Pyocyanin degradation by a tautomerizing demethylase inhibits Pseudomonas aeruginosa biofilms

Pyocyanin degradation by a tautomerizing demethylase inhibits

Pseudomonas aeruginosa

biofilms

Kyle C Costa

Nathaniel R Glasser

Stuart J Conway

, and

Dianne K Newman

1,3,*

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena,

California, USA

Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road,

Oxford, OX1 3TA, UK

Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena,

California, USA

Abstract

The opportunistic pathogen

Pseudomonas aeruginosa

produces colorful redox-active metabolites

called phenazines, which underpin biofilm development, virulence and clinical outcomes. Though

phenazines exist in many forms, the best studied is pyocyanin. Here, we describe pyocyanin

demethylase (PodA), a hitherto uncharacterized protein that oxidizes the pyocyanin methyl group

to formaldehyde and reduces the pyrazine ring via an unusual tautomerizing demethylation

reaction. Treatment with PodA disrupts

P. aeruginosa

biofilm formation similarly to DNase,

suggesting interference with the pyocyanin-dependent release of extracellular DNA into the

matrix. PodA-dependent pyocyanin demethylation also restricts established biofilm aggregate

populations experiencing anoxic conditions. Together, these results show that modulating

extracellular redox-active metabolites can influence the fitness of biofilms.

Bacteria from phylogenetically diverse taxa secrete colorful redox-active metabolites, such

as the well-studied phenazines produced by multiple species, including

Pseudomonas

aeruginosa

(

) (Fig. 1A,B). Phenazines can be toxic to other cells but also benefit their

producers by facilitating extracellular electron transfer and survival in anoxic environments

(

–

). These latter functions support the growth of antibiotic-resistant biofilms, and

aeruginosa

mutants that cannot make phenazines are defective in biofilm development (

Accordingly, we reasoned that selectively manipulating phenazines might present a means to

control biofilms. One way to influence extracellular metabolites is through active

modification or degradation by other organisms (

–

). Recently, we described a group of

Corresponding author: dkn@caltech.edu.

Author contributions.

KCC and DKN conceived the project. KCC, NRG, and DKN designed the experiments. KCC and NRG

performed the experiments. KCC, NRG, SJC, and DKN analyzed and interpreted the results. KCC, SJC and DKN wrote the paper.

Supplementary Materials

Materials and Methods

Figures S1–S8

Tables S1, S2

References # 31–62

HHS Public Access

Author manuscript

Science

. Author manuscript; available in PMC 2017 July 13.

Published in final edited form as:

Science

. 2017 January 13; 355(6321): 170–173. doi:10.1126/science.aag3180.

Author Manuscript

mycobacteria that enzymatically degrade phenazines and identified genes that catalyze

distinct steps in degradation (

). Here, we focus on the structure and function of a previously

uncharacterized protein from

Mycobacterium fortuitum

encoded by MFORT_14352 (NCBI

Accession number: EJZ13467) that catalyzes pyocyanin (PYO) degradation (

To characterize its activity, we purified a heterologously expressed, truncated version of this

protein (lacking a predicted N-terminal, membrane-spanning helix (

)), hereafter referred

to as PodA

30–162

(

demethylase), from

Escherichia coli

(Fig. 1C). PodA

30–162

converts

PYO to 1-hydroxyphenazine (1–OH-PHZ) and formaldehyde (Fig. 1D,E), indicating that it

functions as a demethylase. Generally, enzyme-catalyzed

-demethylations proceed by

oxidation of the methyl group to formaldehyde with electron transfer to a bound cofactor or

iron-sulfur cluster (

). Surprisingly, we found that PodA

30–162

generates 1-OH-PHZ in

the absence of either flavin or 2-oxoglutarate, which are required for most known

demethylases (Fig. S1). Additionally, PodA

30–162

functions under anoxic conditions

suggesting a mechanism distinct from the oxygen-dependent Rieske-iron type demethylases

(

). Kinetic analysis suggests that PodA is a high affinity PYO demethylase (K

< 1 μM

and k

cat

= 1.20 ± 0.07 s

−1

) that operates over a wide regime of pH (<4.9 – 8.7) and salt

concentrations (0 – 400 mM) (Fig. S2) with specificity for

-methylated phenazines (Fig

S3). Under anoxic conditions, PodA

30–162

catalyzes the formation of a reduced phenazine,

suggesting that its substrate serves as the electron acceptor (Fig. 1F,G). This mechanism has

not previously been observed for demethylases (

). We propose the following reaction

for PodA, wherein oxidized PYO (PYO

) is converted to reduced 1-OH-PHZ (1–OH-

PHZ

red

) (Fig. 1A,B):

O (PYO

) + H

→

O (1-OH-PHZ

red

) + CH

To test this model and better understand how PodA

30–162

reduces its substrate, we solved the

X-ray crystal structure at 1.8 Å resolution (Table S1) by molecular replacement using a

search model generated by Robetta (

–

) (Fig. S4). PodA

30–162

crystalized as a trimer in

the asymmetric unit (Fig. 2A). Crystal formation occurred only in the presence of 1-OH-

PHZ, which was visible in a putative solvent-exposed active site (Fig. 2B,C). We found no

evidence of a bound cofactor or metals in the electron density of the active site (Fig. S5)

further supporting the hypothesis that PodA catalyzes a novel demethylation reaction.

Within the active site, there are several charged and polar residues (D68, D72, H121, E154,

and Y156) and a nearby disulfide that are candidate catalytic residues (

); additionally, 1-

OH-PHZ appears to bind via a

stacking interaction with F70 (Fig. 2B).

Based on the active site structure, we propose a mechanism relying on the presence of H121

functioning as an acid and D72, E154 and Y156 collectively functioning as a base (Fig. 2D).

PYO binds in the phenol tautomeric form (Fig. 1A). Deprotonation of the methyl group

results in iminium ion formation and concomitant reduction of the pyrazine ring, with the

second nitrogen atom protonated by H121. H121 is then reprotonated by PYO’s hydroxyl

group (pK

of His ~6 versus PYO ~5). The unfavorable interaction between the negatively

charged phenolate ion and D68 drives product release. We hypothesize that PodA catalyzes

the tautomerization of PYO to an iminium ion that is susceptible to hydrolysis outside of the

enzyme: the structure indicates that the active site cannot accommodate both the substrate

and a water molecule (Fig. 2B–E) (

). D72 and E154 remain protonated in this scenario,

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acidifying the active site, perhaps to ensure that PYO is in the hydroxylated form in

subsequent reaction cycles (Fig. 1A). This scheme highlights the significance of the

hydroxyl group of PYO to recharge PodA for subsequent reaction cycles and to drive

product release. An alternative substrate, methoxyphenazine methosulfate (methoxy-PMS),

which lacks the hydroxyl group, displays an initial burst of activity followed by a slower

steady state (Fig. 3A), which is consistent with our model.

To probe the proposed mechanism, we performed mutagenesis for each of the putative

catalytic residues. H121A, E154A, D68A, and D72N mutants all formed a trimer (Fig.

3B,C) but had <10% wild type activity, consistent with the postulated catalytic mechanism

(Fig. 3D). Y156F formed a trimer but retained ~25% wild type activity, consistent with this

residue facilitating proton transfer to D72 and E154 but not being essential. A possible

alternative mechanism could utilize the disulfide in the active site forming a covalent adduct

with the phenazine, in analogy to some flavoproteins (

). However, our mutagenesis results

indicate that the disulfide bond near the active site is not essential for catalytic activity

although it may be important for structural stability (Fig. 3B–D) (Fig. S6).

Because PodA requires only water and substrate for activity (Figs. 2D,E, S1, and S2), we

hypothesized that it could degrade PYO during active production by

P. aeruginosa

(Fig. S7).

PodA

30–162

addition to

P. aeruginosa

planktonic culture results in the conversion of PYO to

1-OH-PHZ in both rich and minimal medium (Fig. S8). Encouraged by these findings, we

assessed the impact of PodA on biofilm formation. Extracellular DNA (eDNA) comprises

>50% of the

P. aeruginosa

biofilm matrix (

), and recently PYO was shown to drive eDNA

release (

) which is important early in biofilm development (

). We hypothesized that

PodA

30–162

might inhibit

P. aeruginosa

biofilms in part by attenuating DNA release, thereby

removing an important matrix component and changing biofilm architecture. Because PYO

does not completely block DNA release (

), it is also possible that downstream PYO-

DNA interactions may be important. We checked whether PodA

30–162

could access PYO in

the presence of DNA, as PYO is a known DNA intercalator (

); PodA

30–162

catalyzed PYO

demethylation in this context (Fig S8). We grew

P. aeruginosa

biofilms for 5 hours, staining

them with DAPI to image biofilm structure. HPLC analysis of supernatants confirmed the

conversion of PYO to 1-OH-PHZ by PodA

30–162

in these cultures (Fig. 4A). Overall biofilm

formation, as assayed by surface coverage (

), was reduced by PodA

30–162

but not by the

inactive PodA control (Fig. 4B–D), consistent with a role for PYO in early biofilm

development. As previously shown, treatment with DNase independently inhibited biofilms

(

). Interestingly, DNase or PodA

30–162

treatment inhibited biofilms to the same extent,

and dual treatment did not have an additive effect (Fig. 4D).

In addition to impacting early stages in biofilm development, phenazines can expand the

habitable zone within established biofilms (

). As

P. aeruginosa

biofilms mature, cells in

deeper layers of the biofilm begin to experience oxygen limitation and redox stress (

);

these cells are believed to be slow growing and are highly resistant to antibiotics (

Phenazines facilitate anoxic survival and alleviate redox stress (

). Because PYO reacts

with oxygen more efficiently than 1-OH-PHZ (

), we hypothesized that PodA activity

might decrease anoxic fitness by disrupting PYO dependent electron shuttling to oxygen. To

capture key features of

in vivo

biofilm aggregates (

), cells were grown suspended in

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agar blocks at 37 °C for 22 hours to establish an oxygen gradient before a 5 hour treatment

with PodA

30–162

(Fig. 4E,F). Owing to constraints imposed by the incubator, we were

unable to measure oxygen gradients directly, so we used a previously validated model to

estimate the oxic-anoxic transition zone (

). Modeling our aggregate population indicated

that anoxia occurs ~300 μm below the surface as a result of microbial consumption (Fig.

4G), consistent with what is known about oxygen diffusion into

in vivo

biofilms (

). We

observed a decrease in detectable aggregates at depths 300–400 μm below the agar surface;

additionally, cultures treated with PodA

30–162

had a sharper decline in detectable aggregates

below this depth (Fig. 4H). There was no significant difference in aggregate numbers above

the predicted oxic-anoxic transition zone. How PYO demethylation restricts the aggregate

population is unknown.

In conclusion, the discovery of a PYO demethylase that simultaneously catalyzes substrate

reduction shows that redox-active pigments can participate in their own enzyme-catalyzed

modification. Though PodA is the first member of a new class of tautomerizing

demethylases that utilizes an oxidized substrate as the electron acceptor, this reaction is

reminiscent of reduced flavin acting as the electron donor in its own destruction in vitamin

biosynthesis (

). It seems likely that the processing of other redox-active pigments and

cofactors may operate by a similar mechanism where the redox activity of the substrate

enables catalysis. That PodA can inhibit

P. aeruginosa

at different stages of biofilm

development raises the possibility that selective degradation of extracellular electron shuttles

may facilitate treatment of intractable infections.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

The final model and native data set for PodA were submitted to the wwPDB under accession code 5K21. We thank

Elena Perry, Lucas Meirelles, William DePas and Douglas Rees for assistance in experimental design and

interpretation. KCC was supported by a Ruth L. Kirschstein National Research Service Award from the National

Institutes of Health, National Institute of Allergy and Infectious Diseases, Grant no. F32AI112248. NRG was

supported by the National Science Foundation Graduate Research Fellowship, Grant no. 1144469. This work was

further supported by the Howard Hughes Medical Institute (HHMI), NIH (Grant 5R01HL117328-03) and the

Molecular Observatory at the Beckman Institute, California Institute of Technology through the Gordon and Betty

Moore Foundation and the Sanofi-Aventis Bioengineering Research Program at Caltech. Additional support was

provided by the Stanford Synchrotron Radiation Lightsource, which is funded by the US DOE and NIH. SJC thanks

St Hugh’s College, Oxford for research support.

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Figure 1. Biochemical analysis of the PodA reaction

PodA catalyzes the demethylation of PYO (

) to reduced 1-OH-PHZ (

). (

) PodA

30–162

purifies as a trimer by gel filtration chromatography (45.6 kDa). Inset is a denaturing gel

demonstrating the size of monomeric PodA

30–162

. Incubations of PodA

30–162

with PYO

show the conversion of the starting material (

) to 1-OH-PHZ and formaldehyde (

). Data

are average measurements from six reactions and error bars represent one standard deviation

around the mean. Formaldehyde is derivatized to facilitate detection; the derivatization

competes with other compounds in the mixture so stoichiometric production was not

observed. (

) PodA

30–162

is active under anoxic conditions, and the PYO containing

reaction mixture fluoresces under UV illumination. This fluorescent product has an emission

spectrum (250 nm excitation) consistent with a reduced phenazine (

). While both reduced

PYO and 1-OH-PHZ have similar emission maxima (50 μM phenazine), the magnitude of

the peak is consistent with the generation of reduced 1-OH-PHZ by PodA

30–162

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Figure 2. 1.8 Å crystal structure of PodA

30–162

(

) View of the PodA

30–162

trimer with 1-OH-PHZ bound. The PodA active site (

) is

solvent exposed (

) and contains charged and polar residues. There is a nearby disulfide

~3.5 Å from 1-OH-PHZ. (

) A proposed reaction mechanism based on the residues present

in the active site. The model predicts that D72, E154 and Y156 are necessary for methyl

deprotonation. H121 protonates the unmethylated N atom of the pyrazine ring, and D68

reprotonates H121. Formation of the negatively charged phenolate ion promotes product

release due to an unfavorable electrostatic interaction (red dashed circle). D72 and E154

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remain protonated, acidifying the active site to assist in formation of hydroxylated PYO

(Fig. 1A) in the next catalytic cycle. (

) Hydrolysis of the product is spontaneous and occurs

after release from the active site.

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Figure 3. Molecular analysis of the PodA reaction mechanism

(

) An alternative substrate — methoxy-PMS, inset — is demethylated by PodA

30–162

, but

the reaction rate slows significantly after an initial burst, highlighting the importance of the

hydroxyl group of PYO for catalysis and/or driving product release after deprotonation. Data

are averages from triplicate measurements and error bars represent one standard deviation

from the mean. (

) Residues in the PodA active site (Fig. 2B) were mutated and the

resulting proteins purified; H121A, D68A, E154A, D72N, Y156F and the C88A, C102A (C

to A) double mutant all purify as a trimer by gel filtration chromatography. (

) Mutant

proteins were pure as assayed by reducing SDS-PAGE. (

) Activity of mutant proteins

shows that the disulfide bond is not essential for activity. Y156F has ~25% wild type

activity, and all other residues appear essential for catalysis.

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Figure 4. PodA

30–162

inhibits biofilm formation and anoxic fitness of

P. aeruginosa

(

) Phenazines were measured by HPLC in biofilm supernatants after 5 hours of growth.

PCA, phenazine-1-carboxylic acid. (

)

P. aeruginosa

forms a robust biofilm in the presence

of inactivated PodA

30–162

after 5 hours. (

) In the presence of PodA

30–162

, biofilm surface

coverage was decreased. Surface coverage was 43.5 percent compared with 82.7 percent in

the absence of PodA

30–162

in the representative images shown. Scale Bars = 20 μm. (

)

Surface coverage was lower in the presence of PodA

30–162

(p < 10

−6

vs. PodA-inactive, two-

tailed Student’s t-test). DNase addition decreased surface coverage (p < 10

−3

vs. PodA-

inactive), but DNase and PodA

30−162

combined did not have an additive effect (p > 0.05),

consistent with an interaction between PYO and eDNA in supporting biofilms (

). Data are

averages of 12 replicates taken from independent cultures. Error bars represent one standard

deviation around the mean. ∆

phz

, PA14 mutant incapable of making phenazines. (

) Top

down and (

) side views of

P. aeruginosa

grown embedded in 0.5% agar blocks for 27 hours.

Scale bars = 200 μm. Oxygen depletion occurs at lower depths as a result of biological

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consumption outpacing diffusion from the surface, resulting in decreased biomass. (

)

Oxygen diffusion model predicting the shape of the oxycline in agar blocks. Cell densities

were estimated at 10

8.7

cells mL

−1

based on aggregate number and volume. Modeling this

concentration and 2-fold higher and lower densities suggests that oxygen depletion occurs

~300 μm ± 100 μm below the agar surface. Dashed red line indicates the approximate oxic-

anoxic interface. (

) Biofilm aggregates detected at 10 μm increments below the agar

surface. At depths near the oxic/anoxic interface (dashed red line), total biomass begins to

decline. In assays treated for the last 5 hours with PodA

30−162

, there is an apparent biomass

defect specifically at anoxic depths compared to untreated and inactive PodA treated

controls, consistent with the importance of PYO for anoxic survival in

P. aeruginosa

. Data

are averages of six independent experiments and error bars represent one standard deviation

around the mean. Open symbols, p < 0.01, two-tailed Student’s t-test.

Costa et al.

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. Author manuscript; available in PMC 2017 July 13.

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