Identification of Pseudomonas aeruginosa Phenazines
that Kill Caenorhabditis elegans
Brent Cezairliyan1,2, Nawaporn Vinayavekhin3, Daniel Grenfell-Lee1,2¤, Grace J. Yuen2,4,
Alan Saghatelian3*, Frederick M. Ausubel1,2*
1Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America, 2Department of Molecular Biology, Massachusetts General Hospital,
Boston, Massachusetts, United States of America, 3Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of
America, 4Program in Immunology, Harvard Medical School, Boston, Massachusetts, United States of America
Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of
molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse
variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small
molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We
demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a
matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid
and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by
PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay.
Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C.
elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants
deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data
demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches.
Citation: Cezairliyan B, Vinayavekhin N, Grenfell-Lee D, Yuen GJ, Saghatelian A, et al. (2013) Identification of Pseudomonas aeruginosa Phenazines that Kill
Caenorhabditis elegans. PLoS Pathog 9(1): e1003101. doi:10.1371/journal.ppat.1003101
Editor: David S. Schneider, Stanford University, United States of America
Received May 29, 2012; Accepted November 9, 2012; Published January 3, 2013
Copyright: ? 2013 Cezairliyan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grant 1F32AI084318 and an Ann Weinberg Memorial Research Fellowship from the Cystic Fibrosis Foundation
awarded to BC, a DPST scholarship awarded to NV, a Searle Scholar Award and a Burroughs Wellcome Fund Career Award in the Biomedical Sciences awarded to
AS, and NIH grants P01 AI044220 and R01 AI085581 awarded to FMA. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (AS); firstname.lastname@example.org (FMA)
¤ Current address: DSM Nutritional Products, Lexington, Massachusetts, United States of America.
The Gram-negative bacterium Pseudomonas aeruginosa, a patho-
gen of both plants and metazoans, is a prevalent and pernicious
pathogen in persons who are immunocompromised or suffer from
cystic fibrosis (CF) [1,2]. P. aeruginosa employs many mechanisms to
antagonize its hosts, including the production of low molecular
weight toxins [3,4,5]. Identifying toxins, the conditions under
which they are produced, and the mechanisms by which they act,
are of fundamental importance in understanding and combating
the virulence of this clinically-important pathogen.
The nematode Caenorhabditis elegans, which is found in decaying
plants where many pathogenic microbes reside, is a useful model
host for a variety of pathogens including P. aeruginosa [6,7]. PA14 is
a virulent clinical isolate of P. aeruginosa that is capable of killing C.
elegans [8,9]. Previous studies using C. elegans as a model system for
PA14 pathogenesis determined that PA14 can kill C. elegans either
as a consequence of intestinal infection or intoxication, depending
on the media on which the bacteria are grown [8,9]. Nematodes
die in hours when PA14 is grown on a nutrient rich agar
containing peptone, glucose, and sorbitol (PGS agar) . This type
of killing is referred to as ‘fast killing’. In contrast, when the
bacteria are grown on a less rich medium, it takes several days for
C. elegans to die . This type of killing is referred to as ‘slow
killing’. Fast killing is thought to be mediated by diffusible toxins
because exudates of PA14 grown on PGS agar are sufficient to kill
C. elegans; worms need not be in the presence of live PA14 in order
to be killed. In contrast, slow killing requires bacterial growth in
the worm gut to effect pathogenesis. Experiments in mice have
demonstrated that the fast killing toxin-based model is relevant in
plant and mammalian pathogenesis because mutants that are
defective in fast killing are also defective in Arabidopsis thaliana and
murine PA14 infection models .
In this study we focus on identification of the toxins responsible
for fast killing. In a previously published screen for PA14 mutants
that exhibited reduced levels of fast killing, several isolates
displayed reduced levels of the blue-green phenazine pigment
pyocyanin . Phenazines are a class of tricyclic aromatic
molecules produced by P. aeruginosa and several other Gram-
negative and Gram-positive bacteria [10,11,12]. Some phena-
zines, especially pyocyanin, have been shown to act as toxins
against bacteria, fungi, or mammals as a consequence of their
redox activities [12,13,14,15].
Only a subset of the previously identified C. elegans fast killing-
deficient PA14 mutants were found to produce less pyocyanin than
PLOS Pathogens | www.plospathogens.org1January 2013 | Volume 9 | Issue 1 | e1003101
wild-type , suggesting that other phenazines or a different class
of molecules are involved. Moreover, phenazine toxicity has not
been demonstrated directly in C. elegans . In order to better
understand the mechanisms of P. aeruginosa PA14 toxicity, we
sought to identify the toxin molecules produced by PA14 that kill
C. elegans. We demonstrate that three of the phenazines produced
by PA14 can rapidly kill C. elegans: phenazine-1-carboxylic acid
kills C. elegans at acidic pH; pyocyanin, a product of phenazine-1-
carboxylic acid, kills C. elegans at neutral or basic pH; 1-
hydroxyphenazine, a second product of phenazine-1-carboxylic
acid, kills C. elegans in a pH-independent manner. We also show
that under the conditions of the fast killing assay phenazine-1-
carboxylic acid, not pyocyanin, is the primary toxin responsible for
the rapid death of C. elegans in the presence of PA14.
Phenazine production is essential for killing of C. elegans
by P. aeruginosa PA14
As described above, among the previously isolated PA14
mutants that are deficient in toxin-mediated killing of C. elegans
on PGS agar, those with the largest reduction in toxicity were
found to produce less of the phenazine pyocyanin than wild-type
PA14 . Pyocyanin is one of at least four phenazines that are
produced by wild-type PA14 [10,16,17] (Figure 1A). Phenazine-1-
carboxylic acid, the precursor of all other phenazines produced by
P. aeruginosa, is synthesized from chorismate by genes constituting
the redundant phzA1-G1 and phzA2-G2 operons, each of which
encodes a full set of functional phenazine-1-carboxylic acid
biosynthetic enzymes . Phenazine-1-carboxylic acid can be
modified by other enzymes to make 1-hydroxyphenazine,
phenazine-1-carboxamide, or pyocyanin .
To determine the importance of phenazines in the pathogenesis
of an established C. elegans model host system, we tested the killing
ability of a PA14 mutant that does not produce any phenazines
(Dphz). The Dphz mutant is missing both the phzA1-G1 operon and
the phzA2-G2 operon, precluding the production of phenazine-1-
carboxylic acid as well as the other phenazines . Lack of
phenazine production by the Dphz mutant was confirmed by
metabolite profiling (Table 1). Wild-type or Dphz mutant PA14
bacteria were spread on PGS agar plates and allowed to grow for
24 hours at 37uC followed by 24 hours at 23uC. L4 stage worms
were then placed on the agar. Worms were scored as live or dead
based on whether or not movement could be elicited by tapping
their heads gently with a thin wire. We found that the Dphz mutant
is severely compromised in its ability to kill C. elegans compared to
wild-type PA14 (Figure 1B). Transformation of Dphz with a
multicopy plasmid containing either the phzA1-G1 operon or the
phzA2-G2 operon partially complemented the killing-deficient
phenotype (Figure 1B). Chemical complementation of the Dphz
phenotype by the addition of 100 mg/mL of synthetic phenazine-
1-carboxylic acid to the agar prior to bacterial growth restored the
nematode-killing phenotype (Figure 1C), suggesting that phena-
zine-1-carboxylic acid or another phenazine derived from
phenazine-1-carboxylic acid could be a toxin involved in
Figure 1. Phenazine synthesis by P. aeruginosa is essential for
killing C. elegans. (A) Phenazine synthesis pathway of P. aeruginosa.
(B) Killing of wild-type P. aeruginosa PA14 and Dphz mutant and partial
complementation of Dphz killing with plasmids containing either the
phzA1-G1 or phzA2-G2 operon. (C) Complementation of killing in the
Dphz mutant by addition of synthetic phenazine-1-carboxylic acid
(100 mg/mL) to the agar medium prior to plating and growth of the
The bacterium Pseudomonas aeruginosa is a pathogen of a
wide variety of organisms. It has been shown that P.
aeruginosa factors that are critical for its toxicity to the
nematode worm Caenorhabditis elegans are also important
for its pathogenicity in mammals. In this report we show
that phenazines, a class of small molecules produced by P.
aeruginosa, act as lethal toxins against the worm. Under
conditions relevant to mammalian pathogenesis, we
identified one phenazine, phenazine-1-carboxylic acid,
that is primarily responsible for killing worms. We found
that the toxicity of this phenazine and one other
phenazine, pyocyanin, are dependent on the pH of the
media. We also identified a third toxic phenazine, 1-
hydroxyphenazine, whose toxicity is not dependent on pH.
These results show that the diversity of toxic molecules
produced and released by P. aeruginosa may serve the
bacterium to facilitate pathogenicity in a variety of
P. aeruginosa Phenazines Kill C. elegans
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PA14-mediated killing of C. elegans. Alternatively, phenazine-1-
carboxylic acid could function indirectly to regulate the produc-
tion of a toxin that kills C. elegans.
Phenazine-1-carboxylic acid and 1-hydroxyphenazine are
toxic to C. elegans
To determine if phenazines are sufficient to kill worms, we
tested the killing activities of synthetic phenazines directly by
adding them to PGS agar in the absence of bacteria. Under these
conditions, 1-hydroxyphenazine killed worms at concentrations
above 16 mg/mL with kinetics similar to a typical fast killing assay
with PA14, whereas phenazine-1-carboxylic acid, pyocyanin, and
phenazine-1-carboxamide did not kill worms on a relevant time
scale, even at much higher concentrations (Figures 2A, S1A). To
account for potential synergistic effects of phenazines with other
metabolites produced by PA14, we tested the killing activities of
synthetic phenazines added to PGS agar after growth of a lawn of
Dphz bacteria. After growth of the lawn, the bacteria were scraped
off the agar, the agar was melted in a microwave, phenazines were
mixed in at several concentrations, and the agar was allowed to
cool prior to introduction of the worms. We refer to the melted
and cooled agar upon which the Dphz bacteria had been grown as
‘‘Dphz agar’’. Similarly to the data shown in Figure 2A, when 1-
hydroxyphenazine was added to Dphz agar it killed worms rapidly
at concentrations above 16 mg/mL, whereas pyocyanin and
phenazine-1-carboxamide killed worms poorly, even at much
higher concentrations (Figures 2B, S2B). Interestingly, contrary to
its activity on naive PGS agar, on Dphz agar phenazine-1-
carboxylic acid also killed worms at concentrations above 16 mg/
mL. These data, together with the observation that 1-hydro-
xyphenazine appeared to be somewhat more toxic to worms when
added to Dphz agar than when added to naive PGS agar (compare
Figures 2A and 2B), suggested that the Dphz strain produces a
factor or factors that enhance the toxicities of 1-hydroxyphenazine
and phenazine-1-carboxylic acid.
The data in Figure 2B showing that both 1-hydroxyphenazine
and phenazine-1-carboxylic acid kill C. elegans when added to Dphz
agar indicated that either or both of these phenazines could be
responsible for PA14-mediated intoxication of C. elegans in the fast
killing assay. In considering the relative contributions to nematode
death of phenazine-1-carboxylic acid and 1-hydroxyphenazine
produced by PA14, we used metabolite profiling to determine the
levels of phenazine-1-carboxylic acid and 1-hydroxyphenazine in
PGS agar following growth of wild-type PA14. We found that the
amount of phenazine-1-carboxylic acid (53 mg/mL) was greater
than the level required for killing worms, whereas the amount of 1-
hydroxyphenazine (1.4 mg/mL) was insufficient to kill worms to a
significant degree (Table 1). These observations suggested that
phenazine-1-carboxylic acid is at least partially responsible for
nematode killing under the conditions of our intoxication assay.
To further investigate which phenazines are responsible for
worm killing, we tested the killing abilities of PA14 mutants that
are unable to synthesize pyocyanin, 1-hydroxyphenazine, or
phenazine-1-carboxamide. A phzM mutant, which does not
produce pyocyanin, killed worms more rapidly than did wild-type
PA14 (Figure 2C). A phzS mutant, which does not produce
pyocyanin or 1-hydroxyphenazine [17,18,19], did not show a
significant difference in killing from wild-type. A phzH mutant,
which is deficient in production of phenazine-1-carboxamide, also
did not show a significant difference in killing from wild-type. The
lack of production of phenazines in these mutants according to the
previously described biosynthetic pathways shown in Figure 1A
was confirmed by metabolite profiling (Table 1). These data, in
combination with the fact that synthetic pyocyanin and phenazine-
1-carboxamide do not kill worms at the concentrations they are
produced under the conditions of our assay (Figure 2B, Table 1),
are consistent with the conclusion that pyocyanin and phenazine-
1-carboxamide do not play a significant role in fast killing.
Moreover, the facts that the phzS mutant retains the ability to kill
and that the level of 1-hydroxyphenazine produced by wild-type
PA14 is insufficient to kill to a significant degree, are consistent
with the conclusion that 1-hydroxyphenazine is either not
necessary for killing or that it is one of multiple factors that
cooperate to kill worms. We also observed that the phzM mutant,
which kills more rapidly than wild-type PA14, produced 34%
more phenazine-1-carboxylic acid than wild-type (70 mg/mL vs.
53 mg/mL; Table 1), which was the greatest amount of phenazine-
1-carboxylic acid produced among the strains tested. Levels of
phenazine-1-carboxylic acid were also slightly elevated with the
phzH mutant (59 mg/mL). In contrast, the phzS mutant showed
slightly depressed levels of phenazine-1-carboxylic acid (46 mg/
mL). These data are consistent with the hypothesis that worm
death requires phenazine-1-carboxylic acid. Furthermore, we
observed no killing of nematodes on naive PGS agar that was
supplemented with all four synthetic phenazines at concentrations
comparable to those produced by wild-type PA14 (data not
shown), indicating that toxicity is not induced by the combination
of phenazines in the absence of other factors. These data further
support the conclusion that pyocyanin and phenazine-1-carbox-
amide are unlikely to play major roles in worm killing under the
Toxicity of phenazine-1-carboxylic acid is pH dependent
The data in Figure 2 suggested that phenazine-1-carboxylic acid
is most likely the primary phenazine toxin responsible for
nematode killing. Because phenazine-1-carboxylic acid killed
Table 1. Levels of phenazines in agar plated with PA14 mutants (mg/mL).
phenazine-1-carboxylic acid 1-hydroxyphenazinepyocyaninphenazine-1-carboxamide*
Dphzndnd nd nd
phzM 70.1966.93 1.9360.08nd2.0960.16
*phenazine-1-carboxamide was detected in all strains except Dphz. However, levels were below the quantifiable detection limit of 1 mg/mL for wild-type, phzH, and
P. aeruginosa Phenazines Kill C. elegans
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worms when mixed with Dphz agar but not when mixed with naive
agar, we reasoned that at least one additional factor provided by
the bacteria is necessary for the toxicity of phenazine-1-carboxylic
acid. In our attempts to identify this factor, we took into
consideration precedents in the literature for the pH-dependence
of the activity of bacterial toxins [20,21]. We found that the pH of
PGS agar drops from approximately 6 prior to bacterial growth to
between 4 and 4.5 after growth of PA14. We tested the killing
activities of phenazine-1-carboxylic acid and 1-hydroxyphenazine
under different buffer conditions and found that killing by
phenazine-1-carboxylic acid (at 100 mg/mL) was strongly pH
dependent, with low pH supporting killing and neutral or higher
pH preventing killing (Figure 3). In contrast, 1-hydroxyphenazine
did not show pH-dependent toxicity at the same concentration.
DMSO, the solvent for the phenazine solutions, did not kill worms
under any of the buffer conditions tested, demonstrating that the
worms are not dying only as a consequence of exposure to the low
pH buffer. These observations explained why phenazine-1-
carboxylic acid failed to kill worms when added to naive agar
(pH 6), and considered together with the genetic and metabolite
profiling data, suggest that phenazine-1-carboxylic acid is the
primary toxin responsible for PA14-mediated killing of C. elegans
under fast killing assay conditions.
Toxicity of pyocyanin is also pH dependent
If phenazine-1-carboxylic acid is the primary toxic agent and if
phenazine-1-carboxylic acid is only active at low pH, we reasoned
that buffering the agar media to pH$6 after growth of PA14
would block toxin-mediated killing activity of the PA14 agar.
Although we observed a delay in killing when we raised the pH of
the media to 7 with potassium phosphate, a substantial fraction of
the worms died within seven hours (Figure 4A). When we
performed the same experiment with a phzM or phzS strain, we
found that killing on the relevant time scale was abrogated
(Figure 4B & C). The phenotype of the phzH mutant was
indistinguishable from wild-type under these conditions. Similar
results were obtained when the pH was raised to 8 with Tris HCl,
demonstrating that the phenomenon is not specific to potassium
phosphate (Figure S2). Because both phzM and phzS are required
Figure 2. phenazine-1-carboxylic acid and 1-hydroxyphena-
zine are toxic to C. elegans. (A) Killing of C. elegans after four hours of
exposure to synthetic phenazines (4, 8, 16, 32, 64, and 128 mg/mL final
concentrations) added to naive PGS agar plates. Data points for
phenazine-1-carboxylic acid, pyocyanin, and phenazine-1-carboxamide
are overlapping. (B) Killing of C. elegans after four hours of exposure to
synthetic phenazines added to PGS agar plates after growth of Dphz
bacteria. (C) Killing of C. elegans by P. aeruginosa PA14 mutants in the
phenazine synthesis pathway.
Figure 3. Toxicity of phenazine-1-carboxylic acid is pH
dependent. Nematode death after exposure to 100 mg/mL of
phenazine-1-carboxylic acid or 1-hydroxyphenazine in PGS agar plates
buffered at pH 4 (50 mM sodium acetate), 5 (50 mM sodium citrate), 6
(50 mM potassium phosphate), 7 (50 mM potassium phosphate), or 8
(50 mM potassium phosphate). There is no observable killing by
phenazine-1-carboxylic acid at pH 6, 7, or 8, or by DMSO without
phenazines under any of the buffer conditions.
P. aeruginosa Phenazines Kill C. elegans
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for the synthesis of pyocyanin and phzH is not, these data suggested
that pyocyanin might be toxic to worms at pH 7 or pH 8. We
tested the toxicity of pyocyanin by observing worm survival on
PGS agar with synthetic pyocyanin (10 mg/mL) and buffer added
after growth of Dphz (Figure 4D). Addition of pyocyanin caused
worm death at pH 7 and pH 8, but not at pH 6. Moreover, the
kinetics of worm death at pH 7 and 8 were similar to those
observed with media on which wild-type PA14 were grown and
subsequently buffered at pH 7, with the toxic effects not evident
until the 7-hour time point. Surprisingly, when we exposed worms
to pyocyanin (10 mg/mL) at pH 8 (50 mM potassium phosphate)
on PGS agar plates in the absence of bacteria, we observed no
worm death within 7 hours (data not shown), suggesting that there
is an unknown non-phenazine product of P. aeruginosa that
sensitizes C. elegans to pyocyanin. Together, these data indicate
that three of four known phenazines produced by P. aeruginosa
strain PA14 are toxic to C. elegans. Toxicity of the phenazines,
however, varies depending on the pH of the media as well as the
presence of other factors.
Pathogenesis of P. aeruginosa can occur through a variety of
mechanisms including the production and excretion of toxins. In
this study we identified three phenazine toxins produced by P.
aeruginosa PA14 that can kill C. elegans under different environ-
mental conditions. We showed that under the conditions of our
assay phenazine-1-carboxylic acid is the predominant toxic
phenazine produced by P. aeruginosa PA14 that kills C. elegans.
Our work suggests that phenazine-1-carboxylic acid should be
further studied as a potentially important contributor to toxin-
mediated pathogenesis in other metazoan hosts besides C. elegans,
We found that the toxicity of phenazine-1-carboxylic acid to C.
elegans requires an acidic environment. Acidic conditions are not
uncommon, for example in wounds [22,23], in the gut [24,25],
and intracellularly within lysosomes [23,26] and secretory granules
. Phenazine-1-carboxylic acid may act as a toxin in a variety of
circumstances in those locations. The pH dependence of
phenazine-1-carboxylic acid toxicity may be due to the proton-
ation state of its carboxyl group, for which the pKais 4.2 .
This hypothesis suggests that the uncharged acid species may be
toxic and the negatively charged carboxylate benign, as has been
observed for the antimicrobial activity of phenazine-1-carboxylic
acid against Bacillus cereus and the fungus Gaeumannomyces graminis
var. tritici . Because the cytosol is buffered near neutral pH, we
suspect that either the toxic effects of phenazine-1-carboxylic acid
are occurring extracellularly or that the charge state affects the
permeability of phenazine-1-carboxylic acid through the mem-
brane. Neutral molecules typically traverse membranes more
easily than charged molecules. After the uncharged phenazine-1-
carboxylic acid species has diffused through the membrane, the
neutral environment of the cytoplasm would result in its
deprotonation. In its negatively charged carboxylate form it might
be unable to diffuse out of the cell, resulting in its toxic
accumulation within the cell. Phenazine-1-carboxamide, which
Figure 4. Toxicity of pyocyanin is pH dependent. (A, B, C) Nematode death on PGS agar with wild-type PA14, phzM, phzH, phzS, and Dphz. After
bacterial growth, agar was melted and potassium phosphate pH 7 (100 mM final concentration) or equal volume of water was added. Worms were
added after the agar had cooled and solidified. (D) Toxicity of exogenously added pyocyanin (10 mg/mL) in Dphz agar raised to pH 6, 7, or 8 with
100 mM potassium phosphate. Pyocyanin and buffer were added after bacterial growth.
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differs from phenazine-1-carboxylic acid by only an amide group
in place of the carboxyl group, is not toxic to C. elegans in our assay.
The amide and the carboxyl groups should both be uncharged at
acidic pH where phenazine-1-carboxylic acid is toxic and
phenazine-1-carboxamide is not. Although it is possible that the
chemical difference between these phenazines could affect their
behavior due to other properties than their difference in pKa, the
difference in toxicity of these two species suggests that charge state
is not the sole determinant of phenazine toxicity.
The zwitterionic species of pyocyanin is the predominant form
at pH 7 and 8, and the net neutral charge may facilitate its
traversal through the membrane as well . The pKa of
pyocyanin is 4.9 , which does not explain why pyocyanin is
ineffective at killing nematodes at pH 6, where the zwitterionic
form should still predominate. However, as shown in Figure 4,
unlike the killing activities of phenazine-1-carboxylic acid and 1-
hydroxyphenazine, the killing activity of pyocyanin appears to be
dependent on a non-phenazine factor produced by P. aeruginosa. It
is possible that the oxidation state of pyocyanin is altered by
components of P. aeruginosa exudates on PGS agar. Phenazines,
including pyocyanin, exert toxic effects in a variety of mammalian
tissues through the generation of reactive oxygen species
[8,12,13,15,30,31]. Oxidized and reduced phenazines also have
different hydrophobicities , which can affect their ability to
permeate membranes or remain in aqueous solution. The
oxidation state of pyocyanin has been shown to change as a
function of cell density in PA14 liquid culture , and changes in
pH also affect its redox potential . The difference in pyocyanin
toxicity to nematodes in the presence and absence of PA14
exudates could be due to differences in the oxidation states of
pyocyanin. It is also possible that the pH dependence of
pyocyanin-mediated killing is due to pH-dependent activation of
the accessory factor(s) and not of pyocyanin itself. Given the
importance of pyocyanin as a toxin in a variety of systems, it would
be valuable to identify these accessory factors and determine if
they play a role in pyocyanin toxicity in other hosts.
Studies have demonstrated that 5-methyl-phenazine-1-carbox-
ylic acid (5MPCA), another phenazine produced by P. aeruginosa, is
toxic to the fungus C. albicans [18,19]. 5MPCA is an intermediate
in pyocyanin synthesis that is produced by PhzM acting on
phenazine-1-carboxylic acid. Given that the phzM strain is the
most toxic in our assay, and that the phzS strain, which results in
the accumulation of 5MPCA, is no more toxic than wild-type, we
think it is unlikely that 5MPCA produced by PA14 is a toxic
species to C. elegans under the conditions of our assay.
CF patients are highly susceptible to lung infections by P.
aeruginosa [1,2]. CF results in abnormal hyperacidification of
organelles of epithelial cells in the respiratory pathway as well as
increased acidity of airway surface liquid as compared with
healthy individuals . Hyperacidification of the lung airway
surface liquid reduces its antimicrobial effects in a porcine CF
model  and has been speculated to have other effects on the
CF lung, including tissue damage, inflammation, and thickening of
the mucus . Increased acidity could also lead to enhanced
toxicity of phenazine-1-carboxylic acid in the P. aeruginosa infected
The concentrations of pyocyanin we determined that are active
against nematodes are within the concentrations observed in
human CF sputum and that have been shown to have
physiological activity [36,37,38,39,40,41]. In wild-type PA14
grown on PGS agar, we detected 2.38 mg/mL (11.3 mM)
pyocyanin. Pyocyanin has been detected at concentrations of up
to 27.3 mg/mL (130 mM) in the sol phase of CF sputum .
Interestingly, a recent study showed that pyocyanin concentrations
in sputum from CF patients correlated with the degree of severity
of the disease, ranging from 7.7 mM in unobstructed airways to
46.8 mM in severely obstructed airways .
Phenazine-1-carboxylic acid concentrations also correlated with
impairment of lung function in CF, with concentrations ranging
from 5.2 mM in unobstructed airways to 39.9 mM in severely
obstructed airways . The concentrations we detected in PGS
agar were considerably higher, at 52.7 mg/mL (235 mM), but we
demonstrated that phenazine-1-carboxylic acid is toxic to nema-
todes at concentrations as low as 16 mg/mL (71.4 mM). Although
this is still higher than concentrations observed in CF lungs, it is
possible that worms are intrinsically more resistant to phenazine-1-
carboxylic acid than mammalian cells.
Pseudomonas infections are also common in wounds, which
exhibit a variety of different pH conditions [22,23]. During
inflammation, the pH of acute wounds falls below 6, a level at
which phenazine-1-carboxylic acid could exert its toxic effects.
During early stages of healing, the pH increases to between 7 and
8, where phenazine-1-carboxylic acid might be ineffective as a
toxin. In that pH range pyocyanin could act as a toxin. Indeed,
pyocyanin has been shown to inhibit wound repair and induce
cellular senescence in an in vitro model of wound healing .
In agreement with previous observations [8,9], we observed that
L4 worms were more susceptible to killing by phenazines than
adult animals. We also noticed that older L4 worms were killed
more quickly than younger L4 worms (B. Cezairliyan and R.
Feinbaum, unpublished observations). We suspect that proximity
to the L4 to adult molt plays a role in the susceptibility of L4
worms. Perhaps worms are most susceptible when they are first
exposed to phenazines during the molt, when phenazines might be
able to most easily due to the shedding of the cuticle. Earlier
exposure to phenazines might allow worms time to detoxify them
before reaching their most susceptible stage. It is also possible that
specific gene expression patterns unrelated to toxin permeability
during the L4 to adult molt are the cause of increased
susceptibility. Identification of genes that reduce susceptibility to
phenazines at this transition or that alter the developmental stage
at which worms are susceptible to phenazines should help to
elucidate the cause of the stage-dependent susceptibility of C.
elegans to phenazines.
An understanding of the regulation of toxin production in P.
aeruginosa is critical for understanding its interactions with its hosts.
Phenazine biosynthesis in P. aeruginosa is dependent on a variety of
environmental factors in the media and is also largely dependent
on cell density via quorum sensing signals [12,13,42,43,44,45,46].
In P. aeruginosa, the genes that are responsible for producing
phenazine-1-carboxylic acid are present in duplicate in two
differently-regulated operons in the genome. The genes that
regulate production of other phenazines, however, are transcribed
independently of one another and of the phzA1-G1 and phzA2-G2
operons, thus allowing for transcriptional regulation of the
production of particular phenazines depending on the environ-
mental stimuli [17,46,47]. Many phenazine-producing bacteria
make more than one type of phenazine . pH has been shown
to influence the production of phenazine-1-carboxylic acid in
Pseudomonas fluorescens and phenazine-1-carboxamide in Pseudomonas
chlororaphis [48,49]. Moreover, P. aeruginosa grown under different
conditions can produce other low molecular weight toxins that are
lethal to C. elegans, including cyanide [5,50] and quinolone .
Our work suggests that one of the purposes that could be served by
the diversity of phenazines is to have an armamentarium of
molecules available that are active under different environmental
P. aeruginosa Phenazines Kill C. elegans
PLOS Pathogens | www.plospathogens.org6 January 2013 | Volume 9 | Issue 1 | e1003101
Knowledge of the toxins that are effective in our nematode
killing assay will allow for a better understanding of phenazine
toxicity in C. elegans and in other hosts. Although the mutants
identified in the screen for toxin-killing deficiency in PA14 were
also reduced in pathogenesis in a mouse thermal injury model and
an Arabidopsis leaf infiltration model , it is still unclear how the
nematode intoxication assay serves as a proxy for the mammalian
and plant models, which are based on infection and colonization
by live bacteria. It will be important to determine if the same
phenazine toxins that we have identified in the killing of C. elegans
play a role in the murine or plant models. If the toxicity in
mammals is similarly based on phenazines, C. elegans will continue
to serve as a useful tool in the identification of pathogenesis
mechanisms of P. aeruginosa in mammals and plants. Host pathways
in C. elegans can be probed genetically to identify mechanisms by
which phenazine-1-carboxylic acid acts to kill the worm and
whether the three toxic phenazines exert their lethal effects in
similar ways in the host. A combination of proteomics, gene
expression, and mutant analysis should help to shed light on
toxicity mechanisms of phenazines in the host and the ways by
which their production is regulated in the pathogen.
Materials and Methods
Media and chemicals
Worms were maintained on lawns of E. coli OP50 on nematode
growth medium agar (NGM) plates prior to killing assays. Killing
assays were performed on PGS agar (1% bacto-peptone, 1%
glucose, 1% NaCl, 150 mM sorbitol, 1.7% bacto-agar) as
described . 1-hydroxyphenazine was purchased from TCI
America (H0289), pyocyanin from Cayman Chemical Company
(10009594), phenazine-1-carboxylic acid from Apollo Scientific
(OR01490), and phenazine-1-carboxamide from Princeton Bio-
molecular Research (PBMR030086). All phenazine stocks were
dissolved in DMSO.
Bacterial and worm strains
The PA14 Dphz mutant strain lacking both the phzA1-G1 and
phzA2-G2 operons was kindly provided by Lars Dietrich and
Dianne Newman . Other PA14 mutant strains were obtained
from a non-redundant transposon insertion library : phzM
(mutant ID 40343), phzH (39981), phzS (44099). All transposon
insertion strains were sequenced to confirm the location of the
transposon. The phzM and phzS genes are on opposite sides of the
phzA1-G1 operon. phzM and phzS are coregulated to some degree
with phzA1-G1, but they are part of separate transcriptional units
not containing other genes [17,46]. Thus, the transposons in phzM
and phzS are unlikely to exert polar effects.The phzH gene is
elsewhere in the genome and is not known to be cotranscribed
with other genes [17,31]. C. elegans strain Bristol N2  was used
for killing assays.
C. elegans killing assays
Killing assays were performed as previously described . In
order to have fourth larval stage (L4) worms for killing assays, 10
gravid worms were picked to a 60 mm plate with NGM agar and a
lawn of E. coli OP50 as a food source. Gravid worms were kept on
plates for 16 hours at 15uC, after which they were removed. Plates
were returned to 15uC for 8 hours, after which they were
transferred to 20uC. Eggs hatched and grew to L4 stage
approximately 36 hours after transfer. Proper staging of L4
worms was critical to the reproducibility of the assay, as worms
that were younger or older were killed less quickly than L4 stage
Killing agar plates were prepared by spreading 5 mL of
overnight culture of PA14 in LB on a 35 mm petri plate
containing 4 mL of PGS agar (1% bacto-peptone, 1% glucose,
1% NaCl, 150 mM sorbitol, 1.7% bacto-agar). Plates were
incubated for 24 hours at 37uC and then transferred to 23uC for
24 hours. L4 stage worms were put on the plates, which remained
at room temperature until the completion of the assay. Worms
were scored as live or dead based on movement elicited by tapping
their heads gently with a thin wire.
To mix the agars of plates seeded with different PA14 strains,
bacteria were scraped off the surface of the agar with a cell scraper
after which the agar was melted by heating in a microwave. The
hot agars were mixed, repoured into plates, and allowed to cool. In
experiments where phenazines or buffer were added, plate agar
was melted, concentrated buffer and/or phenazine stock solution
(in DMSO) was added and mixed, after which plates were
repoured and allowed to cool.
Absolute quantitation of phenazines
In order to determine absolute amounts of phenazines produced
by different strains, a standard curve was constructed for each of
the phenazines at concentrations of 1, 4, 16, 32, and 64 mg/mL.
Killing agar plates seeded with Dphz were melted and doped with a
range of concentrations of synthetic phenazines. Cool, dry plates
were extracted with chloroform/methanol (CHCl3:MeOH:a-
gar=2:1:1). The organic layer was transferred to another vial
and concentrated under a stream of nitrogen. Samples were
dissolved in 200 mL of CHCl3and stored at 280uC. Prior to LC-
MS analysis, reconstituted samples were diluted 200 fold in CHCl3
and spiked with cytosporone B (csn-B) to the final concentration of
5 mM as a standard for normalization across samples. To obtain
the most accurate concentrations of phenazines, samples from
different strains were prepared in the same manner and at the
same time as the standards. Three experimental replicates were
used for each standard and bacterial sample.
LC-MS analysis was performed using an Agilent 6410 LC-ESI-
QQQ instrument in multiple reaction monitoring (MRM) mode.
Samples were analyzed both in the negative and positive ion
modes as previously described . For the LC analysis in the
negative ion mode, a Gemini (Phenomenex) C18 column (5 mm,
4.6 mm650 mm) was used together with a precolumn (C18,
3.5 mm, 2 mm620 mm). Mobile phase A consisted of a 95/5
water/methanol mixture, and mobile phase B was made up of 60/
35/5 isopropanol/methanol/water. Both A and B were supple-
mented with 0.1% ammonium hydroxide as solvent modifiers. For
the LC analysis in the positive ion mode, a Luna (Phenomenex) C5
column (5 mm, 4.6 mm650 mm) was used together with a
precolumn (C4, 3.5 mm, 2 mm620 mm). Mobile phases A and
B, as well as the gradient, were the same as that used for the
negative ion mode analysis, except that, in this case, both A and B
were supplemented with 0.1% formic acid and 5 mM ammonium
formate as solvent modifiers. The gradient started at 0% B for
3 min at 0.2 mL/min and then linearly increased to 100% B over
the course of 8 min at 0.5 mL/min, followed by an isocratic
gradient of 100% B for 4 min at 0.5 mL/min before equilibration
for 5 min at 0.5 mL/min. The total analysis time, including 3 min
at 0.2 mL/min, was 20 min.
MS analysis was performed with an electrospray ionization
(ESI) source. The capillary voltage was set at 4000 V. The drying
gas temperature was 350uC, the drying gas flow rate was 10 L/
min, and the nebulizer pressure was 45 psi. For both ionization
conditions, data was collected in the profile mode. Conditions for
MRM experiment were optimized using the program called
Optimizer (Agilent). The resulting optimized conditions for each
P. aeruginosa Phenazines Kill C. elegans
PLOS Pathogens | www.plospathogens.org7January 2013 | Volume 9 | Issue 1 | e1003101
transition used to quantitate each phenazines are listed in
Supplementary Table S1. Each run was performed using a 1 mL
injection of extract.
For data analysis, ions corresponding to each phenazine and
csn-B were extracted from the total ion chromatograms to obtain
integrated mass ion intensities (peak area; MSII) for each ion. The
levels of phenazines were normalized by dividing MSII of
phenazines by MSII of csn-B detected in each chromatogram to
give normalized MSII (nMSII). nMSII of the standard samples
was applied to construct standard curves. Finally, phenazine
concentrations in the experimental samples were calculated
according to the standard curves.
Killing of C. elegans after four hours of exposure to synthetic
phenazines (4, 8, 16, 32, 64, and 128 mg/mL final concentrations)
added to naive PGS agar plates. Data points for phenazine-1-
carboxylic acid, pyocyanin, and phenazine-1-carboxamide are
overlapping. (B) Killing of C. elegans after four hours of exposure to
synthetic phenazines added to PGS agar plates after growth of
Phenazine toxicity, replicate experiment. (A)
independent of phosphate buffer. Nematode death on PGS
agar with wild-type PA14, phzM, phzH, and phzS. After bacterial
growth, agar was melted and Tris HCl pH 8 (100 mM final
concentration) was added. Worms were added after the agar had
cooled and solidified.
Toxicity of PA14 exudates at basic pH is
quantitate levels of phenazines.
MRM conditions and transitions used to
We thank members of the Ausubel lab for comments and experimental
advice, as well as J. Irazoqui and B. Sawers for thoughtful discussions. We
thank L. Dietrich and D. Newman for the Dphz strain of PA14. We are
grateful to Mathias Leidl for assistance with data compilation.
Conceived and designed the experiments: BC NV DGL AS FMA.
Performed the experiments: BC NV DGL GJY. Analyzed the data: BC NV
DGL GJY AS FMA. Contributed reagents/materials/analysis tools: BC
NV DGL. Wrote the paper: BC NV AS FMA.
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