This document summarizes research finding that filamentous Pf bacteriophage interact with host and microbial polymers found in biofilms and tissues infected by Pseudomonas aeruginosa to spontaneously assemble liquid crystals. The liquid crystalline structure of biofilms organized by Pf bacteriophage enhances the biofilms' tolerance to cationic antibiotics like aminoglycosides, likely by increasing the binding of these antibiotics to the liquid crystalline matrix. Disrupting liquid crystal formation in biofilms could be a potential therapeutic strategy for treating chronic P. aeruginosa infections.

1. Filamentous bacteriophage organize Pseudomonas aeruginosa biofilms into liquid crystals,
increasing antibiotic tolerance
Lia A. Michaels1
, Patrick R. Secor1
, William C. Parks2
, and Pradeep K. Singh1
1
Departments of Medicine and Microbiology, University of Washington, Seattle, WA 98195
2
Department of Medicine (Pulmonary and Critical Care Medicine), Cedar Sinai Medical Center,
Los Angeles, CA 90048
*This work is part of a larger body of work (currently under peer review at Cell) involving
collaborations with scientists in the Chemistry Department at the University of Washington, the
Benaroya Research Institute, and the Department of Immunology at Stanford University.
See page 15 for an explicit description of my contributions to this work
Abstract
Pseudomonas aeruginosa is an important human pathogen able to cause chronic infections in
many disease settings. In chronically infected tissues, P. aeruginosa forms biofilms, which are
aggregates of bacteria encased in a polymer rich matrix. The biofilm matrix helps protect the
bacteria from environmental stresses such as desiccation, immune defenses, and antibiotic
treatment. Therefore, understanding the structure and function of the biofilm matrix is important
in understanding the pathogenesis of chronic P. aeruginosa infections. Many laboratories have
observed high expression of filamentous Pf bacteriophage by P. aeruginosa biofilms. We find
that Pf bacteriophage interact with host and microbial polymers to assemble highly ordered
liquid crystals. Some of the physical properties of liquid crystals could affect biofilm
phenotypes, such as increased viscoelasticity and reduced diffusion. Biofilms organized into
liquid crystals by Pf bacteriophage display increased tolerance to cationic antibiotics, likely a
consequence of enhanced binding of cationic antibiotics to the liquid crystalline matrix. This
novel organization of the biofilm matrix and resulting antibiotic tolerance could be a therapeutic
target. For example, strategies aimed at disrupting liquid crystal formation within the biofilm
matrix could be used to treat chronic P. aeruginosa infections.

2. Introduction
Pseudomonas aeruginosa is an opportunistic pathogen that infects burns, non-healing
ulcers, and the airways of people with cystic fibrosis (CF). CF airways accumulate thick,
polymer rich secretions, providing a favorable environment for P. aeruginosa to form biofilms
(Singh et al., 2000). Biofilms are aggregates of bacteria within a polymer-rich extracellular
matrix, which protects the bacteria from environmental stresses (Costerton et al., 1999). Notably,
long-term antibiotic treatment almost always fails to eradicate chronic P. aeruginosa infections
of CF airways (Hoiby, 1993).
As P. aeruginosa biofilms develop, many laboratories observe that the most highly
transcribed genes belong to filamentous Pf1-like bacteriophage (Pf bacteriophage) (Webb et al.,
2004; Whiteley et al., 2001; Rice et al., 2009; McElroy et al., 2014). Pf bacteriophage are
integrated into the genomes of many clinical isolates and laboratory strains of P. aeruginosa,
such as PAO1, which contains the Pf bacteriophage Pf4. Pf bacteriophage belong to the genus
Inovirus, which consists of negatively charged filamentous bacteriophage ~2 µm in length and 6-
7 nm in diameter.
When suspended in polymer solutions, filamentous bacteriophage spontaneously align
and assemble liquid crystals (Dogic and Fraden, 2006), a state of matter between that of a liquid
and a solid. Liquid crystals are inherently viscoelastic in nature and viscoelastic materials
generally display reduced rates of diffusion. Altering the viscoelastic and diffusion properties of
the biofilm matrix would likely affect several biofilm phenotypes such as antibiotic tolerance.
Therefore, we hypothesized that the matrix of P. aeruginosa biofilms producing filamentous
bacteriophage will be organized into a liquid crystalline structure, and this organization will
enhance antibiotic tolerance.

3. Here, we present data demonstrating that filamentous bacteriophage interact with a
diverse group of disease relevant polymers to assemble the P. aeruginosa biofilm matrix into a
liquid crystalline structure. Further, biofilms with liquid crystalline matrices display increased
tolerance to aminoglycoside antibiotics. This novel organization of the biofilm matrix and
antibiotic tolerance could be a therapeutic target to combat chronic biofilm infections.
Results and Discussion
Pf bacteriophage spontaneously assemble liquid crystals in the presence of host and microbial
polymers
Within infected tissues, P. aeruginosa biofilms are in contact with host and bacterial
polymers. While growing P. aeruginosa biofilms in the presence of hyaluronan (HA), a
nonsulfated host glycosaminoglycan polymer found in inflamed tissues (Gill et al., 2010), we
observed the formation of biofilms with a complex, interwoven morphology (Figure 1A).
Biofilms grown without HA developed flat, confluent biofilms (Figure 1B). When hyaluronidase
(HA’ase) was added, the observed structures dissolved indicating that HA was critical to their
formation (data not shown). Filtered biofilm supernatants retained the ability to organize purified
HA into similar interwoven structures (Figure 1C) suggesting that the formation of these
structures not only depends on the presence of polymers, but also requires a secreted bacterial
factor.

4. Using mass spectrometry based proteomics to analyze the biofilm supernatants, we found
the most abundant protein to be CoaB, the major coat protein of the filamentous bacteriophage
Pf4. When mixed with HA, purified Pf4 at concentrations > 109
PFU/ml quickly (1-5 minutes)
assembled similar interwoven structures (Figure 2A). This result shows that Pf4 is the secreted
bacterial factor responsible for assembling interwoven structures in the presence of HA. Given
that DNA is a major component of both the biofilm matrix (Whitchurch et al., 2002) and CF
sputum (Brandt et al., 1995), we hypothesized that Pf4 would organize DNA into similar
structures. Indeed, when purified Pf4 was mixed with DNA, similar structures assembled (Figure
2B). Further, purified Pf4 formed similar structures with a wide variety of polymers with
disparate chemical properties (Figure 2). These observations indicate that Pf4 can quickly
assemble large, interwoven structures from many polymers.
Figure 1. Pf4 interacts with polymers to form interwoven structures. A. P.
aeruginosa biofilms form a confluent layer on the bottom of a plastic dish. B. P.
aeruginosa biofilms grown with 5 mg/ml HA develop a complex, interwoven
morphology. C. Filtered biofilm supernatants mixed with HA form similar interwoven
structures.

5. While experimenting with various polymers that assemble structures with Pf4, we
observed polymer size greatly influenced the assembly of these structures. When Pf4 was mixed
with DNA of similar size to that observed in CF sputum (high molecular weight (HMW) DNA
~2 kbp, (Brandt et al., 1995)), structures formed (Figure 2B). However, when Pf4 was mixed
with low molecular weight (LMW) DNA (<0.3 kbp), structures did not form (Figure 2B inset),
suggesting that polymer size is an important factor in determining if these structures assemble.
The formation of these polymer and bacteriophage induced structures is also dependent
upon ionic strength. When prepared in deionized water, structure formation is inefficient (Figure
3). However, when prepared in buffers of increasing ionic strength, longer, more compact
Figure 2. Host and microbial polymers interact with Pf4 to form structures. A-J.
Flourescently labled Pf4 (green, 8.8 x109
PFU/ml) was mixed with 5 mg/ml of the
indicated polymer. K and L. Addtion of fd bacteriophage to HA and DNA also resulted
in structure formation.

6. structures assemble (Figure 3). These observations indicate that ionic strength affects the
morphology of structures assembled from Pf4 and polymers.
Taken together, our observations are consistent with the assembly of particles into higher
order structures by a force called depletion attraction. Depletion attraction occurs between like-
charged particles in molecularly crowded environments where the ionic strength is sufficient to
screen repulsive forces between the particles (Asukara and Oosawa, 1958). Thus, high ionic
strength allows the assembly of longer, more condensed structures, consistent with our
observations (Figure 3). Polymer size and concentration affect the range and magnitude of this
force, respectively, with higher polymer concentration and size favoring the assembly of larger
structures (Poon, 2002). When filamentous particles, like bacteriophage, are bundled due to
depletion attraction, they spontaneously align and become liquid crystals (Dogic and Fraden,
2006).
Due to depletion attraction, we predict that filamentous Pf bacteriophage will
spontaneously assemble into liquid crystals in the presence of disease relevant polymers. To
determine whether the structures formed from Pf4 and polymers were liquid crystals, we took
advantage of the optical properties of liquid crystals. Liquid crystals split passing light into two
polarized beams. This property, called birefringence, is a direct result of the molecular alignment
Figure 3. Ionic strength determines structure morphology in Pf4/polymer mixtures.
Pf4 (8.8 x 10
9
PFU/ml) and HA (5 mg/ml) were suspended in DI water, 1x PBS, or 10x
PBS. Scale bars, 50 µm.

7. within the sample (Wu, 1986). Birefringence was quantified using a custom built device for
measuring birefringent media (Glazer et al., 1996). This device measures the phase difference
between the two polarized beams emerging from a liquid crystal as⏐sin(δ)⏐where
𝛿 =
2𝜋Δ𝑛𝐿
𝜆
and Δn = birefringence, L = optical path, and λ = wavelength.
Purified Pf4 (1011
PFU/ml) and DNA (10 mg/ml) alone were not birefringent while
mixtures of Pf4 and DNA assembled highly birefringent structures (Figure 4). We observed
similar results when Pf4 was replaced with other filamentous bacteriophage such as Pf1 or fd, a
filamentous bacteriophage that infects Escherichia coli (data not shown).
Figure 4. Pf4 and polymer mixtures produce birefringent liquid crystals. Purified Pf4
(10
11
pfu/ml) and indicated polymers (10 mg/ml) alone are not birefringent (quantified
as⏐sin(δ)⏐), represented by the dark panels lacking birefringent structures. Pf4, when
mixed 1:1 with polymer, assembles birefringent liquid crystals. Scale bars 10 µm.

8. To determine if liquid crystals are assembled under physiologically relevant conditions,
we mixed disease relevant concentrations of mucin (8% solids) (Matsui et al., 2006) with DNA
(HMW, 4 mg/ml) (Brandt et al., 1995) and added Pf4. The birefringence of Pf4 and mucin/DNA
mixtures increased in response to increasing concentrations of Pf4 (Figure 5A) suggesting that
bacteriophage concentrations ≥ 108
PFU/ml are capable of assembling liquid crystals in
environments that mimic CF airway secretions. To determine if this was a disease relevant
concentration of Pf4, we quantified the amount of Pf bacteriophage in CF sputum. Sputum
collected from individuals infected with P. aeruginosa contained an average of ~108
Pf
bacteriophage/ml while sputum collected from patients not infected with P. aeruginosa
contained no detectable Pf bacteriophage (Figure 5B). Accordingly, Pf positive CF sputum was
Figure 5. Pf4 and polymers abundant in
CF sputum assemble liquid crystals. 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 Pf4. B. 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).
C. The birefringence (⏐sin(δ)⏐) of sputum
samples described in (B) was quantified.
Birefringence of P. a. (-) sputum could be
augmented by adding of 10
8
PFU/ml Pf4.

9. more birefringent than Pf negative CF sputum (Figure 5C). Further, the birefringence of Pf
negative sputum could be augmented by supplementation with Pf4. Taken together, our findings
suggest that host and microbial polymers interact with bacteriophage at disease relevant
concentrations to spontaneously assemble liquid crystals.
The matrix of bacteriophage producing biofilms show liquid crystalline organization
Since the biofilm matrix likely contains high polymer concentrations and liquid crystal
assembly is favored at high bacteriophage and polymer concentrations, we hypothesized that the
polymer-rich biofilm matrix would likewise be assembled into a liquid crystalline structure by Pf
bacteriophage. Thus, liquid crystal formation would most likely occur in biofilms that produce
high bacteriophage concentrations. High bacteriophage production is found in colony types
associated with robust biofilms and increased antibiotic tolerance (Webb et al., 2004; Haussler et
al., 2003). We examined the biofilm matrix of P. aeruginosa Pf bacteriophage overproducers
(Pf++). We found Pf++ to produce ~5 x 104
fold more Pf4 compared to wild type (WT) biofilms
(Figure 6B), in agreement with previous observations (Webb et al., 2004). Accordingly, Pf++
biofilms were more birefringent than WT colonies (Figure 6A). When the bacterial cells were
washed to remove the biofilm matrix, birefringence was markedly decreased (Figure 6C),
showing that the matrix is the source of birefringence and not the bacterial cells. As a control, we
measured the birefringence of a strain of P. aeruginosa lacking the bacteriophage integrase gene
PA0728, which is essential for the production of Pf4 (Castang and Dove, 2012). Colonies of
ΔPA0728 were modestly birefringent (Figure 6E). When purified Pf4 was added to ΔPA0728,
birefringence increased (Figure 6E). Taken together, these results show that Pf4 organizes the
biofilm matrix into a birefringent, liquid crystalline structure.

10. The liquid crystalline matrix enhances antibiotic tolerance.
Bacterial biofilms are inherently tolerant to antibiotics (Costerton et al., 1999). Given that
liquid crystals are viscoelastic and display reduced rates of diffusion, we hypothesized that the
organization of the biofilm matrix into a liquid crystal might impede the penetration of
antibiotics into the biofilm. We found liquid crystalline biofilms were more tolerant to the
Figure 6. Pf4 organizes the P. aeruginosa biofilm matrix into a liquid crystal. A. WT
and Pf++ colony biofilms showing birefringence (⏐sin(δ)⏐). Scale bars, 250 µm. B. Pf4
production for WT and Pf++ biofilms was enumerated as PFUs/ml and normalized to
bacterial CFUs/ml. C. Birefringence was quantified in WT and Pf++ biofilms after
normalizing for sample thickness and measured again after washing of the bacteria to
remove the extracellular matrix. D. Pf4 produced by ∆PA0728 and ∆PA0728 supplemented
with Pf4 (∆PA0728 + Pf4) were enumerated as PFUs/ml and normalized to bacterial
CFUs/ml. E. Birefringence was quantified in ∆PA0728 and ∆PA0728 + Pf4.

11. aminoglycoside antibiotics tobramycin and gentamicin (Figure 7A and B). However, there was
no significant benefit of liquid crystal organization against the fluoroquinolone ciprofloxacin
(Figure 7C).
The increased tolerance to aminoglycosides could be due to the liquid crystalline matrix
as we hypothesize, or due to physiological changes induced in P. aeruginosa by increased Pf4
production. To address this possibility, we created a strain of P. aeruginosa that can neither be
infected nor produce Pf4 (ΔPA0728/pilA). This strain lacks PA0728 and type IV pili (pilA), the
receptor Pf4 utilizes to infect PAO1 (Castang and Dove, 2012). Because the bacteria are lacking
in the ability to produce Pf4 and ability to become infected, the exogenous effects of Pf
bacteriophage can be examined. When planktonic cultures of ΔPA0728/pilA were added to
unorganized or liquid crystalline mixtures of Pf4 and DNA, we observed some protection against
tobramycin compared to controls. However, liquid crystalline mixtures of Pf4 and DNA offered
the most protection against tobramycin (Figure 7D). When we replaced Pf4 with fd
bacteriophage, a similar trend was observed (data not shown). These data suggest that the
increased tolerance to tobramycin is due to an extracellular influence of Pf4 rather than
physiological effects induced by an active bacteriophage infection.

12. Figure 7. The liquid crystalline matrix enhances antibiotic tolerance by binding
aminoglycosides. 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. D. P. aeruginosa
ΔPA0728/pilA, which is not capable of producing or being infected by Pf4, was suspended
in DNA (2.5 mg/ml), Pf4 (10
10
PFUs/ml), and DNA +Pf4, followed by treatment with
tobramycin. Killing is represented as the log10
reduction of viable cells recovered from
cultures treated (90 min.) compared to untreated controls. E. Tobramycin (0-3 µg/ml) or
ciprofloxacin (0-0.02 µg/ml) were added to DNA (2.5 mg/ml), Pf4 (10
10
PFUs/ml), or
DNA + Pf4 to investigate binding. E. coli was then added and the samples were incubated
overnight. The highest antibiotic concentration at which bacterial growth occurred was
plotted. F. Binding of tobramycin to DNA, Pf4, and DNA + Pf4 was visualized by adding
fluorescently conjugated tobramycin (Cy5-tobramycin, 40 µg/ml). Scale bars, 20 µm.

13. Liquid crystals enhance binding of aminoglycoside antibiotics
Aminoglycoside antibiotics like tobramycin are positively charged and bind to
polyanions such as DNA within the biofilm matrix, reducing their efficacy (Tseng et al., 2013).
Ciprofloxacin does not interact electrostatically with the biofilm matrix and thus penetrates more
efficiently (Tseng et al., 2013). Since Pf4 is negatively charged, it would be expected to bind
aminoglycoside antibiotics. Given that liquid crystalline biofilms are more tolerant to
aminoglycosides, we hypothesized that aminoglycosides might efficiently bind the liquid
crystalline matrix, sequestering the antibiotic away from the bacteria, reducing killing.
To test antibiotic binding, we mixed increasing concentrations of tobramycin or
ciprofloxacin to unordered or liquid crystalline mixtures of Pf4 and DNA. Binding was allowed
to occur for four hours followed by the addition of E. coli to the mixtures. After allowing the
cultures to grow overnight, we recorded the highest concentration of antibiotic for which growth
was observed. Pf4 and DNA did not offer E. coli any protection against ciprofloxacin, even when
liquid crystals were present (Figure 7E). However, Pf4 and DNA did reduce the killing of E. coli
grown in the presence of tobramycin. Interestingly, Pf4 and DNA in the liquid crystalline phase
offered the most protection against tobramycin (Figure 7E) suggesting that liquid crystalline
organization enhanced tobramycin binding.
Since liquid crystalline mixtures contain high concentrations of bacteriophage and
polymer, it reasons that they would be capable of binding higher amounts of aminoglycosides.
To determine the impact of liquid crystal formation and polymer concentration on antibiotic
binding, the HMW DNA in liquid crystalline mixtures was replaced with LMW DNA, which
does not induce liquid crystal assembly (Figure 2B). We found that non-liquid crystalline
mixtures of LMW DNA and Pf4 were not as protective as liquid crystalline mixtures of HMW

14. DNA and Pf4, even though they contained the same concentrations of Pf4 and DNA. Since
HMW DNA and LMW DNA displayed equivalent binding of tobramycin, these results suggest
that increased binding of tobramycin was due to liquid crystal assembly and not altered binding
of tobramycin to different sized DNA (Figure 7E). To further investigate the binding of
tobramycin to liquid crystalline mixtures of DNA and Pf4, we used fluorescently Cy5-
conjugated tobramycin. Fluorescent imaging revealed that tobramycin was sequestered within
liquid crystals (Figure 7F) illustrating that liquid crystal assembly does indeed enhance the
binding of tobramycin. The mechanism behind the observed binding could be due to the highly
ordered liquid crystalline structure producing special binding sites or by changing the structure
of polymers like DNA suspended within liquid crystals (Dogic et al., 2004). Taken together, our
results suggest that liquid crystals formed from Pf bacteriophage and DNA efficiently bind
aminoglycosides antibiotics, enhancing antibiotic tolerance.
Summary
Our findings reveal a mutually beneficial relationship between P. aeruginosa biofilms
and filamentous bacteriophage. We observed that Pf bacteriophage interact with host and
microbial polymers to spontaneously assemble liquid crystals and P. aeruginosa biofilms
producing Pf4 have a liquid crystalline matrix. The liquid crystalline structure of the biofilm
matrix enhances the binding of aminoglycoside antibiotics, resulting in increased tolerance.
Thus, the organization of the biofilm matrix into a liquid crystal could promote persistence in
chronic infections. Given that several species of Gram-negative bacteria harbor filamentous
bacteriophage, including several important pathogens such as E. coli and Vibrio cholerae
(Tinsley et al., 2006), this mechanism of liquid crystalline organized matrices might be applied

15. to other bacterial species that cause disease. New therapeutic strategies might be developed
targeting phage production or the liquid crystalline organization of the biofilm matrix.
Undergraduate Researcher Contributions
In all experiments, I was involved with the routine maintenance and cultivation of P. aeruginosa
broth and biofilm cultures. I also independently utilized standard techniques for the propagation,
isolation, purification, and fluorescent labeling of filamentous bacteriophage. I assisted with
mass spectrometry analysis of bacterial supernatants and birefringence measurements of both
purified polymers and bacterial biofilms. Although I did not perform the experiments that
quantified Pf bacteriophage in CF sputum, I performed similar experiments using qPCR to
quantify Pf phage in bacterial supernatants in related experiments. I independently performed the
antibiotic tolerance assays (see Figure 7A-C) which involved maintaining and treating P.
aeruginosa biofilms followed by the enumeration of viable bacteria after antibiotic treatment. I
also independently performed the binding assays described in Figure 7E. I assisted in the
construction of all mutant strains of bacteria described in this work. This included primer design,
PCR, electroporation, mating, selection of the correct clones, and confirmation of the mutation
by DNA sequencing. Lastly, I was also involved in the interpretation and analysis of all data
presented in this body of work, and I wrote this paper, with assistance of Drs. Secor and Singh.

16. Materials and Methods
Chemicals and reagents
Salmon sperm DNA (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 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.
Bacterial strains, media, and growth conditions
Bacterial strains, plasmids, and PCR primers are listed in Table 1 Unless specified otherwise,
bacteria were grown at 37°C with shaking in Luria-Bertani (LB) medium.
Construction of strains ΔPA0728 and ΔPA0728/pilA
Plasmid pEX-ΔPA0728 (Castang and Dove, 2012) was introduced to PAO1 or ΔpilA to create
strains ΔPA0728 and ΔPA0728/pilA, respectively, by allelic exchange (Hoang et al., 1998).
Deletions were confirmed by sequencing using primers PA0728F and PA0728R.

17. 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. Wild type (WT) and Pf over-
producers (Pf++) were isolated from static biofilms by dipping an inoculating loop into the
biofilm and streaking an LB plate. Bacterial cells were removed from static biofilms by
centrifuging at 9,000g for 5 minutes. Supernatants were collected and then passed through a 0.2
syringe filter. Proteins in the supernatants were analyzed by mass spectrometry as described
previously (Starita et al., 2012). Colony biofilms were prepared as previously described (Walters
et al., 2009). 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 ΔPA0728+Pf4 colony biofilms, overnight cultures of ΔPA0728 infected with 106
PFUs/ml Pf4, resulting in a final Pf4 concentration of 109
PFU/ml, were used for biofilm
inoculation. The plates were inverted and grown at 37°C for 48 h. The biofilms were transferred
to a fresh plate every 24 h. Flowcell biofilms were grown and maintained as described previously
(Zhao et al., 2013).
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 into 1 ml PBS via vortexing and viable CFUs were enumerated. For experiments
investigating the role of extracellular phage and polymers in antibiotic tolerance, planktonic
ΔPA0728/pilA or ΔPA0728 (~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.

18. Tobramycin (10 µg/ml) or PBS was then added and the cells were incubated at 37°C for 90
minutes. CFUs were enumerated.
Antibiotic binding assay
Tobramycin (0-3 µg/ml) or ciprofloxacin (0-0.02 µg/ml) were added to the indicated
concentrations of Pf4 and DNA 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 and 10 µl was added to each well. The cultures were incubated
overnight at 37°C. The highest concentration at which microbial growth was observed after
overnight incubation was plotted. Cy5-tobramycin was a gift from B. S. Tseng.
Bacteriophage
Purification
Bacteriophage were purified by precipitation with polyethylene glycol (PEG) as previously
described (Boulanger, 2009). Briefly, bacterial supernatants containing phage were treated with 1
µg/ml DNase (Sigma-Aldrich Co., St. Louis, MO) for 2 h at 37°C. Next, phage were precipitated
from the supernatant by adding 0.5 M NaCl followed by 10% (w/vol) PEG 8000 (Sigma-Aldrich
Co., St. Louis, MO) followed by an overnight incubation at 4°C. Phage were pelleted by
centrifugation at 12,000 g for 20 minutes at 4°C. The pellet was re-suspended in PBS and
subjected to two additional rounds of PEG precipitation. Finally, phage pellets were re-
suspended in PBS and thoroughly dialyzed against PBS using a 10 kDa molecular weight cutoff
Slide-A-Lyzer dialysis cassettes (Thermo Scientific, Waltham, Massachusetts).
Phage quantification
Plaque assays were performed as described previously (Castang and Dove, 2012) with ΔPA0728
as the recipient strain. Bacteriophage fd was quantified using the same methodologies using E.

19. coli strain ATCC 15669 as the recipient strain. The presence of Pf4 in sterile 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 as described previously
(Webb et al., 2004).
Fluorescent labeling
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).
Birefringence measurements
After 2 days of growth on LB plates, colonies were scraped off using a plastic inoculating loop
and 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 to make contact with the parafilm. Birefringence was
measured using Rotopol as described (Glazer et al., 1996).
Statistical analysis
Statistical analysis was performed using Prism GraphPad software, mean with SEM were
calculated and plotted.

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