelic exchange. Deletions were confirmed by sequencing using primers

[0096] Biofilm experiments. Static biofilms were grown in LB broth (supplemented with polymer where indicated) at 37°C in 6-well culture plates. Static biofilms were inoculated with 50 µl of an overnight culture. The media (2 ml per well) was exchanged every 24 h for up to 14 days. Rough and SCVs were isolated from static biofilms by dipping an inoculating loop into the biofilm and streaking an LB plate. Supernatants were collected from static biofilms and bacterial cells were removed by centrifuging at 9,000g for 5 minutes.

Supernatants were then passed through a 0.2 syringe filter. Proteins in the supernatants were analyzed by mass spectrometry. Colony biofilms were prepared as previously described. Briefly, a 5 µl drop of an overnight culture was placed on top of polycarbonate membrane filters (25 mm diameter, 0.2 µm pore size, GE water & Process Technologies) on LB agar plates. For∆PA0728+Pf4 colony biofilms, overnight cultures of∆PA0728 infected with 10⁶ PFUs/ml Pf4, resulting in a final Pf4 titer of ~10⁹ PFU/ml, were used for biofilm inoculation. The biofilms were maintained at 37°C and transferred to a fresh plate every 24 h. Flowcell biofilms were grown and maintained. [0097] Adhesion. Adhesion of P. aeruginosa to polystyrene microtiter plates was performed as described with modifications. [0098] Evaporation and Desiccation experiments. Evaporation of the indicated mixtures of Pf4 (10¹⁰ PFU/ml) and DNA (2.5 mg/ml) was monitored using a plate reader at 37°C. Sixty µl of each sample were placed into randomly selected wells of a 96-well plate. The plate was placed without a cover into the warm plate reader and absorbance (600 nm) was monitored over time until the samples were completely dried. Absorbance readings were normalized by dividing each time point by the intial absorbance reading for each sample. [0099] For desiccation experiments, 48-h colony biofilms (grown on a 0.2 µm membrane) of PAO1 or a SCV were placed onto either Noble agar lacking carbon and nutrient sources (moisture control) or directly onto a dry plastic Petri dish for 18-h at 37°C in an ambient incubator. Percent water loss was calculated by subtracting the final weight from the initial wet weight of desiccated and moisture control biofilms. Bacteria were then resuspended into 1 ml PBS by thorough vortexing, homogenized by passing the sample through a 20 gauge needle multiple times, serially diluted, and plated onto LB agar for enumeration of viable CFUs. [0100] Antibiotic tolerance. Colony biofilms (48-h) were transferred to fresh LB plates or plates supplemented with antibiotics at the indicated concentration and incubated at 37°C for 18-h. Biofilms were then resuspended and viable CFUs were enumerated as described above. For experiments investigating the role of extracellular phage and polymers in antibiotic tolerance, planktonic∆PA0728/pilA or∆PA0728 (2 x 10⁸ CFUs in 50 µl LB broth) were added to 500 µl of the indicated phage and polymer solutions and incubated for 20 minutes at room temperature. Tobramycin (10 µg/ml) or PBS was then added and the cells were incubated at 37°C for 90 minutes. CFUs were enumerated as described above. [0101] Antibiotic binding assays. Tobramycin (0-3 µg/ml) or ciprofloxacin (0-0.02 µg/ml) were added to the indicated concentrations of Pf4 and DNA (prepared in LB broth) in 96-well plates (100 µl volumes). Samples were allowed to incubate at room temperature for 4-h to allow any binding. An overnight culture of E. coli DH5α was diluted to an OD₆₀₀ of 0.05 in LB broth and 10 µl of this was added to each well. The cultures were sealed with parafilm and incubated overnight at 37°C. The following day, the highest concentration at which microbial growth was observed was plotted. [0102] Cy5 conjugated tobramycin (a gift from B. S. Tseng) was added to the indicated phage and DNA mixtures (Pf4, 10¹⁰ PFU/ml, DNA, 2.5 mg/ml) and incubated at room temperature for four hours. The samples were then placed between a glass slide and coverslip with a parafilm spacer and imaged by fluorescent microscopy. [0103] Phage purification. Bacteriophage were purified by precipitation with polyethylene glycol. [0104] Phage detection and quantification. Plaque assays were performed as described previously with∆PA0728 as the recipient strain. Bacteriophage fd was quantified using the same methodologies using E. coli strain ATCC 15669 as the recipient strain. The presence of Pf4 in filtered bacterial supernatants was confirmed by the amplification of an 839-bp region corresponding to the circularization of the Pf4 genome using the primers Pf4F and Pf4R. [0105] Pf phage in CF sputum for biobanking of the patients’ specimens and subsequent use were quantified by qPCR. Sputum was diluted 1:1 (vol:vol) with PBS containing DNase (100 µg/ml) and DTT (1 mM). Sputum was then incubated at 37°C for 5 hours with occasional vortexing. Debris was removed by centrifuging the sample at 6,000g for 10 minutes. The supernatants were then collected. A 100 µl aliquot was then incubated at 95°C for 15 minutes, killing the DNase and releasing any protected phage DNA. The sample was then added to 250 µl buffer P1 from a Qiagen miniprep kit. DNA extraction was then performed as described by the manufacturer’s protocol. For qPCR, primers specific for a conserved region of the Pf prophage (Pf-ConserveF and Pf-ConserveR or the 16S rDNA gene (rpIU-F and rpIU-R) were used (Table S1). Ten µl reaction volumes contained 5 µl SYBR Select Master Mix (Life Technologies, Grand Island, NY), 100 nM of each primer, and 2 µl template. Cycling conditions were as follows: 50°C 2min, 95°C 2min, (95°C, 15 sec, 60°C 1 minute) x 40 cycles. Template copy numbers were calculated by constructing a standard curve from plasmids containing the amplified sequence. To control for any potential contamination of P. aeruginosa chromosomal DNA that might contain Pf prophage sequences, 16S copy numbers were subtracted from Pf copy numbers. [0106] Fluorescent labeling of phage. Bacteriophage were labeled with Alexa Fluor-488 TFP ester (Molecular Probes, Eugene, OR) following the manufacturers protocol. Following labeling, phage were separated from unincorporated dye using PD10 desalting columns (GE healthcare). [0107] Birefringence measurements. Birefringence was measured using Rotopol. Intact colonies grown on LB agar were visualized by carefully excising a 1-inch by 1-inch agar square containing bacterial colonies and placing it on a glass slide prior to obtaining birefringence measurements. Separate measurements were performed to normalize sample thickness by scraping bacterial colonies off of the agar surface using an inoculating loop. The biomass was placed onto a glass microscope slide. Parafilm was cut and placed in a ring around the bacterial mass to provide a spacer with uniform thickness. A glass coverslip was then placed onto the bacteria and pressed down gently to make contact with the parafilm. Likewise, the birefringence of CF sputum and mucin + DNA mixtures were measured after placing the sample between a glass coverslip and slide with a parafilm spacer, as described above. [0108] Viscosity measurements. Viscosity was measured with a capillary viscometer (Cannon Instrument Company, State College, PA) at room temperature following the manufacturer’s instructions. Briefly, using a stopwatch capable of measuring time to 1/10^th of a second, the amount of time a sample took to travel a premeasured distance through the viscometer was measured. The efflux time was then multiplied by the calibration constant supplied by the manufacturer (1.18 mm²/s²) and then multiplied by the sample density, measured in grams per ml, to obtain the sample viscosity in mPa*sec. [0109] Statistical analysis. Statistical analysis was performed using Prism GraphPad software, mean with SEM were calculated and plotted. Results [0110] Filamentous Pf Phage Interact With Host and Microbial Polymers to Assemble Liquid Crystalline Structures. While performing unrelated experiments that involved supplementing cultures of P. aeruginosa with hyaluronan (HA), a host polymer abundant in the CF lung and other inflamed tissues, the formation of morphologically complex biofilms composed of interlaced structures (Figure 12A and 12B) were observed. The addition of hyaluronidase dissolves these structures (Figure 18A-18C), indicating that HA is essential for their formation. When filtered biofilm supernatants were mixed with HA, DNA, or alginate, similar adherent sheets of interwoven structures assemble (Figure 12C; Figure 18D and 18E). These observations suggest that P. aeruginosa biofilms produce an extracellular factor that assembles host and microbial polymers into well-organized structures. [0111] Proteomic analysis demonstrated that CoaB, the major coat protein of the filamentous bacteriophage Pf4, was abundant in these biofilm supernatants, suggesting that Pf phage might be the factor that interacts with polymers. In support of this hypothesis, purified Pf4 rapidly (~1-5 minutes) and spontaneously forms networks and interwoven structures when mixed with DNA, alginate, HA, or a broad, chemically diverse range of microbial and host polymers, including human serum (Figure 12D-12F; Figure 18F-18M). Other filamentous phage behave similarly; the filamentous phage fd, which infects

Escherichia coli, forms similar structures when mixed with HA or DNA (Figure 18N and 18O). These observations show that filamentous phage derived from different bacterial species can interact with diverse polymers to rapidly assemble well-organized structures. [0112] The formation of these structures can be dependent upon polymer size and concentration. High molecular weight (HMW) DNA polymers of similar length to those found in CF sputum (~2 kbp) form structures with Pf4 (Figure 12D) while low molecular weight (LMW) DNA polymers (<0.3 kbp) do not (Figure 12D inset). Similar results were obtained with HA (Figure 18A-18C). Increasing the concentration of either polymer or Pf4 promotes the assembly of larger structures (Figure 12G). [0113] The morphology of these structures is also dependent upon ionic strength. These structures do not efficiently assemble in deionized water, but are more condensed and extended in buffers of increasing tonicity (Figure 12H). Raman spectroscopy reveals that HA tightly interacts with Pf4, transforming its secondary structure by suppressing the vibrational freedom of amide groups on Pf4 coat proteins (Figure 18P and 18Q). This suppression is lost when salts are washed away, indicating that HA is not as tightly associated with Pf4 at low ionic strength. [0114] Taken together, the observations in Figure 12 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 (Asakura and Oosawa, 1958). Polymer size and concentration are proportional to the magnitude and range of depletion attraction, respectively, favoring the assembly of larger structures. When filamentous particles, like phage, are confined to such structures, they spontaneously align and form liquid crystals, a highly ordered phase of matter between the liquid and solid states. While the physics of these interactions are well established, their relevance to microbiology, biofilms, and disease has heretofore not been considered. [0115] One prediction of this model is that the observed higher-order structures formed from Pf4 and disease relevant polymers are liquid crystals. To confirm this, the optical properties of liquid crystals were tested. Liquid crystals are birefringent; that is liquid crystals 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. To quantify birefringence, an optical imaging system for birefringent media called Rotopol were utilized. This device measures quantitatively the phase difference of the polarized light beams emerging from the liquid crystal (i.e., the optical anisotropy) as | sin(δ) | where

Δn=birefringence, L optical

path, λ=wavelength. In an embodiment of this method, a computer driven rotatable polarizer probes the light intensities of incoming circularly polarized light as it is changed by the birefringence of the sample. Using this tool, it was found that neither Pf4 nor polymers alone are birefringent (Figure 13). However, mixtures of Pf4 and polymers present at sites of infection assemble structures that display intense birefringence, indicating the observed structures are indeed liquid crystals. [0116] Pf Phage Assemble Liquid Crystals in the Presence of Polymers Abundant in CF Sputum, Increasing Sample Viscosity. To test whether Pf bacteriophage could promote liquid crystal formation in more physiologically relevant conditions, Pf4 were added to disease relevant concentrations of mucin (8% solids) mixed with DNA (HMW, 4 mg/ml). Supplementation of this host polymer mixture with Pf4 results in a dose-dependent increase in birefringence (Figure 14A and 14B). Liquid crystal assembly occurred at Pf4

concentrations≥ 10⁸ PFU/ml with filamentous structures forming at higher Pf4

concentrations. [0117] To determine whether the concentrations of Pf4 that promote liquid crystal formation are comparable to those seen in disease, a quantitative PCR (qPCR) method was devised to detect Pf phage levels, as detailed in the methods section, and tested sputum collected from CF patients. Sputum collected from patients infected with P. aeruginosa contained on average ~10⁸ Pf phage per ml (Figure 14C). Conversely, Pf phage were not detected in sputum collected from patients not infected with P. aeruginosa. These data suggest that CF sputum contains sufficient quantities of Pf phage to drive the assembly of physiologic concentrations of mucin and DNA into liquid crystals. [0118] To test whether Pf phage confer liquid crystalline properties to CF sputum, the birefringence of Pf positive and Pf negative CF sputum were measured. It was found that the former to be more birefringent than the later. Moreover, the birefringence of Pf negative sputum could be augmented by supplementation with Pf4 (Figure 14D). These data indicate that Pf phage drive the liquid crystalline organization of CF sputum. [0119] Given that liquid crystals are inherently viscous and impaired clearance of viscous airway secretions contributes to airway obstruction and decreased lung function in CF, the addition of Pf4 might increase the viscosity of mucin + DNA mixtures. To test this, viscosity was measured with a capillary viscometer. The viscosity of mucin + DNA mixtures was found to increase with the addition of Pf4 (Figure 14E), showing that filamentous phage enhance the viscosity of disease relevant polymers. [0120] Taken together, these results suggest that filamentous Pf phage interact with disease relevant polymers at physiologically relevant concentrations to assemble birefringent liquid crystalline structures. Moreover, the assembly of such highly ordered structures contributes to sample viscosity. [0121] Filamentous Bacteriophage Organize the Biofilm Matrix Into a Liquid Crystalline Structure. Along with organizing host polymers and fluids, Pf phage might promote similar liquid crystalline organization within the biofilm matrix of P. aeruginosa small colony variants (SCVs). These are P. aeruginosa isolates with enhanced adherence and antibiotic tolerance that are often implicated in intractable CF pneumonia cases. Compared to larger wild type colonies with rough edges (Figure 19A), SCVs produce ~5 x 10⁴ fold more Pf4 (Figure 15A). Consequently, SCVs are intensely birefringent while rough colonies are minimally birefringent (Figure 15B-15D). Removal of the matrix by washing the bacterial cells results in a substantial decrease in birefringence (Figure 15D), illustrating that the LC nature of the biofilm is manifested primarily from the extracellular matrix and not from the bacterial cells. As a control, the birefringence of a strain of P. aeruginosa lacking the phage integrase gene PA0728, which is essential for Pf4 production (Figure 15E; Figure 19B and 19C), were measured. Colonies of∆PA0728 are minimally birefringent while infection by Pf4 augments colony birefringence (Figure 15). Also observe was increased birefringence in flowcell biofilms that correlated with Pf4 production (Figure 20), showing that birefringence is not specific to colony biofilms. Taken together, these data are consistent with the conclusion that Pf4 assembles the biofilm matrix into a highly ordered liquid crystalline structure. [0122] Liquid Crystalline P. aeruginosa Biofilms Display Enhanced Adhesiveness. Given this structural role for Pf4 in biofilm assembly, the liquid crystalline matrix might contribute to biofilm functionality. A canonical feature of biofilms is their ability to adhere to surfaces. Therefore, the adhesiveness of biofilms with a liquid crystalline matrix were measured. [0123] It was found that the addition of Pf4 promotes biofilm adhesion in PAO1 and ∆PA0728 (Figure 16A). Adhesion is also increased when Pf4 is supplied to P. aeruginosa strain K (PAK), which cannot be infected by Pf4 (Figure 19D). This indicates that infection by Pf4 is not required to promote adhesion, but rather that increased adhesiveness might be due to the structural role Pf4 plays in the biofilm matrix. [0124] The Liquid Crystalline Matrix Retains Water Within P. aeruginosa Biofilms, Enhancing Desiccation Tolerance. In addition to their structural role, biofilms protect bacteria against desiccation, thereby promoting bacterial survival. Given that liquid crystals are inherently viscous and viscous solutions generally display reduced rates of evaporation, the liquid crystalline organization of the biofilm matrix might increase water retention and thus increase the tolerance of P. aeruginosa biofilms to desiccation. [0125] To study water retention in polymers in the isotropic (unordered) or liquid crystalline phases, evaporation rates were measured using a plate reader. Samples were placed into wells of a 96-well plate, which was incubated in the plate reader without a cover at 37°C. Liquid crystals assembled from Pf4 and DNA evaporate at a slower rate compared to Pf4 or DNA alone (Figure 16B), suggesting that the liquid crystalline phase retains water more efficiently than polymers in the isotropic phase. [0126] To differentiate the contributions of polymer concentration and liquid crystal assembly towards evaporation, the HMW DNA in liquid crystalline mixtures was replaced with LMW DNA. The resulting mixtures contain the same concentrations of Pf4 and DNA, but since LMW DNA does not promote liquid crystal assembly at the concentrations tested (Figure 12D), the mixtures remain isotropic. These isotropic mixtures evaporate faster than mixtures in the liquid crystalline phase containing the same concentrations of HMW DNA and Pf4. This indicates that liquid crystal assembly rather than high polymer concentrations increase the retention of water. Consistent with this, when colony biofilms with liquid crystalline matrices were placed on dry plastic dishes and maintained for 18-h in an ambient 37°C incubator, they retained more water (wet cell weight) and displayed increased tolerance to desiccation compared to biofilms lacking liquid crystalline order (Figure 16C and 16D). [0127] As a whole, these results suggest that the assembly of the biofilm matrix into a liquid crystalline structure increases water retention, resulting in increased tolerance to desiccation. [0128] P. aeruginosa Biofilms With Liquid Crystalline Matrices Display Enhanced Tolerance to Aminoglycoside Antibiotics. A hallmark of bacterial biofilms is their tolerance to antibiotics. Hence, the liquid crystal organization of the biofilm matrix might further increase antibiotic tolerance in P. aeruginosa. Relative to biofilms not organized into a liquid crystal, bacteria within liquid crystalline biofilms display increased tolerance to

aminoglycoside antibiotics, but are equally sensitive to the fluoroquinolone ciprofloxacin (Figure 17A-17C). [0129] To confirm that tolerance to aminoglycosides could be due to physiological changes induced by Pf4, a strain of P. aeruginosa (∆PA0728/pilA) that can neither produce nor be infected by Pf4 (Figure 19D-19F) were used. This strain lacks PA0728 and type IV pili (∆pilA), the receptor Pf4 utilizes to infect P. aeruginosa. These bacteria were added to isotropic or liquid crystalline mixtures of Pf4 and DNA, simulating cells within a polymeric matrix. Following tobramycin treatment, mixtures containing liquid crystals offered P.

aeruginosa the most protection (Figure 17D). Further, when Pf4 was replaced with the filamentous E. coli phage fd, similar trends were observed (Figure 21). Together, these results indicate that filamentous phage promote tolerance to tobramycin through an extracellular mechanism. [0130] Liquid Crystalline Phases of Pf Phage and DNA Display Enhanced Sequestration of Aminoglycoside Antibiotics and Prevent Bacterial Killing. Aminoglycosides are cationic and 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 are more tolerant to aminoglycosides, liquid crystal assembly might promote binding and sequestration of aminoglycosides in ways that prevent bacterial killing. [0131] To test this, increasing concentrations of tobramycin or ciprofloxacin were added to isotropic or liquid crystalline mixtures of Pf4 and DNA. After a 4-h incubation to allow binding of antibiotics, E. coli, which cannot be infected by Pf4, was added and the highest concentration of antibiotic at which growth occurred was plotted (Figure 17E). Pf4 and DNA did not offer E. coli protection against ciprofloxacin, even in mixtures containing liquid crystals. In contrast, Pf4 and DNA did provide E. coli protection against tobramycin, suggesting that the drug was sequestered away from the bacteria. [0132] To differentiate the contributions of polymer concentration and liquid crystal assembly towards the binding of antibiotics, liquid crystalline mixtures of Pf4 and HMW DNA were compared to isotropic mixtures of LMW DNA and Pf4, as described for the desiccation experiments above. Isotropic mixtures of LMW DNA and Pf4 also provided protection against tobramycin. However, bacterial growth was permitted at the highest tobramycin concentrations when liquid crystals were present. Differential binding of tobramycin to HMW and LMW DNA does not account for these results (Figure 17E) suggesting that liquid crystal assembly might further enhance tobramycin binding. To confirm this, a fluorescent Cy5-conjugated form of tobramycin (Cy5-tobramycin) were used to study the effects of liquid crystal assembly on tobramycin binding. Fluorescent imaging revealed that tobramycin was sequestered within liquid crystals (Figure 17F) consistent with the conclusion that liquid crystal assembly enhances the binding of aminoglycoside antibiotics. Discussion [0133] These results demonstrate that filamentous Pf bacteriophage, along with diverse, disease relevant, host and microbial polymers, self-assemble into liquid crystals. The organization of the biofilm matrix into a liquid crystalline structure promotes the

fundamental, pathogenic features of biofilms, including adhesion, resistance to desiccation, and antibiotic tolerance. [0134] These results demonstrate an unprecedented form of symbiosis between filamentous Pf phage and P. aeruginosa. While bacteriophage are known to encode antibiotic resistance genes and toxins, the findings described here indicate that Pf phage are an integral structural component of the P. aeruginosa biofilm. Of note, filamentous phage of the genus Inovirus, to which Pf phage belong, typically do not lyse their bacterial hosts, but are continuously produced, although variants of Pf phage capable of inducing cell lysis have been reported. Moreover, genetic elements that govern Pf phage production are regulated by bacterial factors. Together, these data argue for a high level of cooperation between Pf phage and P. aeruginosa biofilms. [0135] These findings are likely to have broad implications across many infectious diseases. In addition to P. aeruginosa, filamentous phage of the genus Inovirus are produced by many Gram-negative bacterial species, including human pathogens such as E. coli, Klebsiella pneumoniae, Vibrio cholerae, Yersinia pestis, and others. Given that E. coli bacteriophage fd likewise assembles liquid crystals in the presence of host and microbial polymers (Figure 21), filamentous bacteriophage may contribute to many human infections. [0136] While these findings are relevant to a wide diversity of hardware and tissue infections, they are particularly relevant to the pathophysiology of CF where the high viscosity and adhesion of CF airway secretions are thought to contribute potently to disease symptoms. Here, it was shown that Pf phage can also enhance the viscosity of polymers abundant in CF airway secretions. [0137] These findings reveal a liquid crystal-mediated mechanism of antibiotic resistance. Liquid crystals assembled from Pf phage sequester aminoglycosides, increasing the tolerance of P. aeruginosa biofilms to these antibiotics. In particular, the results indicate that Pf phage in the liquid crystalline phase bind aminoglycosides more efficiently than unordered polyanions. This is perhaps a consequence of the highly ordered nature of the liquid crystalline phase or through extensive structural rearrangements of polymers such as DNA suspended in liquid crystalline solvents. Thus, liquid crystals might be capable of binding and inactivating other cationic antimicrobials in airway defense. Together, these data on sputum viscosity and antibiotic resistance suggest that Pf phage contribute substantially to the persistence and recalcitrance of P. aeruginosa airway infections. [0138] Pf bacteriophage may also promote the transmissibility of P. aeruginosa. The transmission of P. aeruginosa from one patient to another is thought to occur through aerosols or contaminated surfaces and desiccation tolerance is thought to be associated with this transmission. The results described here suggest that Pf-mediated liquid crystal assembly 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. Conversely, it may be possible to use Pf phage industrially to develop materials resistant to desiccation. [0139] As used herein, the singular terms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a marker can include multiple markers unless the context clearly dictates otherwise. [0140] As used herein, the terms“substantially,”“substantial,” and“about” are used to describe and account for small variations. When used in conjunction with an event or circumstance, the terms can refer to instances in which the event or circumstance occurs precisely as well as instances in which the event or circumstance occurs to a close

approximation. For example, the terms can refer to less than or equal to ±10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. [0141] Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for

convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth. [0142] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scopes of this invention.

Claims

WHAT IS CLAIMED IS:

1. A method for organizing polymers, comprising mixing a first composition comprising purified Inovirus bacteriophage with a second composition comprising one or more polymers.

2. The method of claim 1, wherein the Inovirus bacteriophage are selected from the group consisting of Enterobacteria phage, Pseudomonas phage, Ralstonia phage, Vibrio phage, Propionibacterium phage, Stenotrophomonas phage, and Xanthomonas phage.

3. The method of claim 1, wherein the Inovirus bacteriophage are selected from the group consisting of Enterobacteria phage I2-2, Enterobacteria phage If1, Enterobacteria phage Ike, Enterobacteria phage M13, Enterobacteria phage fd, Enterobacteria phage f1, Propionibacterium phage B5, Pseudomonas phage Pf1, Pseudomonas phage Pf3, Pseudomonas phage Pf4, Pseudomonas phage Pf5, Ralstonia phage p12J, Ralstonia phage PE226, Ralstonia phage RSM1, Ralstonia phage RSM3, Ralstonia phage RSS0, Ralstonia phage RSS1, Stenotrophomonas phage phiSMA9, Vibrio cholerae phage KSF-1phi, Vibrio cholerae phage VGJphi, Vibrio parahaemolyticus phage VfO4K68, Vibrio parahaemolyticus Bacteriophage VfO4K68, Vibrio parahaemolyticus phage VfO3K6, Vibrio phage CTX, Vibrio phage fs1, Vibrio phage fs2, Vibrio phage VCY-phi, Vibrio phage VEJphi, Vibrio phage Vf12, Vibrio phage Vf33, Vibrio phage VSK, and

Xanthomonas phage Cf1c.

4. The method of claim 1, wherein the Inovirus bacteriophage are Pf-family bacteriophage.

5. The method of claim 4, wherein the Pf-family bacteriophage are selected from the group consisting of Pf4 bacteriophage, Pf5 bacteriophage, and Pf1 bacteriophage.

6. The method of claim 4, wherein the Pf-family bacteriophage are selected from the group consisting of Pf4 bacteriophage produced by Pseudomonas aeruginosa strain PAO1, Pf5 bacteriophage produced by Pseudomonas aeruginosa strain PA14, and Pf1 bacteriophage produced by Pseudomonas aeruginosa strain PAK.

7. The method of any of claims 1-6, wherein the polymer is a biological polymer or

synthetic polymer.

8. The method of claim 7, wherein the polymer is a biological polymer selected from the group consisting of DNA, alginate, hyaluronan, collagen, heparin, heparin sulfate, fibronectin, chondroitin sulfate, perlican, and a polymer present in human serum.

9. The method of claim 7, wherein the polymer is a synthetic polymer selected from the group consisting of chitosan, dextran, dextran sulfate, polyacrylic acid, polyethylene glycol, polystyrene sulfate, and polyethylene glycol diacrylate.

10. The method of any of claims 1-9, wherein the weight ratio between the Inovirus

bacteriophage to the polymer is 1:50 to 5:1.

11. The method of any of claims 1-10, wherein the Inovirus bacteriophage have an average length of at least 1,000 nm.

12. The method of any of claims 1-11, wherein the Inovirus bacteriophage have an average electric linear charge density of at least 10 e/nm.

13. The method of any of claims 1-12, further comprising obtaining a liquid crystal

comprising the Inovirus bacteriophage and the polymer.

14. The method of claim 13, wherein the liquid crystal has a birefringence |sin(δ)| of at least 0.3.

15. A liquid crystal formed by the method of any of claims 1-14.

16. A liquid crystal comprising Inovirus bacteriophage and one or more polymers, wherein the liquid crystal is substantially free of any bacterial strain capable of producing the Inovirus bacteriophage.

17. The liquid crystal of claim 16, wherein the Inovirus bacteriophage are Pf-family

bacteriophage, and wherein the liquid crystal is substantially free of any Pseudomonas aeruginosa strain capable of producing the Pf-family bacteriophage.

18. The liquid crystal of claim 17, wherein the Pf-family bacteriophage are selected from the group consisting of Pf4 bacteriophage, Pf5 bacteriophage, and Pf1 bacteriophage.

19. The liquid crystal of any of claims 16-18, wherein the polymer is a biological polymer or synthetic polymer.

20. The liquid crystal of any of claims 16-19, wherein the weight ratio between the Inovirus bacteriophage to the polymer is 1:50 to 5:1, wherein the Inovirus bacteriophage have an average length of at least 1,000 nm, wherein the Inovirus bacteriophage have an average electric linear charge density of at least 10 e/nm, and wherein the liquid crystal has a birefringence |sin(δ)| of at least 0.3.